IJC International Journal of Cancer

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1 IJC International Journal of Cancer A nonrandomized, prospective, clinical study on the impact of circulating tumor cells on outcomes of urothelial carcinoma of the bladder patients treated with radical cystectomy with or without adjuvant Armin Soave 1, Sabine Riethdorf 2, Roland Dahlem 1, Gunhild von Amsberg 3, Sarah Minner 4, Lars Weisbach 1, Oliver Engel 1, Margit Fisch 1, Klaus Pantel 2 and Michael Rink 1 1 Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Department of Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Department of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany To investigate outcomes of urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC) according to the presence of circulating tumor cells (CTC) and the administration of adjuvant (AC). We prospectively enrolled 226 UCB patients treated with RC without neoadjuvant at our institution between 2007 and Blood samples were obtained from all patients preoperatively and analyzed for CTC using the CellSearch VR system. Platinumbased AC was administered in 50 patients (27.0%). Cox regression models evaluated the association of CTC with disease recurrence, cancer-specific and overall mortality according to AC administration. 185 patients were available for analyses. CTC were present in 41 patients (22.2%). Patients with presence of CTC received AC more frequently, compared to patients without CTC (p ). At a median follow-up of 31 months, the presence of CTC was associated with disease recurrence, cancerspecific and overall mortality (p-values < 0.001) in patients without AC administration. In patients who received AC, there was no difference in either endpoint between patients with or without presence of CTC. In multivariable analysis of patients without AC administration, the presence of CTC was an independent predictor for disease recurrence (HR: 4.9; p < 0.001), cancerspecific (HR: 4.2; p ) and overall mortality (HR: 4.2; p ). The may be implemented in decisionmaking regarding AC administration in UCB patients following RC. CTC measurement should be implemented in future UCB studies on systemic to validate our findings. Introduction Urothelial carcinoma of the bladder (UCB) is the sixth most common malignancy and a deadly disease accounting for over 16,000 deaths in 2015 in the USA. 1 Radical cystectomy (RC) with bilateral pelvic lymphadenectomy is the gold standard surgical treatment for patients with muscle-invasive and Key words: urinary bladder cancer, urothelial carcinoma, circulating tumor cell, micrometastasis, radical cystectomy, adjuvant, outcome, survival Additional Supporting Information may be found in the online version of this article. A.S. and S.R. contributed equally to this work Grant sponsor: DISSECT to KP; Grant number: ERC AdG_ DOI: /ijc History: Received 6 May 2016; Accepted 7 Sep 2016; Online 26 Sep 2016 Correspondence to: Michael Rink, University Medical Center Hamburg-Eppendorf, Department of Urology, Martinistraße 52, Hamburg, Germany, Mrink@uke.de; Tel: ; Fax: recurrent high-risk nonmuscle-invasive UCB refractory to intravesical instillation therapy. 2 Despite remarkable advances in UCB biology, surgical technique, imaging, perioperative management, and systemic, little has changed in outcomes over the past few decades. 3 For the majority of patients with organ-confined disease, RC offers durable disease control and long-term survival, while over half of patients with advanced disease or lymph node metastasis face poor outcomes. 2,3 Perioperative systemic is recommended in patients at high-risk for disease recurrence, although its efficacy and toxicity renders it a source of continuing debates. 4,5 Histopathologic parameters alone as well as prediction models based on clinicopathologic variables unfortunately remain imperfect for patient counseling and decision making regarding adjuvant (AC) administration. 6 Indeed, the majority of patients who develop metastases have disease recurrence within the first 2 years following RC, 7 suggesting that disease recurrence may be due to early dissemination of micrometastases that were undetectable using conventional staging techniques prior to surgical therapy. 8 Int. J. Cancer: 140, (2017) VC 2016 UICC

