Biological markers in the diagnosis of recurrent bladder cancer: an overview

Review

10.1586/14737159.8.1.63 © 2008 Future Drugs Ltd ISSN 1473-7159 63www.future-drugs.com

Biological markers in the diagnosis of recurrent bladder cancer: an overviewExpert Rev. Mol. Diagn. 8(1), 63–72 (2008)

Aza Mohammed†, Ziauddin Khan, Ignacio Zamora and Aftab Bhatti†Author for correspondenceUrology Unit, North Tees University Hospital, Hardwick Road, Stockton on Tees, TS19 8P, [email protected]

Over the last years, many biological markers emerged that act as an adjunct for the detectionof early bladder cancer recurrence. Some of these markers had a proven better sensitivity andspecificity for cancer detection than urine cytology. Nevertheless, the majority of thesebiological makers are still in advanced stages of clinical trials and are not used for routineclinical purposes. In this article, we give an overview on the biological markers that can play arole in early bladder cancer detection and can perhaps subsequently reduce the need forfrequent cystoscopies.

KEYWORDS: biological marker • bladder cancer • diagnosis • recurrence • surveillance

Bladder cancer is the second most common gen-itourinary malignancy after prostate cancer. It isthe fourth most prevalent cancer in males andthe eighth most prevalent cancer in females,accounting for 6 and 2% of the newly diag-nosed malignancies in males and females,respectively [1].

Bladder cancer is usually stratified into twobroad categories: papillary superficial and solidinvasive tumors. Superficial tumors are the mostfrequent of all newly diagnosed bladder cancers,comprising 75%. This type is often diagnosed inan early and noninvasive stage (stage Ta), withmild forms of cellular dysplasia (low grade). Pro-gression to muscle-invasive disease occurs in lessthan 5% of patients with Ta and up to 50% ofpatients with T1 bladder cancer [2].

Solid tumors are often of high grade and usu-ally diagnosed in an invasive stage (T1 or worse).In more than half of the cases, muscle invasion isdiagnosed upon presentation (stage T2 orworse). They are believed to arise from flat,highly dysplastic but noninvasive lesions calledcarcinoma in situ (CIS; stage Tis). However, CISis associated with minimal symptoms and conse-quently it is seldom diagnosed as the primarylesion (only 10% of cases). Concomitant CIS ismore common, and may be found in up to 40%of stage T1 cancers and in 50% of the muscleinvasive stages. The 5-year survival of patients isless than 50% for muscle-invasive disease and10% for metastatic disease [3].

In the WHO 2004 classification, urotheliallesions are described as the following [4]:

• Normal urothelium

• Hyperplasia– Flat hyperplasia– Papillary hyperplasia

• Flat lesions with atypia– Reactive (inflammatory) atypia– Dysplasia– CIS

• Papillary neoplasms– Papilloma– Papillary urothelial neoplasms of low malig-

nant potential– Papillary carcinoma (low grade)– Papillary carcinoma (high grade)

Bladder cancers are histologically graded into [5]:

• Grade I: Well-differentiated tumors

• Grade II: Moderately differentiated tumor

• Grade III: Poorly differentiated tumors

Occupational exposure to certain chemicals(β-naphthylamines, xenylamine, 4-nirtobiphenyland benzidine) that are found in the textile,leather and rubber industry and smoking areamong the most common risk factors for bladdercancer. Other risk factors include pelvic irradia-tion, chronic cystitis (including biharziasis) andcyclophosphamide therapy [6].

Genetic factors may play a role in the patho-genesis of bladder cancer. Superficial bladder

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64 Expert Rev. Mol. Diagn. 8(1), (2008)

Review Mohammed, Khan, Zamora and Bhatti

cancer has at least two discrete pathways for development andprogression, which may explain the differences in the invasiveand metastatic potential of the disease. Constitutive activationof the receptor tyrosine kinase (RTK)–Ras pathway and dele-tion of chromosome 9 are mainly responsible for early develop-ment of papillary Ta superficial bladder cancer. The former cat-egory includes mutation of fibroblast growth factor receptor-3(FGFR3), found in 60–70%, and HRAS, in 30–40% of thedisease [3].

The main clinical presentation of bladder cancer is hematuria(>80% of patients). It is usually macroscopic and painless. Itcould occasionally present as asymptomatic microscopic hema-turia or lower urinary tract symptoms (such as frequency,urgency and dysuria). In less than 10% of cases, the disease isdiagnosed incidentally on routine radiological examination [6].The mainstay of diagnosis is the identification malignant cells inurine specimens and direct visualization of the bladder mucosawith cystoscopy and resection of the tumor if identified.

