International Urology and Nephrology 35: 331–343, 2003.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Non-invasive methods of bladder cancer detection

Brian LittleMonklands Hospital, Monkscourt Avenue, Airdrie, ML6 0JS, UK

Abstract. At present, the gold standard for the detection of bladder carcinoma is cystoscopy, often as part ofthe investigation of haematuria. There has been substantial research into the development of a non-invasive testfor voided urine that would obviate the requirement for flexible cystoscopy. If such a test was 100% sensitiveand 100% specific, and was cost-effective, it could represent a way of separating patients who required tumourresection from those with no bladder carcinoma development. This article reviews the development of non-invasivetests for the detection of bladder carcinoma, with emphasis on the most recent developments in fields such asmicrosattellite analysis, telomerase, and matrix metalloproteinase detection, in addition to the more widelyresearched tests such as NMP22 and BTA.

Key words: Bladder carcinoma, Detection, Non-invasive, Transitional cell carcinoma, Urine

Introduction

At present, the gold standard for the detection of de-novo or recurrent carcinoma of the urinary bladder iscystoscopy and thus direct visualisation of the bladdermucosa. Tests of the patient’s urine are also used toadd to the diagnostic process, but as yet none has beensensitive or specific enough to replace this. The idealtest for bladder cancer detection should be 100% sens-itive, 100% specific, cost-effective, non-invasive andproduce results instantaneously. At present, no testfor non-invasive bladder cancer detection fulfils all ofthose criteria.

This article will review the non-invasive teststhat have been developed to date, with particularreference to the sensitivity and specificity of themethods used. The majority of the studies reportedare constructed in a similar way. Urine samples frompatients with bladder tumours are obtained and subjectto analysis using the detection procedure under evalu-ation, and the test is repeated using urine samples frompatients with benign urological complaints, frequentlybenign prostatic hyperplasia. Most of the studies areperformed under blind conditions, i.e., the researcherdoes not know which patients are from the tumourgroup and which are from the benign group.

Follow up of patients is variable. In the majorityof cases, the test used is reported as a point sensitivityand specificity, and no data is supplied regarding thedevelopment of tumours in succeeding months. Thereis therefore little information regarding the signifi-cance of a positive test result and subsequent riskof tumour development. The exceptions to this are486p3/12 monoclonal antibody, Lex antigen, EGF,and the microsattelite based studies [1–6]. A problemwith the interpretation of follow-up data arises when atest result is regarded as positive if a follow up urinetest months or a year later becomes positive. Thismay lead to reporting a spuriously high sensitivity andspecificity, as such a test would not have utility inscreening out patients with bladder tumours requiringresection at the time of a positive test result.

The methods used for detection of bladder cancerinclude the detection of tumour antigens expressedon the tumour surface which are also present in theurine, detection of abnormal blood group antigenexpression, detection of growth factors and receptorsfrom tumours, detection of protein fragments fromtumour activity, detection of chromosomal abnormal-ities in cells from voided urine specimens, detec-tion of tumour mRNA from reverse transcriptasePCR, and microsattellite analysis of sediment fromurinary DNA. The BARD BTA tests and NMP22

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are considered in a separate section because of thewidespread nature of publications regarding these twocommercially produced detection modalities.

Tumour antigens

M344

Bonner et al examined the detection of p300 markerwith M344 antibody, finding that urinary detectionwas 78% sensitive, and 92–94% specific [7]. M344may also be associated with a higher incidence oftumour recurrence at 75%, compared to 20% for thosewith negative M344. 19A211 may also be associatedwith an increased tumour recurrence [8].

Immunocyt

LDQ10 is another monoclonal antibody. Combinedwith M344 and 19A211 it makes the three mono-clonal antibodies of the ‘Immunocyt’ testing kit forbladder carcinoma. The antibodies are directed againsttumour mucins and a high molecular weight form ofglycosylated carcinoembryonic antigen. In the studyby Lodde et al, overall sensitivity is 89%, with littlechange from G1 to G3 carcinomas, and specificity is75%. In the same study, cytology was 50% sensitiveand 98% specific [9]. Olssen et al found sensitivityof 100% and specificity of 69%. Their study had 31tumour patients and 90 controls [10].

