WO2009017475A1 - Procédés pour le diagnostic et le pronostic de cancers épithéliaux - Google Patents

Procédés pour le diagnostic et le pronostic de cancers épithéliaux Download PDF

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WO2009017475A1
WO2009017475A1 PCT/US2007/016955 US2007016955W WO2009017475A1 WO 2009017475 A1 WO2009017475 A1 WO 2009017475A1 US 2007016955 W US2007016955 W US 2007016955W WO 2009017475 A1 WO2009017475 A1 WO 2009017475A1
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cancer
cystatin
epithelial
level
biomarker
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PCT/US2007/016955
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English (en)
Inventor
Adam S. Feldman
Bruce R. Zetter
W. Scott Mcdougal
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Children's Medical Center Corporation
The General Hospital Corporation
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Priority to PCT/US2007/016955 priority Critical patent/WO2009017475A1/fr
Publication of WO2009017475A1 publication Critical patent/WO2009017475A1/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
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • Patent No 6,358,683 high ZNF217 protein levels in blood (WO 98/02539), and differential expression of a newly identified protein in breast cancer, PDEBC 5 which is useful for diagnosis (U.S. patent application No. 20030124543).
  • Cell surface markers such as CEA, CA-125 and HCG are frequently elevated in the serum of patients with locally advanced and metastatic bladder cancer (Izes et al., J Urol. Jun;165(6 Pt l):1908-13, 2001), and studies involving circulating levels of tumor-related proteins such as matrix metalloproteinase-2 (Gohji et al., Cancer Research 56:3196, 1996), hepatocyte growth factor (Gohji et al., J. Clin. Oncol.
  • tissue polypeptide antigen (Maulard-Durdux et al., J. Clin. Oncol. 15:3446, 1997) have shown promise. These biomarkers offer alternative methods of diagnosis: however, they are not widely used. Furthermore, despite the use of a number of histochemical, genetic, and immunological markers, clinicians still have a difficult time predicting which tumors will metastasize to other organs.
  • biomarkers are particularly relevant to improving diagnosis, prognosis, and treatment of the disease. As such, there is need in the art to identify alternative biomarkers that can be quickly, easily, and safely detected. Such biomarkers may be used to diagnose, to stage, or to monitor the progression or treatment of a subject with bladder cancer, in particular, an invasive, potentially metastatic stage of the disease.
  • the present invention is based on the surprising discovery that three proteins, Cystatin B, Chaperonin 10, and Profilin (also referred to as “epithelial cancer markers"), are present in the urine of patients with bladder cancer, a cancer of epithelial origin. Accordingly, the present invention is directed to methods for prognostic evaluation of cancers of epithelial origin and to methods for facilitating diagnosis of cancers of epithelial origin by monitoring the presence of these markers in biological samples. The invention is also directed to markers for therapeutic efficacy, In particular, the amount of Cystatin B detected in urine correlates with disease status such that Cystatin B levels can be used to predict the presence of invasive bladder cancer.
  • measuring the level of Cystatin B, Chaperonin 10, and/or Profilin proteins in urine provides a quick, easy, and safe screen that can be used to both diagnose and prognose bladder cancer in a patient.
  • the absence of these markers can provide an indication that the patient does not have bladder cancer.
  • a method for facilitating the diagnosis of cancer of an epithelial origin in a patient comprises obtaining a biological sample, preferably a voided urine specimen, from a patient and detecting the presence or absence of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin) in the sample, wherein the presence of at least one epithelial cancer biomarker is indicative of cancer of epithelial origin.
  • a biological sample preferably a voided urine specimen
  • Bio samples for example, can be obtained from blood, tissue (e.g. tumor or breast), serum, stool, urine, sputum, cerebrospinal fluid, nipple aspirates and supernatant from cell lysate.
  • tissue e.g. tumor or breast
  • serum e.g., serum, stool, urine, sputum, cerebrospinal fluid, nipple aspirates and supernatant from cell lysate.
  • urine e.g. tumor or breast
  • cancer of epithelial origin refers to cancers that arise from epithelial cells which include, but are not limited to, breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers that effect epithelial cells throughout the body.
  • a method for facilitating the diagnosis of bladder cancer in a patient comprises obtaining a biological sample, preferably a voided urine specimen, from a patient and detecting the presence or absence of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin) in urine sample, wherein the presence of at least one epithelial cancer biomarker is indicative of bladder cancer.
  • a biological sample preferably a voided urine specimen
  • the method for diagnosing a cancer of epithelial origin comprises measuring the level of at least one epithelial cancer biomarker present in a biological sample (test sample) from a patient and comparing the observed level of at least one marker (Cystatin B, Chaperonin 10, or Profilin) with the level of the marker present in a control sample of the same type. Higher levels of markers in the test sample, as compared to the control sample, is indicative of cancer of epithelial origin.
  • the cancer of epithelial cancer may be breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer, stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, skin cancer, prostate cancer, and/or renal cell carcinoma.
  • the methods of the invention are used for early detection of cancer.
  • a patient can be screened by a physician during their physical.
  • a method for diagnosing bladder cancer comprises measuring the level of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin) present in a biological sample (the test sample) from a patient and comparing the observed level of at least one marker with the level of the marker present in a control sample of the same type. Higher levels of markers in the test sample, as compared to the control sample, is indicative of bladder cancer.