Bladder cancer is characterized by the high rates of recurrence(50–80%). The 3-year recurrence-free survival rate is higher inTa disease than in T1 disease (50 vs 30%). The rates are alsoaffected by tumor grade, with 50% in G1 disease, 40% in G2,and 20% in G3. The pathological stage affects the progressionrate of disease to the muscle layer. In Ta disease, the 3-year pro-gression-free survival rate is 96%, whereas survival decreased to70% in T1 disease. The rate of G3 disease (55%) is differentfrom that of G1 (100%) and G2 (89%) disease. Thus, the

pathological stage and grade are factors predictive of recurrenceand progression in short- or intermediate-term follow-up ofsuperficial bladder cancer. Tumor size, multiplicity, CIS andtumor stage and grade are listed as clinical and pathologicalfactors affecting recurrence and progression of the disease [7].

Strict surveillance of patients with bladder cancer is essentialto detect early recurrence. Surveillance is usually by urine cyto-logy and cystoscopy. The frequency of check cystoscopies varyaccording to the stage and grade of the tumor [8]. Patients withlow-risk cancers (TaG1) should have their first cystoscopy at3 months. If negative, then the next cystoscopy is advised at9 months and yearly afterwards for 5 years. High-risk patientsshould have a check cystoscopy every 3 months for the first2 years, then every 4 months in the third year. Later on, theyare checked every 6 months for the following 2 years, andyearly afterwards. Yearly imaging using intravenous urogram orcomputed tomography scan is recommended [9].

This vigilant surveillance makes bladder cancer the mostexpensive in terms of terms of follow-up and treatment of com-plications. Annual direct costs of treating bladder cancer in theUSA alone are estimated at approximately $4 billion, and thecost from diagnosis with bladder cancer to death is estimated tobe $96000–187000 per patient [10,11].

Urine cytology is regarded as the gold standard noninvasivemethod for diagnosing bladder cancer. It has been used eitheras a voided or bladder-washout specimen as an adjunct tobladder cancer surveillance. Urine cytology is a sensitive testfor the detection of recurrence of high-grade, superficial andmuscle-invasive bladder cancer. However, it has low sensitiv-ity (30%) for low-grade tumors. It also requires an experi-enced cytopathologists for interpretion of the results. There-fore, it is of limited use as a reliable means of diagnosing low-grade bladder cancer recurrence. In addition, false-positiveresults could be produced in patients receiving intravesicalchemotherapy [12].

Biological markers of bladder cancer: practical aspects

Several biological markers have emerged in recent years to aidthe early detection of tumor recurrence and prognosis (BOX 1).In order for a test to be suitable for diagnostic or mass screen-ing purposes, it should have a reasonably good sensitivity andspecificity:

• Sensitivity is defined as a statistical measure of how well atest correctly identifies a condition. The results of thescreening test are compared with some absolute (gold stan-dard); for example, for a test to determine whether a personhas a certain disease, the sensitivity to the disease is the prob-ability, that if the person has the disease, the test will be pos-itive. Thus, sensitivity is the proportion of true positives ofall diseased cases in the population:

Box 1. Biological markers in bladder cancer.

• Nuclear matrix protein-22

• Bladder tumor antigen (BTA; BTAStat™ and BTATrack™)

• Microsatellite instability assays

• Fluorescent in situ hybridization (FISH; UroVysion™)

• Immunocytology (ImmunoCyt™)

• Fibrinogen degradation products (Aura Tek FDP Test™)

• Telomerase activity

• Cytokeratins

• Soluble Fas

• Urokinase-type plasminogen activator

• Bladder cancer antigen-4

• Growth factors:- Fibroblast growth factor- Epidermal growth factor

• Hyaluronic acid and hyaluronidase

• Survivin

• E-cadherin

• Matrix metalloproteinases and their tissue inhibitors

• Tumor-associated trypsin inhibitor

• Blood group antigens (Lewis X)

• Mucin 7

• Prostate stem cell antigen-14 Sensivity True positiveTrue positive False negative+------------------------------------------------------------------------=

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• Specificity is a statistical measure of how well a test correctlyidentifies the negative cases, or those cases that do not meetthe condition under study. For example, given a test thatdetermines whether a person has a certain disease, the speci-ficity of the test to the disease is the probability that the testindicates negative if the person does not have the disease.Therefore, the specificity is the proportion of true negativesof all negative cases in the population:

Another important issue to consider is that some novelbiomarkers are still in the developmental process for clinicaluse in the management of bladder cancer patients. There-fore, it is essential to define the process through whichmarkers are developed. While individual studies do notoften clearly define the particular developmental phase ofthe marker under examination, some observers have laid outspecific phases of biomarker development that can give theclinician a sense of the potential clinical applicability forthat test. The phases of the development of a test include thefollowing [13]:

Phase I studies: including preclinical or exploratory studiesIn this phase, initial iterations of assays for candidate biomark-ers are piloted, with the immediate-term goal of evaluating areproducible assay in a representative target population toestablish feasibility for a candidate marker.