The most recent work with Immunocyt by Pfisteret al examined a series of nearly 700 urine samples,with 143 of these from patients with bladder tumours.This study showed Immunocyt to have an overall sens-itivity of 67%, compared to sensitivity for cytology of49%. In the same study, specificity was 84% and 94%respectively [11].

AN43 and BB639

AN43 and BB639 are two monoclonal antibodies.They bind to an antigen that is expressed in urothe-lium, and is detectable in shed urothelial cells. Itis 47% sensitive and 89% specific, in a small studytotalling 19 urine samples [12].

486p3/12 monoclonal antibody

Cells in the voided urine from patients with trans-itional cell carcinoma have a greater than 30% bindingfrequency for 486p3/12, a monoclonal antibody.

Patients without bladder carcinoma have a less than30% binding rate. Using this technique, 90% ofbladder carcinomas could be identified, regardless ofgrade or stage, but specificity is only 53%, usingpatients with previous carcinomas as controls [13].More recent work found a sensitivity of 68% andspecificity of 84% [14].

Interestingly, a study of 55 patients found thatwhen 486p3/12 status remains negative on urinarytesting every 4 weeks following tumour resection,only 9% developed a tumour recurrence. If 486p3/12became positive, the recurrence rate by 2 yearswas 42%, but the development of marker positivitypreceded cystoscopically visible tumour developmentby two to five months [1].

Psoriasin

Psoriasin is a calcium binding protein expressed inthe urine of bladder squamous cell carcinomas, andthose transitional cell carcinomas that are undergoingsquamous differentiation. In 100 urine samples, all 4squamous cell carcinoma samples expressed psoriasin.However, only 16.3% of transitional cell carcinomapatients were positive for this marker. In addition, 17%of control specimens from patients with no urologicaldisease were positive. It may be that female patientswith squamous metaplasia of the trigone expresspsoriasin. Overall sensitivity for bladder carcinoma(Squamous and transitional) was 26%, and specificitywas 85% [15].

DD23

DD23 is a monoclonal antibody that detects a tumour-associated antigen that is expressed in bladder tumourcells in vivo, and is not detected in the absence oftumour cells. In a series of 151 urine samples, ofwhich 64 were from patients with tumour recurrences,the sensitivity of this method was 59.4% and thespecificity was 71%. Combining DD23 with cytologyincreased the sensitivity to 68.8%, without changingspecificity. Using bladder barbotage instead of voidedurine samples also increased sensitivity [16]. Previouswork by Bonner et al, using the more sensitive methodof automated quantitative fluorescence image analysisand fluorescent labelling, rather than staining and lightmicroscopy, produced a higher sensitivity rate of 85%,with 95% specificity [17].

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Urinary basic fetoprotein

The measurement of urinary basic fetoprotein hasbeen reported by Sato et al. This is a cancerrelated foetal protein, which is expressed in urothelialtumours. Measurement of this can be compared tosamples from a normal population. If the normal levelexpressed in urine is 4.5 + –6.1 ng/ml, and the cut-off value for normal is set as below 15ng/ml, then thesensitivity of this method for detection of tumours is54%, and the specificity is 88% [18].

Survivin

Survivin is an inhibitor of apoptosis that may beover-expressed in tumour tissues, but not in normaltissue such as urothelium. Survivin may be detectedin urine by a biodot method, measuring the chemilu-minescence of samples on a nitrocellulose sheet aftertreatment with a polyclonal antibody [19]. Using thismethod, Smith et al examined 46 bladder cancer urinesamples, from group of 138 samples. They found itto be 100% sensitive, and 96% specific. Their fourfalse positives occurred in 3 patients being evaluatedfor haematuria, and one with raised PSA. Survivinnormalised in 91% of patients following treatment oftheir bladder tumour [19].

