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WO1997035871A1 - Detection du cancer de la vessie au moyen de l'activite de la telomerase - Google Patents

Detection du cancer de la vessie au moyen de l'activite de la telomerase Download PDF

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Publication number
WO1997035871A1
WO1997035871A1 PCT/US1997/004888 US9704888W WO9735871A1 WO 1997035871 A1 WO1997035871 A1 WO 1997035871A1 US 9704888 W US9704888 W US 9704888W WO 9735871 A1 WO9735871 A1 WO 9735871A1
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bladder cancer
telomerase activity
cells
telomerase
sample
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PCT/US1997/004888
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English (en)
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Brian Liu
Elizabeth Kaveler
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Mount Sinai School Of Medicine Of The City University Of New York
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Priority to AU25476/97A priority Critical patent/AU2547697A/en
Publication of WO1997035871A1 publication Critical patent/WO1997035871A1/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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to a method for detecting bladder cancer cells in a cytology sample collected from a subject comprising measuring telomerase activity, wherein an increase in telomerase activity bears a positive correlation with the presence of bladder cancer cells in the sample.
  • Olovnikov proposed that cells lose a small amount of DNA at their terminal ends with each replication.
  • the 3' to 5' (leading) strand of the parent DNA is copied in a continuous manner, but the 5' to 3' (lagging) strand of the parent DNA is copied discontinuously as Okazaki fragments.
  • Each fragment is primed with an RNA primer, which is subsequently degraded.
  • the fragments are then ligated by DNA repair enzymes that operate behind the replication fork.
  • the 3' end of the lagging strand is then left incompletely copied and is lost. This piece of nucleotide is called a telo ere (Blackburn, 1991, Trends Biol. Sci. 16 :378) .
  • the cell can only afford to lose a finite number of these telomeres before sequences of the parent DNA are lost, resulting in chromosomal instability and subsequent cell death (Harley, 1991, Mutation Res. 256 :271) . Fifty to two hundred nucleotides are lost with each round of replication (Blackburn, 1991, Nature 3_5_0:569) .
  • telomeres Germ cell telomeres are expressed despite multiple rounds of replication suggesting that they produce an enzyme, telomerase, that maintains their telomere length (Hastie et al . , 1990, Nature 346 :866) .
  • the telomere sequence is synthesized by a ribonucleoprotem called telomerase.
  • telomere length and telomerase activity appear to be markers in the replicative history of a cell. Besides germ cells, telomerase is found n immortal human tumor cell lines. It is not found in normal somatic cells . Telomerase activation may play a key role in transforming a mortal somatic cell into an immortal tumor cell (Haber, 1995, N. Engl. J. Med. 33_ :955) .
  • telomere activity in tissue specimens from a wide variety of different cancers and immortal cell lines; malignant tissues expressing telomerase activity included skin, connective tissue, adipose tissue, breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate, nervous system tissue, and blood cells.
  • United States Patent No. 5,489,508 by West et al. relates to the therapy and diagnosis of conditions related to telomere length and/or telomerase activity. Although a correlation between malignancy and telomerase activity is described, there is no disclo ⁇ sure which suggests that a telomerase assay could be used as a sensitive and specific detector of exfoliated bladder cancer cells m a ur e specimen.
  • telomere activity is measured in exfoliated cells from voided urine of patients with hematuria, whereby an increase in telomerase activity bears a strong correlation to the diagnosis of bladder cancer and is associated with significantly fewer false positive and false negative results relative to conventional cytology.
  • the present invention relates to a diagnostic method whereby a urine sample from a subject being evaluated for the presence of a bladder cancer may be assayed for telomerase activity. It has been discovered that the presence of telomerase activity is a more sensitive and specific indicator of the presence of bladder cancer cells, and particularly low grade bladder cancer cells, compared to conventional cytological analysis. The increased sensitivity and specificity of the telomerase assay results may obviate the performance of unnecessary invasive cystoscopy in patients who exhibit false negative cytology results.
  • FIGURE 1 Sensitivity of assay: Sensitivity of detection involved identification of telomerase activity in cell extracts from invasive (T24) bladder cancer cells. The cell lysates were extracted from a known number of cells and the telomerase repeat amplification protocol (TRAP) assay was performed to amplify the telomeres. With this method, we were able to determine that only 50 invasive tumor cells per sample was needed to identify telomerase activity.
