WO1997028446A1 - Techniques de detection et d'evaluation du cancer de la vessie - Google Patents

Techniques de detection et d'evaluation du cancer de la vessie Download PDF

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WO1997028446A1
WO1997028446A1 PCT/US1997/000546 US9700546W WO9728446A1 WO 1997028446 A1 WO1997028446 A1 WO 1997028446A1 US 9700546 W US9700546 W US 9700546W WO 9728446 A1 WO9728446 A1 WO 9728446A1
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hyaluronidase
hyaluronic acid
sample
bladder cancer
amount
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PCT/US1997/000546
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English (en)
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Vinata B. Lokeshwar
Norman L. Block
Henri T. Pham
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University Of Miami
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Priority to JP9527660A priority Critical patent/JP2000504114A/ja
Priority to EP97905574A priority patent/EP0922222A1/fr
Priority to AU22422/97A priority patent/AU2242297A/en
Publication of WO1997028446A1 publication Critical patent/WO1997028446A1/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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to novel methods for detecting and evaluating bladder cancer, utilizing hyaluronic acid (HA) and hyaluronidase (HAase) .
  • Bladder carcinoma is the most common cancer of the urinary tract, accounting for 51,000 new cases and 11,000 deaths each year in the United States.
  • G2 tumors grade, G2 tumors
  • G3 tumors and carcinoma in si tu (CIS) highly aggressive tumors and carcinoma in si tu
  • the high-grade tumors generally metastasize quickly; indeed, at the time of clinical presentation (e.g., hematuria, irritative voiding symptoms etc.), invasive disease already exists for many patients with high-grade bladder tumors.
  • bladder cancer detection involves cystoscopy, bladder washings, and biopsy. These procedures are invasive and require some form of anesthesia. Urine cytology use is possible but its specificity is low due to its subjective nature. A few other markers such as DNA ploidy, p53 mutations, microsatellite DNA, ⁇ - glucuronidase, basic-FGF levels, autocrine motility factor receptor etc. have been shown to be associated with bladder cancer (Sidransky and Messing, Molecular genetics and biochemical mechanisms in bladder cancer. Urol. Clin. North Am., 19: 629-639, 1992; Mao et al. , Molecular detection of primary bladder cancer by microsatellite DNA.
  • the hematuria home screening test has high sensitivity but low specificity due to the wide spectrum of benign genito-urinary (GU) conditions (kidney stones, benign prostatic hyperplasia etc) which give rise to false positives (Britton et al., A community study of bladder cancer screening by the detection of occult urinary bleeding. J. Urol., 148:788-790, 1992; Messing et al., Hematuria home screening: Repeat testing results. J. Urol., 154: 57-61, 1995) .
  • the second test is the Bard BTA Latex agglutination assay.
  • the inventors have developed non-invasive methods to detect bladder cancer by measuring the levels of certain "molecular determinants" specifically expressed in the biological fluids (such as urine specimens) of bladder cancer patients. More particularly, the methods of the invention are based on the inventors' discovery that levels of hyaluronic acid and hyaluronidase in a sample of biological fluid, especially urine, are associated with the presence and grade of bladder cancer.
  • Hyaluronic acid also known in the art as hyaluronate and hyaluronan, and abbreviated as HA
  • HA is a glycosaminoglycan comprising a straight unbranched polysaccharide chain with alternating units of N-acetyl-D-glucosamine and D-glucuronic acid.
  • HA is present ubiquitously in various types of biological material, including both bacteria and animals. In humans, HA is found in high concentrations in umbilical cords, vitreous humor of the eyes, cartilage and synovial fluid. Small amounts of HA are present in CSF, lymph, blood, serum and urine.
  • HA rheumatoid arthritis
  • liver cirrhosis a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasm originating from a malignant neoplasms, and Wilms' tumor.
  • HA is associated with non-specific tumors in general, but its use has not been applied heretofore to the discovery, therapy and management of particular clinical tumors.
  • HA has been known to play a role in several pathophysiological conditions including cancer.
  • HA levels have been shown to be elevated in certain animal tumor models (e.g., rabbit V2 carcinoma, Knudson et al. , The role and regulation of tumor associated hyaluronan.
  • animal tumor models e.g., rabbit V2 carcinoma, Knudson et al.
  • human cancers e.g., lung, Wilms' tumor, breast, etc., Knudson et al. , ibid.
  • HA expands upon hydration opening spaces for tumor cell migration (Knudson et al., The role and regulation of tumor associated hyaluronan. In: The Biology of Hyaluronan (J. Whelan, ed.) , pp. 150-169, New York, Wiley Chichister (Ciba Foundation Symposium 143) , 1989) . Furthermore, tumor cells migrate on HA matrix by interacting through certain cell surface receptors (e.g., CD44; Thomas et al. , Migration of human melanoma cells on hyaluronate is related to CD44 expression. J. Invest. Dermatol. , 100: 115-120, 1993) .
  • CD44 cell surface receptors
  • HA also forms a halo around tumor cells that protects them against immune surveillance (Hobarth et al . , Topical chemoprophylaxis of superficial bladder cancer by mitomycin C and adjuvant hyaluronidase. Europ. Urol., 21: 206-210, 1992) . More recently, small fragments of HA (- 3-25 disaccharide units) have been shown to promote angiogenesis (West et al . , Angiogenesis induced by degradation products of hyaluronic acid. Science, 228: 1324-1326, 1985; West and Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp.
  • the inventors measure the HA levels in the urine of normal individuals and bladder cancer patients, and in the extracts prepared from normal bladder and tumor tissues, as discussed below. Also as discussed below, the inventors examine the profile of HA species present in the urine of normal individuals, bladder cancer patients, and patients with other genito-urinary (GU) conditions. In addition, they determine whether the HA or HA fragments present in the urine affect the proliferation of human endothelial cells.
  • GU genito-urinary
  • the urinary HA levels of bladder cancer patients with Gl, G2 and G3 tumors are significantly elevated (for instance, 4-9 fold elevation) as compared to those of normal individuals and patients with other genito-urinary (GU) conditions (P ⁇ 0.001).
  • GU genito-urinary
  • the inventors also discovered that a comparison of pre- and post-treatment urinary HA levels can be used to monitor treatment efficacy.
  • elevated post-treatment urinary HA levels are indicative of persistent bladder cancer and possible relapse at later time.
  • the urinary HA levels are also useful to monitor bladder cancer recurrence during follow-up visits subsequent to the initial treatment.
  • the increase in urinary HA concentration is a direct correlate of the elevated tumor-associated HA levels, because the HA levels are also elevated (for instance, 3-5 fold) in bladder tumor tissues (P ⁇
  • the profiles of urinary HA species of normal individuals and bladder cancer patients are different. While only the intermediate size HA species are found in the urine of normal and low- grade bladder tumor patients, the urine of high- grade bladder cancer patients contains both the high molecular mass and the small angiogenic HA fragments.
  • the inventors also disclose in the present application that these urinary HA fragments stimulate a mitogenic response (for instance, 2-4 fold) in primary human microvessel endothelial cells, suggesting that the small HA fragments may regulate tumor angiogenesis by modulating endothelial cell functions.
  • the inventors hypothesized that small HA fragments in the urine of high-grade bladder cancer patients may indicate that a hyaluronidase activity is present in the urine of these patients.
  • Hyaluronidase is an endoglycosidic enzyme that degrades HA by hydrolyzing the N- acetylglucosaminic bonds in HA.
  • the limited degradation of HA by hyaluronidase results in the generation of HA fragments of specific lengths (- 3- 25 disaccharide units) that are angiogenic (West et al . , Angiogenesis induced by degradation products of hyaluronic acid. Science, 228: 1324-1326, 1985) .
  • hyaluronidases can be categorized into two classes, those active at neutral pH (pH optimum 5.0) , and those active at acidic pH (pH 3.5-4.0) (Roden et al . , Enzymatic pathways of hyaluronan catabolism. In: The Biology of hyaluronan, (J. Whelan, ed.), pp. 60-86, New York, Wiley Chichister (Ciba Foundation Symposium 143) , 1989; West et al . , ibid. ; Gold, Purification and properties of hyaluronidase from human liver. Biochem.
  • a hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus layer surrounding the egg. J. Cell Biol., 125: 1157-1163, 1995) .
  • the testicular hyaluronidase is of neutral type whereas the liver hyaluronidase has an acidic pH optimum.