Blood group antigens

The Lewis X antigen is a blood-group related antigen,noted to be increased in expression in urothelialcarcinoma. In 1988 Cordon-Cardo et al demonstratedthat Lewis X antigen was not expressed in normalurothelium, but was expressed in the majority ofinvasive tumours, along with the Ley antigen [20]. In1990, Sheinfeld et al. used an anti-Lewis X mono-clonal antibody and exfoliated urine cells, with adetection sensitivity of 85.4% and 85% specificity[21]. Attempts to use this as a screening test showedthat by using 2 voided urine samples for immunoper-oxidase staining for Lewis X antigen, a sensitivity of97% and a specificity of 85% could be obtained. Inter-estingly, detection was not influenced by tumour grade[22]. The predictive values for Lewis X antigen in anearlier study was 86% sensitivity and 87% specificity,who also found that expression of Lex antigen wasassociated with increased tumour recurrence [2]. In1998, a further study regarding immunostaining of theLewis-X antigen using exfoliated urine cells, foundthat for 1 urine sample sensitivity was 79.8%, and

specificity was 86.4% [23]. Detection was improvedby using two voided urine samples, however this wasat the expense of a fall in specificity to 73%. Themost recent work has found that sensitivity was 75%and specificity was 90%, supporting the relatively highrates found for this method [14].

Growth actors and receptors

EGFR and EGF

Epidermal growth factor receptors have been shown tobe present in transitional cell carcinoma, using mono-clonal antibody EGFR1. Tumours are more likelyto be EGFR positive if they are of high grade orare invasive [24]. Epidermal growth factor is foundin urine in high concentrations, and is thereforein continuous contact with the urothelium. Normalurothelium contains EGF receptors, and urinary EGFcan induce DNA synthesis. Bladder tumour cells differfrom normal urothelium, in that tumour surface cellshave EGF receptors whereas urothelium receptors arein the basal layer [25]. Therefore it follows thatbladder tumour cell growth may be influenced byurinary EGF.

Messing has found EGF in voided urine frompatients with bladder cancer, and finds in to be signifi-cantly reduced, and speculating that it may be causedby EGF binding to the EGF receptor [26]. He issupported by Fuse, who similarly finds that the EGFconcentration in urine is reduced in patients withurothelial tumours, and that the higher stage tumours(T4) had lower urinary EGF levels than tumours oflower stage. Low urinary EGF is therefore associ-ated with the development of muscle invasive bladdercarcinoma [3].

FGF

Urinary basic fibroblastic growth factor has beenshown to present in higher concentrations in patientswith carcinoma of the urinary bladder, however ata cut-off value of 6.0 ng/g creatinine, it was only42% sensitive and 88% specific. Reducing the cut-offvalue did improve sensitivity, but only at the price ofworsening specificity [27].

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Detection of tumour enzymes

Telomerase

Telomeres are the two poles of the chromosome. WithDNA replication, telomeres shorten, and so produceprogrammed cell death once the telomere length fallsbelow a certain critical length. This is known as apop-tosis. Malignant cells produce an enzyme, telomerase,which reconstructs the telomere sequence, making thecell immortal. Telomerase can be detected in urine ofpatients with bladder cancer. Müller et al found thatthe ribosomal component of telomerase, hTR, was themost reliable, and is stable in urine for 3 days. It hada detection sensitivity of 83%, and was 80% specific[28]. A further study found telomerase to be 85% sens-itive and 66% specific in cases of haematuria, althoughall healthy controls were negative [29].

The most recent work on telomerase activity hasalso shown a high sensitivity of 85%, with 80%specificity, however a substantial proportion of condi-tions, which were non-malignant, such as benignprostatic hypertrophy and ureteric colic, were falsepositives (76%). This suggests that its utility in cancerdetection is limited [30]. This finding was supportedby a subsequent study that also found telomeraseto have 70% sensitivity, but only 46% specificity[31]. Others have reported rates of 84% and 93% forsensitivity and specificity [32].

Matrix metalloproteinases

Matrix metalloproteinases are implicated in thedegradation of basement membranes and invasionof the extracellular matrix, and are implicated inthe progression of many cancers. Özdemir et al.examined the expression of MMP-1, MMP-2 andMMP-9 in transitional cell carcinoma of the urinarybladder. They found that only MMP-9 expression rateswere increased with rising grade and stage. MMP-9was also detectable in the urine in all patients withurothelial carcinoma, and so was 100% sensitive. Italso was 100% specific, in a series of 33 tumours and16 controls [33].

More recent work by Gerhards et al found lowersensitivity of 51% for MMP-2, compared to just 31%for MMP-9 [34]. The specificity was not stated in theirpaper, but MMP-2 elevation was associated with 18.5times risk for bladder cancer, compared to 3.9 whenMMP-2 was increased. They suggest that MMP-2should be prospectively tested.