  • FIGURE 2 Sensitivity of assay: Using cell extracts from superficial (RT4) bladder cancer cells, we were able to determine that only 200 superficial tumor cells per sample were needed to identify telomerase activity with the TRAP assay.
  • RT4 superficial
  • FIGURE 3 Exfoliated cells from voided urine specimens were obtained from patients with benign causes of hematuria and from one patient with prostate cancer who presented with hematuria.
  • Lane 1 T24 bladder cancer cells as control.
  • Lane 2 to lane 4 are patients #2 to 4 that have benign cause of hematuria.
  • Lane 5 is the lysis buffer and served as a negative control .
  • FIGURE 4 Exfoliated cells from voided urine samples of patients with various grades of bladder cancer were tested for telomerase activity.
  • the present invention relates to a method for detecting bladder cancer cells in a urine sample of a subject in need of such evaluation, wherein an increase in the level of telomerase activity relative to control levels has a positive correlation with the presence of bladder cancer cells.
  • a "subject in need of such evaluation” includes any sub ect, who may reasonably be tested for the presence of bladder cancer, including, but not limited to, a subject who exhibits hematuria, who suffers from difficulty urinating or from painful, or unduly frequent, urination, or who is at risk for developing bladder cancer.
  • Subjects at risk for developing bladder cancer include those subjects having a history of bladder cancer or toxin exposure, subjects havmg indwelling urinary catheters, smokers, and patients suffering from or having a history of Schistosomiasis infection.
  • Other subjects in need of such evaluation are subjects who have been previously diagnosed and treated for bladder cancer and who need follow-up evaluation for recurrent disease. Alteratively, a "subject in need of such evaluation” may be asymptomatic and may merit evaluation only for routine screening purposes.
  • a “subject”, according to the invention, is preferably a human subject but may also be a non-human mammalian subject.
  • a urine sample may be a voided urine sample or may be obtained by catheteri- zation.
  • the volume of the urine sample is at least 20 ml, and more preferably at least 100 ml.
  • the sample size is such that at least 50-400 exfoliated cells, and more preferably at least 200 exfoliated cells, are present in the sample.
  • exfoliated cell refers to a normal or malignant cell havmg its origin m the mucosa of the bladder.
  • telomerase activity refers to a level of telomerase activity which is increased relative to a negative control, defined herein as the amount of telomerase activity present in a urine sample of essentially the same volume from a subject who does not suffer from any malignant disease.
  • An "increase” preferably refers to a level which is at least 1.5 - times background levels, and more prefer ⁇ ably at least five-times background levels. Because the level of telomerase activity in such a normal sample is essentially zero, the term “increased telomerase activity,” for practical purposes, refers to any telomerase activity above background levels, such that running a control urine sample in parallel with actual test samples is not always necessary.
  • the methods described herein may be used not only to detect the presence or absence of telomerase or telomerase activity but may also be used to measure the amount of telomerase or telomerase activity.
  • the term "measure” as used herein refers to either quantitative measurement or a less rigorous comparative evaluation, such as, for example, the determination that the amount of telomerase or the level of telomerase activity is some multiple of a background amount or level.
  • autoradiographic analysis of gels or blots may be used to measure the amount of telomerase or telomerase activity according to the invention.
  • the level of telomerase activity may be detected and/or measured by any method known in the art, includ ⁇ ing, but not limited to, assays which detect and/or measure the activity of telomerase activity on a nucleic acid primer substrate, as well as assays which measure the amount of telomerase protein or the amount of ribonucleic acid which encodes telomerase protein.
  • assays which detect and/or measure the activity of telomerase activity on a nucleic acid primer substrate
  • assays which measure the amount of telomerase protein or the amount of ribonucleic acid which encodes telomerase protein.
  • antibody polyclonal or monoclonal
  • the lengths of telomeres may be measured and compared to the lengths of telomeres in cells of the same histologic type contained in a urine sample from a subject matched by age, tumor grade, level of invasion, or any other prognostic indicator.
  • telomerase enzyme activity may be measured by a variety of techniques, including, but not limited to, the telomeric repeat amplification protocol (hereinafter, "TRAP") disclosed in Kim et al . , 1994, Science 266:2011-2015.