2 382 Circulating tumor cells and adjuvant after radical cystectomy What s new? The ability to predict disease recurrence in patients with urothelial carcinoma of the bladder (UCB) can potentially overcome current limitations in perioperative management and improve patient outcomes. Accurate blood-based and tissue markers are lacking, though the present study highlights the promise of circulating tumor cells (CTCs). In the study population, CTCs were isolated from about 22% of patients treated with radical cystectomy (RC). Among UCB patients who did not receive adjuvant, CTC presence independently predicted reduced survival and unfavorable outcome. CTC detection may serve a valuable role in informing decisions about adjuvant administration in UCB. Currently, there is a lack of accurate tissue or blood-based biomarkers in UCB for predicting disease recurrence following extirpative surgery with curative intent that can be used to identify those patients, who might safely continue regular follow-up versus those who may benefit from multimodal treatment approaches after surgery. 8,9 In general, there is a principal reluctance of urologists to recommend neoadjuvant or adjuvant, 4,5 when a maximum of only half of patients will experience relapse or respond to systemic. 10,11 Thus, a predictive biomarker may not only help selecting patients best suited for adjuvant, but also in identifying patients who are likely to be cured with local therapy alone, thus reducing the burden of unnecessary care. Circulating tumor cells (CTC) are malignant epithelial cells in the circulatory system, representing a marker for occult but clinically relevant micrometastatic disease burden. 12 The Food and Drug Administration-approved, semiautomated CellSearch VR system allows a reliable and standardized detection and enumeration of CTC, 12 as demonstrated in various malignancies including UCB According to results of previous studies from others and our research group, the presence of CTC is associated with unfavorable outcomes in UCB However, the relevance of CTC in decision making remains unclear. Several studies in other cancer entities have reported that CTC are a valuable marker regarding decision making Therefore, the aim of this nonrandomized, prospective study was to investigate the impact of preoperatively detectable CTC on outcomes and predictive significance in clinically nonmetastatic UCB patients treated with RC with or without AC. Materials and Methods Patient population After written informed consent, we prospectively enrolled 226 UCB patients treated with RC and bilateral pelvic lymphadenectomy without neoadjuvant at the University Medical Center Hamburg-Eppendorf between 2007 and Indications for RC were recurrent Ta, T1, or carcinoma in situ (CIS) refractory to transurethral resection of the bladder (TURB) with or without intravesical immunotherapy or, or muscle invasive UCB. Preoperative staging comprised histopathologic evaluation of TURB specimens, computed tomography (CT) of the abdomen/pelvis and thorax, as well as bone scan and CT of the brain when clinically indicated. Patients with metastatic UCB at preoperative staging (n 5 6), patients with a history of any other malignancy (n 5 4), male patients with known or suspected coincidental prostate cancer (n 5 11), patients with missing histopathologic or clinical data or missing follow-up (n 5 6) were excluded from the present study (Fig. 1). Patients with previous systemic or radiation were ineligible. The study was approved by the local ethics committee (no. PV3779). Pathological evaluation For pathological evaluation, the complete surgical RC specimen was inked, and multiple sections were obtained from the bladder and the tumor in addition to the regional lymph nodes and ureters. Tumor stage and nodal status were assessed according to the tumor, lymph node, and metastasis (TNM) system. Tumor grade was assessed according to the 1998 World Health Organization (WHO) grading system. 19 Concomitant CIS was defined as the presence of CIS in conjunction with another tumor other than CIS alone. Lymphovascular invasion (LVI) was defined as the unequivocal presence of tumor cells within an endothelium-lined space without underlying muscular walls. 20 Microvascular invasion (MVI) was defined as the presence of tumor cells within a vessel with a vascular wall and red blood cells in the lumen. 21 A positive soft tissue surgical margin (STSM) was defined as the presence of tumor at inked areas of soft tissue on the RC specimen. 22 Adjuvant Generally, AC started within 90 days after RC. 23 AC was recommended in patients with extravesical disease (pt 3) and/or lymph node metastasis according to guideline standards. 2 In general, mg/m 2 cisplatin-based AC regimens were administered, consisting of cisplatin combined with gemcitabine protocols, or cisplatin combined with methotrexate, vinblastine, and adriamycin protocols. No age limits were applied, but an adequate performance status, hematological and renal functions, and normal auditory and cardiac functions were required in patients scheduled for cisplatin-based AC. In patients with reduced renal function (i.e., glomerular

