A phase 2 study of TMX-101, intravesical imiquimod, for the treatment of carcinoma in situ bladder cancer

Urologic Oncology: Seminars and Original Investigations 35 (2017) 39.e1–39.e7

Original article

A phase 2 study of TMX-101, intravesical imiquimod, for the treatment of carcinoma in situ bladder cancer Nicholas M. Donin, M.D.a,*, Karim Chamie, M.D., M.S.H.S.a, Andrew T. Lenis, M.D., M.S.a, Allan J. Pantuck, M.D., M.S.a, Madhu Reddy, M.D.b, Dana Kivlin, M.D.b, Johanna Holldack, M.D.c, Rafaella Pozzi, PharmDc, Gil Hakim, B.Scg, Lawrence I. Karsh, M.D.d, Donald L. Lamm, M.D.e, Laurence H. Belkoff, D.O.b, Arie S. Belldegrun, M.D.a, Stuart Holden, M.D.a, Neal Shore, M.D.f a

Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, CA b Division of Urology, Hahnemann University Hospital, Philadephia, PA c Telormedix SA, Bioggio, Switzerland d The Urology Center of Colorado, Denver, CO e BCG Oncology, Phoenix, AZ f Carolina Urologic Research Center, Myrtle Beach, SC g UroGen Pharma, Ra’anana, Israel Received 3 June 2016; received in revised form 28 August 2016; accepted 21 September 2016

Abstract Purpose: Imiquimod is a toll-like receptor agonist with proven antitumor activity as a topical treatment for skin cancer. TMX-101 (Vesimune) is a novel liquid formulation of imiquimod optimized for intravesical delivery. The agent demonstrated safety as an intravesical treatment for non–muscle-invasive bladder cancer in a phase 1 clinical trial. We report the results of a phase 2 prospective multicenter clinical trial assessing the safety and activity of TMX-101. Materials and methods: Patients with non–muscle-invasive bladder cancer containing carcinoma in situ were eligible for inclusion. Enrolled patients received 6 weekly intravesical administrations of 200 mg/50 ml TMX-101 0.4%. End points included rate of adverse events, changes in urinary cytokine levels following treatment, and clinical response at 6 weeks following final instillation, defined as negative posttreatment bladder biopsy and urine cytology results. Results: A total of 12 patients were enrolled, with 10 available for efficacy analysis. Half of the patients (6/12) had received Z2 prior induction courses of bacillus Calmette-Guerin. All patients received all 6 doses of TMX-101 per protocol. Overall, 75% of patients experienced treatment-related adverse events, only 1 of which was 4grade 2 (urinary tract infection). Furthermore, 2 patients demonstrated a negative cytology and biopsy result at 6 weeks following treatment. Significant increases in urinary cytokines, including IL-6 and IL-18, were seen following treatment. Conclusion: In this phase 2 pilot study in patients with carcinoma in situ bladder cancer, intravesical TMX-101 was safe and well tolerated with common, mild genitourinary adverse effects. Clinical activity was suggested by the increase in posttreatment urinary cytokines. Complete responders were seen. Further investigation of the agent is warranted. r 2017 Elsevier Inc. All rights reserved.

Keywords: Imiquimod; Urinary bladder neoplasms; Carcinoma in situ; Administrations, intravesical drug; Immunotherapy

Trial registration—Clincaltrials.gov: NCT01731652. Study sponsor: Telormedix. The research was funded by the European Community’s Seventh Framework Programme, Belgium (FP7-2007-2013) under Grant agreement nHEALTH-F4-2011-281608 (TIMER). * Corresponding author. Tel.: þ1-310-794-2858; fax: þ1-310-794-3513. E-mail address: [email protected] (N.M. Donin). http://dx.doi.org/10.1016/j.urolonc.2016.09.006 1078-1439/r 2017 Elsevier Inc. All rights reserved.

1. Introduction Bladder cancer is a common malignancy, representing 429,800 new cases and 165,100 deaths annually worldwide [1]. Approximately 80% of these patients present with

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non–muscle-invasive bladder cancer (NMIBC), which exists in 2 phenotypic forms: discrete papillary tumors that are generally well demarcated and can be removed via endoscopic resection and carcinoma in situ (CIS), a highgrade multifocal entity with a tendency toward frequent recurrences and progression to muscle-invasive disease [2]. Intravesical bacillus Calmette-Guerin (BCG) is an effective treatment for CIS [3], demonstrating consistent initial response rates of 70% [4]. Unfortunately, BCG fails in a significant proportion of patients, owing to either intolerable adverse effects, serious infections, or refractory disease [5–7]. Patients in whom BCG fails are at high risk for disease progression [8]. Many of these patients may not be candidates for radical cystectomy owing to underlying health status. As such, there is a clinical need for alternative treatments for patients with CIS bladder cancer. Toll-like receptors (TLRs) are a family of highly conserved transmembrane receptors that function in innate and adaptive immunity [9]. Activation of TLR pathways results in expression of various proinflammatory cytokines including chemokines and other inflammatory mediators [10]. These mediators, in turn, result in efficient antigen presentation by mature dendritic cells and enhance production of antigen-specific T cells. Imiquimod is a small molecule of the imidazoquinoline family that acts via agonism toward TLR7 [11,12]. Imiquimod has been successfully used as the active ingredient in Aldara as a topical treatment for various benign and malignant skin conditions. This has prompted interest in imiquimod as a potential intravesical bladder cancer treatment. TMX-101 is a novel liquid formulation of imiquimod that is optimized for intravesical delivery. Intravesical TMX-101 induces local immunological activation, resulting in significant reductions in tumor burden in an orthotopic bladder cancer model [11,13]. A phase 1 dose-escalation study demonstrated doses up to 200 mg (0.4%) were safe, with low systemic uptake and common, mild, adverse effects and no reported grade 3 adverse events (AEs) [14]. Herein, we present the results of a phase 2 single-arm study evaluating the tolerability, safety, and activity of intravesical TMX-101 in patients with NMIBC containing CIS.