  • the concerted actions of both HA and hyaluronidases are known to play important roles during embryonic development, vasculogenesis, vascular remodeling, immune surveillance and tumor progression (McCormick and Zetter, Adhesive interactions in angiogenesis and metastasis. Pharmacol.
  • hyaluronidase levels are elevated in prostate cancer and the increase correlates with the aggressiveness of prostate cancer (Lokeshwar et al . , Association of hyaluronidase, a matrix-degrading enzyme with prostate cancer progression. Cancer Res., 56: 651- 657, 1996) .
  • the inventors disclose in the present application that the urinary HAase levels of bladder cancer patients with G2 and G3 tumors are significantly elevated (for instance, 5-8 fold) as compared to those of normal individuals, patients with Gl tumors and patients with other GU conditions (P ⁇ 0.001) .
  • the inventors also discovered that a comparison of pre- and post- treatment urinary hyaluronidase levels can be used to monitor treatment efficacy. For example, post- treatment elevated urinary hyaluronidase levels are indicative of persistent G2 or G3 bladder tumor and possible relapse at a later time.
  • the urinary hyaluronidase levels are also useful to monitor G2 or G3 bladder tumor recurrence during follow-up visits subsequent to the initial treatment.
  • the increase in urinary hyaluronidase levels is due to the secretion of a tumor-associated hyaluronidase into the urine, as the hyaluronidase levels in G2/G3 tumor tissues are also higher (for instance, about 6-7 fold) than those in normal bladder and Gl tumor tissues (P ⁇ 0.001) .
  • the bladder tumor-associated hyaluronidase activity is distinct from other hyaluronidases, has a pH optimum of 4.3 and is attributed to two proteins of Mr 65 kD (p65) and 55 kD (p55) .
  • HA nor HAase Prior to the invention, neither HA nor HAase have been associated with bladder cancer, nor have they been used for the detection or evaluation of bladder cancer. However, using the assay methods of the present invention, HA and HAase can be used in a non-invasive test to detect bladder cancer and evaluate its particular grade.
  • ELISA-like assays to determine HA concentrations have been described previously.
  • Goldberg et al. U.S. Pat. No. 5,378,637 have described that HA can be measured in a biological sample by coating a solid support with HA, incubating the sample with a cartilage proteoglycan (which is known to bind HA in any biological material) , and then exposing the sample to a coated solid support.
  • the amount of cartilage proteoglycan bound to the solid HA support is determined by anti-keratin sulfate-reactive antibody.
  • a similar method has been described using pig laryngeal cartilage proteoglycan by Fosang et al . (Matrix, 10: 306-313, 1990) . That article describes an ELISA plate-based assay for hyaluronan using biotinylated proteoglycan Gl domain (HA-binding region) .
  • the methods of the present invention are based on the discovery that urinary HA and HAase levels are diagnostic markers for the detection of bladder cancer, evaluation of its grade and monitoring of the efficacy of its treatment.
  • the measurements of HA and HAase levels are technically simple, because these are ELISA-like assays. Both assays require only an HA-binding protein, which can be purified in large quantities using a well established procedure (Tengblad, A. Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage.
  • the ELISA-like assay for HA measurement detects bladder cancer regardless of the tumor grade.
  • the ELISA-like assay for HAase measurement preferentially detects intermediate-grade (G2) to high-grade (G3) bladder tumors. Since urinary hyaluronidase measurement detects CIS (pre-invasive G3 bladder tumors) as well as G2, Ta tumors, the invention is a better non- invasive method for the early detection of G2 and G3 bladder tumors which present with poor prognosis for the patient.
  • bladder cancer is tested for by quantitatively measuring HA in a sample of biological fluid (such as, for instance, a urine specimen) collected from a patient suspected of having bladder cancer.
  • a sample of biological fluid such as, for instance, a urine specimen
  • Any conventional assay methodology can be used to determine the presence and measurement of HA, including radioassays, sandwich assays, inhibition assays and the like.
  • HA is preferably measured by a competitive binding assay. More preferably, the assay of the invention works in the same manner as an ELISA test, but does not make use of antibody complexing mechanisms.
  • bladder cancer can be detected in an assay method comprising the steps of :
  • HA binding protein HABP
  • the coated HA and the HA contained in the sample "compete" to bind with the HABP. Where HA is present in the sample, less HABP will bind to the coated HA, as determined by, for instance, comparison with a standard. In other words, little HABP bound to the coated HA would mean HA present in the sample, which would be indicative of bladder cancer.
  • the preferred way to determine the amount of HABP bound to the HA coated on the solid support, and determine therefrom the amount of HA present in the sample is to detect a signal associated with or produced by the bound HABP.
  • a microtiter plate reader can be used to measure absorbance of colored product as an indirect measure of biotinylated HABP bound to the solid support (avidin-enzyme conjugate and labeled substrate are used to generate the colored product) .
  • the maximum absorbance can be obtained by incubating the HA-coated wells with buffer alone in the absence of any HA or HA-containing sample.
  • a standard graph can then be prepared by plotting absorbance versus ng/ml of HA.
  • the HA concentration (ng/ml) in each dilution of the sample can be calculated. From several such determinations the mean HA concentration in each sample can be determined.
  • Protein concentration (mg/ml) of the sample can be determined, for example, by automated analysis or with a protein assay kit (BioRad, Richmond, CA) .
  • the HA concentrations can be normalized to the protein content and expressed as ng/mg protein.
  • the calculations for determining urinary HA levels can be as follows: A x dilution factor ⁇ mg/ml urinary protein, where A is ng/ml of HA concentration extrapolated from the standard graph.
  • the HA levels are finally expressed as ng/mg total protein.
  • a low absorbance reading would be indicative of a significant amount of HA in the urine sample, which would itself be indicative of bladder cancer in the patient.
  • a calculation of more than about 100 ng/mg HA in the sample is indicative of bladder cancer in the patient.
  • the sensitivity of this method to detect bladder cancer using HA can be about 88% or more (and may be as high as 100%) and the specificity can be about 87% or more (and may be as high as 100%) .
  • specificity is understood to be a measurement of false positives, where a specificity of 100% means there are no false positives (i.e., no suggestion of the presence of bladder cancer when the patient does not in fact have bladder cancer) .
  • Sensitivity is understood to be a measurement of false negatives, where a sensitivity of 100% means there are no false negatives (i.e., no suggestion that there is no bladder cancer when the patient in fact does have bladder cancer) .
  • bladder cancer is tested for and its grade evaluated by quantitatively measuring HAase in a sample of biological fluid (such as a urine specimen) collected from a patient suspected of having bladder cancer.
  • biological fluid such as a urine specimen
  • any conventional assay methodology can be used to determine the presence and measurement of HAase, including radioassays, sandwich assays, inhibition assays and the like.
  • HAase is preferably measured by a competitive binding assay. More preferably, the assay of the invention works in the same manner as an ELISA test, but does not make use of antibody complexing mechanisms.
  • bladder cancer can be detected in an assay method comprising the steps of:
  • HA binding protein HABP
  • the preferred way to determine the amount of HABP bound to the HA coated on the solid support, and determine therefrom the amount of HAase present in the sample, is to detect a signal associated with or produced by the bound HABP.
  • a microtiter plate reader can be used to measure absorbance of colored product as an indirect measure of biotinylated HABP bound to the solid support (avidin-enzyme conjugate and labeled substrate are used to generate the colored product) .
  • the maximum absorbance can be obtained by incubating the HA-coated wells with buffer alone in the absence of any HAase or HAase-containing sample.
  • a standard graph can be prepared by plotting absorbance versus mU/ml of Streptomyces HAase. Using this standard graph, the HAase concentration (mU/ml) in each dilution of the sample can be calculated. From several such determinations the mean HAase concentration in each sample can be determined.
  • Protein concentration (mg/ml) of the sample can be determined by automated analysis or with a protein assay kit (BioRad, Richmond, CA) .
  • the HAase concentrations can be normalized to the protein content and expressed as mU/mg protein.
  • the calculations for determining urinary HAase levels can be as follows: A x dilution factor ⁇ mg/ml urinary protein, where A is mU/ml of HAase concentration as extrapolated from the standard graph. The HAase levels are finally expressed as mU/mg total protein. A low absorbance reading would indicate a high amount of HAase present in the urine sample, which itself would indicate intermediate- or high-grade bladder cancer in the patient.
  • a calculation of more than about 10 mU/mg HAase is indicative of intermediate or high grade bladder cancer in the patient.