3 Soave et al. 383 magnetic resonance imaging) were conducted at the discretion of the treating physician when clinically indicated. Disease recurrence was defined as local failure in the operative site, regional lymph nodes, or distant metastasis. Upper tract urothelial carcinoma was not considered disease recurrence but metachronous tumor. Cancer-specific mortality was defined as death from UCB. Overall mortality was defined as death from any cause. The cause of death was determined by the treating physician, by chart review corroborated by death certificates, or by death certificates alone. 25 Perioperative mortality (i.e., death within 30 days of surgery) was censored at time of death for bladder cancer-specific survival analyses. Figure 1. Study flow chart. filtration rate <60 ml/min) or inadequate auditory function, carboplatin-based AC (area under the curve 4.5) was administered. Patients received four cycles of AC, unless AC had to be stopped due to toxicity or patients desire. More than four cycles were administered at the clinicians decision based on the tumor and health status. The was not considered for AC decision-making. Rather, AC was administered at the clinicians decision based on the tumor and health status as well as patients desire. Circulating tumor cell measurement The CellSearch VR system (Janssen, Raritan, NJ, USA) was used for analysis as described previously. 13,14 Preoperative blood samples (7.5 ml) were usually collected on the day prior to RC in CellSave tubes (Janssen Diagnostics), at least 14 days after the preceding TURB. In brief, all blood samples were analyzed within 96 hr after collection (median: 24 hr; interquartile range (IQR): 24; 48 hr). Antibodies directed against keratin 8, 18, and 19 were used to detect epithelial cells among the cells captured by epithelial cell adhesion molecule (EpCAM) antibodies. A cluster of differentiation (CD)245 antibody was used to exclude leukocytes. Nuclei were counterstained with 4,6-diamino-2-phenylindole. After enrichment and immunochemical staining, immunomagnetically labeled cells were kept in a strong magnetic field and scanned using the CellSpotter TM analyzer (Janssen Diagnostics). Image galleries were manually evaluated for CTC according to criteria reported previously. 24 Follow-up regimen Patients were usually seen every 3 months for the first year after RC, every 6 months from the second to fifth years, and annually thereafter. Follow-up included a history, serum chemistry evaluation and physical examination. Diagnostic imaging of the abdomen including the urinary tract (e.g., ultrasonography and/or intravenous urography, CT of the abdomen/pelvis with intravenous contrast) and chest radiography were performed at least annually or when clinically indicated. Additional radiographic evaluations (i.e., bone or brain scans and Statistical analysis The coprimary endpoints of the present study were recurrence-free, cancer-specific, and overall survival, according to the and AC administration. The indicator variables (i.e., CTC and AC status) were analyzed as categorical variables. Associations between categorical variables were assessed using the Fisher exact and v 2 tests. Differences in continuous variables were analyzed using the Mann Whitney U test (two categories) and the Kruskal Wallis test (three or more categories). Recurrence-free, cancer-specific, and overall survival probabilities were estimated using the Kaplan Meier method and differences between groups were assessed using the log rank statistic. Uni- and multivariable Cox regression models assessed time to disease recurrence, cancer-specific and overall mortality. All tests were two-sided and a p values of <0.05 was set to be statistically significant. All analyses were performed with SPSS 20 (IBM Corporation, Armonk, NY). Results Clinicopathologic characteristics according to the adjuvant status and CTC were found in 41 patients (22.2%). Overall, CTC were present in 15 patients (36.6%) with localized UCB without lymph node metastases. The median CTC count per 7.5 ml blood was 1 [mean: 10.49; standard deviation (SD): 33.79; range: 1 163]. In patients with localized and advanced diseases, the median number of CTC was 1 (mean: 2.07; SD: 2.43; range: 1 10) and 1 (mean: 15.35; SD: 41.90; range: 1 163) (p ), respectively. AC was administered in 50 patients (27.0%), with cisplatin-based regimens in 41 patients (82.0%). The median cycle number was 4 (mean: 3.89; SD: 1.15; range: 1 6), and 42 patients (84.0%) and 31 patients (62.0%) completed 3 and 4 cycles, respectively. In total, 17 patients (41.5%) with the presence of CTC received AC, compared to 33 patients (22.9%) without CTC (p ). The administration of AC was associated with an increased number of CTC (p ). Table 1 presents the descriptive characteristics of the study cohort. The presence of CTC and the administration of AC, respectively, were associated with presence of LVI, MVI and a positive STSM (all p-values 0.024). AC was also associated with advanced tumor stage, higher tumor grade, lymph node metastasis, and younger age (all p-values 0.027).

