Prognostic Role and HER2 Expression of Circulating Tumor Cells in Peripheral Blood of Patients Prior to Radical Cystectomy: A Prospective Study

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1 EUROPEAN UROLOGY 61 (2012) available at journal homepage: Bladder Cancer Prognostic Role and HER2 Expression of Circulating Tumor Cells in Peripheral Blood of Patients Prior to Radical Cystectomy: A Prospective Study Michael Rink a,e, *,y, Felix K. Chun a, *,y, Roland Dahlem a, Armin Soave a, Sarah Minner b, Jens Hansen a, Malgorzata Stoupiec c, Cornelia Coith c, Luis A. Kluth a, Sascha A. Ahyai a, Martin G. Friedrich d, Shahrokh F. Shariat e,f, Margit Fisch a, Klaus Pantel c, Sabine Riethdorf c a Department of Urology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany; b Department of Pathology, University Medical Centre Hamburg- Eppendorf, Hamburg, Germany; c Department of Tumor Biology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany; d Department of Urology, HELIOS Hospital Krefeld, Krefeld, Germany; e Department of Urology, Weill Medical College of Cornell University, New York Presbyterian Hospital, New York, NY, USA; f Division of Medical Oncology, Weill Medical College of Cornell University, New York Presbyterian Hospital, New York, NY, USA Article info Article history: Accepted January 10, 2012 Published online ahead of print on January 18, 2012 Keywords: Bladder cancer Circulating tumor cells CTC Outcome Survival CellSearch System HER2 Abstract Background: Preliminary research has suggested the potential prognostic value of circulating tumor cells (CTC) in patients with advanced nonmetastatic urothelial carcinoma of the bladder (UCB). Objective: Prospectively analyze the clinical relevance and human epidermal growth factor receptor 2 (HER2) expression of CTC in patients with clinically nonmetastatic UCB. Design, setting, and participants: Blood samples from 100 consecutive UCB patients treated with radical cystectomy (RC) were investigated for the presence (CellSearch system) of CTC and their HER2 expression status (immunohistochemistry). HER2 expression of the corresponding primary tumors and lymph node metastasis were analyzed using fluorescence in situ hybridization. Intervention: Blood samples were taken preoperatively. Patients underwent RC with lymphadenectomy. Measurements: Outcomes were assessed according to CTC status. HER2 expression of CTC was compared with that of the corresponding primary tumor and lymph node metastasis. Results and limitations: CTC were detected in 23 of 100 patients (23%) with nonmetastatic UCB (median: 1; range: 1 100). Presence, number, and HER2 status of CTC were not associated with clinicopathologic features. CTC-positive patients had significantly higher risks of disease recurrence and cancer-specific and overall mortality ( p values: 0.001). After adjusting for effects of standard clinicopathologic features, CTC positivity remained an independent predictor for all end points (hazard ratios: 4.6, 5.2, and 3.5, respectively; p values 0.003). HER2 was strongly positive in CTC from 3 of 22 patients (14%). There was discordance between HER2 expression on CTC and HER2 gene amplification status of the primary tumors in 23% of cases but concordance between CTC, primary tumors, and lymph node metastases in all CTC-positive cases (100%). The study was limited by its sample size. Conclusions: Preoperative CTC are already detectable in almost a quarter of patients with clinically nonmetastatic UCB treated with RC and were a powerful predictor of early disease recurrence and cancer-specific and overall mortality. Thus CTC may serve as an indication for multimodal therapy. Molecular characterization of CTC may serve as a liquid biopsy to guide individual targeted therapy in future clinical trials. # 2012 European Association of Urology. Published by Elsevier B.V. All rights reserved. y Both authors contributed equally to the manuscript. * Corresponding author. Department of Urology, University Medical Centre Hamburg-Eppendorf, Martinistr. 