  • the sensitivity of this method to detect the intermediate- to high-grade bladder cancer can be about 85% or more (and may be as high as 100%) and the specificity can be about 88% or more (and may be as high as 100%) .
  • a combination of HA and HAase tests may be used.
  • a calculation of less than about 10 mU/mg HAase (corresponding to a higher amount of HABP bound to the coated HA) is indicative of either low grade bladder cancer or no bladder cancer at all.
  • This embodiment of the invention utilizing an HAase assay does not distinguish between the presence of low grade bladder cancer or no bladder cancer at all . Consequently, it is preferred that the HA assay be used in conjunction with the HAase assay to test a patient, because the HA assay can detect the presence of even low grade bladder tumors, although it does not distinguish between particular grades.
  • both the HA and the HAase assays should be run.
  • the HA assay would give a positive result (i.e., levels of HA exceeding about 100 mg/mg, indicating the ' presence of a tumor)
  • the HAase assay would give a negative result (i.e., levels of HAase less than about 10 mU/mg, indicating that there is no intermediate or high grade tumor present) .
  • the invention relates to diagnostic kits for testing and evaluating bladder cancer.
  • the kit comprises HA and/or HAase, HABP and a marker or HABP conjugated to a marker, and ancillary reagents suitable for use in detecting the presence of HA and/or HAase in a biological sample.
  • An example of a diagnostic kit contemplated by this invention is a conventional dipstick test device.
  • FIGURES Pig. 1 Determination of treatment efficacy for bladder cancer by determining pre- and post ⁇ operative urinary HA levels. Urinary HA levels are expressed as ng/mg protein (Mean + SEM) .
  • RC radical cystectomy
  • PC partial cystectomy
  • TURBT Transurethral resection of bladder tumor
  • IC intravesical chemotherapy. Following RC, patient number 2 and patient number 9 received a neo-bladder and an ileal conduit, respectively.
  • the intestinal 19 Determination of treatment efficacy for bladder cancer by determining pre- and post ⁇ operative urinary HA levels. Urinary HA levels are expressed as ng/mg protein (Mean + SEM) .
  • RC radical cystectomy
  • PC partial cystectomy
  • TURBT Transurethral resection of bladder tumor
  • IC intravesical chemotherapy.
  • patient number 2 and patient number 9 received a neo-bladder and an ileal conduit, respectively.
  • mucosa used to make ileal conduit and neobladder may have caused this increase, as intestinal mucosa is rich in glycosaminoglycans, including HA.
  • the HAase levels of these two patients showed a « 5- fold decrease (See Fig. 2: Patient numbers 1 and 7) . Therefore, a combination of two methods is more useful to monitor the treatment efficacy.
  • Fig. 2 Determination of treatment efficacy for high-grade bladder cancer by determining pre- and post-operative urinary HAase levels.
  • HAase levels are expressed as mU/mg protein (mean ⁇ SEM) .
  • the abbreviations used are the same as described in Fig. 1 legend.
  • Fig. 3 An example of a typical standard graph for HA ELISA-like assay. The dilutions of urine specimens which yield an absorbance reading in the linear range of the graph are chosen for calculations. HA used to prepare the standard graph is from the human umbilical cord.
  • Fig. 4 An example of a typical standard graph for HAase ELISA-like assay. The dilutions of urine specimens which yield a absorbance reading in the linear range of the graph are chosen for calculations. HAase used to prepare the standard graph is from the Streptomyces sp.
  • Fig. 5A and B Measurement of urinary HA levels by an ELISA-like assay.
  • the urinary HA levels among different groups of individuals were measured as described in EXAMPLE 2 below.
  • A The scatter diagram of individual HA levels. In each category, a dot represents the urinary HA level of each individual and "n" represents the number of individuals tested.
  • G3 category includes 34 patients with G3 tumor (stages T1-T4) and 9 CIS patients. The dash line represents a minimum cut-off limit of 100 ng/mg HA concentration.
  • Fig. 6A and B Determination of HA levels in bladder tissue extracts. The concentration of HA in tissue extracts was determined by the ELISA-like assay as described in EXAMPLE 2 below.
  • A The scatter diagram of individual HA levels.
  • Gl and G2+G3 tumors are referred to as low-grade and high-grade TCCs respectively.
  • Fig. 7 Examination of the urinary HA profiles of normal individuals and bladder cancer patients.
  • I-V represent HA peaks present in the urine of high-grade TCC patients.
  • Fig. 8A and B Effect of HA and HA fragments on HMVEC-L mitogenic response.
  • HMVEC-L cells were incubated with HA or HA fragments in "Endothelial cell basal medium" at 37° C for 18 h, followed by incubation with [ 3 H] -thymidine for 2 h, as described in EXAMPLE 2 below.
  • A Effect of HA or HA fragments, generated in vitro on HMVEC-L mitogenic response.
  • Control represents the incorporation of [ 3 H] -thymidine in DNA, in the absence of any added HA or HA fragments .
  • the [ 3 H] -thymidine (dpm 1031 ⁇ 87) incorporated in control samples is designated as 100%.
  • the results are an average of triplicate determinations.
  • HA and HA fragments (peaks I-V) isolated from high-grade TCC patients' urine (Fig. 7) on the mitogenic response of HMVEC-L cells.
  • a single concentration (2 ⁇ g/ml) of HA present in various peaks was used to test the mitogenic response.
  • Control represents [ 3 H] -thymidine incorporation in the absence of any added HA.
  • the dpm 838 ⁇ 49 incorporated in the control samples are designated as 100%.
  • the results are mean ⁇ s.d. of triplicate determinations.
  • Fig. 9 Detection of urinary hyaluronidase activity by substrate (HA) -gel assay.
  • HA SDS-PAGE substrate
  • Fig. 10A and B Quantitative determination of urinary hyaluronidase activity by an ELISA-like assay. The hyaluronidase activity was measured as described in EXAMPLE 3 below.
  • A The scatter diagram of individual hyaluronidase activities. In each category, a dot represents the urinary hyaluronidase activity of each individual and "n" represents the number of individuals tested.
  • Fig. 11A and B Determination of hyaluronidase activity in bladder tissue extracts. The hyaluronidase activity in tissue extracts was determined by the ELISA-like assay as described in EXAMPLE 3 below.
  • A The scatter diagram of individual hyaluronidase activities.
  • Fig. 12 Determination of the pH activity profile of bladder tumor-associated hyaluronidase.
  • a urine specimen and a tumor tissue extract obtained from a patient with a high-grade bladder tumor were incubated on HA-coated wells at different pH. Following incubation, HA remaining on the wells was estimated as described in EXAMPLE 3 below. The results are calculated as described in EXAMPLE 3 below.
  • Fig. 13A, B and C Molecular mass determination of bladder-tumor derived hyaluronidase by substrate (HA) SDS-PAGE.
  • A Substrate (HA) SDS-PAGE analysis of urine specimens. Urine specimens (- 40 ⁇ g protein) were separated on a 9% SDS-PAGE minigel together with the BioRad broad range prestained molecular weight markers. Following electrophoresis the gel was processed as described in EXAMPLE 3 below. Lane 1: urine specimen from a patient with Gl tumor; lane 2 urine specimen from a patient with G2 tumor; lane 3 urine specimen from a patient with G3 tumor; lane 4 urine specimen from a normal individual. B: SDS-PAGE analysis of total urinary proteins. Urine specimen (- 20 ⁇ g protein) from a patient with G3 tumor were separated by 12% SDS-PAGE together with BioRad prestained broad range molecular weight markers. Following electrophoresis, the gel was silver stained.
  • HA Substrate
  • HA Substrate SDS-PAGE analysis of tissue extracts. Extracts (40 ⁇ g protein) prepared from a G3 tumor and a normal bladder tissue specimens were separated on a 12% substrate (HA) SDS-PAGE together with BioRad prestained broad range molecular weight markers. Following electrophoresis the gel was processed as described in EXAMPLE 3 below. Left lane: G3 tumor tissue extract; right lane: normal bladder tissue extract.
  • the HA assay of the invention shows a sensitivity of about 88% or more (although it may be as high as 100%) to detect bladder tumors and specificity of about 87% or more (although it may be as high as 100%) .
  • cut-off limits of HA concentration may vary, and the population spread must be taken into consideration.
  • Setting the cut ⁇ off limit of HA concentration to arrive at appropriate determinations of bladder cancer status may involve considering factors such as, for example: age, diet, concentration of protein in the sample, environmental influence, genetic background, hydration status, medical history, physical condition, sex, weight, or the like.