  • PCR polymerase chain reaction
  • red blood cells, white blood cells, necrotic tissues, and/or cellular debris be decreased or eliminated prior to measurement of telomerase activity.
  • These elements may be decreased or eliminated by filtration, centrifugation, precipita ⁇ tion, chromatography, any combination thereof or any other method known m the art.
  • red blood cells, white blood cells, necrotic tissue and cellular debris may be substantially removed, where the term "substantially removed” refers to the removal of at least 50 percent of the total amount of red blood cells, white blood cells, necrotic tissue and cellular debris present in the urine sample.
  • a voided urine sample having a volume of approximately 100 ml, may be evaluated for telomerase activity as follows: The sample, soon after collec ⁇ tion, may be applied to a .45 micron Millipore filter, such that exfoliated cells containing telomerase activity may be retained by the filter, and other elements, including red blood cell components, may be removed. Cells retained on the filter may then be washed in ice-cold phosphate-buffered saline, pelleted at 10,000g for 30 min.
  • ice-cold wash buffer 10 mM HEPES-KOH (pH 7.5) , 1.5 mM MgCl,, 10 mM KCI, ImM dithiothreitol] , pelleted again, and then resuspended at 50 to IO 4 cells per 5 to 100 microliters of ice-cold lysis buffer [10 mM tris- HC1 (pH 7.5) , 1 mM MgCl 2 , ImM EGTA, 0.1 mM phenylmethyl ⁇ sulfonyl fluoride, 5mM /3-mercaptoethanol, 0.5% CHAPS (Pierce) , 10% glycerol] .
  • the suspension may then be Potter-Elvejhem homogenized and incubated for 30 min. on ice and then may be centrifuged for 10 min. in a microcentrifuge (14,000g, 4°C) . The supernatant may then be recovered for further assay and the pellet discarded. If the supernatant is cloudy, it may further then be microultracentrifuged for 30 min. (100,000g, 4°C) , after which the resulting pellet may be discarded and the supernatant used for telomerase assay.
  • the cellular extract prepared as set forth above, including any supematants collected, i.e.
  • sample extract the material to be assayed for telomerase activity
  • the sample extract may be quick-frozen on dry-ice, and stored at -70°C until assay.
  • oligonucleotides may be used: a first oligonucleotide onto which telomerase may synthesize telomeric repeats (for example, but not limited to, the "TS" oligonucleotide, 5' -AATCCGTCGAGCAGAGTT-3' ; SEQ ID N0:1) ; and a second oligonucleotide which may serve as a primer for amplification of telomerase products (for example, but not limited to, the "CX" oligonucloetide 5' - (CCCTTA) 3 CCCTAA-3 ; SEQ ID NO:2) .
  • the TRAP assay may be performed as follows. Assay tubes may be prepared by lyophilizing 0.1 microgram of CX primer onto the bottom of at least a 0.25 ml Eppendorf assay tube and then sealing the resulting pellet with about 7-10 microliters of molten wax (A pliwax, Perkin-Elmer) heated at 65°C for about 2 min. After the wax has been allowed to solidify at room temperature, the tubes may be stored at 4°C.
  • molten wax A pliwax, Perkin-Elmer
  • TRAP reaction mixtures may be produced above the wax which are 20mM tris-HCl (pH 8.3) , 1.5rruM MgCl,, 63 mM KCI, 0.005% Tween-20, ImM EGTA, 0.1 mg/ml of bovine serum albumin and 50 micromolar deoxynucleoside triphosphates, and which contain 0.1 microgram of TS oligonucleotide, 1 microgram of T4g32 protein (Boehringer Mannheim) , 2 Units of Taq DNA polymerase (Boehringer Mannheim), .15 microliters of [ ⁇ - 32 P] dCTP and 1-15 microliters (preferably 15 microliters) of the sample extract prepared as set forth above; wherein the final reaction volume may be adjusted to 50 microliters with distilled, sterilized autoclaved water.
  • Extension of TS oligonucleotide by any telomerase present may then be allowed to proceed for about 10 minutes at approximately 23°C, and then the assay tube may be transferred to a thermal cycler for about 27 rounds at 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1.5 minutes.