52, D Hamburg, Germany. Tel ; Fax: address: mrink@uke.uni-hamburg.de (M. Rink) /$ see back matter # 2012 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 EUROPEAN UROLOGY 61 (2012) Introduction Urothelial carcinoma of the bladder (UCB), the fifth most common cancer in the United States, with an estimated new cases and deaths in 2010, is an important cause of morbidity and mortality worldwide [1]. Radical cystectomy (RC) with bilateral pelvic lymphadenectomy is the standard of care for patients with high-risk non muscleinvasive UCB and those with muscle-invasive UCB [2]. However, despite advancements in surgical technique, imaging, perioperative management, and chemotherapy, up to 50% of patients develop metastasis and die from their disease [2 4]. In these patients, disease progression is often due to early dissemination of microscopic metastatic disease that remains undetectable prior to definitive therapy. Unfortunately, conventional staging modalities such as imaging techniques (ie, ultrasound, computed tomography [CT], magnetic resonance imaging, positron emission tomography, and bone scanning) and histopathologic procedures have a limited role in staging these patients because of their poor performance in detecting early low-volume occult UCB metastasis [5,6]. Therefore, new markers that can detect clinically relevant occult metastatic disease, which is associated with a high likelihood of eventual disease progression, may be helpful for selecting patients best suited for multimodal therapy (ie, neoadjuvant or adjuvant chemotherapy) and for sparing those patients who are likely to be cured with local therapy alone from the toxicity associated with unnecessary systemic therapy [7]. Circulating tumor cells (CTC) in the bloodstream constitute the seed for metastasis and have prognostic relevance in patients with occult and detectable metastasis [8]. On the basis of a series of prospective trials, the CellSearch assay received US Food and Drug Administration (FDA) clearance as an aid to the monitoring of disease status in patients with metastatic breast, colorectal, and prostate cancer [9 11]. In bladder cancer, preliminary research has suggested the potential prognostic value of CTC in patients with localized [12 15] and metastatic disease [16,17]. The presence of biologically and clinically relevant CTC would allow the identification of patients who could benefit from neoadjuvant or adjuvant chemotherapy in patients planned to be treated with RC for advanced nonmetastatic bladder cancer. Despite level 1 evidence that supports the use of neoadjuvant cisplatin-based combination chemotherapy for clinical T2 4aN0M0 stage bladder cancer [3], it is seldom used [18]. This is largely due to the reluctance of urologists to recommend neoadjuvant chemotherapy when a maximum of only half of patients will experience relapse [19]. Therefore, there is an urgent need for biomarkers allowing more accurate identification of patients who are likely to experience disease recurrence after RC. To address this unmet need, we set out to assess the prognostic value of preoperatively detectable CTC in a prospective cohort of wellcharacterized patients treated with RC for clinically nonmetastatic bladder cancer. In addition to providing prognostic information and serving as a surrogate marker of response to therapy, CTC have the potential to reveal a snapshot of the molecular makeup of an individual patient s tumor and to profile for determinants that predict for sensitivity or resistance to treatment. One such determinant is human epidermal growth factor receptor (EGFR) 2 (HER2), a tyrosine kinase in the EGFR family that can promote oncogenesis and has been found to be associated with biologically aggressive bladder cancer when assessed in the primary tumor [20 22]. HER2 expression status has been shown to identify tumors that respond to a targeted monoclonal antibody [23]. Therefore, we also assessed the prognostic role of HER2 expression in the CTC with cancer-specific outcomes as well as with HER2 expression in corresponding primary tumors and lymph node metastasis. 2. Material and methods 2.1. Study population Between July 2007 and September 2010, 100 consecutive patients with histopathologic-confirmed UCB, who underwent RC with bilateral lymphadenectomy, were prospectively enrolled. Exclusion criteria included a history of other malignant disease, male patients with known or suspected coincidental prostate cancer, patients treated with cystectomy for reasons other than UCB, and metastatic disease at staging. None of the patients received neoadjuvant chemotherapy or radiotherapy. Staging studies includedstandardlaboratorytests aswellasct ofthorax, abdomen, and pelvis and bone scan as well as brain CT when clinically indicated. A total of22 patients (22%) received adjuvant treatment (20 chemotherapy, 1 radiation therapy, 1 both) based on postoperative pathologic features, overall health status, and individual patient preferences. The study was conducted under a protocolapprovedbythe institutional reviewboard, and all patients signed written informed consent Circulating tumor cell measurement Preoperative blood samples (7.5 ml) were collected on the day prior to surgery