  • the ability to detect even low-grade and low- stage bladder tumors (i.e., Gl and Ta tumors) using HA assays represents another important feature of this embodiment of the invention.
  • the three known non-invasive urinary tests mentioned above--namely, the hematuria home screening test, the Bard BTA test, and the NMP22 test--do not detect these tumors with high sensitivity.
  • Such tumors also go undetected for a long period of time due to lack of any clinical manifestations.
  • urinary HA levels detect low-grade and low- stage (88-90%) , as well as high-grade and high-stage tumors (88-96%) , with similarly high sensitivities.
  • any conventional assay system can be employed with the invention to detect bladder cancer, as long as it measures levels of HA in the biological fluid sample.
  • the assays of this invention are not limited to urine, but also may be used to test biological fluids such as blood, serum, plasma, ascitic fluid, peritoneal fluid, bile, seminal fluid and cerebrospinal fluid.
  • the method of the invention starts with adsorbing HA onto the surface of a solid phase.
  • the HA can be derived from any convenient source, such as human umbilical cord.
  • the solid phase can be any conventional solid phase, including nitrocellulose and the like, and preferably microtiter wells.
  • the surface of the solid phase is preferably washed using conventional buffer(s) . Because the solid phase still has sites left on its surface which are capable of coupling with the HA or other molecules, it is preferred that prior to addition of the sample a blocking substance be added so as to cover any part of the solid phase on which the HA has not been adsorbed. Examples of suitable blocking substances include ⁇ -globulin and albumin derived from cows or other animals. Bovine serum albumin is preferred. After blocking the free sites of the solid phase, the surface of the solid phase is preferably washed using conventional buffer (s) .
  • HA binding protein HA binding protein
  • the incubation time and conditions can vary within wide limits, but an incubation time of about 4 to about 16 hours, and an incubation temperature of about 4° C to about 37° C is satisfactory. Longer or shorter incubation times and higher or lower incubation temperatures are also possible, as would be understood by someone of ordinary skill in this art.
  • HABP suitable for use with the assays of this invention can be readily purified from a number of sources, such as bovine nasal cartilage (Tengblad, Biochim. Biophys. Acta, 578: 281-289, 1979) , pig laryngal cartilage (Fosang et al., Matrix, 10: 306- 313, 1990) .
  • the surface of the solid phase is preferably washed using conventional buffer (s) .
  • the amount of HABP bound to the HA coated on the solid support is determined.
  • the HABP is biotinylated, and the bound HABP is visualized following incubation with an avidin-enzyme conjugate and any substrate for the enzyme which generates a colored product.
  • an avidin-enzyme conjugate i.e., avidin-enzyme conjugate
  • any substrate for the enzyme which generates a colored product i.e., enzyme molecules
  • the signal i.e., colored product
  • any conventional marker system may be used in conjunction with the HABP.
  • Suitable marker systems include enzymes, fluorescence, chemiluminescence, enzyme-substrate, isotope markers, radiolabels and the like.
  • the determination of the amount of HABP bound to the HA coated on the solid support is via an avidin-biotin detection system.
  • Another useful marker system employs keratin sulfate and keratin sulfate-reactive antibodies.
  • the urinary HA levels can usefully be determined using a microtiter plate reader, and can be extrapolated from a standard graph.
  • the amount of HABP coupled with the coated HA can then be correlated with the existence of bladder cancer in the patient from whom the sample of biological fluid was collected.
  • purified hyaluronic acid is preferably used as a standard.
  • Hyaluronidase (HAase) Determination (HAase test)
  • HA is adsorbed onto the surface of a solid phase in the same fashion as described above for the HA assay.
  • Any conventional assay system can be employed with the invention to detect bladder cancer, as long as it measures levels of HAase in the sample of biological fluid.
  • the surface of the solid phase is preferably washed using conventional buffer(s) .
  • a sample of biological fluid e.g., urine
  • a sample of biological fluid e.g., urine
  • a person suspected of having bladder cancer is added to the coated solid support, and incubated under conditions such that the HAase present in the sample (if any is present) is permitted to degrade the HA coated on the solid support.
  • the degraded HA is preferably removed by washing using conventional buffer(s) .
  • a blocking substance e.g., serum albumin
  • the surface of the solid phase is preferably washed using conventional buffer(s) .
  • the solid phase is exposed to HABP, under conditions such that the HABP is permitted to bind to any non-degraded HA coated on the solid support.
  • the incubation time and conditions can vary within wide limits, but an incubation time of about 30 minutes to about one hour, and an incubation temperature of about 37° C is satisfactory. Longer or shorter incubation times and higher or lower incubation temperatures are also possible, as would be understood by someone of ordinary skill in this art.
  • the surface of the solid phase is preferably washed using conventional buffer(s) .
  • the amount of HABP bound to the HA coated on the solid support is determined.
  • the HABP is biotinylated, and the bound HABP is visualized following incubation with an avidin-enzyme conjugate and any substrate for the enzyme that generates a colored product .
  • an avidin-enzyme conjugate and any substrate for the enzyme that generates a colored product i.e., avidin-enzyme conjugate and any substrate for the enzyme that generates a colored product.
  • a detection system does not use radioactivity as a label, multiple markers (i.e., enzyme molecules) are immobilized for every HABP bound to the solid support, and the signal (i.e., colored product) is amplified through turnover of the enzyme.
  • any conventional marker system may be used in conjunction with the HABP.
  • suitable marker systems include enzymes, fluorescence, chemiluminescence, enzyme-substrate, isotope markers, radiolabels and the like.
  • the determination of the amount of HABP bound to the HA coated on the solid support is via an avidin-biotin detection system.
  • Another useful marker system employs keratin sulfate and keratin sulfate-reactive antibodies.
  • the urinary HA levels can usefully be determined using a microtiter plate reader, and can be extrapolated from a standard graph.
  • the amount of HABP coupled with the coated HA can then be correlated with the existence of bladder cancer and the grade of the cancer in the patient from whom the sample of biological fluid was collected. A calculation of more than about 10 mU/mg is indicative of intermediate or high grade bladder cancer.
  • cut-off limits of HAase concentration may vary, and the population spread must be taken into consideration. Setting the cut ⁇ off limit of HAase concentration to arrive at appropriate determinations of bladder cancer status may involve considering factors such as, for example: age, diet, environmental influences, hydration, physical condition, sex, weight, or the like.
  • purified hyaluronidase is preferably used as a standard.
  • the sensitivity of the HAase method to detect the intermediate- to high-grade TCCs is about 85% or more (although it may be as high as 100%) and the specificity is about 88% or more (although it may be as high as 100%) .
  • the HAase assay of the invention is useful both to detect intermediate and high grade bladder tumors and to evaluate their grade.
  • the HA and HAase assays of the invention can be used separately or, preferably, in conjunction with each other.
  • the HAase assay alone does not positively detect and/or evaluate low grade bladder cancers (i.e., calculation of less than about 10 mU/mg) .
  • low grade bladder cancer can be detected when both the HA and HAase assays are used to test a patient's biological fluid sample.
  • HA and HAase are stable after incubation at room temperature for > 8 h (up to about 16 h) .
  • the inventors have analyzed the spot urine specimens, and consequently it is established that special conditions such as first morning void are not required. Since the tests of the invention include normalization of the HA (ng/ml) and HAase (mU/ml) levels in the sample to the protein concentration (mg/ml) , hydration status of the patient does not influence the result.
  • the normalization to total protein rather than to total creatinine is preferred because the former is less influenced by hematuria, 32
  • the assays for HA and HAase of this invention have numerous applications.
  • the invention covers methods for screening individuals to detect bladder cancer and evaluating its grade. This is particularly useful to detect early onset of bladder cancer, especially for those who are at a high risk (e.g. , smokers, and workers in paint, dye and leather industries) .
  • the invention also contemplates methods to evaluate the efficacy of treatment for bladder cancer (i.e., by testing the patient pre- and post- treatment. Similarly, the invention is useful as a method for long-term follow-up of bladder cancer patients to monitor tumor recurrence.
  • the assays of the invention are useful to screen, evaluate treatment efficacy and follow-up of all other urological malignancies
  • the assays are useful to screen other tumors where the tumor comes in contact with any body fluids or normal saline which may be squirted into the tumor to solubilize HA and HAase.