Phase IIPhase II studies provide a greater depth of evaluation for clini-cal utility, with definition of standards and refinement ofhypotheses to facilitate the design of studies of greater rigor forsubsequent evaluation. In addition, the performance character-istics of a test (sensitivity and specificity) are usually furthercharacterized.

Phase IIIPhase III studies search to confirm and replicate Phase II find-ings, testing developmental hypotheses with larger numbers ofpatients and, hence, greater statistical power. These are often per-formed in more defined clinical settings, typically utilizing thelarge number of samples available in retrospective longitudinalrepository studies.

Phase IVPhase IV studies represent the culmination of the develop-ment process, where large prospective studies are designedwith several immediate goals. First, results from earlier phasestudies are validated and, second, the practical technologytransfer aspects of the application are defined in a broader set-ting, with the ultimate goal of incorporating a new markerinto clinical practice. It is at this stage that the test is submittedfor US FDA approval.

Bladder cancer markers in clinical practiceNuclear matrix protein-22Nuclear matrix protein-22 (NMP22) is responsible for chro-matoid regulation and cell separation during duplication. It isreleased from the nuclei of tumor cells during apoptosis and itsexpression is significantly higher in bladder cancer tissue thannormal bladder urothelium. NMP22 is a quantitative immuno-assay performed by referring the urine specimen to a referencelaboratory. A cut-off point of 6–12 IU/ml is usually essentialfor optimizing the test performance [14]. NMP22 gained FDAapproval for use in screening for bladder cancer recurrence in1996. In a multicenter randomized controlled trial by Gross-mand and coworkers, the sensitivity and specificity of NMP22alone for the detection of bladder cancer were 55.7 and85.7%, respectively [15]. Later on, another study by the samegroup to evaluate the role of NMP22 for the detection ofrecurrent bladder cancer showed the sensitivity and specificityto be 49.5 and 87.3%, respectively. In addition, the sensitivityof the combination of NMP22 and cystoscopy for the detec-tion of bladder cancer was superior (99%) to the sensitivity ofthe combination of cytology and cystoscopy. In two separatestudies by Grossman and coworkers, NMP22 increased theaccuracy of cystoscopy in detecting both new and recurrentbladder cancers [16,17].

Shariat and coworkers developed nomograms that canaccurately predict the probabilities of disease recurrence andprogression in patients with Ta, T1 and Tis bladder cancer.They incorporated urinary NMP22 levels, urine cytology,patient age and gender in their nomograms, which wereinternally validated and proved accuracy of over 80% [18].

Bladder tumor antigen (BTAStat™ & BTATrack™)The original bladder tumor antigen (BTA) test is a latex agglu-tination test that detects basement membrane degradationcomplexes. Two new versions of this test emerged recently(BTA-Stat™ and BTA-Track™), which measure the humancomplement factor H in urine. BTA-Stat test is a qualitativeassay performed as a point-of-care test at the bedside and BTA-Track is a quantitative ELISA test that requires sending theurine sample into a reference laboratory. In a series of studies,the sensitivity of BTA ranged from 32 to 74% while its speci-ficity was 40–96%. The sensitivity and specificity of the origi-nal BTA test was comparable to urine cytology [11]. However,BTA-Stat showed slightly higher sensitivity, but much lowerspecificity, than urine cytology [19].

Microsatellite instability assaysMicrosatellites are short tandem repeats of DNA that are dis-tributed throughout the genome. They are unique for eachindividual. Tumors with microsatellite instability (MSI) tend toaccumulate errors at a much higher rate than other sequences inthe genome because of the defective DNA repair. These errorsappear as mutational copy errors and deletion of gene loci

Specificity True negativeTrue negative False postive+-----------------------------------------------------------------------=



known as loss of heterozygosity (LOH). MSI can be detected byexamining microsatellite repeat fragments, as the numbers ofrepeats in some of the microsatellites can change due to thedefective DNA repair [20]. MSI is a key factor in several cancers,including bladder, colorectal, endometrial, ovarian and gastriccancers. Since bladder cancer is associated with abnormalities inchromosome 9, MSI has been identified as LOH near theP region of chromosome 9.