in CellSave tubes (Veridex, Raritan, NJ, USA) at least 14 d after transurethral resection (TUR). The CellSearch system (Veridex) was used as previously described [10,15,24]. All blood samples were measured within 96 h (median: 24 h) after collection. Epithelial cells among the cells captured by anti-epcam antibodies were detected by binding of antibodies directed against cytokeratins 8, 18, and 19. An anti-cd45 antibodywas used toexclude leukocytes. Nuclei were counterstainedwith DAPI. After enrichment and immunocytochemical staining, immunomagnetically labeled cells were kept in a strong magnetic field and scanned using the CellSpotter Analyzer (Veridex). Image galleries were manually evaluated for CTC according to criteria reported earlier [10,24] Determination of circulating tumor cell HER2 expression status HER2 expression of the CTC was assessed using the fluorescein-labeled anti-her2 antibody (CellSearch tumor phenotyping reagent HER2/neu, Veridex) as described earlier [25]. HER2 status was categorized as negative (0), weakly positive (1+), equivocal (2+), or strongly positive (3+) [25] Determination of HER2 expression status on paraffinembedded tissue by fluorescence in situ hybridization Fluorescence in situ hybridization analysis was performed using the PathVysion Kit (Vysis, Abbott) or the HER2/Cen17 probes (Zytomed, Berlin, Germany). Chromosome 17 centromeric signals (CEP17) and HER2 gene-specific signals were counted within at least 60 nuclei of

3 812 EUROPEAN UROLOGY 61 (2012) invasive cancer cells from primary tumors or lymph node metastases. Ratios of both signals were subsequently determined. HER2 to CEP17 ratios <1.8 were categorized as negative without HER2 amplification, ratios of were categorized as equivocal, and ratios >2.2 were indicative for amplification and categorized as HER2 positive [25,26] Statistical analyses All statistical analyses were performed using PASW Statistics 18 (SPSS, IBM Corp, Armonk, NY, USA). Descriptive analyses were performed using chi-square and Fisher exact tests for categorical variables. Differences in variables with a continuous distribution across categories were assessed using the Mann-Whitney U test (two categories) and Kruskal-Wallis test (three and more categories). Probabilities of progression-free, cancerspecific, and overall survival were calculated using the Kaplan-Meier method. Differences among both groups were assessed using the log-rank test. Univariable and multivariable Cox regression models addressed time to disease recurrence and cancer-specific and all-cause mortality after RC. In all models, proportional hazards assumptions were systematically verified using the Grambsch-Therneau residual-based test. All tests were two sided, and a p value <0.05 was considered statistically significant. 3. Results 3.1. Association of circulating tumor cells with clinicopathologic features and clinical outcomes CTC were detectable in 23 of 100 patients (23%) with clinically nonmetastatic UCB. The mean number of CTC was 2/7.5 ml blood (range: 1 100; median: 1): 16 patients had 1 CTC/ 7.5 ml, three patients had 2 4 CTC/7.5 ml, and four patients had 5 CTC/7.5 ml, respectively. Table 1 summarizes patients characteristics according to their CTC status. CTC status (present vs absent) and number (not shown) was not associated with any clinical or pathologic features. During a median follow-up of 16 mo (range: 1 45), 14 CTC-positive (52%) and 10 CTC-negative (13%) patients experienced disease recurrence. At the time of censor, 28 patients (26.9%) died and 19 patients (18.3%) died of UCB. Presence of CTC was associated with an increased risk of disease recurrence, cancer-specific mortality, and all-cause mortality (all p 0.001; Fig. 1A 1C; Table 2a). From the 10 patients who experienced disease recurrence and were CTCnegative, 9 had pt3 or pt4 tumors, 5 had lymph node metastases, and 4 had soft tissue surgical margin involvement. Number of CTC stratified by one per 7.5 ml blood was not associated with outcomes in this restricted cohort (Fig. 2A 2C; all p > 0.05). Other cut-offs also did not stratify patients into statistically different risk groups. In multivariable Cox proportional hazards regression analyses that adjusted for the effects of pathologic stage and grade, lymph node status, surgical soft tissue margin status, and adjuvant treatment, CTC status was an independent predictor of disease recurrence (HR: 4.60; p < 0.001), cancer-specific mortality (HR: 5.22; p = 0.002), and allcause cause mortality (HR: 3.50; p = 0.003) (Table 2b). Table 1 Descriptive characterization of the study cohort and stratification by