  • a method for diagnosing invasive bladder cancer in a patient is provided.
  • the method comprises measuring levels of Cystatin B epithelial cancer biomarker present in a biological sample obtained from the patient (test sample) and comparing the level of Cystatin B in the test sample with the level of Cystatin B present in a non-invasive cancer control sample. A higher level of Cystatin B in the test sample as compared to the level of Cystatin B in the control sample is indicative of invasive bladder cancer.
  • control sample refers to a biological sample (e.g. blood, urine, tumor) obtained from a "normal” or “healthy” individual(s) that is believed not to have cancer. Controls may be selected using methods that are well known in the art. Once a level has become well established for a control population, array results from test biological samples can be directly compared with the known levels.
  • biological sample e.g. blood, urine, tumor
  • non-invasive control sample refers to a biological sample obtained from a individual(s) that has a non-invasive form of cancer. Once a level has become well established for a control population, array results from test biological samples can be directly compared with the known levels.
  • test sample refers to a biological sample obtained from a patient being tested for a cancer of epithelial origin.
  • the present invention also contemplates the assessment of the level of epithelial cancer biomarker present in multiple test samples obtained from the same patient, where a progressive increase in the amount of the marker over time indicates an increased aggressiveness (e.g. metastatic potential) of the cancer tumor.
  • the levels of the epithelial cancer biomarker serve as a predictor of disease status and stage.
  • the present invention iurther contemplates the assessment of epithelial cancer biomarker/s to monitor the therapeutic efficacy of a treatment regime designed to treat a patient having a cancer of epithelial origin (e.g. bladder cancer).
  • a cancer of epithelial origin e.g. bladder cancer
  • epithelial cancer biomarker levels e.g. (Cystatin B, Chaperonin 10, or Profilin
  • epithelial cancer biomarker levels present in a test biological sample are measured by contacting the test sample, or preparation thereof, with an antibody-based binding moiety that specifically binds to the epithelial cancer biomarker, or to a portion thereof.
  • the protein level of an epithelial cancer biomarker or a level of cystatin B is measured by a method comprising contacting a test sample, or a preparation thereof, with an antibody based binding moiety which specifically binds the epithelial cancer biomarker or to Cystatin B to form an antibody-epithelial cancer biomarker complex, and detecting the presence of the complex, thereby measuring the level of epithelial cancer biomarker present.
  • the antibody-based binding moiety is labeled with a detectable label.
  • the label may be a radioactive label, a hapten label, a fluorescent label, and an enzymatic label.
  • the antibody-based binding moiety is an antibody.
  • the antibody is a monoclonal antibody.
  • Antibody-based immunoassays are the preferred means for measuring levels of biomarkers. However, any means known to those skilled in art can be used to assess biomarker levels. For example, biomarker levels can be assessed by mass spectrometry, including SELDI mass spectrometry.
  • kits that comprise means for measuring at least one epithelial cancer biomarker in a biological sample.
  • Such kits optionally contain directions for use.
  • the kit comprises a container for holding a biological sample (e.g. urine sample), and at least one antibody that specifically binds an epithelial cancer biomarker.
  • the kit comprises a container for holding the urine sample.
  • the kit comprises two antibodies that specifically bind to an epithelial cancer biomarker.
  • one antibody is immobilized on a solid phase and one antibody is detectably labeled.
  • the kits can comprise anti-Cystatin B 5 anti- Chaperonin 10, and/or anti-Profilin antibodies.
  • a method for assessment of a cancer of epithelial origin comprises measuring the level of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profllin) present in a biological sample (the test sample) from the subject and determining whether the level of the marker is higher than a predetermined level. Higher levels of the marker in the test sample, as compared to the predetermined level, is indicative of an increased risk of cancer progression. Any of the assay and measurement methods described herein are applicable to this method of assessment.
  • the sample from the subject is a blood, tissue, serum, plasma, stool, sputum, cerebrospinal fluid, nipple aspirate, or tissue sample.
  • the biological sample is a urine sample.
  • the cancer of epithelial origin is bladder cancer, breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, Hp cancer, mouth cancer, esophageal cancer, small bowel cancer, stomach cancer, colon cancer, liver cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, skin cancer, prostate cancer, and renal cell carcinoma.
  • the cancer of epithelial origin is bladder cancer.
  • the level of the epithelial cancer biomarker (e.g., Cystatin B) is compared to a predetermined level.
  • the predetermined level may be based on the level of the epithelial cancer biomarker normally found in biological samples of healthy subjects. In some embodiments, the predetermined level may be based on a prior measurement of the subject's epithelial cancer biomarker. In other embodiments, the predetermined level may be based on the subject's level of the biomarker prior to treatment. In some embodiments, the subject's epithelial cancer biomarker may be monitored over time (e.g., monthly, quarterly, bi-yearly, yearly, every 2 years, every 5 years). [0030] In some embodiments, the determination of an epithelial cancer biomarker
  • the cancer progression is a recurrence of cancer, an increase of metastatic activity, and/or a progression in cancer grade or stage.
  • the epithelial cancer biomarker measured is Cystatin B.
  • Cystatin B protein may be measured using an immunoassay as described herein.
  • cystatin B protein may be measured by mass spectrometry.
  • Other aspects of the invention are disclosed infra.
