bladder cancer: natural history, tumor markers, and early detection strategies

© 1997 Wiley-Liss, Inc.

Seminars in Surgical Oncology 1997; 13:299–306

Bladder Cancer: Natural History, TumorMarkers, and Early Detection Strategies

WILLIAM H. FORESMAN, MD* AND EDWARD M. MESSING, MDUniversity of Rochester Department of Urology, Strong Memorial Hospital,

Rochester, New York

Transitional cell bladder carcinoma is characterized by a dichotomous, multichronotopic naturalhistory. Low and moderate grade Ta lesions frequently recur, yet rarely invade, and carry anexcellent prognosis with currently available treatments. High grade Ta lesions, tumors withlamina propria invasion (T1), and carcinoma in situ often progress to invasive disease, atwhich time overall prognosis is significantly decreased, despite various treatment alterna-tives. Although early detection of bladder tumors, prior to muscle invasion, should vastlyimprove our ability to save both bladders and lives, current methods of detection are neithersufficiently sensitive nor specific. Tumor marker analysis is an exciting new frontier in blad-der cancer evaluation, and may have important applications to early detection strategies, incombination with simple hematuria testing and other selected noninvasive screening meth-ods. Semin. Surg. Oncol. 13:299–306, 1997.© 1997 Wiley-Liss, Inc.

KEY WORDS: bladder neoplasms; carcinoma in situ; transitional cell carcinoma; hematuria;urothelium; tumor suppressor genes; p53 gene; retinoblastoma protein;neoplasm invasiveness; antigens; biological tumor markers; oncogenes

INTRODUCTION

Transitional cell bladder carcinoma (TCC) representsan intriguing entity in many ways. Yearly incidence con-tinues to rise despite improved awareness of cancer-relatedcarcinogens, both at home and in the workplace. An esti-mated 54,500 Americans will be diagnosed with bladdercancer in 1997, with a 22% overall mortality rate of 11,700patients [1]. Recent advances in molecular and genetic tech-niques for the identification and characterization of “tu-mor markers” have contributed greatly to our currentunderstanding of the many intricacies of bladder tumori-genesis. Unlike traditional models of tumorigenesis, whichare based upon a steady progression from low grade, su-perficial disease to high-grade, invasive disease, bladderTCC appears to exhibit a distinctly dichotomous nature.Low- and moderate-grade superficial (Ta) lesions fre-quently recur yet rarely invade. High-grade lesions, de-spite originating within the urothelium, most often initiallyare diagnosed as invasive tumors, with significantly moreominous implications. Tumor marker data suggest that thetwo major forms of bladder cancer may arise from distinctgenetic pathways [2,3].

The multichronotopic nature of bladder cancer is well-known, with multifocal tumor occurrences throughout theurothelium, both concurrently and along a time continuum.This phenomenon has long been attributed to a “field

change,” presumably arising from endogenous or exog-enous carcinogen exposure. Recent molecular genetic stud-ies of bladder tumors, however, support the alternativenotion that in at least some individuals, separate tumorsare genetically related, arising by clonal migration/reimplantation and proliferation of mutated cancer cellswith selective growth advantages [4].

The observation that bladder cancer is rarely found asan incidental finding on autopsy implies a short preclini-cal latency, indicating that early detection is unlikely to beharmful (since the disease will almost always cause symp-toms, requiring diagnostic work-up and therapy) [5]. Mo-lecular “marker” evaluation is a potentially powerfuladjunct to standard histopathologic and cytopathologicanalyses in the early detection, prognostic stratification,and prevention of bladder TCC. This review correlatespresent knowledge regarding the natural history of blad-der TCC using traditional histological and cytological pa-rameters with current understanding of molecular eventsas reflected in detectable molecular markers. Additionally,potential applications to early detection strategies are dis-cussed. A formal discussion of upper tract TCC, which may

*Correspondence to: William H. Foresman, MD, Department of Urology,Box 656, University of Rochester Medical Center, 601 Elmwood Avenue,Rochester, NY 14642. E-mail: [email protected]

300 Foresman and Messing

obviously serve as an additional source of “positive” uri-nary tumor markers, is beyond the scope of this review.