circulating tumor cell status Parameter Entire cohort (n = 100) CTC positive (n = 23) CTC negative (n = 77) p value Preoperative, clinical findings Age, mean SD Sex (%) Male 71 (71.0) 19 (82.6) 52 (67.5) 0.19 Female 29 (29.0) 4 (17.4) 25 (32.5) Time between last TUR and CTC measurement, d Mean SD Range Clinical stage (%) cta 2 (2.0) 0 (0.0) 2 (2.6) ctis 2 (2.0) 0 (0.0) 2 (2.6) ct1 29 (29.0) 9 (39.1) 20 (26.0) 0.61 ct2 63 (63.0) 13 (56.6) 50 (64.9) ct3 4 (4.0) 1 (4.3) 3 (3.9) Grading of TUR bladder specimens (%) G2 8 (8.0) 2 (8.7) 6 (7.8) 0.89 G3 92 (92.0) 21 (91.3) 71 (92.2) Postoperative findings Final pathology after cystectomy (%) pt0 8 (8.0) 0 (0.0) 8 (10.4) pta 8 (8.0) 3 (13.0) 5 (6.5) ptis 3 (3.0) 1 (4.3) 2 (2.6) pt1 15 (15.0) 3 (13.0) 12 (15.6) 0.45 pt2 20 (20.0) 5 (21.8) 15 (19.5) pt3 32 (32.0) 6 (26.1) 26 (33.7) pt4 14 (14.0) 5 (21.8) 9 (11.7) pn positive 22 (22.0) 6 (26.1) 16 (20.8) 0.82 Margin positive 11 (11.0) 4 (17.4) 7 (9.1) 0.27 Pathologic grading after cystectomy (%) No grading (pt0) 8 (8.0) 0 (0.0) 8 (10.4) G2 8 (8.0) 2 (8.7) 6 (7.8) 0.50 G3 84 (84) 21 (91.3) 63 (81.8) CTC = circulating tumor cells; SD = standard deviation; TUR = transurethral resection.

4 [(Fig._1)TD$FIG] EUROPEAN UROLOGY 61 (2012) [(Fig._2)TD$FIG] Fig. 1 Kaplan-Meier plots of (A) recurrence-free, (B) cancer-specific, and (C) overall survival stratified according to circulating tumor cells (CTC) status (positive vs negative) in 100 patients treated with radical cystectomy and bilateral lymphadenectomy for urothelial carcinoma of the bladder. SD = standard deviation. Fig. 2 Kaplan-Meier plots of (A) recurrence-free, (B) cancer-specific, and (C) overall survival stratified according to numbers of circulating tumor cells (CTC; 1 CTC vs I2 CTC/7.5 ml) in 23 CTC-positive patients treated with radical cystectomy and bilateral lymphadenectomy for urothelial carcinoma of the bladder.

5 814 EUROPEAN UROLOGY 61 (2012) Table 2 (a) Univariable and (b) multivariable Cox regression analyses predicting disease recurrence, cancer-specific death, and overall death of 100 patients treated with radical cystectomy for urothelial carcinoma of the bladder Variable Disease recurrence Cancer-specific death Overall death HR 95% CI p value HR 95% CI p value HR 95% CI p value (a) CTC status (positive vs. negative) < < Pathologic T stage (pt2 vs. pt3) Pathologic N stage (positive vs. negative) < < <0.001 Tumor grade (continuous) Margin status (positive vs negative) < < <0.001 Adjuvant treatment (yes vs no) < Variable Disease recurrence Cancer-specific death Overall death HR 95% CI p value HR 95% CI p value HR 95% CI p value (b) CTC status (positive vs negative) < Pathologic T stage (pt2 vs pt3) Pathologic N stage (positive vs negative) <0.001 Tumor grade (continuous) Margin status (positive vs negative) Adjuvant treatment (yes vs no) HR = hazard ratio; CI = confidence interval; CTC = circulating tumor cells. [(Fig._3)TD$FIG] Fig. 3 Determination of human epidermal growth factor receptor 2 (HER2) status by (A) fluorescent in situ hybridization and (B) immunofluorescence using the CellSearch system. (A) Primary tumor with strong HER2 gene amplification (clusters of HER2 signals: red; CEP17 signals: green); (B) circulating tumor cells detected with the CellSearch system (0: HER2 negative; 1+: weak; 2+: moderate; 3+: strong intensity of HER2-specific immunofluorescence) Comparison of HER2 expression status between circulating tumor cells, primary tumors, and lymph node metastasis Comparisons of HER2 expression of CTC (Fig. 3B) and the corresponding primary tumors (Fig. 3A) were performed in 22 CTC-positive cases (Table 3). Overall, 14 of 22 cases (64%) exhibited similar HER2 expression status between CTC and corresponding primary tumors (Table 3, bold roman). Although 13 of 16 patients (81%) with HER2- negative primary tumors also had HER2-negative CTC, only 25% (n = 1) of patients with HER2-positive primary tumors (n = 4) also had HER2-positive CTC. The HER2 status of lymph node metastases from five CTC-positive patients was identical to that of the corresponding primary tumors as well as that of the CTC (Table 3, bold roman). Two primary tumors with a heterogeneous HER2 status had only HER2-negative (0, 1+) CTC. In addition, in two cases HER2 expression showed heterogeneity among the identified CTC (Supplemental Table 1; eg, patient 20: 1 CTC/7.5 ml was strongly HER2 positive, but the other CTC was HER2 negative). Correlation of HER2 status of the CTC (continuous and categorical [HER2 positive vs negative]) was not associated with any clinicopathologic features or clinical outcomes (all p > 0.05; not shown).

