Articles

Urine Markers for Detection and Surveillance of Non–Muscle-Invasive Bladder Cancer

By: Derya Tilkia lowast , Maximilian Burgerb, Guido Dalbagnic, H. Barton Grossmand, Oliver W. Hakenberge, Juan Palouf, Oliver Reichg, Morgan Rouprêth, Shahrokh F. Shariati and Alexandre R. Zlottaj

Published online: 01 September 2011

Keywords: Detection, Biomarkers, Prediction, Diagnosis, Urothelial carcinoma, Bladder neoplasms

Abstract Full Text Full Text PDF (770 KB)

Abstract

Context

Bladder cancer diagnosis and surveillance includes cystoscopy and cytology. The limitation of urinary cytology is its low sensitivity for low-grade recurrences. As of now, six urine markers are commercially available to complement cystoscopy in the detection of bladder cancer. Several promising tests are under investigation.

Objective

In this nonsystematic review, we summarize the existing data on commercially available and promising investigational urine markers for the detection of bladder cancer.

Evidence acquisition

A PubMed search was carried out. We reviewed the recent literature on urine-based markers for bladder cancer. Articles were considered between 1997 and 2011. Older studies were included selectively if historically relevant.

Evidence synthesis

Although different studies have shown the superiority of urine markers regarding sensitivity for bladder cancer detection as compared with cytology, none of these tests is ideal and can be recommended unrestrictedly.

Conclusions

Urine markers have been studied extensively to help diagnose bladder cancer and thereby decrease the need for cystoscopy. However, no marker is available at present that can sufficiently warrant this. Several urinary markers have higher but still insufficient sensitivity compared with cytology. Urinary cytology or markers cannot safely replace cystoscopy in this setting. To identify an optimal marker that can delay cystoscopy in the diagnosis of bladder cancer, large prospective and standardized studies are needed.

Take Home Message

No bladder cancer urine marker is available that can safely decrease the need for cystoscopy. Several urinary markers have higher but still not sufficient sensitivity compared with cytology. For identification of an optimal marker, prospective and standardized studies are needed.

Keywords: Detection, Biomarkers, Prediction, Diagnosis, Urothelial carcinoma, Bladder neoplasms.

1. Introduction

Bladder cancer is one of the most common malignancies of the urinary tract. It is the fourth most common cancer in men and results in significant morbidity and mortality [1]. Approximately 70% of patients have cancers confined to the epithelium or subepithelial connective tissue at initial diagnosis. The recurrence rate for these tumors ranges from 50% to 70%, and 10–15% progress to muscle invasion over a 5-yr period, necessitating lifelong surveillance in many cases [2]. Diagnosis and surveillance of bladder cancer consists of cystoscopy and cytology [3]. Cystoscopy identifies most papillary and solid lesions but is invasive.

Urine cytology has reasonable sensitivity and specificity for the detection of high-grade bladder cancer, but sensitivity for detection of low-grade tumors ranges only from 4% to 31% [4]. Cytology misses up to 60% of high-grade tumors. This number might be an extrapolation of the results from series in which the cytology standard was poor, however.

An ideal urine marker for bladder cancer should reliably detect bladder cancer. It might also reduce the number of cystoscopies in surveillance of non–muscle-invasive bladder cancer (NMIBC).

To date, six urine markers have been approved for clinical use in the detection of bladder cancer. We summarize the existing data on commercially available and promising investigational urine-based bladder cancer marker systems that can detect differences in the presence of cell surface antigens on exfoliated cells, soluble proteins, nuclear morphology, or gene expression.

2. Evidence acquisition

We reviewed the literature nonsystematically using the National Library of Medicine database (http://www.pubmed.gov). A Medline search was performed with special emphasis on urothelial malignancies and urine markers using combinations of the following terms: urinary tract cancer, bladder carcinomas, urothelial carcinomas, detection, bladder, recurrence, and urine markers. Articles were considered between 1997 and 2011. Older studies were included selectively if historically relevant or in the case of scanty data in more recent publications. No evidence level 1 information from prospective randomized trials was available.