  • Figure 1 is a flow diagram showing the approach to epithelial cancer biomarker discovery.
  • Figure 2 shows comparative 2D-P AGE of invasive bladder tumor tissue and normal bladder tissue reveals many potential spots. The circled spot was identified by mass spectroscopy to be Cy statin B.
  • Figure 3 shows a graph depicting the results of a semi-quantitative Western Blot analysis of Cystatin B detected in voided urine specimens.
  • Figure 4 shows a series of 2D-P AGE depicting the proteomic analysis of bladder cancer patient urine and tissue.
  • 2D SDS-PAGE was used to separate proteins across a charge range of pi 3-10, and through a 4-20% acrylamide gradient.
  • High grade bladder cancer patients' urine were compared to urine from normal age-matched controls ( Figure 4B).
  • Bladder cancer tissue was compared to normal patient matched urothelial tissue ( Figure 4D).
  • the arrow points to the peptide spot corresponding to cystatin B.
  • Figure 5 shows pictures depicting the immunohistochemical analysis of cystatin B expression in bladder cancer tissue. Immunohistochemical staining using cystatin B monoclonal antibody showed increased cystatin B levels in bladder cancer compared to normal bladder tissue ( Figure 5 A and 5B). Additionally, percent high intensity cystatin B staining for normal and TCC tissue sections are shown ( Figure 5C). Scale bars indicate 200 ⁇ m.
  • Figure 6 provides a picture and graphs depicting the analysis of cystatin B levels in urine from bladder cancer patients and normal age-matched controls.
  • Figure 6A shows a representative western blot of urine samples using monoclonal cystatin B antibody.
  • Lane 1 (+) is MGH-Ul total cell Iy sate (positive control) used to normalize signals between individual western blots.
  • Lane 2 (++) represents protein isolated from pooled urine specimens and TCC tissue. Cystatin B levels in patient urines were quantified by chemiluminscence image scan using a ProXPRESS imaging system. It should be noted that an image acquisition artifact was present in all images obtained with this system, as seen in lane 7. Significantly higher levels of cystatin B were found in TCC with increasing tumor grade (Figure 6B), and stage (Figure 6C).
  • Figure 7 provides graphs depicting that urinary cystatin B level is predictive of time to TCC disease recurrence and disease grade/stage progression.
  • Kaplan Meier analysis was used to estimate (Figure 7A) disease recurrence-free survival time, and (Figure 7B) grade/stage progression-free survival time, for patients with high (cutoff >0.54) or low urinary cystatin B levels.
  • Invasive cancer refers to the proclivity of a rumor for expanding beyond its boundaries into adjacent tissue (Darnell, J. (1990), Molecular Cell Biology, Third Ed., W. H. Freeman, NY). Invasive cancer can be contrasted with organ-confined cancer wherein the tumor is confined to a particular organ.
  • the invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, that degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the capsule, and beyond confines of the particular tissue in which that tumor is located.
  • Invasive bladder cancer includes invasive into Muscularis Propria and/or Lamina Propria.
  • tumor metastasis refers to the condition of spread of cancer from the organ of origin to additional distal sites in the patient.
  • the process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location (Fidler, et al., Adv. Cancer Res. 28, 149-250 (1978), Liotta, et al., Cancer Treatment Res. 40, 223-238 (1988), Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988) and Zetter, N. Eng. J. Med.
  • a "biological sample” refers to a urine sample obtained from a patient.
  • Biological samples for example, can be obtained from blood, tissue (e.g. tumor or breast), serum, stool, urine, sputum, cerebrospinal fluid, nipple aspirates and supernatant from cell lysate.
  • One preferred biological sample is urine.
  • the biological sample is treated as to prevent degradation of epithelial cancer biomarkers. Methods for inhibiting or preventing degradation include, but are not limited to, treatment of the sample with protease, freezing the sample, or placing the sample on ice. Preferably, prior to analysis, the samples are constantly kept under conditions as to prevent degradation of the markers.
  • a tumor sample refers to a portion, piece, part, segment, or fraction of a tumor, for example, a tumor which is obtained or removed from a subject (e. g., removed or extracted from a tissue of a subject), preferably a human subject.
  • Cystatin B refers to the protein of Genebank accession NM_000100.2, NP_000091 (Homosapiens). The term also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof.
  • Chaperonin 10 refers to the protein of Genebank accession, protein, AAA50953 (Homosapiens). The term also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof.
  • Profilin refers to the protein of Genebank accession, protein, A28622 (Homosapiens). The term also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof.
  • the present invention is directed to methods for facilitating diagnosis of cancers of epithelial origin in a patient.
  • the method comprises obtaining a biological sample from a patient and detecting the presence or absence of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin) in the sample, wherein the presence of at least one marker is indicative of the presence of cancer of epithelial origin.
  • epithelial cancer biomarker Cerstatin B, Chaperonin 10, or Profilin
  • the methods involve measuring levels of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Profilin) in a test sample obtained from a patient being tested for cancer, and comparing the observed levels to the levels of the epithelial cancer biomarker found in a control sample, for example a sample obtained from an individual patient or population of individuals that do not to have cancer.
  • a control sample for example a sample obtained from an individual patient or population of individuals that do not to have cancer.
  • Levels of at least one epithelial cancer biomarker higher than levels that are observed in the normal control indicate the presence of cancer of epithelial origin.