NATURAL HISTORYSuperficial Transitional Cell Carcinoma

Because bladder cancer is virtually never recognized asan incidental finding on autopsy, information regarding itsnatural history largely reflects that of “treated” disease.The most common mode of presentation of bladder canceris moderately to well-differentiated, superficial, papillaryTCC, accounting for an estimated 55–85% of tumor pre-sentations [6,7]. The National Bladder Project Collabora-tive Group A (NBCP) has accumulated information on acohort of patients entered into index-case bladder cancersurveillance protocols limiting treatment to fulguration/resection alone, thereby mimicking the natural course ofdisease as closely as possible. Prout Jr et al. [8] reportedon 178 such patients with stage Ta, grade 1 (TaG1) lesions,followed for a median of 58 months, documenting recur-rences in 61% of patients over the study period. Although27% of patients exhibited grade progression upon recur-rence, stage progression occurred in only 4.5%; tumor sizeand multiplicity correlated with risk of recurrence. Heneyet al. [4] reported on 249 NBCP patients with stage Ta andT1 disease for a median period of 39 months. Only 2% ofgrade 1 tumors progressed to muscle-invasive/metastaticdisease as compared with 11% of grade 2 and 45% of grade3 superficial tumors. Progression occurred within 24months in most cases. 4% of those with Ta disease pro-gressed, as opposed to 30% of those with T1 tumors. Pa-tients with high-grade lamina propria invasion (T1G3) hada nearly 50% risk of progression over this interval.

Thus, conservatively treated low grade Ta lesions tendto recur but rarely invade, while higher grade Ta lesionsand those with lamina propria invasion (especially high-grade, stage T1) are significantly more aggressive whenthey recur.

Carcinoma In Situ (CIS)

The natural history of bladder CIS, which is essentiallygrade 3 nonpapillary TCC that is only a few cell layersthick, has been significantly altered by the widespread useof intravesical chemotherapy and immunotherapy. MostCIS is diagnosed in patients with previously identifiedTCC; fewer than 10% of cases are diagnosed as isolatedlesions in patents without prior or concomitant TCC [9].The risk of subsequent progression is very high in the set-ting of CIS that is managed exclusively by endoscopicsurgery. Prout Jr et al. [10] reviewed the NBCP experi-ence in managing CIS during the pre-Bacille Calmette-Guérin (BCG) era, documenting a dismal long-termprognosis for patients treated conservatively. For example,11 of 12 patents managed with resection alone developedprogression to muscle invasion within 60 months. These

findings have been confirmed in numerous studies, and itseems clear, from clinical evidence alone, that bladder CISis a precursor of invasive disease.

Muscle Invasive Transitional Cell Carcinoma

Muscle invasion is already present in approximately 15–25% of patients with newly diagnosed TCC [6]. The vastmajority of these tumors are of high histologic grade [7].Although roughly 10% of patients with low- and moder-ate-grade superficial tumors, and 25–45% with high-gradesuperficial ones eventually progress to muscle invasivedisease, the majority of patients with muscle invasive TCC(84–90%) have this at their first presentation [11,12]. Thetendency of invasive TCC to be present at the initial tumordiagnosis is especially disturbing, since nearly one-half ofall patients with muscle invasive disease have concomi-tant occult distant metastases. Aggressive surgical inter-vention for regional metastatic disease “cures” only30–35% of patients at best. While this outlook may besomewhat improved by the addition of adjuvant chemo-therapy [13–15], many of these patients eventually expe-rience systemic progression. Once this occurs, the ultimateprognosis is even worse. Indeed, the Intergroup phase IIImethotrexate, vinblastine, adriamycin, cisplatin (MVAC)vs. cysplatin trial involving patients with distant metastases,as well as the more recent Eastern Cooperative OncologyGroup (ECOG) Escalated MVAC trial have demonstratedcomplete remission rates in the MVAC arms of only 13%and 17%, respectively [16]. Corresponding overall survivalwith MVAC therapy was only 12.5 and 9.4 months. In sum-mary, invasive and metastatic tumors currently have a veryguarded prognosis. Because all invasive TCC must beginat the urothelium, it follows that improved methods forearly detection should identify the majority of de novo in-vasive lesions at superficial and highly treatable points intheir natural history. Tumor marker analysis may provideimportant assistance in this regard.