Hyaluronic acid and hyaluronidase

Urinary Hyaluronic acid and hyaluronidase have alsobeen tested. Hyaluronic acid is a glycosaminoglycan,present in body fluids. Their concentrations rise in thepresence of tumour, and are degraded by hyaluroni-dase. In a study of 513 urine specimens, the hyaluronicacid and hyaluronidase levels were measured relativeto a diagnostic level of 500 ng/mg. A sensitivity of81.5%, and a specificity of 90.1% were found [35].

A follow-on study of 225 prospectively collectedurine samples, of which 178 showed tumour, and 47did not, found that the hyaluronic acid-hyaluronidasetest had sensitivity of 91%, and specificity of70% [36]. This part of their study was specific-ally examining the follow-up of patients with bladdercarcinoma.

Urokinase-type plasminogen activator and receptor

This is a serine protease that has a role in cell invasionand metastasis. It produces a breakdown in laminin,a major basement membrane component, and permitsactivation of metalloproteinases. In a sample of 122patients with bladder cancer and 107 controls, signifi-cantly higher levels of urokinase-type plasminogenactivator (UPA) and receptor (UPAR) were found inthe group with bladder cancer. It has not been formallyevaluated as a screening test [37].

Detection of protein fragments

Fibrin degradation products

Fibrin degradation products are protein fragmentsgenerated by the action of the fibrinolytic systemon fibrin and fibrinogen. Bladder tumours producevascular endothelial growth factor, which increasesvascular permeability, leading to leakage of plasmaproteins such as fibrin and fibrinogen into urine. Astudy of 197 patients could only produce a sensitivityof 68% overall, rising in invasive disease. A specificityof 86.2% was also found [38]. Fibrin degradationproducts were further evaluated by Johnston et al., andcompared to BTA and cytology. FDP’s were found tobe 81% sensitive and 75% specific. This comparedvery favourably to BTA, which had a detection rateof only 28%, but BTA was more specific at 87%.Cytology was the most specific test, at 90% [39].

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UBC antigen

UBC is an enzyme-linked immunoassay that detectsfragments of cytokeratin 8 and 18. In a study of 107patients, 59 of whom had bladder carcinoma, UBChad a sensitivity of 64% and a specificity of 73%, ifa value above 1 µg/l is regarded as positive. Sens-itivity improved significantly with worsening tumourgrade, stage and size [40]. Similar work by Eissaand co-workers had shown 61% sensitivity and 81%specificity, in a series of 168 patients, of whom 100had bladder carcinoma [41]. An earlier, smaller seriesfrom Sánchez-Carbayo had shown that UBC was 77%sensitive and 83% specific [42].

CYFRA 21-1

CYFRA 21-1 measures fragments of cytokeratin 19. Itcan be problematic comparing results for this methodfrom different centres, as different threshold values areused to define tumour positivity.

CYFRA 21-1 immunoassay has been tested in 226urine samples by Sánchez-Carbayo et al., who foundthat it has a sensitivity of 81%, and 97.2% specificity,at a threshold value of 5.7 µg/l [43]. Pariente et al.also examined this method, finding 96.9% sensitivitybut only 67.2% specificity, using a threshold valueof 4 µg/ml [44]. The most recent study of 325 urinesamples by Nisman et al., using a diagnostic thresholdvalue of 4.9 µg/ml found a sensitivity of 79% and aspecificity of 89% [45].

Tissue Polypeptide antigen (TPA)

Tissue polypeptide antigen is found in the cytoskel-eton of non-squamous epithelium and is present in theproteolytic fragments of cytokeratin 8, 18 and 19 thatare released by cell death. It is a marker of cell differ-entiation. Serum TPA levels are elevated in 20–25%of patients with invasive TCC bladder, and falls in theserum level are associated with responses to treatment[46]. TPA levels greater than 70 UI/ml were associatedwith a two-fold risk of recurrence [47].

Boman et al. have recently studied its use as aurinary marker for bladder cancer detection, in a seriesof 260 urine samples, of which 134 were from patientswith bladder cancer [48]. They found that TPA wassignificantly elevated in the urine of patients withbladder tumours, compared to controls. There wasalso a significant correlation between TPA level andtumour grade, stage and size. They used absorbanceat 450 nm of 42 as their diagnostic threshold, above

which samples were considered positive for tumour.This gave an overall sensitivity of 59%, and specificityof 66%.