3. Evidence synthesis

3.1. Commercially available urine markers

3.1.1. Bladder tumor antigen tests

The bladder tumor antigen (BTA) stat is a qualitative point-of-care test with immediate results, whereas BTA TRAK is a quantitative test requiring trained personnel and a reference laboratory. These assays detect human complement factor H-related protein in the urine of patients with bladder cancer [5].

The reported overall sensitivity and specificity for the BTA stat test are 57–83% [6], [7], and [8] and 60–92% [9] and [10], respectively. Many of the studies excluded patients with benign genitourinary problems. In healthy persons, the specificity is 97%, but in patients with benign genitourinary conditions, the specificity is only 46% [9]. Hematuria from other causes than bladder cancer can lead to false-positive results [11] and [12]. In a recent prospective multicenter trial of 501 patients, the reported sensitivity of BTA stat to monitor for bladder cancer recurrence was greater than that of cytology, particularly in grade 1 lesions (48% vs 13%) [13]. However, intravesical treatment, benign prostatic hyperplasia, kidney stones, and urinary tract infections caused a high false-positive rate, and the BTA stat test would have missed 47% of tumors detected on cystoscopy.

The BTA TRAK test is a quantitative sandwich immunoassay [14]. The cut-off limit of human complement factor H-related protein to detect bladder cancer is 14U/ml [15]. Using this cut-off, the reported overall sensitivity is 62–91% [15], [16], [17], [18], [19], [20], [21], and [22]. However, with a set sensitivity of 90%, the specificity of BTA TRAK was only 25% [17]. As with the BTA stat test, benign genitourinary conditions may lead to false-positive results [15], [19], and [21].

Both tests are approved by the US Food and Drug Administration (FDA) only in combination with cystoscopy for the monitoring of bladder cancer. Because of their high false-positive rate, they cannot be recommended without cystoscopy.

3.1.2. ImmunoCyt

ImmunoCyt (Scimedx Corp, Denville, NJ, USA) combines cytology with an immunofluorescence assay [23]. It detects cellular biomarkers for bladder cancer in exfoliated urothelial cells using fluorescent monoclonal antibodies for a high molecular weight form of carcinoembryonic antigen and two bladder tumor cell–associated mucins. The test requires trained personnel, it is expensive, and a large number of exfoliated cells are necessary to perform an accurate test [24] and [25]. ImmunoCyt has a reported overall sensitivity of 50–100% [26], [27], and [28]. Its specificity has been reported as 69–79%, with a higher false-positive rate in patients with benign prostatic hyperplasia or cystitis [27]. It has an improved sensitivity when compared with cytology, especially in low-grade tumors, but this is accompanied by a lower specificity and reduced positive predictive values (PPVs) [29]. The test may be helpful as an adjunct to cystoscopy and should be used only to monitor patients with bladder cancer.

3.1.3. Nuclear matrix protein 22 tests

The two marker tests for bladder cancer detecting nuclear mitotic apparatus protein 22 (NMP22) in voided urine are the original NMP22 bladder cancer test kit (Matritech Inc, Newton, MA, USA), a laboratory-based, quantitative, sandwich-type, microplate, enzyme immunoassay, and the NMP22 BladderChek (Matritech), a qualitative point-of-care test cartridge containing the NMP22 detecting and reporter antibodies. Both are FDA approved for use in bladder cancer surveillance. The NMP22 BladderChek test is also approved as a screening test for individuals who have symptoms of or are at risk for bladder cancer.

The sensitivity of the original NMP22 immunoassay has ranged from 47% to 100% and its specificity from 60% to 90% depending on the cut-off value [6], [7], [8], [9], [19], [21], [30], [31], [32], [33], and [34]. The specificity is significantly decreased in the presence of benign inflammatory or infectious diseases, renal or bladder calculi, a foreign body, bowel interposition, other genitourinary cancer, or instrumentation [7]. Intravesical bacillus Calmette-Guérin does not alter the performance characteristics of NMP22 [35]. Despite the promising data, the quantitative enzyme NMP22 immunoassay has not been widely used due to a high false-positive rate.