CHROMOSOMAL ANALYSIS AND TUMORSUPPRESSOR GENES

One of the fundamental mechanisms in the process ofcarcinogenesis is the functional loss of tumor suppressorgenes. Many suppressor genes encode proteins necessaryfor DNA repair and regulation of key points in the cellcycle, including those involving cell cycle progression anddirected cell death (apoptosis). Tumor suppressor gene in-activation occurs through mutations or deletions, and re-quires alterations in both chromosomal alleles of putativesuppressor genes. Alterations which occur in a nonrandomfashion most likely reflect a selective growth advantage,either allowing the cell to proliferate more rapidly thanother epithelial cells, to survive homeostatic processes thatusually prevent excessive proliferation, survival of DNAdamaged cells, or induction of cellular sennescence. Clonal

Bladder Cancer: Tumor Marker Applications 301

expansion of these mutated cells occurs. Because many ofthe affected genes involve DNA repair mechanisms or di-rection of cells with damaged DNA toward apoptosis orcell cycle arrest, abrogation of the functions of these genesresults in further genetic alterations and overall geneticinstability in progeny cells. Modern molecular genetic,immunohistochemical, and chromosomal analysis tech-niques have identified many potential tumor suppressorloci and associated gene products which appear related tobladder cancer tumorigenesis. The most well-characterizedand closely associated of these are reviewed below.

Chromosome 9 and p16 (CDKN2)

Nonrandom loss of part or all of chromosome 9 is foundin TCC of all grades and stages [2,6], occurring in 60% ofbladder tumors [17]. Chromosome 9 deletions occur in ahigh proportion of superficial tumors [2,3, review 6], sug-gesting inactivation of a tumor suppressor gene(s) as animportant event in the initiation of tumorigenesis. Thesearch for a tumor suppressor gene has centered aroundthe 9p21 region, where loss of heterozygosity (LOH) ofone of the two inherited alleles of the genes in this regionhas been identified in many tumor types, including breast,lung, and prostate carcinomas and melanoma [18]. Initialfocus on the p16 (p16 INK4, MTS-1, CDKN2) locus withinthis region was hampered by the high frequency of ho-mozygous p16 deletions (i.e., both alleles were lost) in blad-der tumors. The inability to detect homozygous allelicdeletions resulted in detection of only normal cell con-taminants, which had retained the p16 region in bothchromosomal alleles, thereby suggesting retention of het-erozygosity [18]. Microsatellite analysis and deletion map-ping has now definitively placed the putative tumorsuppressor gene at the p16 locus, with exclusion of theclosely positioned and related p15 (MTS2) gene [17,18].The p16 protein product plays a critical role in cell cycleregulation, through the binding and inhibition of cyclin-dependent protein kinases, as detailed below. Chromosome9 may harbor additional bladder cancer suppressor genes,including long arm (9q) foci [5], although these are as yetundefined.

Sidransky et al. [19], analysed X-chromosome inacti-vation and 9q allelic loss in multifocal high grade tumors,most of which were invasive, and found inactivation ofthe same X-chromosome and 9q allele in topologicallydistinct tumors in each of four women. The finding of ge-netically related tumors at different sites supports the con-cept of clonality in at least some cases of bladder cancertumorigenesis. (The aforementioned study involved ahighly select population of patients, most with prior blad-der TCC, which represents only 10–15% of all invasiveTCCs and roughly 4–5% of all TCCs.) Inactivation of tu-mor suppressor genes may promote a selective advantageover normal urothelium, with repopulation initiated either

through cell migration or intravesical seeding. Subsequentadditional genetic events would then transform the cell intoa more invasive state with resultant progression of diseasein some cases [19].