Fibronectin

Fibronectin is an extracellular matrix component, andfibronectin fragments can be measured in the urineusing a murine monoclonal antibody kit. Using avalue of 198ng of fibronectin /mg creatinine as acut-off value, above which a sample was consideredpositive for bladder carcinoma, sensitivity was 83%and specificity was also 83% [41].

BTA and NMP22

Bard BTA test

The Bard BTA test (Bladder Tumour Antigen) is alatex agglutination test that qualitatively detects base-ment membrane complexes in voided urine. Theseare produced when tumour cells become invasiveand produce proteolytic degradation of transitionalepithelium basement membrane. The test procedureis simple; Latex particles coated with human IgG areadded to a voided urine sample, followed by the testsolution, which consists of a water-soluble yellowdye and a blue dye suspension. The latex particlesagglutinate in the presence of basement membranecomplexes, trapping the blue dye suspension. Whentest filter paper is dipped into the sample, bothdyes travel up it if there are no basement membranecomplexes, so it turns green (blue + yellow). However,if basement membrane complexes are present, the bluedye travel is impaired, and pure yellow is seen at thetop of the paper [49].

When the sensitivity of the Bard test is comparedwith that of conventional cytology, an identical detec-tion rate for both of 0% for G1 tumours, 50% detectionfor both at G2, but a higher detection rate of 100% forBARD when compared to 14% for urinary cytologyof G3 disease [49]. Nasuti et al. found BTA STATto have a very low positive predictive value, of only16% [50]. A large multi-center study of 499 patients in1995 found BTA to have 40.4% sensitivity comparedto 16.6% for cytology. BTA specificity was 95.9%,and detection was improved at low disease stage [51].Other studies similarly found BTA STAT to have ahigher sensitivity overall, especially when comparedto cytology in low-grade disease [52].

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Comparison between BTA stat and the laboratorytest BTA TRAK has shown that BTA TRAK is moresensitive than BTA stat, with rates of 77.6 and 65.3%respectively [53].

A full description of the reported sensitivities andspecificities for the BTA tests is shown in Table 2.

NMP22

Urinary NMP22 represents an alternative marker forrecurrent disease. Its main practical disadvantagecompared to the Bard BTA test is that it takes 4–5hours to complete, and requires an expert technicianto perform it because of its complexity [54] NMP22has been exhaustively evaluated in bladder cancer anda summary of the sensitivities and specificities of thevarious studies is shown in Table 2.

In studies where NMP 22 has been compared toBTA, NMP22 has generally had a higher detection ratefor bladder tumours, and a slightly better specificity,with the exception of the study by Ramakumar et al.[55].

NMP22 was compared to BTA and cytology, usinga cut-off value of 6U/ml for NMP22 by Del Nero et al.For pTa disease detection, BTA, NMP22 and cytologyhad detection rates of 27%, 83% and 20% respectively.For pT1 tumours, the rates were 67%, 98% and 64%respectively. BTA and NMP22 had false positives of30% and 23%, whereas cytology was never incor-rect. All had high pick-up rates for high-grade disease,but NMP22 was positive in 86% of G1 tumours,compared to 38% for both other methods [54]. Sözenalso compared NMP22, BTA stat and cytology, findingboth tests to be more sensitive than cytology (73%,69% and 35% respectively), and specificities of 85,68 and 90% respectively [56]. Hughes finds only 47%sensitivity [57]. Sharma’s exclusion criteria (Benigninflammatory/infective conditions, Calculi, Recentforeign body or instrumentation, bowel interpositionsegment or another genitourinary cancer) increases thespecificity of NMP22 to 95.6%, and of BTA STATto 91.5%. Sensitivity of NMP22 was 82%, and BTA68% [58]. However, it could be argued that a non-invasive test should not require such exclusions toimprove diagnostic accuracy. Specifically looking atthe predictive value for recurrence, Soloway looked atNMP22 status 10 days after TURBT. He found thatNMP22 could predict tumor status at follow-up with asensitivity of 70%, rising to 100% for invasive disease[59].