More recently, the point-of-care test NMP22 BladderChek was introduced. A multi-institutional trial revealed that the addition of the NMP22 BladderChek test to cystoscopy improved the detection rate of bladder cancer in patients with risk factors for bladder cancer [36]. The NMP22 BladderChek test sensitivities were 50% and 90% for noninvasive and invasive cancer, respectively, with an overall sensitivity of 56%. In contrast, cytology in this study performed poorly, with comparable sensitivities of 17% and 22% in noninvasive and invasive bladder cancer, respectively, with an overall sensitivity of 15.8%, which casts doubt on the accuracy of reflection on the method. Overall specificity was still higher for cytology at 99% compared with 86% for NMP22 BladderChek.

In a recent study, the clinical benefit of NMP22 in the surveillance of patients with NMIBC and negative cytology was assessed by a decision-curve analysis [37]. The authors observed that NMP22 can help in making decisions about immediate versus delayed cystoscopy depending on the clinician's risk threshold for conducting a cystoscopy. For a risk-averse clinician who would perform a cystoscopy even at a low risk of recurrence or progression, NMP22 would not add any clinical benefit, and the optimal strategy would be to offer cystoscopy to every patient who is at risk according to the European Association of Urology (EAU) guidelines.

3.1.4. UroVysion

UroVysion (Abbott Molecular, Inc, Des Plaines, IL, USA) is a multitarget fluorescence in situ hybridization (FISH) assay that detects aneuploidy in chromosomes 3, 7, and 17 as well as loss of the 9p21 locus of the P16 tumor suppressor gene [38]. This test has been approved by the FDA both for monitoring patients with a history of bladder cancer and for bladder cancer detection in patients with hematuria.

The FISH test combines assessment of the morphologic changes of conventional cytology with molecular DNA changes. Each probe is a fluorescently labeled, single-stranded DNA fragment complementary to specific target sequences of cellular DNA that are denatured to allow hybridization with the probe. A minimum of 25 morphologically abnormal cells is viewed. If ≥4 cells exhibit polysomy of 3, 7, or 17 or ≥12 cells exhibit loss of 9p21, the case is considered positive for tumor. However, no uniform criteria exist for a positive UroVysion assay at this time.

In most comparative studies, FISH outperformed cytology across all stages and grades of bladder cancer [39], [40], and [41]. The overall sensitivity of FISH was superior to cytology (74% vs 48%), especially in high-grade disease such as carcinoma in situ (100% for FISH vs 67% for cytology) [42]. A meta-analysis of FISH reported that the overall performance of FISH was better than that of cytology (area under the curve: 0.87 vs 0.63) [42]. This difference, however, was almost entirely attributable to the difference in performance in diagnosing Ta patients.

It has been suggested that some patients with a positive FISH result and negative cystoscopy might eventually develop urothelial cancer because several studies have found that 85–89% of patients with a false-positive test had a positive bladder biopsy within 12 mo of the test [43] and [44]. However, others have found that the recurrence rate after a positive FISH result and negative cystoscopy can be <50% [45]. The real role of a positive FISH result remains unclear because most patients with NMIBC experience disease recurrence.

Combining morphology with FISH may prove an alternative modality of cancer detection [46] and [47]. Using the Duet automatic scanning system (BioView, Ltd, Rehovat, Israel), which examines both morphology and FISH scoring, an examination of voided urine specimens from 115 patients with negative or atypical cytology found that 44% of these patients did not have a prior diagnosis of bladder cancer. The combination of morphology and FISH resulted in 100% sensitivity and 65% specificity [46].

The high cost and the need for large urine volume or tumor burden as well as exfoliation of tumor cells have prevented a wider use of this test. Another limitation is that the FISH assay does not detect diploid cells without 9p21 deletions.