  • the HA and/or HAase can then be assayed.
  • the invention contemplates any dipstick test applications using HA and/or HAase to detect bladder and/or other urological cancers and evaluate their grade. For example, using conventional methodology a solid phase in the form of a dipstick can be used to 34
  • Voided urine samples were collected from all subjects and stored at -20° C until assayed. At the time of assay, the samples were thawed and centrifuged at 4° C for 10 min at 2,000 rpm to remove sediments.
  • 96-well microtiter wells (Corning, Corning, NY) were coated with human umbilical cord HA (Sigma Chemical Co., St. Louis, MO; 25 ⁇ g/ml) dissolved in 0.1 M sodium bicarbonate solution, pH 9.2. The coating was performed at 4° C for « 16 h (overnight) . (This reaction was also run quite successfully at 37° C for 4 h.)
  • the HA-coated wells were washed 3 times in phosphate buffered saline (PBS) .
  • the non-specific sites on the wells were blocked by incubating the wells in 1% bovine serum albumin (BSA) solution prepared in PBS containing 0.05% Tween 20 (PBS+Tween) , at 37° C for 1 h.
  • BSA bovine serum albumin
  • HABP biotinylated bovine nasal cartilage HA-binding protein
  • PBS plus 0.1% Tween 20 and reagents A and B from an Elite Vectastain ABC kitTM (Vector Laboratories, Burlingame, CA) (4 drops of reagents A and B per 10 ml of PBS plus 0.1% Tween 20 solution) at room temperature ( «- 23° C) for 30 min as per manufacturers instructions.
  • Elite Vectastain ABC kitTM Vector Laboratories, Burlingame, CA
  • Burlingame, CA Burlingame, CA solution prepared by mixing 4 drops each of buffer pH 5.3, ABTS and hydrogen peroxide solutions in 10 ml distilled water as per manufacturers instructions. The microtiter plate was incubated at room temperature in darkness until the color (green) develops.
  • the absorbance of the colored product was measured at 405 nm using a microtiter plate reader. In each assay, the maximum absorbance (A max ) 405 nm was 36
  • Urinary protein concentration (mg/ml) was determined by automated analysis or with a protein assay kit (BioRad, Richmond, CA) .
  • the HA concentrations were normalized to the protein content and expressed as ng/mg protein.
  • the calculations for determining urinary HA levels were as follows: A x dilution factor ⁇ mg/ml urinary protein, where A is ng/ml of HA concentration extrapolated from the standard graph.
  • An example of a standard graph obtained using human umbilical cord HA is shown in Fig. 3.
  • Urinary HA levels were determined in a total of 139 patients. These included normal individuals
  • the mean urinary HA levels in all TCC patients are 4-7 fold higher than those in normal 37
  • urinary HA levels are also elevated in a statistically significant manner in patients with low-grade and low-stage (Gl, Ta) bladder tumors.
  • the pre- and post-treatment urinary HA levels were determined in 14 patients. Post-treatment levels were measured 1-3 weeks following the treatment. As shown in Fig. 1, the pre-operative elevated HA levels show a decrease in the majority of patients following treatment suggesting that urinary HA level determinations are also useful in monitoring treatment efficacy.
  • Table 1 Determination of urinary HA levels in various groups of individuals by ELISA-like assay. Results are expressed as mean ⁇ SEM and "n" represents number of individuals in each category.
  • 96-well microtiter wells (Corning, Corning, NY) were coated with human umbilical cord HA (Sigma Chemical Co. St. Louis, MO; 200 ⁇ g/ml) dissolved in O.I M sodium bicarbonate solution, pH 9.2. The coating was performed at 4° C for ⁇ 16 h (overnight) . (This reaction was also run quite successfully at 37° C for 4 h.)
  • HA-coated wells were washed 3 times in phosphate buffered saline (PBS) and incubated with several concentrations of urine specimens (0.5 - 10 ⁇ l) or purified Streptomyces hyaluronidase (0.02 - 40 mU/ml) (Calbiochem, San Diego, CA) in 100 ⁇ l (although different volumes can be used, as would be appreciated by someone skilled in this art) of
  • HAase-ELISA buffer 0.1 M sodium formate, 0.1-0.15 M NaCI and 0.2 mg/ml bovine serum albumin (BSA) , pH 4.3) , at 37° C for - 16 h (overnight) .
  • BSA bovine serum albumin
  • HABP biotinylated bovine nasal cartilage HA-binding protein
  • HAase levels were as follows: A x dilution factor ⁇ mg/ml urinary protein, where A is mU/ml of HAase concentration as extrapolated from the standard graph. An example of the standard graph obtained using Streptomyces HAase is shown in Fig. 4.
  • the urinary HAase levels in patients with either G2 or G3 + CIS tumors are 5- 8 fold higher than those in patients with Gl tumors, patients with GU conditions and normal individuals (P ⁇ 0.001).
  • the ELISA-like assay at 10 mU/mg cut ⁇ off limit, has a sensitivity of 100% and a specificity of 88% to detect intermediate- to high- grade TCCs.
  • pre- and post- treatment HAase levels were measured in 17 patients with high-grade TCCs (G3 tumor or CIS) .
  • TCCs G3 tumor or CIS
  • the pre-operative urinary hyaluronidase levels show a decrease in the majority of patients following treatment showing that measurement of 42
  • urinary HAase levels is useful in monitoring treatment efficacy.
  • Table 2 Determination of urinary HAase levels in various groups of patients by ELISA-like assay. The results are expressed as mean ⁇ SEM and "n" represents number of individuals in each category.
  • BPH benign prostate hyperplasia
  • the G3 subcategory of bladder cancer patients included 34 individuals with G3 tumor (stages T1-T4) and 9 with carcinoma in situ (CIS) .
  • CIS is a sub-class of high- grade bladder tumors that are flat and superficial (confined to the urothelium) . All specimens were collected and stored at -20° C until assayed.
  • Neoplastic bladder tissues were obtained from patients (41-72 years) undergoing cystectomy or transurethral resection of the bladder tumor.
  • Tissue specimens (- 0.5-1 g) were homogenized in 5 mM HEPES buffer pH 7.2, containing 1 mM benzamidine-HCl .
  • the homogenates were clarified by centrifugation at 40,000 x g for 30 min and the clear extracts were assayed.
  • ELISA-like assay for HA level determination The concentration of HA in urine specimens and tissue extracts was determined by an ELISA-like assay described by Fosang et al . (An ELISA plate based assay for hyaluronan using biotinylated proteoglycan Gl domain (HA-binding) region. Matrix, 10: 306-313, 1990) with the following modifications.
  • 96-well microtiter plates coated with human umbilical cord HA 25 ⁇ g/ml were incubated with serial dilutions of urine specimens, tissue extracts or human umbilical cord HA (Sigma Chemical Co., St. Louis, MO) , in phosphate buffer saline (PBS) + 0.05% Tween 20 (PBS+Tween) , and a biotinylated bovine nasal cartilage HA-binding protein (1 ⁇ g/ml) . Following incubation at room temperature for 16 h, the wells were washed in PBS+Tween.
  • PBS phosphate buffer saline
  • Tween 20 PBS+Tween
  • the HA-binding protein bound to these wells was quantitated using an avidin-biotin detection system and ABTS (2,2' azino-bis (3-ethyl-benzthiazolin-6-sulfonic acid) ) substrate (Vector Laboratories, Burlingame, CA) .
  • a standard graph was prepared by plotting absorbance (405 nm) versus human umbilical cord HA concentrations (ng/ml) . Using this graph, the HA 46
  • the mean HA concentration in each sample was determined and then normalized to the protein concentration (mg/ml) in the sample, where the sample could be urine or a tissue extract.
  • the total protein concentration in each clinical sample was determined with a protein detection kit (BioRad, Richmond, CA) .
  • Human umbilical cord HA ( ⁇ 500 mg) was digested with 20,000 units of testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) , at 37° C for different time intervals. The HA fragments generated were separated on a Sephadex G- 50 column (1.5 x 120 cm) . Ten ml fractions were collected and assayed for the uronic acid content (Bitter and M ⁇ ir, A modified uronic acid carbazole reaction. Anal. Biochem., 4: 330-334, 1962) . The fractions were combined to give three preparations, FI, F2 and F3. The number of reducing ends in each fraction was determined by the Dygert assay (Dygert et al .