  • the levels of biomarkers can be represented by arbitrary units, for example as units obtained from a densitometer, luminometer, or an Elisa plate reader.
  • a higher level of at least one epithelial cancer biomarker in the test sample as compared to the level in the control sample refers to an amount of at least one biomarker that is greater than an amount of the same biomarker present in a control sample.
  • the term “higher level” refers to a level that is statistically significant or significantly above levels found in the control sample.
  • the “higher level” can be for example 1.2 fold to 1.9 fold higher.
  • the "higher level” is at least 2 fold greater, or even 3 fold greater.
  • test sample and control sample are of the same type, that is, obtained from the same biological source.
  • the control sample can also be a standard sample that contains the same concentration of the epithelial cancer biomarker that is normally found in a biological sample that is obtained from a healthy individual.
  • a secondary diagnostic step can be performed. For example, if a level of at least one epithelial cancer biomarker is found to indicate the presence of cancer, then an additional method of detecting the cancer can be performed to confirm the presence of the cancer. Any of a variety of additional diagnostic steps can be used, such as ultrasound, PET scanning, MRI 5 or any other imaging techniques, biopsy, clinical examination, ductogram, or any other method. [0057] The present invention further provides for methods of prognostic evaluation of a patient suspected of having, or having, cancer of epithelial origin.
  • the method comprises measuring the level of at least one epithelial cancer biomarker (Cystatin B, Chaperonin 10, or Prof ⁇ lin) present in a test biological sample obtained from a patient and comparing the observed level with a range of at least one epithelial cancer biomarker levels normally found in biological samples (of the same type) of healthy individuals.
  • a high level for example is indicative of a greater potential for metastatic activity and corresponds to a poor prognosis, while lower levels indicate that the tumor is less aggressive and correspond to a better prognosis.
  • disease progression can be assessed by following the levels of at least one epithelial cancer biomarker in an individual patient.
  • changes in the patients condition can be monitored by comparing changes expression levels of Cystatin B, Chaperonin 10, or Prof ⁇ lin in the patient over time.
  • Progressive increases in the levels of at least one epithelial cancer biomarker is indicative of increased potential for tumor invasion and metastasis.
  • the prognostic methods of the invention also are useful for determining a proper course of treatment for a patient having cancer.
  • a course of treatment refers to the therapeutic measures taken for a patient after diagnosis or after treatment for cancer. For example, a determination of the likelihood for cancer recurrence, spread, or patient survival, can assist in determining whether a more conservative or more radical approach to therapy should be taken, or whether treatment modalities should be combined. For example, when cancer recurrence is likely, it can be advantageous to precede or follow surgical treatment with chemotherapy, radiation, immunotherapy, biological modifier therapy, gene therapy, vaccines, and the like, or adjust the span of time during which the patient is treated.
  • the methods of the invention are suitable to diagnose or prognose any cancer of epithelial origin, including but not limited to , breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers that effect epithelial cells throughout the body.
  • any cancer of epithelial origin including but not limited to , breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell cancers, prostate cancer, renal cell
  • the cancer of epithelial origin is bladder cancer.
  • the levels of at least one epithelial cancer biomarker can be measured by any means known to those skilled in the art. In the present invention, it is generally preferred to use antibodies, or antibody equivalents, to detect levels of at least one epithelial cancer biomarker protein in biological samples.
  • levels of at least one epithelial cancer biomarker protein are measured by contacting the biological sample with an antibody-based binding moiety that specifically binds to at least one epithelial cancer biomarker, or to a fragment of at least one epithelial cancer biomarker. Formation of the antibody- epithelial cancer biomarker complex is then detected as a measure of the epithelial cancer biomarker levels.
  • antibody-based binding moiety or “antibody” includes immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, e.g., molecules that contain an antigen binding site which specifically binds (immunoreacts with) to the epithelial cancer biomarker to be detected, e.g. Cystatin B, Chaperonin 10, or Profilin.
  • antibody-based binding moiety is intended to include whole antibodies, e.g., of any isotype (IgG, IgA 5 IgM, IgE, etc), and includes fragments thereof which 'are also specifically reactive the epithelial cancer biomarker protein. Antibodies can be fragmented using conventional techniques.
  • the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein.
  • proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, dAbs and single chain antibodies (scFv) containing a VL and VH domain joined by a peptide linker.
  • the scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites.
  • antibody-base binding moiety includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.
  • antibody-base binding moiety is further intended to include humanized antibodies, bispecific antibodies, and chimeric molecules having at least one antigen binding determinant derived from an antibody molecule.
  • the antibody-based binding moiety detectably labeled.
  • Labeled antibody includes antibodies that are labeled by a detectable means and include, but are not limited to, antibodies that are enzymatically, radioactively, fluorescently, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G 5 HSV, FLAG, V5, or HIS.
  • a detectable tag such as c-Myc, HA, VSV-G 5 HSV, FLAG, V5, or HIS.
  • the level of - at least one epithelial cancer biomarker present in the biological samples correlate to the intensity of the signal emitted from the detectably labeled antibody.
  • the antibody-based binding moiety is detectably labeled by linking the antibody to an enzyme.
  • the enzyme when exposed to it's substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- Vl-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Chemiluminescence is another method that can be used to detect an antibody-based binding moiety.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • radioactively labeling an antibody it is possible to detect the antibody through the use of radioimmune assays.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H 3 131 I, 35 S, 14 C, and preferably 125 I.