3.2. Investigational urine markers

3.2.1. AURKA

The Aurora kinase A (AURKA) gene encodes a serine/threonine kinase associated with aneuploidy and chromosome instability. This gene has been explored in urine sediment by FISH. A training set was used to establish test conditions. A separate testing set of 100 patients with bladder cancer, 92 healthy individuals, and 56 patients with benign urologic disease reported a test sensitivity of 87% and a specificity of 97% [48]. A higher degree of gene amplification was associated with increasing grade. These preliminary data need additional validation in another data set.

3.2.2. BLCA-1 and BLCA-4

BLCA-1 and BLCA-4 are nuclear transcription factors present in bladder cancer. BLCA-1 is not expressed in nonmalignant urothelium [49], whereas BLCA-4 is expressed in both the tumor and adjacent benign areas of the bladder but not in nonmalignant bladders [50]. BCLA-4 is measured in the urine using an enzyme-linked immunosorbent assay (ELISA); its reported sensitivity ranges from 89% to 96% and its specificity reaches 100% [51] and [52]. Similarly, in a small study, BLCA-1 demonstrated good performance with 80% sensitivity and 87% specificity [49]. Tumor grade did not affect their expression. However, up to 19% of patients with spinal cord injuries have elevated BCLA-4 levels [53]. These biomarkers need assay refinement and validation.

3.2.3. CEACAM1

Bladder tumor growth and progression depend on angiogenesis. Human carcinoembryonic antigen-related cell adhesion molecule (CEACAM)1 is a cell adhesion molecule with proangiogenic activity. It has previously been observed that CEACAM1, which is ubiquitously expressed in the luminal surface of normal bladder urothelium, is downregulated in bladder cancer cells while it is concurrently upregulated in endothelial cells of adjacent blood vessels [54]. This differential switch in CEACAM1 expression is accompanied by an upregulation of proangiogenic and prolymphangiogenic factors. Based on these findings, it was assessed whether CEACAM1 was detectable in urine and whether its levels could help differentiate bladder cancer patients from healthy subjects. Tilki et al. constructed an ELISA for CEACAM1 and measured CEACAM1 levels in voided urine specimens from patients with bladder cancer and control subjects without cancer, with common nonmalignant urologic pathologies or with a past history of bladder cancer without present disease [54]. It was found that CEACAM1 forms can be detected in human urine. More importantly, higher urinary levels of CEACAM1 were associated with bladder cancer presence and advanced stage. Within bladder cancer patients, higher CEACAM1 levels were associated with invasive tumor stage. Using receiver operating characteristics analyses, a biologic and clinically relevant cut-off for bladder cancer detection was set. Using the cut-off of 110 ng/ml, a sensitivity of 74% and a specificity of 95% were calculated. Larger studies are needed to determine the potential role of urinary CEACAM1 in the management of patients who are at risk for bladder cancer.

3.2.4. Epigenetic urinary markers

Analysis of gene methylation has been shown to be feasible from voided urine [55] and [56]. Friedrich et al. analyzed the methylation status of different markers in urine samples of patients with bladder cancer and found that methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in most of the samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals [56]. Rouprêt and colleagues compared nine microsatellite markers and the methylation status of 11 gene promoters and found that microsatellite markers (area under curve [AUC] 0.819) have better performance characteristics than promoter hypermethylation (AUC: 0.448) for detecting bladder cancer recurrence [57]. Hoque et al. analyzed the promoter hypermethylation pattern of nine key genes methylated in bladder cancer analyzing paired samples of primary tumor DNA and urine sediment DNA [58]. They compared the pattern of methylation using urine sediment DNA samples from bladder cancer patients and control subjects. For all 15 patients with paired DNA samples, the promoter methylation pattern in urine matched that in the primary tumors. Sensitivity of the methylated gene panel was >80%; specificity was >90%. In urine, hypermethylation of DAPK, RARbeta, E-cadherin, and p16 has been shown to have a good sensitivity and specificity for bladder cancer detection [59]. Renard et al. initially unveiled candidate methylated genes using DNA extracted from noncancerous and bladder cancer tissue and subsequently analyzed the genes of interest in urine [60]. They identified TWIST1 and NID2 to be frequently methylated in urine samples collected from bladder cancer patients with sensitivity and specificity of a two-gene panel >90%; sensitivity and specificity of cytology were 48% and 96%. Future studies are necessary to validate these findings.