  • the fractions were assayed for HA by the ELISA-like assay as described above. Since the standard globular protein markers and linear polysaccharides such as HA and HA fragments have different shapes, the column was calibrated using human umbilical vein HA (Mr ⁇ 2 x IO 6 D) and the HA fragments, FI, F2 and F3. Alternatively, to test the effect of HA and HA fragments isolated from urine on endothelial cell proliferation, the specimens were precipitated with trichloroacetic acid (5% v/v) at 4° C for 4 h. The precipitation step was included in order to denature and remove any protein growth factors (e.g., basic-FGF) present in the urine.
  • protein growth factors e.g., basic-FGF
  • HMVEC-L human lung microvessel endothelial cells
  • urinary HA levels were found to be influenced by the hydration status and urine output, these levels were normalized to urinary protein content (mg) .
  • the urinary HA levels were normalized to total protein rather than to creatinine because the normalization to protein was found to be less influenced by hematuria, a condition commonly found in bladder cancer patients (Soloway, The management of superficial bladder cancer. Cancer, 45: 1856- 1865, 1980).
  • the enzyme levels of patients with GU conditions other than bladder cancer were also measured in this study.
  • the distribution of urinary HA levels among normal individuals and patients with other GU conditions is very similar and the HA levels of most of the individuals included in these two groups are ⁇ 100 ng/mg.
  • the urinary HA levels are uniformly elevated in bladder cancer patients, regardless of the tumor grade (e.g., Gl, G2 and G3) , and for most patients, these levels are > 100 ng/mg (Fig. 5 A) .
  • Fig. 5 B The comparison of the mean urinary HA levels among various groups is shown in Fig. 5 B.
  • Table 3 Dunn's multiple comparisons test for comparing the mean urinary HA levels among normal individuals, patients with other GU conditions or bladder cancer. The data presented in Fig. 5 were analyzed using the Dunn's multiple comparisons test.
  • the data on urinary HA levels were further analyzed to determine the specificity and sensitivity of the ELISA-like assay for detecting bladder cancer. As shown in Table 4, the overall specificity of this assay using 100 ng/mg as a minimum cut-off limit, was 92.8%. At the same cut ⁇ off limit, the sensitivity of this assay to detect bladder cancer was 91.9%. The analysis shows that the false positive and the false negative outcomes from this assay were 7.2 % and 8.1% respectively.
  • Table 4 Determination of the sensitivity and specificity for the ELISA-like assay to detect bladder cancer.
  • a The specificity for each group of individuals such as normals and patients with other GU conditions are 96% and 91.1% respectively
  • b The sensitivity of the ELISA-like assay for detecting the low-grade (Gl) and low-stage (Ta) 53
  • tumors was 88.7%.
  • the sensitivity of the assay to detect high-grade ( ⁇ G2) and high-stage ( ⁇ Tl) tumors was 92.7%.
  • the inventors examined HA levels in the tissue extracts prepared from normal bladder, low-grade TCCs (Gl tumor) and high-grade TCCs (G2 + G3 tumors) using the ELISA-like assay. As shown in Fig. 6 A, the HA levels are elevated in bladder tumor tissues, regardless of the tumor grade, as compared to those in normal bladder tissues.
  • the mean HA levels present in the low-grade TCC tissues (5.8 ⁇ 2.1 ⁇ g/mg) and high-grade TCC tissues (9.3 ⁇ 3.3 ⁇ g/mg) are 3- and 5-fold higher than those present in the normal bladder tissues (1.8 ⁇ 0.4 ⁇ g/mg) respectively.
  • the differences in tissue HA levels among normal bladder specimens and bladder tumors (low-grade or high- grade TCC) are statistically significant (P ⁇ 0.001) .
  • the differences in the HA levels present in the low-grade and high-grade TCCs tissues are not statistically significant (P > 0.05, Table 5) .
  • Table 5 Dunn's multiple comparisons test for comparing mean HA levels among normal and bladder tumor tissues. The data presented in Fig. 6 was analyzed by the Dunn's multiple comparisons test. The low-grade and high-grade TCCs indicate the groups of individuals with either Gl or G2+G3 tumors respectively. 57
  • the sizes of urinary HA species were determined by calibrating the column with high molecular mass HA (Mr ⁇ 2 x IO 6 dalton) and HA fragments of known lengths (FI (10-15 disaccharide units), F2 (2-3 disaccharide units) , and F3 ( ⁇ 2 disaccharide units) .
  • the FI fragment has been shown to modulate various functions of bovine aortic endothelial cells (Banarjee and Toole, Hyaluronan binding protein in endothelial cell morphogenesis.
  • the urine of normal individuals contains a small amount of HA and its size was intermediate between the high molecular mass HA and the FI fragment.
  • the urine of low-grade TCC patients contains a small amount of high molecular mass HA and a broad second peak of intermediate size HA (Fig. 7) .
  • the second peak appears to contain some amount of FI fragment (Fig. 7) .
  • the HA profile of the high-grade TCC patients urine shows a complicated pattern. The profile consists of two large peaks, corresponding to the high-molecular mass HA and the FI fragment . These two peaks are separated by a peak of the 58
  • HA intermediate size HA
  • the high-grade TCC patients urine contains two small HA peaks that correspond approximately to the F2 and F3 HA fragments (Fig. 7) .
  • HMVEC-L human lung microvessel endothelial cells
  • the F2 and F3 fragments are not mitogenic to these cells (Fig. 8 A) .
  • peak I high-molecular mass HA
  • peak II intermediate size HA
  • peak III which corresponds to the FI fragment
  • ⁇ 2.4-fold mitogenic response in HMVEC-L cells
  • urinary HA levels are significantly elevated (4-9 fold; Fig. 5) in all bladder cancer patients and show that urinary HA levels are useful as a marker for bladder cancer.
  • the increase in the urinary HA levels of bladder cancer patients may be due to the direct secretion of tumor-associated HA in urine (Fig. 6) .
  • the differences in the urinary profiles of normal, low-grade TCC and high-grade TCC patients indicate that size determination of urinary HA may be another indicator of the prognosis for bladder cancer.
  • the profile of HA species in bladder cancer is different from those found in the urine or sera of children with renal cancer (Lin et al. , Urinary hyaluronic acid is a Wilms' tumor marker. J. Ped. Surg., 30: 304-308, 1995; Kumar et al. , Sera of children with renal tumors contain low molecular mass hyaluronic acid.
  • the size of HA species and the pattern of their distribution may be different in tumors of different orgins as well as grade.
  • Voided (clean-catch) urine specimens were collected from 139 individuals under a protocol approved by the Institutional Review Board of
  • Group 1 normal 60
  • GUI genito-urinary
  • the G3 subcategory included, 34 individuals with G3 tumors (stages T1-T4) and six individuals with carcinoma in situ (CIS) .
  • CIS is a subclass of high-grade tumors that are flat and superficial (confined to the urothelium) . All specimens were collected and stored at -20° C until assayed.
  • Tissue specimens ( « 0.5-1 g) were homogenized in a buffer containing 5 mM HEPES pH 7.2 and 1 mM benzamidine-HCl .
  • the homogenates were clarified by centrifugation at 40,000 x g for 30 min and the clear extracts were assayed.
  • a hyaluronidase assay buffer 0.1 M sodium formate, 0.15 M NaCI, pH 4.3
  • the proteins electrophoresed on a denaturing gel were renatured by incubating the gel in a 3% Triton X-100 solution prior to incubation in the hyaluronidase assay buffer. Following incubation, the gels were stained sequentially with 0.5% Alcian blue and 0.15% Coomassie blue solutions, and destained with 10% methanol/10% acetic acid solution. The presence of hyaluronidase was inferred from the unstained (clear) area(s) in the gel, as described previously (Lokeshwar, V.B., Lokeshwar, B.L., Pham, H.T., and Block, N.L.
  • SDS-Polyacrylamide gel electrophoresis SDS-PAGE
  • silver staining SDS-PAGE
  • Urine specimens ( ⁇ 20 ⁇ g protein) were analyzed by 12% SDS-PAGE and then silver stained to reveal the total urinary protein profile .
  • 96-well microtiter plates coated with 200 ⁇ g/ml HA were incubated with serial dilutions of urine specimens, tissue extracts or Streptomyces hyaluronidase (CalBiochem, San Diego, CA) in hyaluronidase assay buffer containing 0.2 mg/ml BSA at 37° C for 16-18 h.
  • the degraded HA was washed off and HA remaining in the wells was quantitated using a biotinylated cartilage HA-binding protein (Tengblad, Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage. Biochim. Biophys.