A comparative study of BTA stat, NMP22, urinarycytology, Fibrin degredation products, telomerase,chemiluminescent haemoglobin and dipstick haemo-globin found sensitivity of 74, 53, 44, 52, 70, 67and 47% respectively. Specificity was 73, 60, 95,91, 99, 63 and 84% respectively. They conclude thattelomerase had the highest combination of sensitivityand specificity, at 70% and 99% respectively [55].

Neural networks

Parekattil et al constructed a neural network basedaround three markers of urinary malignancy, andtested it on a series of 253 patients, including 27 withbladder cancer [61]. The markers used were: Urinarycellular adhesion molecule 1, NMP22, and monocytechemoattractant protein 1. The results of the analysisshowed a sensitivity of 100%, with a specificity of76%. The test was found to be cheaper and moresensitive than cytology (67% sensitive, 81% specific).

Chromosomal abnormality detection

Flow cytometry

Flow cytometry is a method of determining cellularchromosomal content. It allows the identificationof cells with greater or fewer numbers of chromo-somes than normal (aneuploidy), such as can befound in tumours, but is time-consuming to under-take. Sánchez-Carbayo et al. have analysed the DNAcontent of bladder cells from centrifuged voided urinespecimens. If a urine sample yields bladder cellswhere more than 11% are hyperdiploid, it is 77% sens-itive and 83% specific. Hyperdiploid cells are thosethat contain more than 50 chromosomes, comparedto the normal karyotype of 46 chromosomes. DNAaneuploidy was only 36% sensitive, but 93% specific[42].

H-Ras

Mutation converts H-ras into an oncogene in 10% ofbladder tumours, the majority at codons 12 and 61.Saito et al have looked at the overall frequency ofH-ras mutation at these sites, finding an incidenceof 12%. He has looked at the urinary detection ofH-ras, finding it to be possible despite the presenceof mutated DNA being as low as 1%. However, the

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overall low incidence of ras mutations rather limits itsclinical application [61].

Fluorescent in-situ hybridization

Fluorescent in-situ hybridisation (FISH) wascompared to cytology by Junker et al. [62]. Theyfound that FISH had the same detection rate for bothvoided urine and for bladder washings. They furtherfound that FISH had a sensitivity of 68% overall,compared to 49% for urinary cytology. A furtherstudy of FISH, using probes for chromosomes 9 and17, found a sensitivity of 85%, and was 95% specific[63]. This was a small study of 44 patients and 37controls.

More recently, the development of the VysisUrovision assay, a multi-target FISH assay has hadreported sensitivity of 81% and specificity of 96%, ina series of 280 urine samples [31]. Vysis Urovisionutilises probes for the pericentromeric regions of chro-mosomes 3, 7, 17 and the 9p21 locus. The findingmore than 4 urinary cells with gains of 2 or morechromosomes, or more than 9 cells with gains of onechromosome on the slide was scored as positive forurothelial carcinoma. Homozygous deletion of 9p21in more than 20% of the epithelial cells was alsoconsidered diagnostic for tumour [31].

Reverse transcriptase polymerase chain reaction(RTPCR) methods

HMSH2

The human mismatch repair gene hMSH2 is a DNArepair gene. In bladder washes subjected to reversetranscriptase PCR, hMSH2 mRNA was detected in81% of bladder carcinoma cases. Specificity was 80%[64].

CGβ

Messenger RNA for chorionic gonadotrophin β wastested by Hotakainen et al. [65]. Elevation of CGβ

was only found in 40% of TCC patients, although allcontrols tested negative. It is therefore not sensitiveenough for screening purposes.

Mucin 7 and cytokeratin 20

Retz et al. found the lowest sensitivity and specificityfor cytokeratin 20. They assessed mRNA from in a

series of 50 patients with bladder tumours, and 80controls, including some controls with other urolo-gical malignancies (Renal and prostate carcinoma).The sensitivity of cytokeratin-20 mRNA expressionwas 78%, but it was only 36% specific. Mucin-7 mRNA expression was 66% sensitive and 80%specific for bladder carcinoma [66].

Other authors have found sensitivities of 82–91%and specificities of 67–97% for cytokeratin 20 RTPCR[30, 67–69]. Detection was improved in higher tumourgrades. False positive results in these studies oftenshow atypical histology, raising the possibility that thistest detects precursor lesions also.