  • fluorescent labeling compounds include CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • An antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • levels of at least one epithelial cancer biomarker protein can be detected by immunoassays, such as enzyme linked immunoabsorbant assay (ELISA), radioimmunoassay (RIA), Immunoradiometric assay (IRMA), Western blotting, or immunohistochemistry, each of which are described in more detail below.
  • Immunoassays such as ELISA or RIA, which can be extremely rapid, are more generally preferred.
  • Antibody arrays or protein chips can also be employed, see for example U.S. Patent Application Nos: 20030013208A1; 20020155493A1; 20030017515 and U.S. Patent Nos; 6,329,209; 6,365,418, which are herein incorporated by reference in their entirety.
  • Radioimmunoassay is a technique for detecting and measuring the concentration of an antigen, biomarker to be detected, using a labeled (e.g.. radioactively labeled) form of the antigen.
  • a labeled labeled e.g.. radioactively labeled
  • radioactive labels for antigens include 3 H, . 14 C, and 125 I.
  • the concentration of antigen in a biological sample is measured by having the antigen in the biological sample compete with the labeled (e.g. radioactively) antigen for binding to an antibody that specifically binds the antigen.
  • the labeled antigen is present in a concentration sufficient to saturate the binding sites of the antibody. The higher the concentration of antigen in the sample, the lower the concentration of labeled antigen that will bind to the antibody.
  • the antigen-antibody complex In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be separated from the free antigen.
  • One method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum.
  • Another method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with formalin-killed S.
  • aureus Yet another method for separating the antigen- antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibody is linked (e.g., covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells. By comparing the concentration of labeled antigen bound to antibody to a standard curve based on samples having a known concentration of antigen, the concentration of antigen in the biological sample can be determined.
  • a solid-phase radioimmunoassay where the antibody is linked (e.g., covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells.
  • a "Immunoradiometric assay” is an immunoassay in which the antibody reagent is radioactively labeled.
  • An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein e.g., rabbit serum albumin (RSA).
  • the multivalent antigen conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient distance apart to allow binding by at least two antibodies to the antigen.
  • the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere.
  • sample antigen and antibody to antigen which is radioactively labeled are added to a test tube containing the multivalent antigen conjugate coated sphere.
  • the antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites.
  • the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined.
  • the amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.
  • ELISA Immunosorbent Assay
  • an antibody e.g. anti-cystatin B, anti-chaperonin 10, or anti-profilin
  • a solid phase i.e. a microtiter plate
  • antigen e.g. cystatin B 5 chaperonin 10, and/or profilin.
  • a labeled antibody e.g. enzyme linked
  • Examples of enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and B-galactosidase.
  • the enzyme linked antibody reacts with a substrate to generate a colored reaction product that can be measured.
  • antibody is incubated with a sample containing antigen (i.e. at least one epithelial cancer biomarker).
  • antigen i.e. at least one epithelial cancer biomarker.
  • the antigen-antibody mixture is then contacted with a solid phase (e.g. a microtiter plate) that is coated with antigen (i.e., at least one epithelial cancer biomarker).
  • a solid phase e.g. a microtiter plate
  • antigen i.e., at least one epithelial cancer biomarker
  • a labeled (e.g., enzyme linked) secondary antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.
  • tissue microarrays can be used in methods of the invention.
  • Other techniques may be used to detect at least one epithelial cancer biomarker, according to a practitioner's preference, based upon the present disclosure.
  • One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter.
  • Detectably labeled anti-biomarker antibodies can then be used to assess the levels of at least one epithelial cancer biomarker, where the intensity of the signal from the detectable label corresponds to the amount biomarker present. Levels can be quantitated, for example by densitometry.
  • At least one epithelial cancer biomarker may be detected using Mass Spectrometry such as MALDI/TOF (time-of-fiight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC- MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.).
  • MALDI/TOF time-of-fiight
  • SELDI/TOF SELDI/TOF
  • LC-MS liquid chromatography-mass spectrometry
  • GC- MS gas chromatography-mass spectrometry
  • HPLC-MS high performance liquid chromatography-mass spectrometry
  • capillary electrophoresis-mass spectrometry
  • Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such as proteins (see, e.g., Li et aL (2000) Tibtech 18:151-160; Rowley et al. (2000) Methods 20: 383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8: 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).
  • a gas phase ion spectrophotometer is used.
  • laser-desorption/ionization mass spectrometry is used to analyze the sample.
  • Modern laser desorption/ionization mass spectrometry (“LDI-MS”) can be practiced in two main variations: matrix assisted laser deso ⁇ tion/ionization (“MALDI”) mass spectrometry and surface-enhanced laser desorption/ionization (“SELDI”).
  • MALDI matrix assisted laser deso ⁇ tion/ionization
  • SELDI surface-enhanced laser desorption/ionization
  • MALDI Metal-organic laser desorption ionization
  • the substrate surface is modified so that it is an active participant in the desorption process.
  • the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest.
  • the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser.
  • the surface is derivatized with molecules that bind the protein of interest and that contain a photo lytic bond that is broken upon application of the laser.
  • the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied. See, e.g., U.S. Pat. No. 5,719,060 and WO 98/59361.