Chromosome 17 and p53

Loss of heterozygosity at 17p is the second most fre-quently detected chromosomal abnormality in bladder can-cer [6], and is identified far more frequently in invasivethan superficial lesions [2,3]. Mutations in the p53 genelocus are observed in over 50% of muscle-invasive blad-der tumors and in CIS, but infrequently in well- and mod-erately-differentiated superficial lesions (a statisticallysignificant finding) [2]. The p53 gene encodes a nuclearphosphoprotein (p53) which is critical for both DNA re-pair of genetically abnormal cells and direction towardapoptotic pathways for selected cells. Alteration of bothp53 alleles results in loss of p53 protein function. In mostcases, the altered p53 protein is not only nonfunctional,but is also degraded far more slowly than wild-type p53.Nuclear accumulation of mutant p53 protein is immuno-histochemically detectable, and generally reflects loss oftumor suppressor function. Nakopoulou et al. [20] foundnuclear accumulation of p53 significantly more often ininvasive tumors on transurethral biopsies than superficialtumors (P < 0.05), although p53 positivity was not an in-dependent predictor of overall survival. Esrig et al. [21],in an evaluation of predominantly high grade, advancedstage cystectomy specimens, found an independent corre-lation between p53 protein accumulation and both recur-rence and overall survival (P < 0.001). Level of mutantp53 expression was an independent predictor of stage pro-gression and survival in 18 patients with CIS evaluated byHudson et al. over a 10-year period [9], and may help de-termine which patients should forego intravesical chemo-therapy and proceed directly to cystectomy.

The role of p53 overexpression as a prognostic indica-tor in patients with superficial bladder cancer is not estab-lished [5]. Nonetheless, chromosome 17/p53 data supportthe concept of alternate pathways of bladder tumorigen-esis. Normal p53 protein function in low- to moderate-grade superficial lesions enables these tumors to maintaingenetic stability through appropriate DNA repair and cell-directed apoptosis, thereby preserving their relatively be-nign course. Absence of functional p53 protein in certainhigh-grade superficial tumors, many CIS lesions, and mostinvasive TCCs presumably promotes genetic instability,continued mutation, increased aggressiveness, and rapidprogression.

Chromosome 13 and pRb

Nonrandom LOH at chromosome 13q14, site of the re-tinoblastoma (Rb) gene, does occur in bladder tumors al-though with a wide range in reported incidences [6]. These


mutations and/or deletions correlate with loss of Rb nuclearphosphoprotein (pRb) expression less closely than 17plosses do with p53 gene mutations and abnormal p53 ex-pression. They can occur in both superficial and invasivetumors, and appear most common in advanced stage le-sions [22]. Although much of the literature supports anassociation between absence of Rb expression and pro-gression/prognosis, findings are conflicting. Of course,since malfunction of this protein removes a normal cellcycle regulator and should therefore encourage prolifera-tion (see below), it would not be surprising if Rb lossesand mutations were associated with TCC. The majority ofinvasive lesions, however, have normal Rb expression.Many tumors with identified 13q LOH maintain normalsecond Rb alleles and produce functional wild-type pRB,casting doubt on the significance of Rb mutations in blad-der cancer [5]. The presence of an additional tumor sup-pressor gene(s) on chromosome 13 to explain thediscordance between cytogenetic and molecular sequenc-ing studies remains unknown.

Tumor Suppressor Genes and Cell Cycle Regulation;Cyclins and Cyclin Dependent Protein Kinases

An appreciation of the complex interactions betweenthe aforementioned tumor suppressor genes and gene prod-ucts, as they relate to the cell cycle and neoplastic growth,is critical to properly understand their role in the develop-ment and progression of bladder cancer. Cordon-Cardo hasintegrated current concepts in a recent review on this topic[23]. The binding of regulatory cyclins, proteins synthe-sized and promptly degraded at specific points in the cellcycle, to cyclin-dependent protein kinases (Cdk) is funda-mental to the process of growth regulation. These cyclin-Cdk complexes are responsible for the phosphorylation ofserine and theonine residues on key substrates, again atspecific points in the cell cycle. In this process, pRB is acentral target. Underphosphorylated pRb may inhibit cellgrowth by binding to growth-promoting DNA binding pro-teins such as the E2F transcription factor. pRb bound E2Fis unable to bind with the promoting regions of genes con-trolling DNA synthesis. Phosphorylation of pRb via acti-vation of cyclin-Cdk complexes occurs in late G1/early Sphase, prompting release of transcription activators suchas E2F, resulting in DNA synthesis, cell cycle inductionand growth.