6 EUROPEAN UROLOGY 61 (2012) Table 3 Comparison of immunocytochemical human epidermal growth factor receptor (HER2) expression on circulating tumor cells (CTC) and HER2 gene amplification status of primary tumors and corresponding lymph node metastases detected by fluorescent in situ hybridization analysis in CTC-positive patients Primary tumors Lymph nodes HER2 negative HER2 positive Heterogeneous HER2 expression HER2 positive HER2 negative CTC No. of analyzed CTC-positive cases HER2 negative, 0, 1+ (%) 13 (81.3) * 3 (75.0) ** 2 (100) 0 (0) 5 (100) HER2 questionably positive, 2+ (%) 1 (6.3) 0 (0) 0 (0) 0 (0) 0 (0) HER2-positive, 3+ (%) 2 (12.5) 1 (25.0) 0 (0) 0 (0) 0 (0) * Bold roman: Concordant results between primary tumors and CTC. ** Bold italic: Discordant results between primary tumors and CTC. 4. Discussion We found that patients with clinically nonmetastatic UCB treated with RC who harbored detectable CTC were at a significantly increased risk of early disease recurrence and death. The CTC detection rate was 23%, which is close to that of previously reported studies [12,14]. Similarly to Guzzo et al. [14], we did not find any association between CTC status and clinicopathologic features of patients with advanced UCB. However, although an association with clinicopathologic features is interesting, prediction of clinical outcomes is crucial in the management of UCB patients. Therefore, we performed a prospective study to investigate the biologic and clinical significance of preoperative CTC in the peripheral blood of RC patients with clinically nonmetastatic UCB using the CellSearch system. Previous studies primarily focused on the validity and reliability of the CellSearch assay in nonmetastatic [14] and metastatic [16,17] UCB patients. In the current study, we found that CTC presence is a strong independent predictor of poor outcomes in patients treated with RC after controlling for standard clinicopathologic features such as tumor stage, grade, lymph node, and soft tissue margin status and adjuvant treatment. This is in accordance with a previous study that used a different technology to identify CTC. Using the CELLection Dynabeads assay, Gradilone et al. found that T1G3 non muscle-invasive UCB patients harboring survivin-expressing CTC in the peripheral blood had significantly worse outcomes [13]. In contrast to this study, we used the only FDA-approved device, the CellSearch system, which has been shown to be analytically valid and works semiautomatically [11,24,27]. This approach is based on a highly standardized technology validated in external quality assurance studies [28]. This assay is based on well-done studies in metastatic breast [10], prostate [11], and colorectal cancer [9] that have shown that CTC presence confers worse survival. We found that even a single CTC conferred a worse prognosis in CTC-positive patients undergoing RC. This is in contrast to metastatic colon, breast, and prostate cancer studies that identified three to five or more CTC to provide an optimal prognostic cut-off value [9 11]. These differences may be due to differential biology underlying these malignancies as well as the disease stage at which CTC are measured. Recent reports in breast and prostate cancer have underlined the importance of a single CTC [29] and even EpCAM+, CK+, and CD45- events that do not meet strict definitions for CTC [30] for tumor progression and survival. Tibbe et al. developed a model describing the statistics of the different processing steps that are needed for the isolation and detection of CTC [31]. For metastatic patients they demonstrated that a decrease in the variability between readers of the CTC results might reduce the current cut-off value of 5 to 1 per 7.5 ml of blood. In this study, we did not observe any statistical difference with regard to outcomes based on number of CTC regardless of the cut-off used. Another reason underlying this lack of significance could be the small sample size, underlining the necessity of pragmatic clinical trial designs with integrated biomarker correlates. We furthermore analyzed the HER2 status of the CTC and compared it with that of the corresponding primary tumors and lymph node metastases. Molecular characterization of the detected CTC offers the potential to get insight into their biologic makeup and guide individualized targeted therapies. We found a high concordance rate in the HER2 status of CTC and the corresponding primary tumors (64%) and lymph node metastases (100%). This not only supports the relationship of the CTC to tumor of origin but also suggests that blood and lymphatic spread might derive from the same primary tumor. In addition, the overall rate of strongly HER2- expressing CTC (14%; n = 3) is similar to that reported in large studies on primary tumor tissue in muscle-invasive/advanced UCB [21,22], suggesting that both sources could serve to identify patients who are possible candidates for anti- HER2 targeted therapy. However, discrepancy in target presence between primary tumor and CTC (ie, the detection of HER2-negative CTC in patients having primary tumors with heterogeneity concerning HER2 gene amplification) may explain in part the lack of efficacy of targeted therapies. Similar differences have been reported between primary tumors and lymph node metastases [32]. We also detected HER2-positive CTC in 12.5% of patients with HER2-negative primary tumors, implying that HER2 status may need assessment of CTC instead or in addition to the primary tumor to be able to improve prediction of HER2 status. Further larger studies are necessary to assess the validity of HER2 expression of CTC. These might help stratify patients with metastatic disease by substituting determination of the HER2 status of unresectable overt metastases that are not available for further analysis.