3.2.5. FGFR3 mutations

Mutations in the fibroblast growth factor receptor (FGFR)3 occur in 50% of primary bladder tumors and might be associated with good prognosis [61]. FGFR3 mutations are especially prevalent in low-grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases [61]. Van Oers et al. described a simple assay for the simultaneous detection of nine different FGFR3 mutations in bladder cancer and voided urine [61]. In urine samples from patients with a mutant tumor, the sensitivity of mutation detection was 62%. Zuiverloon et al. evaluated FGFR3 mutation in voided urine to detect recurrences during surveillance in patients with low-grade NMIBC with a FGFR3-mutant tumor. The sensitivity (58%) of the assay for detection of recurrences was higher than urinary cytology only but still far from perfect [62].

3.2.6. Hyaluronic acid and hyaluronidase

Urine hyaluronic acid (HA), a nonsulfated glycosaminoglycan, has been shown to yield 92% sensitivity and 93% specificity for bladder cancer detection [63]. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small fragments that promote angiogenesis [64]. There is a positive correlation between the secretion of HAase by bladder cancer cells and their invasive potential. A five- to eightfold elevation of HAase in the urine of patients with grade 2 or 3 bladder cancer could be detected [65].

The levels of HA and HAase are combined in the HA-HAase test [66]. In a study of 225 urine samples from 70 patients with known bladder cancer, the HA-HAase test performed better than the BTA stat test, with a reported sensitivity >90% across all tumor grades [66]. However, the accuracy of HA-HAase for detecting low-grade tumors was poor and lower than that of voided urine cytology [67]. Further refinement of the assay and evaluation in larger clinical trials would help define the clinical applicability of this marker.

3.2.7. Microsatellite analysis

One of the most common genetic changes in bladder cancer is loss of heterogeneity in chromosome 9 [68]. Chromosomes 4p, 8p, 9p, 11p, and 17p also often display loss of heterogeneity in patients with bladder cancer [69] and [70]. Several studies have analyzed voided urine with 17–20 microsatellite biomarkers [68] and [71]. The overall sensitivity from these studies ranged from 72% to 97%, and overall specificity ranged from 80% to 100%. Microsatellite biomarkers outperformed cytology in low-grade, low-stage tumors. In a study of 228 patients, a sensitivity of 58% and a specificity of 73% were reported for this test [72]. Microsatellite analysis can predict recurrences of low-grade tumors in up to 80% of the cases but lacks sensitivity [57].

3.2.8. MicroRNA markers

MicroRNAs (miRNAs) are noncoding RNAs that posttranscriptionally regulate gene expression [73]. They might serve as an ideal bladder biomarker because they are stable within urine and require little handling care [74], and they are more stable against nuclease degradation due to their small size. Urine contains many nucleases, and assays to examine mRNA expression often fail due to target degradation or require stringent prelaboratory handling of the urine sample. Recently, urinary miRNA expression was reported and the upregulation of miRs-126/182/199a was found to discriminate bladder cancer patients from disease-free controls [75]. The combination of miR-126 and -182 identified up to 77% of bladder cancer cases, despite a lack of differential expression for any of these miRNAs in malignant and normal urothelium [76]. Larger clinical trials are necessary to further define these markers.