Wild-type p53 protein induces G1 cell cycle arrest andrepair of “injured” DNA, and directs cells with irreparableDNA abnormalities toward programmed cell death(apoptosis). This function is dependent upon the ability ofwild type p53 to bind to the involved DNA. Mutations inp53’s DNA binding site result in replication of even ab-normal DNA, causing clonal expansion of mutated cells,further genetic mutations, and ultimately genetic instabil-ity [24].

p21, p15, and p16 are members of a newly recognizedfamily of negative cell cycle regulators referred to as Cdkinhibitors. These proteins bind to and inactivate cyclin-Cdk complexes, thereby inhibiting pRb phosphorylationand associated release of transcription activators neces-sary for progression through the cell cycle. For example,activation of the wild-type p53 G1 arrest/apoptotic path-way stimulates production of p21, which in turn inhibits avariety of cyclin-Cdk complexes necessary for Rb phos-phorylation, ultimately halting Rb-mediated cell cycle pro-gression. Various growth factors (discussed below) provideexternally-mediated stimulation of p21 activity with simi-lar effects. p16 binds specifically to Cdk4 and 6, again in-hibiting pRB phosphorylation. p15 and p27 help mediateTGF-b-induced cell cycle arrest. The intricate interrela-tionship of these cell cycle regulatory molecules reflectsthe complexity of tumorigenesis, and is but a sampling ofthe overall process. It is clear, however, that our under-standing of these molecules, which can serve as markersfor several malignancies including bladder cancer, providesa wealth of information about the biology of bladder car-cinogenesis and is of considerable clinical value.

TUMOR MARKERS AND EARLY DETECTIONSTRATEGIES

Overview and Rationale for Early Detection

The unique natural history of bladder TCC lends itselfwell to potential early detection strategies. The paucity ofbladder cancer diagnosed at autopsy suggests that nearlyall patients will eventually present clinically during theirlifetime [5]. All bladder tumors arise at the urothelial sur-face, providing a “window of opportunity” for surgical cureprior to invasion. Low- and intermediate-grade Ta lesionsare likely to recur, but rarely invade. High-grade Ta tu-mors (TaG3), tumors with lamina propia invasion (T1),and CIS are the precursors to invasive TCC, having beendetected early in their natural history. The main challengeof early detection lies in the rapid identification and ag-gressive extirpation of these pre-invasive lesions. Fortu-nately, high-grade superficial TCC and CIS are fairlyeffectively managed with endoscopic resection and BCG.Use of transurethral resection (TUR) alone or with intra-vesical chemotherapy is associated with a 75–90% 3–5 yearprogression rate, while combined TUR and BCG reducesprogression to 25% for CIS and 45% for T1G3 lesions[25,26]. With close follow-up and appropriate cystoscopicsurveillance, ultimate mortality from high-grade TCC/CISis reduced from 50% (once muscle invasion has occurred—T2-T4, NX) to 12–15% (without systemic chemotherapy).Moreover, bladder loss (assuming all those with invasivedisease undergo cystectomy) is reduced from 100% in thosewith pre-existing muscle invasion to 25–45% in thosewith detection prior to muscle invasion. Theoretically, then,early detection and management decreases morbidity and


mortality from bladder TCC three to four times using es-timates from current clinical data. Unfortunately, as notedbeforehand, the majority of high-grade tumors are cur-rently detected only after muscle invasion has alreadyoccurred [11,12].

Messing et al. [7] carried out studies in which healthymen, age 50 and over, tested their urine repeatedly at homewith a chemical reagent strip for hematuria. They demon-strated a reduction in de novo muscle-invasive tumor pre-sentation from 55% of all high-grade bladder cancer (inan age- and geography-matched unscreened population)to 10% of all high-grade bladder cancers detected in thescreened population. Most importantly, disease-specificmortality decreased from 16.4–0%, a statistically signifi-cant finding. Although hematuria screening is highly sen-sitive, only 8–10% of those with positive tests actually hadbladder cancer. This poor specificity has raised doubtsabout the application of this screening regimen for the gen-eral population. However, even as it currently stands, he-maturia screening has been found to be cost-effective bythe standards of screening for chronic diseases [27]. Anadditional noninvasive, sensitive, and specific test or panelof tests could further decrease the frequency (hence mor-bidity and cost) of nontumor-related hematuria evaluationsif only those patients with combined microhematuria andpositive additional noninvasive test(s) were to undergosubsequent cystoscopic evaluation. Unfortunately, the tra-ditional methods of voided urinary and bladder wash cy-tological evaluation are not sufficiently sensitive for thedetection of low- and moderate-grade tumors. Specificityis also limited, with a 12% false-positive rate [5]. Conven-tional flow cytometry and fluorescent image analysis havesimilar drawbacks [28]. Fortunately, awareness of tumormarkers has spurred important advances in the use of thesetechniques, with concomitant improvement in both sensi-tivity and specificity of detection. Tumor marker-basedanalyses hold promise not only as tools for early detectionof primary lesions, but also as a means of surveillance andprognostic stratification following treatment. The mostpromising applications are briefly described below, withonly limited reference to technical aspects and method-ological limitations.