4 384 Circulating tumor cells and adjuvant after radical cystectomy Table 1. Descriptive characteristics of 185 urothelial carcinoma of the bladder patients treated with radical cystectomy and bilateral lymphadenectomy stratified by the administration of adjuvant status and the circulating tumor cell status No administration of adjuvant (n 5 135) Administration of adjuvant (n 5 50) p values negative (n 5 144) positive (n 5 41) p values All Parameter (n 5 185) Age [year; 68.0 (59; 75) 69.0 (61; 75) 65.0 (56; 72) (60; 75) 71.0 (57; 75) median (IQR)] Gender (n; %) Male 135 (73.0) 98 (72.6) 37 (74.0) 103 (71.5) 32 (78.0) Female 50 (27.0) 37 (27.4) 13 (26.0) 41 (28.5) 9 (22.0) ECOG-PS (85.9) 115 (85.2) 44 (88.0) 126 (87.5) 33 (80.5) 1 26 (14.1) 20 (14.8) 6 (12.0) 18 (12.5) 8 (19.5) Days between TURB and RC [median (IQR)] 24 (17; 34) 24 (17; 33) 24 (15; 34) (17; 33) 24 (18; 36) Pathologic tumor stage (n; %) < pt0 18 (9.7) 18 (13.3) 0 (0) 18 (12.5) 0 (0) pta 11 (5.9) 10 (7.4) 1 (2.0) 8 (5.6) 3 (7.3) ptis 10 (5.4) 8 (5.9) 2 (4.0) 9 (6.2) 1 (2.4) pt1 18 (9.7) 16 (11.9) 2 (4.0) 16 (11.1) 2 (4.9) pt2 45 (24.3) 40 (29.6) 5 (10.0) 32 (22.2) 13 (31.7) pt3 59 (31.9) 33 (24.4) 26 (52.0) 46 (31.9) 13 (31.7) pt4 24 (13.0) 10 (7.4) 14 (28.0) 15 (10.4) 9 (22.0) Combined tumor stage (n; %) < Localized (pt2) 102 (55.1) 92 (68.1) 10 (20.0) 83 (57.6) 19 (46.3) Advanced (pt3) 83 (44.9) 43 (31.9) 40 (80.0) 61 (42.4) 22 (53.7) Combined disease stage (n; %) < pt2 and pn0 90 (48.6) 85 (63.0) 5 (10.0) 75 (52.1) 15 (36.6) pt3 or pn (51.4) 50 (37.0) 45 (90.0) 69 (47.9) 26 (63.4) Pathologic tumor grade (n; %) No grading (pt0 18 (9.7) 18 (13.3) 0 (0) 18 (12.5) 0 (0) disease) G2 10 (5.4) 10 (7.4) 0 (0) 7 (4.9) 3 (7.3) G3 157 (84.9) 107 (79.3) 50 (100.0) 119 (82.6) 38 (92.7) Concomitant carcinoma in situ (n; %) Absent 121 (65.4) 90 (66.7) 31 (62.0) 96 (66.7) 25 (61.0) Present 64 (34.6) 45 (33.3) 19 (38.0) 48 (33.3) 16 (39.0) Lymph node metastasis (n; %) < Negative 132 (71.4) 116 (85.9) 16 (32.0) 106 (73.6) 26 (63.4) Positive 53 (28.6) 19 (14.1) 34 (68.0) 38 (26.4) 15 (36.6) Lymphovascular invasion (n; %) < Absent 130 (70.3) 105 (77.8) 25 (50.0) 107 (74.3) 23 (56.1) Present 55 (29.7) 30 (22.2) 25 (50.0) 37 (25.7) 18 (43.9)

5 Soave et al. 385 Table 1. Descriptive characteristics of 185 urothelial carcinoma of the bladder patients treated with radical cystectomy and bilateral lymphadenectomy stratified by the administration of adjuvant status and the circulating tumor cell status (Continued) No administration of adjuvant (n 5 135) All Parameter (n 5 185) Microvascular < invasion (n; %) Absent 160 (86.5) 124 (91.9) 36 (72.0) 129 (89.6) 31 (75.6) Present 25 (13.5) 11 (8.1) 14 (28.0) 15 (10.4) 10 (24.4) Outcomes according to the adjuvant status and The median follow-up of cancer survivors was 31.0 months (IQR: 13; 46). Actuarial 2- and 5-year recurrence-free, cancer-specific, and overall survival estimates were % (standard error) and %, % and %, and % and %, respectively. In Kaplan Meier analyses patients with presence of CTC had reduced recurrence-free, cancer-specific, and overall survival, compared to patients with absence of CTC (p-values <0.001; Fig. 2). In CTC-positive patients, there was no difference in all endpoints according to the number of CTC (stratified 1 CTC vs. >1 CTC/7.5 ml, and 1 CTC vs. 2 4 CTC vs. 5 CTC/7.5 ml; figure not shown). Patients receiving AC also had reduced recurrence-free, cancer-specific and overall survival, compared to patients not receiving administration of AC (p-values <0.001; Fig. 2). There was no difference in either endpoint according to AC regimen or the number of AC cycles, when stratified by <4 versus 4 cycles (Fig. S1). Administration of adjuvant (n 5 50) p values negative (n 5 144) positive (n 5 41) p values Soft tissue surgical margin status (n; %) Negative 159 (85.9) 123 (91.1) 36 (72.0) 129 (89.6) 30 (73.2) Positive 26 (14.1) 12 (8.9) 14 (28.0) 15 (10.4) 11 (26.8) Number of lymph nodes 15 (11; 21) 15 (11; 20) 19 (11; 28) (11; 22) 16 (11; 21) removed [median (IQR)] Administration of adjuvant (n; %) No 135 (73.0) 135 (100.0) 111 (77.1) 24 (58.5) Yes 50 (27.0) 50 (100.0) 33 (22.9) 17 (41.5) Adjuvant regimen (n; %) Cisplatin based 41 (22.2) 41 (82.0) 27 (81.8) 14 (82.4) Carbolatin based 9 (4.9) 9 (18.0) 6 (18.2) 3 (17.6) (n; %) Negative 144 (77.8) 111 (82.2) 33 (66.0) 144 (100.0) Positive 41 (22.2) 24 (17.8) 17 (34.0) 41 (100.0) CTC count in patients with presence of CTC [median (range)] 1 (1 163) 1 (1 163) 2 (1 150) (1 163) Statistical significant results are highlighted in bold letters. Abbreviations: CTC, circulating tumor cells; ECOG-PS, Eastern Cooperative Oncology Group performance score; IQR, interquartile range; RC, radical cystectomy; TURB, transurethral resection of the bladder. Outcomes of patients without administration of adjuvant CTC-positive patients who did not receive AC had inferior recurrence-free, cancer-specific, and overall survival, compared to patients without CTC (all p-values 0.001; Fig. 3). In the subgroup of localized UCB patients without lymph node metastasis, CTC-positive patients demonstrated reduced recurrence-free, cancer-specific, and overall survival, compared to patients without CTC (all p-values 0.003; Fig. S2). In univariable analyses, the presence of CTC was associated with all three endpoints (p-values 0.001). In multivariable Cox regression analysis that adjusted for established clinicopathologic UCB risk factors, the presence of CTC remained an independent predictor for reduced recurrence-free, cancerspecific, and overall survival (p-values 0.003; Table 2). Outcomes of patients with administration of adjuvant In patients who received AC, there was no difference in recurrence-free, cancer-specific and overall survival according

6 386 Circulating tumor cells and adjuvant after radical cystectomy Figure 2. Kaplan Meier plots of recurrence-free (A and D), cancer-specific (B and E), and overall survival (C and F) stratified by the circulating tumor cell status (A C) and adjuvant status (D F) in 185 urothelial carcinoma of the bladder patients treated with radical cystectomy with and without adjuvant. [Color figure can be viewed at wileyonlinelibrary.com] to the (Fig. 3). In univariable Cox regression analysis, the presence of CTC was not associated with inferior recurrence-free, cancer-specific, and overall survival (p-values: n.s.). Discussion This is the first prospective study investigating the potential role of the regarding decision making on AC administration in UCB patients following RC. We previously demonstrated that CTC are associated with poor outcomes in UCB. 13,14 In the present study, we found that particularly in UCB patients treated with RC without AC administration, the presence of CTC was an independent predictor for reduced recurrence-free, cancer-specific, and overall survival, when adjusted for established risk factors of unfavorable UCB outcomes. Although CTC are relatively rare events among more than a million blood cells, 26 they have already demonstrated their potential identifying patients at risk in other tumor types and, thus, are a suggested biomarker for decision-making on systemic In the present study, the presence of CTC was also associated with unfavorable outcomes in patients with localized UCB without lymph node metastasis, despite the small patient number in this subgroup. Therefore, further prospective, randomized studies are necessary to elucidate the promising role of CTC for decision making on AC administration in patients, who otherwise would not be counseled regarding AC based on their pathologic UCB features. Considering current controversies on AC administration in UCB, especially its debatable efficacy, which has mainly been assessed in retrospective and underpowered studies, its under-utilization and insufficient patient selection, 4,23 our findings emphasize the clinical importance of CTC as a potential biomarker for the decisionmaking on AC. Although different tools (e.g., nomograms) improved the estimation of the risk of disease recurrence, 27 clinical application of the majority of models remains sparse due to an imperfect prediction of the true tumor biology and course of disease. In addition, various biomarkers have been