3.2.9. Survivin

Survivin, a novel member of the inhibitor-of-apoptosis gene family, is prominently reexpressed in many types of cancer. Survivin messenger ribonucleic acid (mRNA) is overexpressed in human cancers and can be detected in urine using a bio-dot immunoassay incorporating a rabbit polyclonal antisurvivin antibody [77]. Urinary levels of survivin gene activation, both at the protein and the mRNA level, are associated with bladder cancer presence, higher grade, and advanced pathologic stage [78], [79], [80], and [81]. In the first study to evaluate the diagnostic potential of survivin in bladder cancer, survivin protein and mRNA were detected in all of 47 patients with bladder cancer but in only 3 of 35 patients with negative cystoscopic evaluation [80]. Another study showed a correlation of urinary levels of survivin with increased risk of bladder cancer presence and higher grade, but not tumor invasion [79]. In this study, survivin sensitivity was 64% and specificity was 93%. More recently, Horstmann et al. evaluated the potential of survivin mRNA measurement in a prospective pilot study in voided urine samples of 50 patients with suspicion of new or recurrent bladder cancer before transurethral resection [82]. They found survivin to be a reliable biomarker for high-grade urothelial bladder cancer (sensitivity 83%) but not for low-grade (sensitivity 35%) urothelial bladder cancer with a high specificity (88%).

Although the results are promising, the lack of assay standardization and cut-off value has to be resolved before possible clinical use [79].

3.2.10. Telomerase

Telomerase is a ribonucleoprotein enzyme that acts in chromosomal instability by synthesizing telomeres [83], [84], [85], and [86]. Malignant neoplasms, including bladder cancer [87], have been shown to produce telomerase and thus to regenerate telomeres and prevent cell death [88]. The standard technique to measure telomerase activity is the telomeric repeat amplification protocol (TRAP) assay [88]. Another telomerase-based assay detects the catalytic subunit of telomerase, human telomerase reverse transcriptase, using polymerase chain reaction. When compared with TRAP, human telomerase reverse transcriptase polymerase chain reaction has higher sensitivity than the TRAP assay, ranging between 75% and 100% [89] and [90]. The reported overall sensitivity of telomerase testing for the detection of bladder cancer is between 7% and 100%, mostly in the range of 70–86% [6], [32], [91], [92], [93], [94], [95], [96], [97], [98], and [99]. The reported overall specificity of telomerase for bladder cancer varies from 24% to 90%, mostly in the range of 60–70% [6], [32], [91], [93], [94], [95], [96], [98], [99], [100], and [101]. In 2005, researchers who examined a range of TRAP cut-offs found good overall performance characteristics with a reported sensitivity of 90% and specificity of 88% for the arbitrary cut-off of 50 enzyme units [102]. However, because many bladder cancer patients have other comorbidities, the clinical applicability of the telomerase assay may be limited.

3.2.11. Urinary UBC test

IDL Biotech AB (Bromma, Sweden) developed the UBC, a point-of-care qualitative assay, and the UBC ELISA, a quantitative assay that measures cytokeratins 8 and 18 in the urine [103] and [104]. A study measuring UBC Rapid in the urine of 180 patients found an overall sensitivity of 66% and a specificity of 90% [104]. In a comparative study, however, BTA stat and BTA TRAK outperformed the UBC Rapid test, particularly with regard to sensitivity [105]. Similarly, cytology showed a better sensitivity and specificity than either UBC or UBC II ELISA [106]. More recently, investigators compared the sensitivity and specificity of cytology (20% and 99%), BTA (54% and 84%), and UBC (12% and 97%) [17]. For carcinoma in situ, UBC had a higher sensitivity (100%) compared with cytology (67%) and BTA (0%). The overall performance of the UBC test is not superior to cytology or other current biomarkers.

4. Conclusions

The high rate of disease recurrence of bladder cancer requires lifelong surveillance in many patients consisting of cystoscopy, which is invasive and expensive, and cytology, which shows low sensitivity for low-grade recurrences. Noninvasive urine markers might therefore significantly improve the management of NMIBC.

The comparison of urine markers in different studies and their clinical use are problematic. Marker performance can vary widely depending on the population that is tested, how the samples were stored, how the test was run, and how the results were analyzed. Therefore, the comparison of biomarkers is imprecise, except within a given study. Furthermore, translation to the clinic is difficult. Seemingly, positive predictive value (PPV) and negative predictive value (NPV) are most useful to clinicians because they indicate whether a test result is likely to be true. However, PPV and NPV depend on the prevalence of disease in the tested population. Urine markers are ordered to indicate the presence of disease. Because clinical care is an ongoing effort and the disease prevalence is not known, it is impossible to calculate the PPV and NPV accurately in any clinical practice.