Most of the studies demonstrated the sensitivity of MSI forthe detection of tumor recurrence to be in the range of 83–95%and a specificity of 100% using bladder washouts and urinecytology specimens [11].

MSI could be performed on stored urine specimens and it hasthe ability to detect tumor recurrence months before it becomespositive on cystoscopic examination [21]. However, inflamma-tory conditions of the bladder could produce false-positive MSIresults [22].

Fluorescent in situ hybridization (UroVysion™)Fluorescent in situ hybridization (FISH) detects urothelialtumors based on chromosomal alteration, as opposed to themorphologic changes identified by cytology. FISH consists offluorescent probes to centromeres on chromosomes 3, 7, 17and 9p21 to identify chromosomal anomalies (i.e., aneuploidy)known to be associated with bladder cancer. Because thismethod relies upon chromosomal abnormalities and not cellmorphology, this technology should allow improved discrimi-nation of malignant cells from cellular changes associated withinflammatory processes. This test requires intact urothelial cellsfor analysis and specially trained laboratory staff. It gained FDAapproval in 2005 [14].

FISH was found to have a sensitivity 84% (range: 73–92%)and specificity of 95% (range: 92–100%). The cut-off for posi-tive results in various studies was found to be a over 10 or over20 aneupolid cells [9]. Its sensitivity was found to be superior tocytology and at least equivalent to BTAStat™ for the detectionof recurrent bladder cancer [23].

FISH could be of great value in patients with confirmed tumorrecurrence and equivocal or negative urine cytology. In a studyby Skacel and coworkers, 60% of patients with biopsy-provenbladder cancer and negative urine cytology were positive forFISH. More significantly, false-positive FISH results in patientswith negative bladder biopsies may represent an early geneticalteration predictive of later bladder cancer development [24].

FISH also has value in monitoring patients with superficialbladder cancer who receive intravesical chemotherapy. Thepresence of positive results by the end of the treatment periodindicate high risk of progression into muscle-invasive bladdercancer [25].

Immunocytology (ImmunoCyt™)ImmunoCyt™ is a panel of three fluorescent monoclonal anti-bodies (19A211, M344 and LDQ10). The test is FDAapproved and is used in conjunction with urine cytology [13].

ImmunoCyt has a sensitivity and specificity for the detection ofrecurrent bladder cancer of 86.1 and 79.4%, respectively [26]. Itwas found to increase the sensitivity of urine cytology from 23to 81% and it is more sensitive to low-grade tumors than cyto-logy [27]. In a different study, the sensitivity for combined cyto-logy and ImmunoCyt was 79.3, 90.9 and 98.9% for Grade 1, 2and 3 tumors, respectively [28]. Pfister and coworkers similarlyshowed an increase in the sensitivity of combined urine cytologyand ImmunoCyt in the detection of low- as well as high-gradebladder tumors [29].

Fibrinogen degradation products (Aura Tek FDP Test™)The Aura Tek FDP Test™ is based on the principles that bloodcells as well as plasma proteins such as fibrin are constantly shedfrom bladder tumors. This is caused by the effect of vasculargrowth factors produced by the tumor cells. FDP levels tend tobe proportional to the tumor grade and stage. Aura Tek FDPTest is an ELISA test that detects FDPs in the urine with a sen-sitivity and specificity of 81 and 75%, respectively [30]. It isFDA-approved for the detection of bladder cancer recurrence.

Telomerase activityTelomerase is ribonucleoprotein with reverse transcriptaseactivity. It consists of tandem TTAGGG repeats located at theends of chromosomes, which often undergo degradation at theend of each cell cycle. The gradual loss of these repetitivesequences causes chromosomal instability and subsequent celldeath. The enzyme telomerase is involved in the synthesis ofthese telomeric sequences, thereby protecting chromosomalends from mitotic senescence. Telomerase appears to be activein transforming normal somatic cells into immortalized tumorcells. Telomerase activity is tested by using telomerase repeatamplification protocol (TRAP).

Telomerase was found to have sensitivity and specificity of 75and 86%, respectively [9]. In a different Phase II study by San-chini and coworkers, the sensitivity and specificity of telo-merase activity were 90 and 88%, respectively [31]. Eissa andcoworkers compared the effectiveness of telomerase againstmatrix metalloproteinase-9 and cytology in both voided urineand bladder-wash specimens. The overall sensitivity and speci-ficity of telomerase were 83 and 88.6%, respectively, when usedalone. However, when combined with cytology and matrixmetalloproteinase-9, sensitivity improved to 95% and negativepredictive value improved to 91%. Of interest in that study wasthat bladder cancers in patients with past history of bilharziasisdemonstrated higher positive values for telomerase than thenonbilharziasis counterparts [32].