  • the data are presented as either mean hyaluronidase activity for individual patients or mean + SEM for each group of patients. The differences between groups were assessed by the Tukey-Kramer multiple comparisons test since the mean hyaluronidase levels showed a parametric distribution.
  • Hyaluronidase activity in urine samples was detected using a sensitive substrate (HA) -gel technique (Lokeshwar et al . , Association of hyaluronidase, a matrix-degrading enzyme with prostate cancer progression. Cancer Res., 56: 651- 64
  • the hyaluronidase levels were normalized to urinary protein concentrations rather than creatinine since the normalization to protein was found to be less influenced by hematuria, a condition commonly found in bladder cancer patients (Soloway, The management of superficial bladder cancer. Cancer, 45: 1856-1865, 1980) .
  • the enzyme levels of patients with GU conditions other than TCC e.g., BPH, prostate cancer, kidney stones, 65
  • bacterial infections and cystitis, renal trauma, prostatitis, and epididymitis, n 48) were also included in the study.
  • Fig. 10 A the distribution of urinary hyaluronidase levels among normal individuals and patients with either low- grade (Gl) bladder tumors or other GU conditions is very similar.
  • the enzyme levels of most of the individuals included in these three groups are ⁇ 10 mU/mg.
  • the hyaluronidase levels are elevated among all patients with intermediate
  • the comparison of the mean urinary hyaluronidase levels among various groups is shown in Fig. 10 B.
  • the mean urinary hyaluronidase levels among normal individuals (4.2 + 1.2 mU/mg) , those with other GU conditions (7.4 ⁇ 1.4 mU/mg) , or Gl tumors (6.5 + 1.7 mU/mg) do not vary significantly. However, the levels are significantly elevated among patients with G2 tumors (32 ⁇ 6.1 mU/mg) or G3 tumors (34.3 ⁇ 3.1 mU/mg) .
  • the mean urinary hyaluronidase levels of all patients with G2 or G3 tumors combined (33.4 ⁇ 4.5 mU/mg) are 5-9 fold higher than those in normal individuals and patients with other GU conditions or Gl bladder tumors. It is important to note that the urinary hyaluronidase levels are elevated 6-11 fold (46 + 5.9 mU/mg) in all patients with CIS (a subclass of high-grade bladder TCCs that are superficial and flat) . These results show that all high-grade bladder cancer patients examined here have elevated urinary hyaluronidase levels prior to the occurrence of invasive disease. 66
  • Table 6 Tukey-Kramer multiple comparisons test for comparing mean urinary hyaluronidase levels in normal individuals and bladder cancer patients. The data presented in Fig. 10 were analyzed statistically using the Tukey-Kramer multiple comparisons test. If the q value is greater than 3.916, then the P value is less than 0.05. TABLE 3
  • the data on urinary hyaluronidase levels were further analyzed to determine the specificity and sensitivity of the ELISA-like assay for detecting high-grade TCCs.
  • Table 7 the overall specificity of this ELISA-like assay, using 10 mU/mg as a minimum cut-off limit, was 88.8%.
  • the sensitivity of this assay to detect high-grade TCCs was 100% (i.e., not a single high-grade tumor was missed) .
  • the analysis shows that false positive and false negative outcomes from this assay were 11.2% and 0% respectively.
  • Table 7 Determination of sensitivity and specificity for the ELISA-like assay to detect high- grade bladder cancer.
  • the data on urinary hyaluronidase levels shown in Fig. 10 were analyzed for sensitivity and specificity calculations using 10 mU/mg as a minimum cut-off limit for an enzyme level.
  • Sensitivity true positive results/total number high-grade TCC patients.
  • Table 8 Tukey Kramer multiple comparisons test for comparing mean tissue hyaluronidase levels in normal individuals and bladder cancer patients. The data presented in Fig. 11 were analyzed statistically using the Tukey Kramer multiple comparisons test. If the q value is greater than 2.655, then the P value is less than 0.05.
  • the pH activity profile of the bladder tumor- associated hyaluronidase activity was determined using the ELISA-like assay. As shown in Fig. 12, the hyaluronidase activity present in the urine and tumor (G3) tissue of a high-grade TCC patient has a distinct pH optimum, 4.3, for HA degradation.
  • the pH optimum for bladder tumor-associated hyaluronidase activity is different from those reported for hyaluronidases from other sources such as serum, liver, kidney, testis and prostate (Gold, Purification and properties of hyaluronidase from human liver. Biochem. J. , 205: 69-74, 1982; Stern et al .
  • Fig. 13 A two hyaluronidase proteins of relative Mr 65 kD (p65) and 55 kD (p55) are present in the urine of patients with G2 (lane 2) , G3 (lane 3) tumors. However, no hyaluronidase bands are detected in the urine of a patient with Gl tumor (lane 1) or a normal individual (lane 4) at the same total urine protein concentration.
  • a urine specimen from a G3 bladder cancer patient was analyzed by SDS-PAGE and silver staining to reveal the total protein profile.
  • the high-grade bladder cancer patient urine contains several proteins.
  • tissue extracts ( ⁇ 40 ⁇ g protein) prepared from these sources were analyzed by substrate (HA) SDS-PAGE. As shown in Fig. 13, both p65 and p55 are present in G3 tumor tissue extract (left lane) , but as expected, not in the normal bladder tissue (right lane) .
  • the sensitivity and specificity of the test to detect bladder cancer patients were calculated using 100 ng HA/mg protein as a minimum cut-off limit (Table 9) .
  • the urinary HAase levels expressed as mU/mg protein were found to be elevated 4-7 fold in G2 and G3 bladder cancer patients (31.4 +, 3.6) as compared to those in Gl bladder cancer patients (6.3 +, 1.1) , other GU patients (7.4 ⁇ 1.4) and normals (4.4 ⁇ 0.8) (P ⁇ 0.001) .
  • the sensitivity and specificity of the HAase test were calculated using 10 mU/mg protein as a minimum cut-off limit (Table 9) .
  • Table 9 Determination of the sensitivity, specificity, false-positive and false-negative rates and accuracy of the HA-HAase test in Study A. The data were obtained from Study A in which a total of 191 individuals were tested. The HA test detects all bladder cancer patients but the HAase test detects 78
  • the inventors conducted a blinded study involving 165 individuals.
  • the subjects included 51 bladder cancer patients, 10 normals and 114 patients with other GU conditions.
  • the bladder cancer category of patients included 41 patients with the disease and 10 individuals with a history of bladder cancer but no disease at the time of testing.
  • the data are presented in Table 10.
  • Table 10 Determination of the sensitivity, specificity, false-positive and false negative rates and accuracy of the HA-HAase test in Study B. This was a blinded study involving 165 individuals. It is noted that both tests when combined missed only two of the 41 bladder cancer patients, for an overall sensitivity of 95.1%.
  • the bladder cancer patients include 121 and 36 TCC patients from Study A and Study B respectively.
  • the Gl category of bladder cancer (BCa) patients includes 6 patients with Gl-2.
  • the G3 category includes 25 CIS patients.
  • Table 12 Sensitivity of HA test by tumor stage.
  • the HA test detected Gl, G2 and G3 bladder tumors with similar sensitivity, i.e., 90% (Gl) , 96% (G2) and 88% (G3) .
  • the sensitivity values for HA test to detect bladder tumors of various stages are also very similar.
  • the HA test detected tumors of stages Ta, Tl, T2+ and CIS with sensitivity values of 90%, 88%, 92% and 96% respectively.
  • the bladder cancer patients include 121 and 36 TCC patients from Study A and Study B respectively.
  • Table 13 Sensitivity of the HAase test by tumor grade.
  • Five Gl bladder cancer (BCa) patients had HAase levels > 10 mU/mg. These patients were considered as false positives and were included in the specificity determination.
  • 2 GlTa patients had urinary HAase values the same as the cut-off limit (10 mU/mg protein) .
  • the Gl category of patients included 40 GlTa patients and one G1T1 patient.
  • Table 14 Sensitivity of the HAase test by tumor stage. The sensitivity values are calculated for high-grade tumors (G2+G3) except for CIS which is a sub-class of G3 tumors. TABLE 13
  • the HAase test detected Gl, G2 and G3 bladder tumors with sensitivity values of 12.1%, 88% and 91.3%. This suggested that the HAase test preferentially detected intermediate- to high-grade bladder tumors ( ⁇ G2) .