7 Soave et al. 387 Figure 3. Kaplan Meier plots of recurrence-free (A and D), cancer-specific (B and E), and overall survival (C and F) stratified by the circulating tumor cell status in 135 urothelial carcinoma of the bladder patients treated with radical cystectomy without adjuvant (A C), and in 50 urothelial carcinoma of the bladder patients treated with radical cystectomy plus administration of adjuvant (D F). [Color figure can be viewed at wileyonlinelibrary.com] Table 2. Multivariable Cox regression analysis predicting disease recurrence, cancer-specific and overall mortality of 135 urothelial carcinoma of the bladder patients treated with radical cystectomy and bilateral lymphadenectomy without administration of adjuvant Parameter Disease recurrence Cancer-specific mortality Overall mortality HR 95% CI p values HR 95% CI p values HR 95% CI p values Gender (female vs. male) Age (continuous) Tumor stage (localized vs. advanced) Tumor grade (continuous) Carcinoma in situ (presence vs. absence) Lymph node metastasis (presence vs. absence) Soft tissue surgical margin (positive vs. negative) Lymphovascular invasion (presence vs. absence) Microvascular invasion (presence vs. absence) (positive vs. negative) < Statistical significant results are highlighted in bold letters. Abbreviations: CI, confidence interval; CTC, circulating tumor cells; HR, hazard ratio.

8 388 Circulating tumor cells and adjuvant after radical cystectomy investigated regarding their capacity improving outcome prediction and selection of UCB patients for multimodal treatments. 9 However, currently no blood-based biomarkers succeeded in daily clinical routine. Proof of an early micrometastatic disease, undetectable using standard staging techniques, may have the encouraging potential to finally overcome this difficulty and, therefore, should be included in the planning of multimodal treatment in UCB patients. We did not find differences in outcomes according to in patients who received AC. Not surprising, patients receiving AC had substantial, unfavorable pathologic features, including advanced tumor stage and lymph node metastasis, which may certainly explain the association of the administration of AC with inferior outcomes. Thus, a missing statistical difference according to the may be due to the general worse outcome in this patient subgroup. Alternatively, AC may also abrogate the adverse impact of CTC on disease recurrence and survival. However, our study did not implicitly demonstrate that administration of AC to patients with presence of CTC impacts their likelihood of disease recurrence. Although it has been demonstrated that the half-life of CTC in the peripheral blood is limited due to mechanical shear forces and the activity of the immune system, 12 AC might have a direct impact on CTC that have escaped these mechanisms, for example, by promoting apoptosis or inhibiting the deposition of these cells at distant sites. Correspondingly, it has been suggested that the CTC status and changes in CTC count might correlate with the biologic UCB behavior during systemic. 28 Further studies are warranted to verify these hypotheses. In our study, 27% of patients received AC with cisplatincontaining protocols in the majority of patients, which is inline with current rates and regimens in the peer-reviewed literature. 3 5 The decision-making on AC administration was independent of the. This may have influenced our results, as AC was administered according to currently established pathologic risk factors as well as patients health status and desire. Only 47% of patients who were eligible based on their pathologic UCB characteristics received AC, which might be due to reduced health status, patients preferences or other undetermined reasons. However, our results reflect the real world clinical scenario, and other authors investigating the delivery of in UCB found even lower AC administration rates. 5 Although this was a nonrandomized study, our study population was homogenously distributed regarding ECOG-PS. In contrast, AC consisted of different cisplatin-containing and carboplatincontaining protocols, thus introducing considerable treatment heterogeneity and a possible source of bias and also reflecting a real-world clinical scenario. 