  • the two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix -containing liquid to the captured analyte to provide the energy absorbing material.
  • Detection of the presence of a marker will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of a polypeptide bound to the substrate. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g., visually, by computer analysis etc.), to determine the relative amounts of particular biomolecules.
  • Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.
  • any of the components of a mass spectrometer e.g., desorption source, mass analyzer, detect, etc.
  • varied sample preparations can be combined with other suitable components or preparations described herein, or to those known in the art.
  • a control sample may contain heavy atoms (e.g. 13 C) thereby permitting the test sample to mixed with the known control sample in the same mass spectrometry run.
  • a laser desorption time-of-flight (TOF) mass spectrometer is used.
  • TOF time-of-flight
  • a substrate with a bound marker is introduced into an inlet system.
  • the marker is desorbed and ionized into the gas phase by laser from the ionization source.
  • the ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of-flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge ratio.
  • the relative amounts of one or more biomolecules present in a first or second sample is determined, in part, by executing an algorithm with a programmable digital computer.
  • the algorithm identifies at least one peak value in the first mass spectrum and the second mass spectrum.
  • the algorithm compares the signal strength of the peak value of the first mass spectrum to the signal strength of the peak value of the second mass spectrum of the mass spectrum.
  • the relative signal strengths are an indication of the amount of the biomolecule that is present in the first and second samples.
  • a standard containing a known amount of a biomolecule can be analyzed as the second sample to provide better quantify the amount of the biomolecule present in the first sample.
  • the identity of the biomolecules in the first and second sample can also be determined.
  • At least one epithelial cancer biomarkerlevels are measured by MALDI-TOF mass spectrometry.
  • the antibodies for use in the present invention can be obtained from a commercial source. Alternatively, antibodies can be raised against the epithelial cancer biomarker polypeptide, or a portion of the epithelial cancer biomarker polypeptide.
  • Antibodies for use in the present invention can be produced using standard methods to produce antibodies, for example, by monoclonal antibody production (Campbell, A.M., Monoclonal Antibodies Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, the Netherlands (1984); St. Groth et al., J. Immunology, (1990) 35: 1-21; and Kozbor et al., Immunology Today (1983) 4:72).
  • Antibodies can also be readily obtained by using antigenic portions of the protein to screen an antibody library, such as a phage display library by methods well known in the art.
  • an antibody library such as a phage display library
  • U.S. patent 5,702,892 U.S.A. Health & Human Services
  • WO 01/18058 Novopharm Biotech Inc. disclose bacteriophage display libraries and selection methods for producing antibody binding domain fragments.
  • the present invention is also directed to commercial kits for the detection and prognostic evaluation of bladder cancer and for the diagnosis of invasive bladder cancer.
  • the kit can be in any configuration well known to those of ordinary skill in the art and is useful for performing one or more of the methods described herein for the detection of at least one epithelial cancer biomarker.
  • the kits are convenient in that they supply many if not all of the essential reagents for conducting an assay for the detection of at least one epithelial cancer biomarker in a biological sample.
  • the assay is preferably performed simultaneously with a standard or multiple standards that are included in the kit, such as a predetermined amount of at least one epithelial cancer biomarker protein or nucleic acid, so that the results of the test can be quantitated or validated.
  • kits include a means for detecting at least one epithelial cancer biomarker levels such as antibodies, or antibody fragments, which selectively bind to at least one epithelial cancer biomarker protein.
  • the diagnostic assay kit is preferentially formulated in a standard two-antibody binding format in which one at least one epithelial cancer biomarker- specific antibody captures the biomarker in a patient sample and another epithelial cancer biomarker-specific antibody is used to detect captured at least one epithelial cancer biomarker.
  • the capture antibody is immobilized on a solid phase, e.g., an assay plate, an assay well, a nitrocellulose membrane, a bead, a dipstick, or a component of an elution column.
  • the second antibody i.e., the detection antibody, is typically tagged with a detectable label such as a calorimetric agent or radioisotope.
  • the kit comprises a means for detecting levels of at least one epithelial cancer biomarker in a sample of urine.
  • the kit comprises a "dipstick" with at least one anti-epithelial cancer biomarker antibody or fragments, immobilized thereon, which specifically bind a epithelial cancer biomarker protein.
  • a epithelial cancer biomarker protein can then be detected using, for example, a second antibody that is detectably labeled with a calorimetric agent or radioisotope.
  • the assay kits may employ (but are not limited to) the following techniques: competitive and non-competitive assays, radioimmunoassay (RIA) , bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA 5 microtiter plates, and immunocytochemistry.
  • RIA radioimmunoassay
  • bioluminescence and chemiluminescence assays fluorometric assays
  • sandwich assays sandwich assays
  • immunoradiometric assays sandwich assays
  • dot blots enzyme linked assays including ELISA 5 microtiter plates
  • enzyme linked assays including ELISA 5 microtiter plates
  • immunocytochemistry enzyme linked assays including ELISA 5 microtiter plates
  • the above described assay kits would further provide instructions for use and a container to hold the urine sample.
  • Urine can serve as an excellent medium for epithelial cancer biomarker discovery and analysis.
  • Proteomic analysis by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is one effective tool to analyze the proteome of human specimens. 2D PAGE was used to analyze voided urine, human bladder tumor and normal tissue, and human derived bladder cancer cell lines as a method for biomarker discovery.