7 816 EUROPEAN UROLOGY 61 (2012) Our study has several limitations. First and foremost are limitations due to the limited sample size and heterogeneity in treatment regimen. Different factors such as positive soft tissue surgical margin status or adjuvant therapy regimes might have influenced clinical outcomes. We did not perform a power calculation before study initiation due to a lack of data regarding CTC findings in nonmetastatic UCB, and therefore some of our analyses may have been underpowered (ie, the association of number of CTC with outcomes). We also did not stain the primary tumors and lymph node metastases of CTC-negative patients for HER2 and thus could not control for any difference between both groups. Finally, we cannot exclude any false-positive CTC findings, although the short half-life time of CTC (1 3 h) [33,34] makes it very unlikely that putative cytokeratinpositive cells from the last TUR bladder might have influenced the results. 5. Conclusions The presence of preoperative CTC is a powerful predictor of disease recurrence and cancer-specific mortality in patients treated with RC for clinically nonmetastatic UCB. Therefore, these patients could be considered for systemic therapy such as neoadjuvant or adjuvant chemotherapy because they are unlikely to be cured with local therapy alone. With the advent of targeted therapies, CTC status and molecular makeup additionally may serve as a liquid biopsy to guide the choice and efficacy of select therapies. Determination of HER2 status in CTC and primary tumor as well as lymph node metastases may help identify patients for HER2- directed targeted therapy. Author contributions: Michael Rink 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: Rink, Riethdorf. Acquisition of data: Rink, Soave, Minner, Riethdorf, Kluth, Stoupiec, Coith. Analysis and interpretation of data: Rink, Chun, Riethdorf. Drafting of the manuscript: Rink, Chun, Riethdorf. Critical revision of the manuscript for important intellectual content: Rink, Chun, Dahlem, Soave, Hansen, Minner, Stoupiec, Coith, Kluth, Ahyai, Friedrich, Shariat, Fisch, Pantel, Riethdorf. Statistical analysis: Rink, Shariat, Chun. Obtaining funding: None. Administrative, technical, or material support: Minner, Riethdorf, Stoupiec, Coith. Supervision: Rink, Fisch, Dahlem, Riethdorf. 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: Michael Rink receives honoraria from Pfizer Pharma for lecturing and is supported by The Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust. Shahrokh F. Shariat is coinventor of four patents (Method to Determine Prognosis After Therapy for Prostate Cancer, US ; Methods to Determine Prognosis After Therapy for Bladder Cancer, US ; Prognostic Methods for Patients with Prostatic Disease, US ; Soluble Fas Urinary Marker for the Detection of Bladder Transitional Cell Carcinoma, US ) and is on the advisory board of Ferring Pharma. The other authors have nothing to disclose. Funding/Support and role of the sponsor: None. Acknowledgment statement: The authors acknowledge Susanne Hoppe, Heike Hoop, and Oliver Mauermann for excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.eururo References [1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, CA Cancer J Clin 2010;60: [2] Stein JP, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. J Clin Oncol 2001;19: [3] Grossman HB, Natale RB, Tangen CM, et al. Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 2003;349: [4] Shariat SF, Karakiewicz PI, Palapattu GS, et al. Outcomes of radical cystectomy for transitional cell carcinoma of the bladder: a contemporary series from the Bladder Cancer Research Consortium. 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