The catalytic subunit of the telomerase complex (humantelomerase reverse transcriptase [hTERT]) was analyzed byMellissourgos and coworkers using a reverse transcriptase PCRassay. The test was performed on spontaneously voided urinesamples from patients with bladder cancer and controls andcompared with the results of standard urine cytology. Expres-sion of hTERT mRNA was detected in 92% of the bladder



cancer patients, which was significantly better than the sensitiv-ity of cytology in this study (44%). This advantage in sensitiv-ity was most pronounced in the detection of low-grade bladdercancer [33].

CytokeratinsCytokeratins are intermediate filament keratins found in theintracytoplasmic cytoskeleton of epithelial tissue. There are twotypes of cytokeratins: low-molecular-weight (Type I or acidic)and high-molecular-weight (Type II or basic) cytokeratins. Thesubsets of cytokeratins that an epithelial cell expresses dependmainly on the type of epithelium, the moment in the course ofterminal differentiation and the stage of development. This testis performed using ELISA techniques. In a study by Parienteand coworkers, the use of cytokeratin-19 (CYFRA 21-1)achieved a sensitivity and specificity of 96 and 74%, respec-tively [34]. Cytokeratin (CK)20 has a superior sensitivity andspecificity to urine cytology: 65 and 91% versus 54 and 86%,respectively [35]. A strong correlation between recurrent superfi-cial bladder cancer and the level of CK20 was found by Chris-toph and coworkers. This suggests a possible use for the plan-ning of check cystoscopies for patients, because those withlower risk of tumor recurrence will have a longer intervalbetween cystoscopies [36].

Soluble FasSoluble Fas (sFas) is one of the major regulators of programmedcell death or apoptosis. Fas is a member of the TNF receptor(TNF-R) family and can occur as both a cell-surface and a sol-uble protein (sFas) [37]. The interaction between Fas ligand(FasL) and cell surface Fas induces apoptosis in sensitive cellsthrough the activation of an intracellular Fas-associated deathdomain, the recruitment of caspase-8 and the activation of acascade of proteases, resulting in proteolysis and degradation ofchromosomal DNA. sFas is usually generated by alternativemRNA splicing and lacks a transmembrane domain. By bind-ing to FasL, sFas is thought to inhibit cell-surface Fas–FasL sig-naling and downregulate Fas-mediated apoptosis. Bladder can-cer is characterized by increased tissue and serum levels of sFas.Urinary sFas was recently found to be an independent predic-tor of bladder cancer recurrence and invasiveness, with a sensi-tivity range of more than 75% [38]. In a different study con-ducted in Japan by Mizutani and coworkers, elevated serumlevels of sFas and sFasL were found to be predictive for bladdercancer recurrence in patients with Ta disease [39].

Urokinase-type plasminogen activatorUrokinase-type plasminogen activator (uPA) is a serine pro-tease enzyme that catalyzes the conversion of plasminogen toplasmin, which in turn facilitates basement membrane diges-tion. The enzyme also activates matrix metalloproteases andgrowth factors involved in tumor invasion and metastasis [40].In a study by Shariat and coworkers, uPA was added toNMP22 and cytology, and was found to substantially improve

their ability to predict bladder cancer. In addition, the intensityof urokinase plasminogen activator receptor expression was sta-tistically more pronounced in high-grade bladder cancers. Thisfinding could potentially make uPA a good prognostic factorfor bladder cancer [41].

Bladder cancer antigen-4This is nuclear structural protein expressed in bladder cancer.Sequence analysis of BLCA-4 reveals that it is a member of theETS transcription factor family and that it seems to associatewith transcription factors. BLCA-4-overexpressing clonesexhibit a 4.3-fold greater proliferation rate than vector-onlycontrols or untransfected cells. Microarray analysis comparinggene expression patterns between overexpressing clones andvector-only controls revealed that numerous genes were upreg-ulated in cells that overexpress BLCA-4. Upregulated genesincluded IL-1α, IL-8 and thrombomodulin, and the proteinexpression of these genes was confirmed by immunoblots. Thisinformation has provided a potential model of BLCA-4 action.Overexpression of BLCA-4 appears to increase the growth ratein cells and also causes cells to express a more tumorigenic pheno-type. Immunohistochemical studies revealed overexpression innormal cells adjacent to bladder cancer, but not in urothelialcells in a normal bladder [42]. Van Le and coworkers studiedthe use of second-generation sandwich immunoassay BLCA-4in the detection of recurrent bladder cancer and found thesensitivity and specificity to be 89 and 95%, respectively [43].