  • bladder tumor tissue and normal urothelium were harvested from the cystectomy specimen of a patient with stage T3 Nl MO transitional cell carcinoma. Tissue specimens were immediately frozen in liquid nitrogen and total protein was then isolated and quantified. 40 ng of protein from each tumor and normal tissue were analyzed and compared by 2D PAGE.
  • MGH-Ul cultured from high grade transitional cell carcinoma of the bladder and highly tumorigenic in nude mice
  • MGH-U4 cultured from a patient with severe urothelial atypia and non-tumorigenic in nude mice.
  • 40 ng of each cytoplasmic, nuclear and membrane protein fractions from each cell line were analyzed and compared by 2D PAGE.
  • unique protein spots were isolated and analyzed by liquid chromatography mass spectroscopy-mass spectroscopy (LCMS-MS).
  • Tissue was incubated with mouse monoclonal anti-cystatin B/Stefin B antibody, clone A6/2 (GeneTex, Inc, San Antonio, TX) 5 followed by anti-mouse biotinylated secondary antibody, amplified using ABC kit, and developed using DAB. Tissue was counterstained using Gill's Hematoxylin #3 (Sigma-AIdrich, St. Louis, MO), and blued using Tacha's Bluing Solution (Biocare, Concord, CA). All reagents were purchased from Vector Laboratories, Burlingame, CA, except where noted. AU images were captured at equal exposure time. Results
  • Bladder cancer is the second most common genitourinary malignancy in the United States.
  • 2006 there were an estimated 61,420 newly diagnosed cases of bladder cancer and an estimated 13,060 deaths due to cancer of the bladder (Jemal, A. et al. CA Cancer J Clin 2006;56: 106-30).
  • Tumor suppressor genes such as p53 and Rb have been extensively studied in bladder cancer; however, both of these markers have demonstrated variable predicative value in assessing the risk for disease progression and survival (Habuchi, T. et al. Urology 2005 ;66: 64-74).
  • Cell cycle regulatory proteins p27 and Ki-67 may have some prognostic value for predicting recurrence and disease progression; however, further studies are necessary, and these markers are not yet clinically applicable (Kamai, T. et al. Br J Cancer 2001; 84: 1242-51; Korkolopoulou, P. Hum Pathol 2000;31: 751- 60; Liukkonen, T. et al. Eur Urol 1999;36: 393-400).
  • cystatin B a cathepsin protease inhibitor
  • cystatin B can be used as a bladder cancer biomarker in both patient tissue and urine. It should be understood that these results are contemplated to be indicative of the usefulness of cystatin B obtained from a variety of biological samples in all types of cancers of epithelial origin. Thus, these results provide evidence that cystatin B is useful to assess risk of cancer progression (for example, recurrence of cancer, increase of metastatic activity, and/or progression in cancer grade or stage) in cancers of epithelial origin, such as bladder cancer.
  • Voided urine specimens were immediately cooled to 4°C and then transferred for storage at -80°C within hours.
  • samples were thawed on ice, diluted in 1 volume of 1OmM ammonium bicarbonate buffer pH8, supplemented with protease inhibitors (ImM PMSF, 5mM phenanthroline and 5mM benzamidine (Sigma- Aldrich)), and centrifuged at 3000 x g to remove insoluble material. Samples were concentrated by centrifugation using Centricon Plus-20, 5000 MWCO devices (Millipore) and aliquots lyophilized.
  • protease inhibitors ImM PMSF, 5mM phenanthroline and 5mM benzamidine (Sigma- Aldrich)
  • Bladder tumor tissue and normal urothelium from the grossly normal contralateral bladder wall were harvested from the cystectomy specimen of individual patients with known stage T3 N 1 MO transitional cell carcinoma, under an IRB approved protocol. Tissue specimens were immediately frozen in liquid nitrogen. For processing, frozen tissue specimens were ground to powder and suspended in 1 OmM ammonium bicarbonate buffer/pH8, supplemented with protease inhibitors (ImM PMSF, 5mM Phenanthroline and 5mM Benzamidine (Sigma-Aldrich)). Samples were homogenized and fractionated into water- soluble and 2% CHAPS detergent-soluble fractions, concentrated as above, and aliquots lyophilized.
  • ImM PMSF protease inhibitors
  • Protein concentrations of all specimens were determined using the BCA protein assay (Pierce, Rockford, IL). Equivalent protein aliquots (lO ⁇ g) from individual urine specimens were pooled into Ta, high-grade and normal control groups. Eight patients were included in each of the pooled groups. Forty micrograms of protein from each of the pooled groups were resuspened in isoelectric focusing buffer and loaded onto isoelectric focusing gel- strips of pi 3-10 (Biorad Laboratories, Hercules, CA). Forty micrograms of protein from each of the tissue specimens, tumor and normal urothelium were prepared for isoelectric focusing in a similar manner. Proteins were separated using a linear voltage ramp from 0-8000V.
  • Immunohistochemical staining was performed using a commercially available bladder cancer tissue microarray BL801 (US Biomax Inc, Rockville, MD) consisting of 36 TCC specimens and 33 normal urothelial specimens.
  • the patient population included 26 male and 10 female TCC specimens with a mean patient age of 59.4 years (range 37-88), and 26 male and 7 female normal control specimens from patients of mean age 61.4 years (range 40-88). Of the TCC specimens, 8 were grade 1, 16 were grade 2, and 12 were grade 3. No staging data was available.