For most of the investigated urine markers, equal or higher sensitivities for bladder cancer detection have been reported than for cytology, certainly for low-grade disease. None of these tests, however, meets the criteria of an ideal urine marker and thus can facilitate reliable bladder cancer detection.

Some of the FDA-approved bladder cancer urine markers have been incorporated into the follow-up schedule in selected patients with early low-grade bladder cancer with a reduced frequency of cystoscopies [107] and [108]. It remains to be answered, however, if reduction of cystoscopies is safe and/or if this reduction in cystoscopies in selected patients could also be facilitated without an additional urine marker. A substantial number of investigational urine markers have been described. A part of these has only been described by single groups and not been tested further, and another part showed very promising results, which, however, could not be confirmed by other groups. For these reasons, improved standardization of the methods for urine marker studies is required, as well as prospective validation of urine markers in heterogeneous patient populations [109].

Although urine markers are intended to provide an accurate noninvasive alternative for bladder cancer detection, markers with a high false-positive rate will by necessity lead to more invasive tests and will be harmful to patients.

Until reliable urine marker systems, which may also be a combination of markers [110], have been found and validated, cystoscopy remains the gold standard for early bladder cancer detection and, according to the EAU guidelines, cystoscopy and cytology for surveillance [3].

Author contributions: Derya Tilki had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Tilki, Burger, Dalbagni, Grossman, Hakenberg, Palou, Reich, Rouprêt, Shariat, Zlotta.

Acquisition of data: Tilki, Burger, Dalbagni, Grossman, Hakenberg, Palou, Reich, Rouprêt, Shariat, Zlotta.

Analysis and interpretation of data: Tilki, Burger, Dalbagni, Grossman, Hakenberg, Palou, Reich, Rouprêt, Shariat, Zlotta.

Drafting of the manuscript: Tilki, Burger, Dalbagni, Grossman, Hakenberg, Palou, Reich, Rouprêt, Shariat, Zlotta.

Critical revision of the manuscript for important intellectual content: Tilki, Burger, Dalbagni, Grossman, Hakenberg, Palou, Reich, Rouprêt, Shariat, Zlotta.

Statistical analysis: None.

Obtaining funding: None.

Administrative, technical, or material support: None.

Supervision: Tilki, Shariat.

Other (specify): None.

Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/ affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: H. Barton Grossman is a patent holder on the Aurora kinase A test, which is licensed to Abbott Molecular. Juan Palou is a company consultant and receives speaker honoraria from Sanofi-Pasteur, General Electric. He has participated in trials for General Electric.

Funding/Support and role of the sponsor: None.

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Footnotes

a Department of Urology, Ludwig-Maximilians-University, Klinikum Grosshadern, Munich, Germany

b Department of Urology, Caritas-St. Josef Medical Centre, University of Regensburg, Regensburg, Germany

c Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

d Department of Urology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

e Department of Urology, Rostock University, Rostock, Germany

f Department of Urology, Fundació Puigvert, Universitat Autònoma de Barcelona, Spain

g Department of Urology, Klinikum Harlaching, Munich, Germany

h The Academic Department of Urology of La Pitié-Salpétrière, Assistance-Publique Hôpitaux de Paris, Faculté de Médecine Pierre et Marie Curie, University Paris VI, Paris, France

i Department of Urology and Division of Medical Oncology, Weill Cornell Medical College, New York-Presbyterian Hospital, New York, NY, USA

j Division of Urology, Department of Surgical Oncology, Princess Margaret Hospital and the University Health Network, Mt. Sinai Hospital, University of Toronto, Ontario, Canada

lowast Corresponding author. Department of Urology, University Hospital Grosshadern, Ludwig-Maximilians-University Munich, Marchioninistr. 15, 81377 Munich, Germany. Tel.: +49 89 7095 0.