Growth factorsFibroblast growth factorFibroblast growth factor is composed of two subtypes. Theacidic subtype (aFGF) is associated with cell proliferation, dif-ferentiation, angiogenesis and motility. Patients with bladdercancer have increase urinary levels of aFGF and it can be uti-lized as a prognostic test for low grade tumors [44]. Basic fibro-blast growth factor (bFGF) promotes endothelial cell prolifera-tion and the physical organization of endothelial cells intotube-like structures. It thus promotes angiogenesis. bFGF isalso found in increasing levels in patients with bladder cancerthan normal subjects with a sensitivity that is superior to urinecytology. Van Oers and coworkers developed a fast assay for thesimultaneous detection of FGFR mutations, which can be usedfor follow-up of patients with bladder cancer as an alternativeto cystoscopy. The sensitivity for the detection of mutationswas 62% [45].

Epidermal growth factorEGF plays an important role in the regulation of cell growth,proliferation and differentiation. It acts by binding with highaffinity to epidermal growth factor receptor (EGFR) on the cellsurface and stimulating the intrinsic protein tyrosine kinaseactivity of the receptor. The tyrosine kinase activity in turninitiates a signal transduction cascade that results in a varietyof biochemical changes within the cell: a rise in intracellular



calcium levels, increased glycolysis and protein synthesis. Inaddition, it increases the expression of certain genes includingthe gene for EGFR, that ultimately lead to DNA synthesis andcell proliferation. A study by Politi and coworkers foundincreased expression of EGF antigen receptors in higher stagebladder cancer. However, these results are yet to be confirmedin further studies [46].

Hyaluronic acid & hyaluronidaseHyaluronidase is a class of extracellular matrix-degradingendoglycosidases that regulate tumor invasion, metastasis andangiogenesis. It degrades hyaluronic acid (HA), a free non-sulfated glycosaminoglycan. The limited degradation of HA byhyaluronidase results in the generation of HA fragments of spe-cific lengths that are angiogenic. Urinary HA and hyaluro-nidase are measured using ELISA-like techniques and have82% sensitivity and 90% specificity [47].

SurvivinSurvivin belongs to a family of proteins that serve as inhibitors ofapoptosis. Survivin inhibits apoptosis by blocking activation ofeffector caspases in both extrinsic and intrinsic pathways ofapoptosis. Expression of survivin has been demonstrated in sev-eral malignant neoplasms and is generally associated with adverseprognosis. In bladder cancer, survivin is expressed in the neo-plastic epithelium, but not in the normal mucosa. Survivin ismeasured using immunoassay techniques. A study by Smith andcoworkers investigated the potential suitability of survivin detec-tion in urine as a novel diagnostic molecular marker of bladdercancer. Survivin was detected in the urine samples of all patientswith new or recurrent bladder cancer. It was not detected in theurine samples of patients treated for bladder cancer with negativecystoscopy results. In addition, none of the healthy volunteers orpatients with prostate, kidney, vaginal or cervical cancer haddetectable survivin in urine samples. Patients with low-gradebladder cancer had significantly lower urine survivin levels thanpatients with CIS (p = 0.002) [48]. A recent study by Shariat andcoworkers further evaluated the levels of urinary survivin in com-parison with urine cytology and NMP22, and it was found tohave higher sensitivity and specificity of 64 and 93%, respec-tively. It was also found that survivin has a strong associationwith high-grade disease, including CIS [49].

E-cadherinE-cadherin is a calcium-dependent cell-to-cell adhesion mole-cule expressed exclusively by epithelial cells. E-cadherin is theprimary mediator of calcium-dependent cell–cell adhesion inepithelial tissues. Loss of E-cadherin-mediated cell adhesioncorresponds to the transition from well-differentiated adenomato invasive carcinoma, supporting its role as a rate-limiting stepin the progression to invasive disease.

Soluble E-cadherin (sE-cadherin) is detected as a proteolyticcleavage product of the E-cadherin peptide from the cell surface.sE-cadherin is found in the circulation of normal individuals,

but it is particularly elevated in patients with various malignan-cies, including transitional cell carcinoma of the urinary bladder[50]. Loss of or decreased expression of E-cadherin in bladdercancer tissue has been associated with advanced stage, highergrade, metastasis and a higher probability of cancer progressionand death.

In a study by Shariat and coworkers, urinary sE-cadherinlevels were significantly higher in patients with bladder cancerthan in normal controls. In addition, higher urinary levels ofsE-cadherin were associated with positive urinary cytology assayresults and muscle-invasive bladder cancer stage. sE-cadherinwas more sensitive but less specific than cytology (71.3 and65% for sE-cadherin vs 53.8 and 89.8% for urine cytology,respectively) for the detection of recurrent bladder cancer [51].