  • the tissues were deparaffinized, endogenous peroxide blocked in 3% hydrogen peroxide in methanol, and microwave antigen retrieval performed using Antigen Unrnasking Solution.
  • Blocking was performed using 5% normal horse serum and endogenous biotin blocked using Avidin/Biotin kit.
  • Tissue was incubated with mouse monoclonal anti- cystatin B/Stefin B antibody, clone A6/2 (GeneTex, Inc, San Antonio, TX), followed by anti- mouse biotinylated secondary antibody, amplified using ABC kit, and developed using DAB.
  • Tissue was counterstained using Gill's Hematoxylin #3 (Sigma- Aldrich, St. Louis, MO), and enhanced using Tacha's Bluing Solution (Biocare, Concord, CA).
  • AU reagents were purchased from Vector Laboratories, Burlingame, CA 5 except where noted.
  • Urinary cystatin B correlates with TCC grade and stage
  • Grade/stage progression was defined as an increase in tumor grade or stage at the time of subsequent recurrence, or development of locally advanced disease ( ⁇ T3) or metastatic disease. Patients with high cystatin B levels were found to have a higher risk of earlier grade/stage progression, compared to patients with a low cystatin B level (Log Rank Test, p ⁇ 0.001). Kaplan Meier survival curves indicated the mean disease progression-free time for patients with low levels of cystatin B was 41.4 months (95%CI 39.4 - 43.4 months) compared to those with high levels of cystatin B with mean time to stage progression of only 30.0 months (95% CI 25.1 - 34.8) (Figure 7B). Median progression time was not reached for patients with low cystatin B levels over the maximum follow-up time (43 months), as only 10% underwent grade or stage progression, whereas 65% of patients with high cystatin B levels progressed.
  • Multivariate Cox regression analysis was used to model the risk for disease recurrence adjusted for grade, stage and cystatin B.
  • High grade and stage were both found to increase the hazard ratio of TCC recurrence in univariate analysis, but neither of these parameters reached statistical significance and were not retained as recurrence predictors in the final multivariate model.
  • Cystatin B (Stefin B) was identified as a urinary and tissue biomarker for
  • Cystatin B is an inhibitor of cathepsin proteases (Turk, V. and Bode, W. FEBS Lett 1991;285: 213-9). Many cathepsin proteases are increased in cancer (reviewed in (Kos, J. and Lah, T.T. Oncol Rep 1998;5: 1349-61)), and as their activity is controlled by cysteine protease inhibitors, such as cystatin B 5 it is becoming clear that the balance of the protease/inhibitor axis may be important. Cystatin B protein levels have been shown to correlate with tumor presence and stage in other types of cancer. In ovarian, lung and laryngeal cancers, cystatin B is upregulated (Ebert, E.
  • cystatin B may be used as a tissue and urine biomarker for TCC of the bladder, associated with grade, stage, recurrence and progression.
  • Clinically-associated CIS increases the likelihood of progression to high-grade invasive disease.
  • CIS is also genetically consistent with invasive TCC than with superficial TCC (Rosin, M.P. et al. Cancer Res 1995;55: 5213-6; Spruck, CH. 3 rd et al Cancer Res 1994;54: 784-8).
  • the elevated expression of cystatin B in those with CIS further supports this protein as a marker associated with invasive TCC and risk of invasion.
  • cystatin B was an independent predictive variable for disease progression, outperforming standard pathologic data, such as grade and stage. Such predictive information regarding time to recurrence and risk of progression would benefit the ability to effectively treat and monitor patients with TCC.
  • MMPs matrix metalloproteinases

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Abstract

La présente invention est basée sur la découverte du fait que trois protéines, la Cy statine B, la Chaperonine 105 et la Profiline, sont présentes dans l'urine de patients ayant un cancer de la vessie, un cancer d'origine épithéliale. En conséquence, la présente invention porte sur des procédés pour une évaluation de pronostic de cancers d'origine épithéliale et sur des procédés pour faciliter le diagnostic de cancers d'origine épithéliale par la surveillance de la présence de ces marqueurs dans des échantillons biologiques. L'invention porte également sur des marqueurs pour une efficacité thérapeutique.
PCT/US2007/016955 2007-07-27 2007-07-27 Procédés pour le diagnostic et le pronostic de cancers épithéliaux WO2009017475A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8685659B2 (en) 2005-01-28 2014-04-01 Children's Medical Center Corporation Method for diagnosis and prognosis of epithelial cancers
US9562905B2 (en) 2009-07-29 2017-02-07 Radox Laboratories Limited Method for detection of, or the risk of, bladder cancer
WO2014060753A1 (fr) * 2012-10-16 2014-04-24 Randox Laboratories Ltd. Diagnostic et stratification de risque du cancer de la vessie
US9892229B2 (en) 2012-10-16 2018-02-13 Randox Laboratories Ltd. Diagnosis and risk stratification of bladder cancer
WO2020047636A1 (fr) * 2018-09-04 2020-03-12 Centro Nacional De Pesquisa Em Energia E Materiais Méthode de pronostic métastatique du cancer de la bouche, biocapteur, kit de pronostic, biomarqueurs et utilisation correspondante

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