Loss of heterozygosity studies (Polymerase chainreaction)

This method of detection uses voided urine samplesto extract DNA from, and then to try and detect dele-tions that are known to occur in bladder carcinoma.Mao et al performed the first study, and found thatmicrosattellite analysis of the urinary sediment wasmore sensitive than urinary cytology, for detectionof bladder carcinoma [4]. Overall, it was found thatconventional cytology detected cancer cells in 50% ofthe samples, but microsatellite analysis showed 95%sensitivity, at one third of the cost, with no false posit-ives. Continuing this work, Steiner et al. [5] followed21 patients with urine samples analysed with a panelof 20 microsatellite markers, finding that recurrencewas detected in 10 of 11 patents, with no positivecontrols. The one false negative was in a patient witha solitary small pTaG1 tumour. Shigyo et al. foundthat LOH on chromosome 9 could be detected in urinewith 70% frequency, compared to 71% for tumour[70]. Friedrich et al. have used PCR to detect p53 loss,by comparing urinary sediment to tumour samples.They found that 34% of tumours showed LOH forthe variable number tandem repeat (VNTR) region ofintron 1 of p53, and that all of these could be detectedurine samples [71]. Detection of p53 mutation has alsobeen described by Prescott et al., using urinary sedi-ment analysis, with a detection rate of 4–19% mutantclones. The low rate may be due to contaminationfrom epithelial cells from normal mucosa [72]. Linnretrospectively looked at LOH in the urine of patientswith TCC, but did not use any control specimens. Hisdetection rate was 87% [73] Mourah’s use of microsat-telite analysis in the detection of bladder cancer finds,

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Table 1. Reported sensitivity and specificity results for microsattelite analysis. The headline rate is the reportedresults. The ‘Actual’ rate excludes patients subsequently found to have bladder tumours after a period of follow-up

Study Number of Number of Headline Actual

patients with control patients Sensitivity Specificity Sensitivity Specificity Positive

tumours % % % % predictive

value%

[4] 20 10 95 100 95 80 86[5] 10 11 91 100 72 100 80[6] 12 47 83 100 83 76 48

[70] 33 20 70 100 70 100 100[73] 15 N/A 87 N/A 87 N/A N/A[74] 28 10 93 100 93 100 100[75] 109 34 89 100 89 100 91[76] 24 69 74 82 74 82 72

Mean 31 29 85 97 83 91 82

once again, that specificity is 100%, with an 83%detection rate [6].

Most recently, Sourvinos et al. have found thatmicrosattellite analysis using markers at the p16,Retinoblastoma and a p53 locus had a sensitivity of93% and was 100% specific. Their study size was28 patients with bladder cancer, and 10 controls [74].In a larger study totalling 153 patients, Zhang et alreported a sensitivity of 89%, and a specificity of100%. Positive predictive value was 91%. False posit-ives in this group included patients with cystitis andhaematuria of unknown origin [75].

However, when the sensitivity and specificityfigures for the urinary detection of bladder cancer util-ising LOH analysis are examined in more detail, theresults are less impressive. Often, the ‘headline’ rateof detection includes tumours that are not found onthe initial cystoscopy, but rather on cystoscopies overa year later. It could be considered that the urinaryepithelium is simply exhibiting chromosomal aber-rations that make tumour development more likely,rather than predicting the development of a tumour denovo.

Table 1 shows the results of the currently publishedpapers on LOH analysis. This includes not only theinitial papers discussed above, but also the subsequentmore recent works. The cited ‘headline’ sensitivityand specificity is recorded, and the actual sensitivityand specificity obtained once tumours that are detectedon the subsequent cystoscopy are excluded. If thestudy did not prospectively examine urine specimensfrom normal controls then ‘N/A’ is recorded.

A frequent finding in the papers is that results,which are initially considered false positives, are thenfollowed up for a period of 12 months, and if a tumouris subsequently detected then the result is consideredto have in fact been correct. As can be seen, theaverage number of patients per study is only sixty.Once tumours that are detected at times other than the1st follow-up cystoscopy after LOH analysis becomespositive are excluded, the mean sensitivity is 81%,specificity is 89% and positive predictive value is 78%.