Matrix metalloproteinases & their tissue inhibitorsTissue inhibitors of metalloproteinase (TIMPs) are zinc-depen-dent endopeptidases that belong to a larger family of proteasesknown as the metzincin family. Collectively they are capable ofdegrading all kinds of extracellular matrix proteins, but can alsoprocess a number of bioactive molecules. They are known to beinvolved in the cleavage of cell surface receptors, the release ofapoptotic ligands (e.g., FasL), and chemokine inactivation.MMPs are also thought to play a major role in cell behaviorssuch as cell proliferation, migration (adhesion/dispersion), dif-ferentiation, angiogenesis, apoptosis and host defense. The bal-ance of secreted MMPs and some endogenous proteins that actas their specific inhibitors (TIMPs) plays an important role inmaintaining connective tissue homeostasis in normal tissues. Intumors, the imbalance of MMPs and TIMPs leads to an excessof degradative activity, which causes the invasive character oftumor cells [52]. MMPs are divided into several families accord-ing to their target protein. MMP1 (collagenase), MMP2 (gelati-nases), MMP3 (stromelysin) and MMP9 are described as beingassociated with bladder cancer [53–56]. Activated MMP1,MMP3 and latent forms of MMP2 and MMP9 bind to and areinhibited by TIMP1 and TIMP2. Elevated concentrations ofMMP2 and MMP3 were found in patients with advanced blad-der cancer in comparison with the serum of patients with super-ficial tumors [57]. In their study, Staack and coworkers con-cluded that MMP2 is a statistically significant plasma markerfor bladder cancer detection with an increased diagnostic valuein combination with MMP9 and TIMP1. They also found thatthe markers with the highest diagnostic values do not reach thesensitivity and specificity that other markers would show in acombination. The clinical value of separately used MMPs andTIMPs as tumor markers in blood plasma is limited [58].

Tumor-associated trypsin inhibitorTumor-associated trypsin inhibitor (TATI) is a peptide thatinhibits trypsin and acrosin. It is expressed at lower concentra-tions in several healthy tissues, such as gastrointestinal, pancre-atic, lung, hepatic, breast, renal and bladder epithelium.Cancers originating from these tissues have been shown to



express increased tissue, urinary and serum levels of TATI [59].Urinary TATI levels are elevated in patients with bladder can-cer, even in the presence of normal urine cytology. It is associ-ated with decreased survival in patients with superficial andinvasive bladder cancer. In addition, higher urinary levels ofTATI were associated with a positive urine cytology assay resultand higher urinary NMP22 levels. Moreover, patients withinvasive tumor stage (>T1) had higher TATI levels than thosewith Ta disease or CIS. Patients with advanced bladder cancer,the serum levels of TATI are markedly reduced following par-tial or complete response to systemic chemotherapy. UrinaryTATI was found to have a sensitivity that is comparable toMNP22, but with a higher specificity. However, serum TATIlevels have been shown to have a stronger association with blad-der cancer survival than urine levels. In addition, renal functionand the presence of inflammation affect urinary TATI levelssince it is freely filtered by the glomerulus and may also act asan acute-phase reactant [60].

Blood group antigens (Lewis X)Lewis X is a tetrasaccharide carbohydrate that is usuallyattached to O-glycans on the surface of the cells, and is knownto play a vital role in cell–cell recognition processes. Lewis X isone of the most important blood-group antigens and is dis-played on the terminus of glycolipids that are present on thecell surface. Lewis X determinant, E-selectin ligand carbo-hydrate structure is constitutively expressed on granulocytesand monocytes and mediates inflammatory extravasation ofthese cells. Resting T and B lymphocytes lack its expression andare induced to strongly express Lewis X upon activation.Lewis X assays are performed using immunoassay techniques.

Lewis X antigen has sensitivity and specificity of 75 and 85%,respectively [9]. However, a study by Friedrich and coworkerscompared the effectiveness of Lewis X as a screening test againstcommercially available tests such as NMP22 and BTAStat.Lewis X was found to have a higher sensitivity (94.4%) than theother tests, but the specificity was lower (39.9%) [61].