  • the results presented in Table 14 show that the HAase test detects high-grade bladder tumors of all stages with very similar sensitivity.
  • the HAase test detected high-grade bladder tumors of stages Ta, Tl, T2+ and CIS with a sensitivity of 85%, 87.6%, 91% and 88% respectively.
  • the HAase test is capable of detecting high-grade tumors prior to invasion (stages Ta or Tl) with high sensitivity and can be used for the early detection of these class of bladder tumors.
  • the HA-HAase test can be used as a screening test to detect bladder cancer patients and to evaluate its grade.
  • the HA test detects bladder cancer patients regardless of the tumor grade; the HAase test preferentially detects high-grade bladder cancer patients.
  • the key to improve the survival of bladder cancer patients is to detect high-grade bladder cancer patients early prior to invasion.
  • the HAase test is equally sensitive in detecting both the high-grade low-stage and high-grade high-stage tumors.
  • the HAase test also detects CIS, a subclass of G3 tumors which is pre-invasive but highly aggressive, with high sensitivity.
  • the combined use of the HA-HAase test will allow the screening of bladder cancer patients and early detection of high-grade tumors in a simple inexpensive and non-invasive manner. Such a screening may then improve the prognosis of bladder cancer patients in general and high-grade bladder cancer in particular.
  • the HA-3- -.ase test may also be useful in detecting other rare malignancies of the bladder.
  • study A 70 individuals were included in the specificity trial. These 70 individuals included, 22 normals and 48 with GU conditions other than bladder cancer.
  • the specificities of the HA and HAase test were 88.6% and 88.7% respectively.
  • the specificity trial included 124 individuals (15 normals, 99 patients with GU conditions other than bladder cancer and 10 patients with a history of bladder cancer but no cystoscopic evidence of the disease at the time of testing) .
  • the NMP22TM detected all bladder cancer patients with invasive disease.
  • HA-HAase test The comparison of HA-HAase test with BTA and NMP22TM is shown in Tables 15 and 16. As shown in Table 15 and 16, the HA-HAase test is better than either the BTA or NMP22TM test with respect to sensitivity, specificity, and prevalence related parameters in detecting bladder cancer.
  • Table 15 Comparison of BTA, NMP22TM and HA- HAase tests with respect to sensitivity and specificity.
  • the BTA study "a” included 499 patients and study "b” included 60 patients.
  • the NMP22TM study included 90 patients and the HA-HAase study included 356 patients. a Sarosdy et al . , Results of a multicenter trial using the BTA test to monitor for and diagnose recurrent bladder cancer, J. Urol., 154: 379-384, 1995. 90
  • Table 16 Comparison of BTA, NMP22TM and HA- HAase tests with respect to prevalence related parameters.
  • the BTA study included 499 patients, the NMP22TM study included 90 patients, and the HA-HAase study included 356 patients.
  • PPV positive predictive value
  • NPV negative predictive value.
  • a Sarosdy et al. Results of a multicenter trial using the BTA test to monitor for and diagnose recurrent bladder cancer, J. Urol., 154: 379-384, 1995.
  • b Soloway et al. Use of a new tumor marker NMP22 in the detection of occult or rapidly recurring transitional cell carcinoma of the urinary tract following surgical treatment. J. Urol., 156: 363- 367, 1996.
  • the HA-HAase test detected all six recurrences (overall sensitivity 100%) in this study. In fact, the HA- HAase test detected four recurrences 3-6 mos. prior to the cystoscopic detection of the tumor. Thus in addition to being a screening test, the HA-HAase test can be used to monitor tumor recurrence.
  • TCCs comprise the majority (90-95%) of bladder tumors.
  • the squamous and adenocarcinomas make up the remaining bladder tumors.
  • the inventors analyzed three samples from adenocarcinoma and one from the squamous cell carcinoma patients. All of these tumors were detected by the HA-HAase test. This is indicative that the HA-HAase test of the inventions can detect all types of bladder tumors.
  • EXAMPLE 5 Monitoring of treatment efficacy and recurrence of bladder cancer by HA-HAase urine test
  • the inventors also monitored 10 patients with a history of bladder cancer to evaluate the usefulness of HA-HAase test in monitoring recurrence.
  • the HA-HAase test identified all six patients who had bladder cancer recurrence.
  • the HA-HAase test detected recurrence of cancer in four patients 3-6 months prior to the cystoscopic detection of tumor.
  • HA-HAase test (false negative rate 12%) and an accuracy of 84.5%.
  • the accuracy of the HA-HAase test was further evaluated by monitoring 18 bladder cancer patients for three months to one year following an initial bladder sparing treatment. In this group of patients, the HA-HAase test detected 39 out of 42 recurrence episodes for an overall sensitivity of 92.9% (false negative rate 7.1%) . Of the five false positives, recurrence was eventually confirmed in three instances 3-6 months later.
  • the HA-HAase test is useful to monitor treatment efficacy and tumor recurrence.
  • the HA-HAase test is a highly sensitive and specific non-invasive method to monitor both bladder cancer occurences and recurrences.

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Abstract

Cette invention a trait à de nouvelles techniques de détection et d'évaluation du cancer de la vessie. Les techniques selon cette invention se fondent sur la découverte que des quantités normalisées d'acide hyaluronique (HA) et d'hyaluronidase (Haase) constituent des marqueurs diagnostiques permettant de déceler un cancer de la vessie, d'évaluer son degré, de contrôler l'efficacité du traitement et de détecter une récurrence tumorale.
PCT/US1997/000546 1996-02-01 1997-01-31 Techniques de detection et d'evaluation du cancer de la vessie WO1997028446A1 (fr)

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JP9527660A JP2000504114A (ja) 1996-02-01 1997-01-31 膀胱癌の検出及び評価方法
EP97905574A EP0922222A1 (fr) 1996-02-01 1997-01-31 Techniques de detection et d'evaluation du cancer de la vessie
AU22422/97A AU2242297A (en) 1996-02-01 1997-01-31 Methods for detection and evaluation of bladder cancer

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WO2000052204A2 (fr) * 1999-02-22 2000-09-08 Orntoft Torben F Expression genique dans les tumeurs de la vessie
US8056395B2 (en) 2009-06-02 2011-11-15 Panasonic Corporation Method for detecting a chemical substance

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KR100475449B1 (ko) * 2002-06-10 2005-03-10 (주)프로테옴텍 유방암 진단용 마커
JP4521809B2 (ja) * 2004-04-16 2010-08-11 生化学工業株式会社 歯周疾患の検定方法および検定用キット
JP2005328752A (ja) * 2004-05-19 2005-12-02 Seikagaku Kogyo Co Ltd グリコサミノグリカン分解酵素の活性測定方法
KR102163550B1 (ko) * 2004-07-23 2020-10-12 퍼시픽 에지 리미티드 방광암 검출용 소변 표지
US8501489B2 (en) 2008-09-26 2013-08-06 University of Pittsburgh—of the Commonwealth System of Higher Education Urinary biomarkers to predict long-term dialysis
JP5711877B2 (ja) * 2009-06-08 2015-05-07 愛知県 癌の悪性度の検知方法及び癌の悪性度の診断剤
KR101439981B1 (ko) * 2012-01-03 2014-09-12 국립암센터 유방암 진단 장치
KR101439977B1 (ko) * 2012-01-03 2014-09-12 국립암센터 위암 진단 장치
KR101439975B1 (ko) * 2012-01-03 2014-11-21 국립암센터 대장암 진단 장치
WO2013103197A1 (fr) * 2012-01-03 2013-07-11 국립암센터 Dispositif de diagnostic du cancer
KR102567842B1 (ko) * 2021-06-16 2023-08-17 을지대학교 산학협력단 남성 난임 자가 진단용 정자 히알루로니다제 활성도 측정 키트

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000052204A2 (fr) * 1999-02-22 2000-09-08 Orntoft Torben F Expression genique dans les tumeurs de la vessie
WO2000052204A3 (fr) * 1999-02-22 2001-04-26 Torben F Orntoft Expression genique dans les tumeurs de la vessie
US6335170B1 (en) 1999-02-22 2002-01-01 Torben F. Orntoft Gene expression in bladder tumors
US6936417B2 (en) 1999-02-22 2005-08-30 Aros Applied Biotechnology Aps Gene expression in bladder tumors
US8056395B2 (en) 2009-06-02 2011-11-15 Panasonic Corporation Method for detecting a chemical substance

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