Chromosomal Analysis

Image analysis of bladder tumor tissue with fluor-escently-labeled DNA probes (fluorescent in situ hybrid-ization, or FISH) is a method for detecting chromosomalaberrations that correspond to known tumor marker geneabnormalities. Available chromosomal probes are large,however, limiting detection to chromosomes with multiplegene deletions or duplications. Indeed, a detectable abnor-mality may require deletion of an entire chromosomal arm.The use of cosmid-directed probes has allowed for detec-tion of smaller abnormalities, although significant limita-

tions remain. Other drawbacks include variable chromo-somal polysomy related to normal cell division (tetrasomy),and heterogeneous/incomplete chromosomal representa-tion related to sectioning of individual cells during tissue/slide preparation. The recent use of FISH for analysis ofexfoliated urothelial cells from bladder irrigation speci-mens is an important improvement in technique [29]. Chro-mosome 9 monosomy has been detected in bladder TCCpatients with no apparent clinical evidence of disease, pos-sible indicating “subclinical recurrence” or urothelial sus-ceptibility to recurrence [29]. Use of probes complementaryto known chromosomal tumor marker loci, such as 9p21,may improve the sensitivity of this approach. Evaluationof other chromosomal aberrations, including 17p deletions,is presently underway.

Phenotypic Antigen Expression

It is possible to detect tumor antigens expressed on ex-foliated urothelial cells through the use of monoclonal an-tibody staining. The combination of immunologicalstaining and cytological evaluation increases the detect-ability of pre-malignant and malignant cells. The Lewis X(LeX) blood-group-related antigen is one example of animmunocytologically detectable tumor marker. This anti-gen is not normally expressed on benign urothelial cells inadults, except for occasional umbrella (superficialurothelial) cells. Bladder tumor cells, however, exhibitenhanced LeX expression, independent of secretor status,grade, and tumor stage [review 30]. Immunocytologicaldetection on two voided urine specimens using monoclonalantibodies to LeX (if either the first or second has five ormore positive cells per slide) has a reported sensitivity of97%, specificity of 85%, positive predictive value of 76%,and negative predictive value of 98% in predicting the pres-ence of bladder TCC [30]. Although suggestions that thistechnique may ultimately replace cystoscopic evaluationin the workup of patients with microhematuria are prema-ture, if the high sensitivity and specificity are confirmedin large, blinded, prospective studies, LeX antigen detec-tion may prove useful in early detection regimens and post-treatment monitoring.

The M344 antigen is expressed on roughly 70% of su-perficial bladder tumor specimens. Detection of M344expression in exfoliated urothelial cells may improve uponthe poor sensitivity of conventional cytological evaluationin the detection of low-grade superficial lesions [31]. TheDD23 antigen is absent from normal urothelium but foundin 80% of bladder tumors regardless of stage and grade.This antigen is present on histologically normal urotheliumin some patients’ radical cystectomy specimens, suggest-ing possible detection of early recurrence or increased neo-plastic susceptibility [32]. The T138 Ag is detectible inboth bladder irrigation and tumor specimens in associa-tion with advanced stage lesions as well as aggressive Ta/


T1 lesions, and may improve the detectibility of near-dip-loid tumors missed on conventional flow cytometric analy-sis [33].

Soluble Growth Factors and Receptors

Epidermal growth factor (EGF) is a protein mitogenexcreted in the urine in a biologically active form [34],with proven potent tumor enhancing properties [6]. TheEGF receptor (EGFR) distribution is limited to the basalcell layer in normal urothelium, whereas it is present in allcell layers in both low-grade superficial and high-grade/invasive bladder TCC. The finding of EGFR on cellsthroughout normal-appearing mucosal cell layers in pa-tients with previous bladder TCC or with multiple bladdertumors lends credance to the mucosal “field change” theoryfor multichronotopic disease [34]. Receptor density inmalignant and distant normal urothelium correlates withtumor grade and aggressiveness [34]. EGFR status is anindependent predictor of both survival and stage progres-sion [35]. Urinary EGF concentrations are significantlylower in patients with TCC than in normal controls, possi-bly because of increased binding of urinary EGF byurothelial EGFRs during tumorigenesis [36]. EGF andEGFR are clearly involved in bladder tumorigenesis, andmeasurement of both urinary EGF and urothelial EGFR islikely to provide important prognostic information.