Mucin 7Mucins are a family of large, heavily glycosylated proteins syn-thesized by glandular epithelial cells lining the urinary tract,which provide a protective barrier for the urothelial-linedmucosa. Mucin genes encode mucin monomers that are syn-thesized as rod-shape apomucin cores that are post-translation-ally modified by exceptionally abundant glycosylation. Thedense sugar coating of mucins gives them considerable water-holding capacity and also makes them resistant to proteolysis,which may be important in maintaining mucosal barriers [62].Mucins are secreted as massive aggregates of proteins. Withinthese aggregates, monomers are linked to one another mostlyby noncovalent interactions, although intermolecular disulfidebonds may also play a role in this process. Upregulation ofmucin genes (MUCs), especially MUC7, may cause an abnor-mal glycosylation pattern that may enhance tumor invasion

and metastatic potential. The overall sensitivity and specificityfor MUC7 in a study by Okegawa and coworkers were foundto be 68 and 87%, respectively [63].

Prostate stem cell antigen-14Prostate stem cell antigen (PSCA)-14 is a homologue of theLy-6/Thy-1 glycosylphosphatidylinositol family of cell surfaceantigens. PSCA was found to be overexpressed in a majority ofbladder cancers. A study by Cheng and coworkers was per-formed using immunocytochemical analysis on stored voidedurine samples from various groups with positive cytology and/orpositive biopsies and negative controls. Sensitivity and specific-ity for PSCA alone were 80 and 85.7%, respectively, whichcompared favorably against the results obtained from cytologyalone (46.7 and 100%, respectively). However, sensitivity andspecificity were improved to 83.3 and 85.7%, respectively, whenthe two tests were combined, illustrating its role as an adjunct tourine cytology for the detection of recurrent bladder cancer [64].

Expert commentary

As a result of the high recurrence rate of superficial bladdercancers, scientists try to develop new tests that could reduce theneed for frequent cystoscopic examinations. The majority ofthese tests demonstrate sensitivities that are superior to urinecytology in detecting low-grade bladder cancers. They also havethe advantage of the being easy to perform without the need forexperienced personnel. NMP22, BTA, MSI and FISH haveproved to have good sensitivity and specificity in the detectionof recurrent bladder cancer. However, more controlled trials areneeded to decide the efficiency of the other biological markersfor use in routine clinical practice.

Five-year view

In 5 years, a single or a group of new tests may emerge thatwould accurately diagnose recurrent bladder cancer. The adventof protein mass spectrometry would enable a new open-endedapproach to biomarker discovery, in which putative proteinbiomarkers can be identified by large-scale profiling of the pro-tein complement of urine. Large-scale proteomics profiling ofnormal human urine samples has revealed the presence of atleast 1000 different protein gene products and many more pep-tide fragments of larger proteins [65]. These could aim to reducethe cost and discomfort related to follow-up of bladder cancer.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement withany organization or entity with a financial interest in or financial conflictwith the subject matter or materials discussed in the manuscript. Thisincludes employment, consultancies, honoraria, stock ownership or options,expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.



References

Papers of special note have been highlighted as:• of interest•• of considerable interest

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•• Provides a systematic review on the most commonly used biological markers for bladder cancer. It shows the effectiveness of microsatellite instability, MNP22, ImmunoCyt™, CYFRA21-1, Lewis X and FISH as potential diagnostic tools.

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Key issues

• Bladder cancer is considered to be one of the most common genitourinary cancers. It is also an expensive cancer for follow-up as a result of the high recurrence rates.

• The first line of management of bladder cancer is the combination of cystoscopy to find papillary lesions or other irregularities, and urine cytology to help identify abnormalities that may not be endoscopically identifiable.

• Complete resection of the tumor is performed endoscopically thereafter with curative and staging intent.

• The frequent use of cystoscopies (which are both uncomfortable and inconvenient to the patients) and the low sensitivity and specificity of urine cytology led to the extrapolation of new means for detection of recurrent bladder cancer.

• Biological markers for bladder cancer could be used alone or as an adjunct to urine cytology for the detection of bladder cancer.

• Few of these markers have been approved by the US FDA for use in clinical practice. The rest of the biological markers are still in various stages of clinical trials.



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Affiliations

• Aza Mohammed, MBChB, MRCSUrology Unit, North Tees University Hospital Hardwick Road, Stockton on Tees TS19 8PE, [email protected]

• Ziauddin Khan, Dip. Urology, FRCS (Urol)Urology Unit, North Tees University Hospital Hardwick Road, Stockton on Tees TS19 8PE, UK

• Ignacio Zamora, LMS, MD, PhDUrology Unit, North Tees University Hospital Hardwick Road, Stockton on Tees TS19 8PE, UK

• Aftab Bhatti, FRCS (Urol)Urology Unit, North Tees University Hospital Hardwick Road, Stockton on Tees TS19 8PE, UK

Documents

Biological markers in the diagnosis of recurrent bladder cancer: an overview