  • the CX primer should be liberated by melting of the wax barrier at elevated temperature during the cycling process, permitting amplification to occur.
  • the results of the TRAP reaction may then be analyzed by electrophoresis in 0.5X tris-borate EDTA on 12% polyacrylamide denaturing gels, and then subjected to autoradiography.
  • the skilled artisan may appreciate that the foregoing specific embodiment may be modified in a variety of ways to measure telomerase activity.
  • a detectably labeled probe complementary to the TS oligonucleotide may be used to hybridize to a DNA blot of unlabeled reaction products of TRAP in order to detect those products.
  • the TS oligonucleotide may be modified or replaced with an analogous sequence with a unique portion (that may be hybridized in detection methods) as well as a portion suitable for telomerase extension; telomerase is capable of extending oligonucleotides of nontelomeric sequence (Morin, 1991, Nature 353:454) .
  • the oligonucleotide to be extended may lack such a unique portion, particularly where the product of the extension reaction or an amplification thereof is directly labeled.
  • the CX oligonucleotide may be modified, although at least one iteration of (CCCTTA; SEQ ID NO:3) should be included, as well as a portion that hybridizes to a portion of the oligonucleotide used as a substrate for telomerase extension.
  • any detectable label may be used, including nonradioactive labels which may be detected by enzymatic, chemical, or fluorescence-based methods.
  • the assay need not include an amplification step, but rather, the products of telomerase extension may be measured directly.
  • telomerase assay refers to a substantial probability that where a urine sample is found to contain telomerase activity exceeding back- ground levels, there are likely to be bladder cancer cells present. Based on currently available data, the percentage of true positive results by telomerase assay is greater than about 75 percent and the percentage of false negatives is less than about 25 percent.
  • Cell line lysates Cells were washed once in phosphate-buffered saline, pelleted at 10,000 X g for 10 min. at 4°C, resuspended in ice-cold wash buffer
  • sample extract The supernatant was removed, quick-frozen, and stored at -70°C until use.
  • Patient samples 20 - 100 ml voided urine samples were obtained from patients with hematuria. The samples were freshly filtered on a 0.45 micron filter unit (Nalgene) . Cells retained on the filter were washed in ice-cold phosphate-buffered saline, pelleted at 10,000g for 30 min.
  • ice-cold wash buffer 10 mM HEPES-KOH (pH 7.5) , 1.5 mM MgCl,, 10 mM KCI, ImM dithiothreitol] , pelleted again, and then resuspended at 50 to IO 4 cells per 5 to 100 microliters of ice-cold lysis buffer [10 mM tris-HCl (pH 7.5) , 1 mM MgCl 2 , ImM EGTA, 0.1 mM phenylmethyl ⁇ sulfonyl fluoride, 5mM /3-mercaptoethanol, 0.5% CHAPS (Pierce) , 10% glycerol] .
  • the resulting suspension was then Potter-Elvejhem homogenized and incubated for 30 min. on ice, and then was centrifuged for 10 min. in a microcentrifuge (14,000g, 4°C) , and the supernatant collected. Then, if the supernatant appeared cloudy, it was further microultracentrifuged for 30 min. (100,000g, 4°C) , after which the resulting pellet was discarded and the supernatant used for telomerase assay.
  • the supernatant following microcentrifugation and/or microultracentrifugation, which was then used in TRAP assay, is henceforth referred to as the "sample extract" .
  • Telomeric repeat amplif cation protocol (TRAP) assay Assay tubes were prepared by lyophilizing 0.1 ug of CX primer (see below) onto the bottom of the tube and sealing it with Ampliwax (Perk -Elmer) . Fifty microliter TRAP reactions above the wax barrier contained 20 mM tris-HCl (pH 8.3) , 1.5 mM MgCl 2 , 63 mM KCI, 0.005% Tween-20 ImM EGTA, 50 uM dNTP, 0.1.
  • TS primer see below
  • 2U AmpliTaq DNA polymerase Perkin-Elmer
  • T4g32protem Boehringer Mannheim
  • bovine serum albumin 0.1 mg/ml
  • sample extract 1 to 15 ul of sample extract
  • 0.2 ul of ⁇ -32P[dCTP] was added as described by (Kim et al . , 1994, Science 266.:2011) .