The angiogenic peptide basic fibroblast growth factor(bFGF) is responsible for much of the capillary endothe-lial cell migration activity intrinsic to the urine of patientswith bladder carcinoma [37]. Subjects with bladder can-cer have higher voided urinary bFGF concentrations thannormal controls, although stage and grade correlations havenot been addressed [38]. The bFGF assay is possibly moresensitive than cytology, although less specific.

Urinary autocrine motility factor (AMF) is a solublecytokine capable of inducing pseudopod protrusion andresultant cell motility. AMF is present in urine in higherconcentrations in patients with bladder cancer than inhealthy controls, and levels appear to correlate with stageand recurrence [39]. AMF receptors (AMFR) are foundboth on exfoliated tumor cells and as soluble urinary prod-ucts. In a recent study, Korman et al. [40] found AMFR inall patients with muscle invasive and 80% of patients withsuperficial bladder TCC as compared to only 25% of nor-mal controls. Moreover, AMFR levels appeared to corre-late with tumor grade.

Miscellaneous Early Detection Markers

Urokinase-Type Plasmin Activator (UPa), an importantenzyme involved in tumor invasion and metastasis, ispresent in high quantities in bladder tumor tissue. Althoughassays to detect UPa in voided urine specimens are not yetavailable, preliminary studies involving a small numberof bladder tumor resection specimens have indicated a sta-

tistically significant correlation between high tumor UPacontent and poor survival in patients with superficial TCC.This correlation was independent of stage, grade, multi-plicity, and tumor size in a multivariate analysis [41].

Two bladder tumor antigens are worthy of mention be-cause their detection with commercially available tests ispotentially promising. The Bard Bladder Tumor Antigentest (BTA-Bard) measures the urinary complexes formedby the aggregation of proteolytically fragmented basementmembranes, presumably from sites of tumor involvement.In the most recently reported study, 65% of 60 patientswith Ta, grade 1–3 and CIS lesions were detected with theBTA-Bard test on voided urine as compared with a 32%detection rate for conventional bladder wash cytology [42].Although neither method is sensitive for detecting low-grade superficial lesions, the BTA-Bard test sensitivity forgrade 2 and grade 3 superficial lesions was 71% and 100%,respectively, comparing favorably to the 21% and 60%sensitivity of bladder wash cytology. Both methods werehighly sensitive for detection of CIS. The major drawbackof the BTA-Bard test is the high incidence of false-posi-tive results in the setting of inflammation/pyuria or uri-nary tract trauma from instrumentation or formal biopsy/resection.

The high levels of nuclear matrix protein 22 in the voidedurine of subjects with active urothelial TCC, compared tonormal levels in those subjects without disease, forms thebasis for the Nuclear Matrix Protein 22 test (NMP22-Matritech). This immunoassay may be performed on home-collection urine samples, and is unaffected by the presenceof pyuria and traumatic changes. Soloway et al. [43] per-formed the NMP22-Matritech test on post-TUR voidedurine samples in 90 patients with pathologically confirmedbladder TCC, with a median post-surgical sampling inter-val of 22 days. Test results were correlated with followupcystoscopic/biopsy results (performed at a median of 119days). A highly statistically significant difference betweentest values in those with and without recurrent disease wasfound (P < 0.00005). The NMP22-Matritech test was ab-normally elevated in 70% of all patients with recurrent dis-ease, including all six patients with muscle-invasiverecurrences. A high negative predictive value (86%) wasfound, using a reference value of 10 units/ml. This testappears promising for both detection of disease and post-resection surveillance. A positive post-TUR result mayprompt cystoscopic evaluation sooner than the acceptedthree-month period, with potentially more timely use ofintravesical therapy or repeat resection.