A possible explanation for the disparity betweenLOH analysis becoming positive and tumour devel-opment was demonstrated in a recent paper. Theyshowed that from a group of 24 patients who under-went transurethral resection of a bladder tumour, thosewho had detectable allelic losses in their urine pre-operatively had a higher recurrence rate than thosewithout detectable LOH [70]. Most interestingly, fourof the six patients tested post-operatively still had lossof heterozygosity. This would be more in keepingwith a field change effect, with the urothelium exhib-iting chromosomal changes that make recurrence morelikely to occur, than a tumour recurring immediatelyafter an operation.

Conclusions

The challenge remains to develop a test that is non-invasive, and 100% sensitive for bladder tumourcompared to endoscopy, with a low false positive rate,and is inexpensive, quick and simple to perform. So

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Table 2. Overall specificities and sensitivities of methods for detection of bladder carcinoma using voided urinespecimens, excluding microsattellite analysis

Study type Reference Sensitivity % Specificity % Mean sensitivity Mean specificity

486p 3/12 [13] 90 52 63 63[14] 36 75

AN43, BB639 [12] 47 89 47 89

bFGF [27] 42 88 42 88

BTA [36] 61 74 59 80[39] 28 87 59 80[50] 100 84

[51] 41 96

[52] 65 x

[54] 51 70

[55] 44 73

[56] 69 68

[58] 68 82

[63] 64 80

[76] 56 79

[77] 56 86

CGβ [65] 42 x 42 x

Cytokeratin 19 [43] 81 97 86 84[44] 97 67

[45] 79 89

Cytokeratin 20 [30] 82 67 86 68[66] 78 36

[67] 87 97

[68] 91 74

[69] 91 67

Cytokeratin 8 and 18 [UBC] [40] 64 73 67 79[41] 61 81

[42] 77 83

Cytology [38] 34 97 41 91[39] 35 90

[41] 44 100

[50] 100 100

[51] 16 x

[52] 32 x

[54] 47 100

[55] 44 95

[56] 35 90

[57] 60 25

[58] 29 93

[63] 32 100

[75] 51 91

[76] 22 95

[77] 19 98

[9] 50 98

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Table 2. Continued

Study type Reference Sensitivity % Specificity % Mean sensitivity Mean specificity

DD23 [16] 59 71 72 83[17] 85 95

Dipstick [38] 41 87 44 86[55] 47 84

FDP’s [38] 68 86 67 84[39] 81 75

[55] 53 91

Fibronectin [41] 83 83 83 83

FISH [31] 81 86 80 94[32] 84 93

[62] 68 100

[63] 85 95

Flow cytometry [42] 77 79 77 79

HMSH2 [64] 81 80 81 80

Hyaluronidase [35] 91 84 91 77[36] 91 70

Immunocyt [10] 100 69 85 76[11] 67 84

[9] 89 75

Lewis X antigen [2] 86 87 87 86[21] 85 85

[22] 97 85

[23] 80 86

M344 [7] 78 92–94 78 92–94

MMP-9 [33] 100 100 100 100

Mucin-7 [66] 66 80 66 80

NMP22 [41] 85 91 75 79[54] 92 77

[55] 74 60

[56] 73 85

[57] 47 79

[58] 82 82

[59] 70 78

Psoriasin [15] 26 85 26 85

Survivin [19] 100 96 100 96

Telomerase [28] 83 80 75 75[29] 85 66

[30] 85 80

[31] 70 46

[55] 52 99

TPA [48] 59 66 59 66

Urinary basic fetoprotein [18] 54 88 54 88

341

far, no test has fulfilled these criteria. The overallresults of the sensitivity and specificities for themethods are shown in the Table 2.

The tests that appear to show average sensitivitiesin reported studies of greater than 90% are Survivin,MMP-9, and hyaluronidase, but the total number ofstudies regarding these methods is small. The tests thathave specificities greater than 90% are Microsattelliteanalysis, M344, survivin, MMP-9, cytology and FISH.The most promising test appears to be microsatteliteanalysis, which has produced consistently high sens-itivity and specificity results in a number of papers.Survivin and MMP-9 have also produced excellentresults, but only in one and two publications each, soprospective studies are required to validate them.

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Address for correspondence: Brian Little, Urology Department,Monklands Hospital, Monkscourt Avenue, Airdrie, ML6 0JS, UKPhone: 0044 1236 748748; Fax: 0044 1236 713136E-mail: b.little@ntlworld.com