  • oligonucleo ⁇ tide TS After 10 min at 23°C for extension of oligonucleo ⁇ tide TS by telomerase, tubes were transferred to a thermal cycler for 27 rounds at 94°C for 30 seconds, 50°C for 30 second, and 72°C for 1.5 min as previously described (Kim et al . , 1994, Science 266 :2011) .
  • the reaction was analyzed by electrophoresis in 0.5X tris- borate EDTA on 12% polyacrylamide nondenatu ⁇ ng gels and autoradiographed as previously described (Ki et al., 1994, Science 266 :2011) .
  • the TS primer is: 5' -AATCCGTCGAGCAGAGTT-3' ; SEQ ID NO: 1
  • the CX primer is: 5' - (CCCTTA) 3 CCCTAA-3' ; SEQ ID NO:2
  • Sensitivity of assay involved identification of telomerase activity in cell extracts from known superficial (RT4) and invasive (T24) bladder cancer cell lines.
  • the cell lysates were extracted from a known number of cells and the telo erase repeat amplification protocol (TRAP) assay was performed to amplify the telomeres .
  • TRAP telo erase repeat amplification protocol
  • Negative telomerase expression in patients without bladder cancer Exfoliated cells from voided urine specimens were obtained from 13 patients with benign causes of hematuria and from one patient with prostate cancer who presented with hematuria. The patient with prostate cancer had a false positive result on cytologic examination of voided urine, with a cystoscopic exam that was negative for bladder tumor. None of these 14 specimens tested had a positive telomerase activity with our assay (Fig. 3 and Table 1) . In particular, four patients with benign and one with malignant prostate disease, all of whom presented with hematuria, were found to be negative for telomerase activity (Table 1) .
  • telomerase activity in patients with bladder cancer: Exfoliated cells from voided urine samples of twenty three patients with various grades of bladder cancer were tested for telomerase activity (see Table 2 and Figure 4) . In particular, in samples
  • telomerase activity would have avoided an unnecessary preoperative cystoscopy. All (4/4) grade I tumors and 87.5% (7/8) of grade II tumors were diagnosed by the telomerase assay.
  • Raab et al performed stepwise logistical regression analysis on various cytologic variables to try to identify key criteria for diagnosing low grade transitional cell carcinomas via cytology alone. They identified 3 features: increased nuclear/cytoplasm ratio, irregular nuclear borders, and cytoplasmic homogenicity. If all these features are present, they claim that cytology could yield a sensi ⁇ tivity of 45% and a specificity of 98% (Rabb et al . , 1994, Cancer 74:1621) . However, if only 2 of these features are used, the sensitivity increased to 85%, but the specificity declines (Rabb et al . , 1994, Cancer 24:1621) .
  • urinary cytology is limited in its usefulness in detecting low grade bladder tumors. Furthermore, conditions that could contribute to false positive results (atypia) are hyperplastic lesions, radiation effects, infections, and urinary stones.
  • Trott and Edwards compared bladder washing specimens with voided urine cytology (Trott and Edwards, 1973, J. Urol. 110:664) .
  • the theory behind the study was that low grade lesions slough off very few cells into the urine. Fresh bladder washing may increase the number of cells per volume and may make the diagnosis more accurate.
  • cystoscopy only 9 patients had true lesions. The other 19 patients had either chronic cystitis, changes due to radiation, or healthy tissue (Trott and Edwards, 1973, J. Urol. 110:664) .
  • Flow cytometry is an alternative to urine cytology. It measures DNA content m an objective and quantitative manner. Neoplastic cells display nuclear enlargement and hyperchromatism, reflecting an increased DNA conten . Samples are compared to normal control cells and the amount of DNA is displayed as a histogram as diploid, tetraploid, or aneuploid (Badalament et al . , 1987, Cancer £0:1423) .
  • Flow cytometry can be performed using voided urine samples. Reports have shown that flow cytometry will detect approximately 80% of all bladder cancers. For example, Badalament et al . (1987, Cancer £0:1423) showed that when bladder washings, from 70 patients with bladder cancer confirmed via cystoscopy, were analyzed by flow cytometry, 83% of the bladder cancers were detected. As with cytology, the detection rates are better for lesions of higher grade and stage: grade 1 papillomas will be detected in only 50% of patients; Ta, Tis, and invasive lesions in approxi ⁇ mately 82%, 89% and 90% of patients (Badalament et al. , 1988, Sem Urol £:22) .