4,5 Interestingly, we found that the administration of AC was associated with the presence of CTC, a known risk factor for unfavorable outcomes in UCB. 13,14 While AC administration is related to aggressive UCB, in accordance with previous reports, the presence of CTC was not significantly associated with advanced disease stages, 13,14 although we found a trend. A major point of discussion therefore remains whether the CellSearch VR system detects all CTC. Particularly the most aggressive and dedifferentiated tumors may be missed due to loss of EpCAM and/or keratin expression in the epithelial mesenchymal transition, a crucial step in the metastatic cascade. 8 A very recent study reported superior CTC detection rates in UCB using the IsoFlux TM device compared to the CellSearch VR system. 29 Other methods of CTC capture were not considered in the present study, including density gradient centrifugation, isolation by size of epithelial tumor cells and dielectrophoretic cell separation. 26 In addition, molecular identification of CTC by targeting transcripts or genomic aberrations of urothelial cancer cells and cancer-associated DNA was not performed, although it may give evidence of the biologic origin of the detected CTC. 8,26 However, to date the CellSearch VR device remains the best-validated technology for CTC analyses. 26 In addition, it remains unclear, if all epithelial cells detected are truly viable and malignant CTC with a metastasis-initiating potential. 12 In vitro culturing and detailed characterization of viable CTC have been described very recently, offering the opportunity to reveal the biologic origin and make-up of these cells. 30 Importantly, molecular profiling of CTC (e.g., whole genome sequencing) may not only enhance the understanding of the metastatic cascade, but also facilitate the identification of patients benefiting most from systemic and emerging targeted therapies. Previously, other authors reported promising findings on alterations in different DNA repair-associated genes for predicting the response to and thus potential selection of patients benefitting from due to reasonable response. 31 In addition, biomarker panels have been suggested for predicting prognosis and selecting patients for more aggressive forms of treatment, especially platinum containing protocols. 32 The present study has some further limitations. First and foremost are limitations inherent to the sample size and nonrandomized study design. We cannot exclude false-positive CTC findings, especially since the release of CTC into the blood stream by TURB was reported. 33 However, the short half-life of CTC in the circulatory system (1 3 hr) 12 makes it unlikely that keratin-positive cells from the preceding TURB might have influenced our results. In addition, the study did not consider patients treated with neoadjuvant, although it is currently recommended in some cases due to a proven, but moderate survival benefit, as demonstrated in various large prospective trials. 2 Nevertheless, our findings may have essential implications on the decision-making regarding neoadjuvant. Therefore, the inclusion of CTC in future clinical studies on systemic for UCB is highly desirable, and a randomized study is urgently warranted to validate our findings. Conclusion The may be useful for counseling and decisionmaking on administration of AC in UCB patients following RC. Further studies are warranted to clarify the ability of

9 Soave et al. 389 CTC identifying patients, who probably benefit from multimodal treatment, particularly those who would not be selected for AC based on established UCB risk factors. Therefore, CTC measurement should be implemented in future clinical and randomized studies on systemic in UCB to validate our findings. Acknowledgement We thank Richard K. Lee, M.D., Department of Urology, Weill Cornell Medical College, New York, NY, USA, for proof reading and linguistic support. Conflict of Interest Statement The authors have declared no conflicts of interest. REFERENCES 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, CA Cancer J Clin 2015;65: Witjes JA, Comperat E, Cowan NC, et al. EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol 2014;65: Stein JP, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. 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