Recent experience with a rapid urine dipstick immunoas-say test for detection of elevated urinary fibrin/fibrinogendegradation products (AuraTek FDP test) in patients un-der surveillance for bladder cancer appears quite promis-ing. In a prospective, blinded, multicenter study, Schmetteret al. found an overall test sensitivity of 68% in 79 sub-


jects with biopsy-confirmed bladder cancer, with a statis-tically significant improvement over that of cytologicalevaluation of urine (34%) or a single hematuria dipsticktest (41%) alone [unpublished observations]. Sensitivityremained significantly higher among 67 subjects with su-perficial disease (63%, vs. 27% and 31% for cytology andhematuria testing respectively). Most importantly, sensi-tivity was maintained for low-grade superficial disease(62%, vs. 8% and 15% for cytology and a solitary hema-turia test, respectively). All 12 patients with muscle-inva-sive disease had positive test results; seven of 24 patients(29%) with false-positive AuraTek FDP test dipstick re-sults developed biopsy-confirmed bladder tumors at a meanfollow-up of 8.8 months. Test specificity was 96% in 73healthy subjects, and remained above 75% in subgroupsof 232 subjects with various non-bladder cancer urologi-cal diseases, including urinary tract infection, nonbladderurological cancers, and benign prostatic and renal disease.In summary, the AuraTek FDP test may allow for efficient,cost-effective detection of bladder tumors independent ofgrade, stage, or benign urological disease.

Microsatellite Analysis and Future Directions

Microsatellite analysis is possibly the most powerfulrecent development in bladder tumor marker research, andhas far-reaching implications for early detection strategies.Microsatellites are stably inherited short tandem repeatDNA sequences that are unique to each individual and havevery low inherent mutation rates [44]. Because mechanismsof DNA repair and apoptotic pathways are often disruptedin malignantly transformed cells, DNA mutations and/ormiscopies are often both tolerated and encouraged. Mi-crosatellite analysis allows rapid comparison of polymerasechain reaction (PCR)-amplified peripheral white blood cell,urine, and tumor DNA with a preselected panel of micro-satellite loci that may, for example, include known tumorsuppressor genes or other tumor marker gene loci. Muta-tional copying errors in normally conserved microsatelliterepeats are detectable in exfoliated tumor cell DNA, andwill correspond to those found in tumor tissue DNA, bothdiffering from the genetically stable normal tissue/bloodDNA. Deletions of genetic loci, seen as LOH by molecu-lar analyses, are also readily detected by microsatelliteanalysis. Mao et al. [45] utilized this method with a 10-microsatellite locus panel, including four dinucleotidemarkers from the 9p21 region, to compare urine, tumor,and blood DNA in 25 patients with cystoscopically suspi-cious bladder lesions. Identification of both LOH and mi-crosatellite alterations with this technique resulted indetection of 95% of the 20 histologically confirmed blad-der cancers in the study group as compared to a 50% de-tection rate by routine cytological analysis. Further workin this area holds great promise for tumor detection andsurveillance.

CONCLUSIONS

Recent advances in bladder tumor marker research havevastly improved our awareness of the natural history ofbladder tumorigenesis, and present the clinician with pow-erful tools for early detection, prognostic stratification,post-treatment surveillance, and perhaps new treatmentstrategies. Chromosome 9 alterations are important in theinitiation of noninvasive, low-grade bladder tumors. p53is associated with CIS and invasive progression, while RB,oncogenes, and other tumor markers appear to occupy morecentral roles, as yet not well defined. The crucial role ofcell cycle regulation in tumorigenesis is well-known, andrecent work has linked various tumor marker substancesto specific points in the cell growth continuum. The natu-ral history of bladder cancer—which is both dichotomousand multichronotopic—lends itself well to the use of tu-mor markers in early detection protocols. Investigators havedemonstrated the benefits of early detection strategies, al-though not in a randomized, prospective fashion. Refine-ments in marker detection assay technique, cost, andavailability are sure to prompt widespread use of the mostpromising methods of detection. Microsatellite analysisappears to hold great potential for efficient tumor markerdetection, and likely will play an important role in futuretumor marker research. With a combination of screeningmethods, perhaps initiated with hematuria testing and fol-lowed by one or several other selected tests, all bladderTCCs can be diagnosed prior to the occurrence of muscleinvasion, leading to a much greater chance of saving bothbladders and lives.

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bladder cancer: natural history, tumor markers, and early detection strategies

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