  • Newer techniques to identify tumor cells in exfoliated cells include the use of monoclonal antibodies (Long, 1995, Infect Urol 8.:103) .
  • the use of a new monoclonal antibody, 486P3-12 was associated with a sensitivity at least twice that of conventional cytology (90% versus 43%) (Huland et al . , 1987, J Urol 137:654; Walker et al. , 1989, J Urol 14_2:1578) .
  • studies have shown good results for high grade lesions, but the diagnosis of low grade lesions continues to be a problem.
  • the above results, which use telomerase activity as a marker for the presence of tumor cells show higher sensitivity and specificity than any of the other current assays.
  • the telomerase activity bears a strong positive correla ⁇ tion with the presence of tumor cells.
  • the number of cells and the method of obtaining the specimen used in our assays were similar to those employed in voided urine cytology studies.
  • the diagnostic yield of the telomerase assay for early grade tumors far exceeds any known non-invasive methods of tumor detection.
  • telomere activity As few as 200 tumor cells present in any volume of urine, we were able to detect the presence of telomerase activity (Fig. 1 and Fig. 2) . All tumor grades were found to express telomerase activity (Table 2 and Fig. 4) . All four patients with grade 1 disease had a positive telomerase activity in their exfoliated cells (Table 2 and Fig. 4) . Furthermore, in several samples, we were able to detect the presence of telomerase activity despite a negative urinary cytology result (Table 2 and Fig. 4; 50% for cytology versus 100% for telomerase) .
  • telomerase activity by the TRAP assay is illustrated in patients with non-cancer causing hematuria. Patients with stone disease, benign stricture disease, benign prostatic hyperplasia, and inflammation all demonstrated negative telomerase activity in their exfoliated cells (Table 1 and Fig. 3) .

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Abstract

L'invention concerne un procédé de détection de cellules cancéreuses de la vessie dans un échantillon d'urine recueillie auprès d'un sujet, qui consiste à mesurer le niveau d'activité de la télomérase, une augmentation de ce niveau ayant une corrélation positive avec la présence de cellules cancéreuses de la vessie dans l'échantillon.
PCT/US1997/004888 1996-03-27 1997-03-26 Detection du cancer de la vessie au moyen de l'activite de la telomerase WO1997035871A1 (fr)

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EP0926245A2 (fr) * 1997-12-22 1999-06-30 Roche Diagnostics GmbH Procédé pour la détection d'un carcinome de la vessie dans un échantillon d'urine
WO2001086288A3 (fr) * 2000-05-08 2002-05-16 Macrochip S R L Methode et dispositif permettant un diagnostic precoce d'une tumeur de la vessie dans des prelevements d'urine
CN106461666A (zh) * 2014-02-17 2017-02-22 希艾娜癌症诊疗有限公司 检测癌症的方法
US10179332B2 (en) 2013-06-19 2019-01-15 Brightwake Limited Filtration device

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EP1866647B1 (fr) * 2005-04-01 2015-04-22 Medvet Science Pty. Ltd. Procédé pour le diagnostic et le traitement et agents utiles pour celui-ci

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0926245A2 (fr) * 1997-12-22 1999-06-30 Roche Diagnostics GmbH Procédé pour la détection d'un carcinome de la vessie dans un échantillon d'urine
EP0926245A3 (fr) * 1997-12-22 2002-09-18 Roche Diagnostics GmbH Procédé pour la détection d'un carcinome de la vessie dans un échantillon d'urine
WO2001086288A3 (fr) * 2000-05-08 2002-05-16 Macrochip S R L Methode et dispositif permettant un diagnostic precoce d'une tumeur de la vessie dans des prelevements d'urine
US10179332B2 (en) 2013-06-19 2019-01-15 Brightwake Limited Filtration device
CN106461666A (zh) * 2014-02-17 2017-02-22 希艾娜癌症诊疗有限公司 检测癌症的方法
US11391738B2 (en) 2014-02-17 2022-07-19 Sienna Cancer Diagnostics Ltd Method of detecting cancer

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