2. Materials and methods 2.1. Study design We performed a prospective, nonrandomized, openlabel, fixed-dose, phase 2 trial investigating the safety, tolerability, and biological activity of intravesical TMX-101 0.4% in patients with CIS bladder cancer at 4 centers in the United States. End points included clinical response at 6 weeks following the final instillation, safety, and the tolerability of TMX-101, as well as biological activity of TMX-101, as measured by pharmacodynamic markers in predosing and postdosing urine specimens. After informed

consent, patients were evaluated for study eligibility during the screening period (days -28 to -1). Screening evaluations included a medical history, physical examination, vital signs, Eastern Cooperative Oncology Group performance status determination, complete blood count, serum chemistry, microscopic urinalysis, urine culture, and urine sampling for pharmacodynamic parameters. Screening procedures also included cystoscopic examination of the bladder with mapping and biopsies of all suspicious areas between days-28 and days-14, followed by bladder washing for cytology. Patients who had undergone such examinations as part of their treatment outside of the study protocol were not required to repeat the procedures, provided that examinations took place during the specified timeframe, and pathologic samples were available for central review. Patients with histologically confirmed CIS and who fulfilled all other eligibility criteria were enrolled in the study. The study planned to enroll up to 12 patients to obtain 6 evaluable patients (patients with confirmed positive CIS on screening histology who received all 6 TMX-101 doses and underwent 6-week evaluation with cystoscopy with interpretable histologic and cytologic sampling). Patients provided informed written consent. Institutional review board approval was obtained at each participating site. 2.2. Patient inclusion Inclusion criteria included age Z18 years, Eastern Cooperative Oncology Group performance status 0 to 2, with adequate bone marrow, hepatic, and renal function, and with histologically proven CIS. CIS lesions could be primary, secondary, or recurrent and could be concomitant to Ta or T1 lesions, provided that these lesions had been fully resected. BCG-naive patients were allowed to enroll in the study, provided they undergo close follow-up after the end of the study period and were allowed to undergo additional treatments following treatment with TMX-101, as dictated by their referring urologist. Women of childbearing age were required to have a negative pregnancy test result at screening and to avoid pregnancy by use of adequate contraceptive methods. Exclusion criteria included the inability to maintain intravesical treatments within the bladder, a history of pelvic radiation, perioperative intravesical chemotherapy, a history of either muscle-invasive urothelial carcinoma of the bladder or urothelial carcinoma of the ureter or renal pelvis, immunocompromised state, an active infection at the time of screening, a history of hypersensitivity to any of the study drug components, or participation in any other investigational trials in the 3 months before screening. Patients who had received any intravesical therapy in the 3 months before screening were also excluded. 2.3. Study drug Intravesical TMX-101(Vesimune) is a sterile liquid dosage form of imiquimod, suitable for intravesical

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instillation. All patients were scheduled to receive intravesical TMX-101 0.4% (200 mg in 50 ml) once weekly for 6 weeks. In the phase 1 study, doses ranging from 0.05% to 0.4% were well tolerated [14], and no relationship was noted between study drug doses and AEs. Because 0.4% is the highest feasible concentration owing to solubility limitations of TMX-101, 0.4% (200 mg/50 ml) was chosen as the concentration for evaluation in this study. Doses were administered intravesically by gravity using a standard urethral catheter. Patients were instructed to maintain the solution in their bladder for 1 hour, based on findings in a preclinical swine model demonstrating a submucosal lymphocytic inflammatory infiltrate within the bladder following a 1-hour dwell time [15].

2.4. Study assessments Patients underwent evaluation before each instillation, consisting of vital signs, urine analysis, urine culture, assessment of AEs, and biochemical and hematological laboratory testing. Urine was obtained before and after each instillation for evaluation of urinary cytokines. At 6 weeks after the last study drug dose was administered, cystoscopy with bladder biopsy and urine cytology were performed. During cystoscopy, any suspicious lesions were biopsied. In cases without suspicious lesions, at least 1 biopsy was taken at the prior positive site, with at least 1 additional random biopsy. All specimens were evaluated locally for the determination of patient response. All slides were sent for assessment to the central pathologist for the confirmation of patient response to treatment. Findings from this 6-week evaluation were used to determine treatment response. Following this final assessment, study patients were referred back to their treating physician for ongoing management. The study protocol allowed for the collection of follow-up information pertaining to bladder cancer in the 6-month period after the last dose of TMX-101 from the patient’s treating physician. Such information was to include any bladder cancer–specific treatments administered as well as any applicable cystoscopy, urine cytology, or biopsy findings.

2.5. End point determination Responders were defined as patients with no evidence of disease (negative histology on biopsy and negative urine cytology results) 6 weeks after the last TMX-101 instillation based on the central evaluation. Safety and tolerability were determined by documentation of AEs, evaluated and graded according to the National Cancer Institutes Common Terminology Criteria for Adverse Events, version 4.03. For evaluation of change in urinary cytokines, the mean difference from pretreatment to posttreatment was calculated, with a t test used to evaluate for differences.

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Table 1 Demographic and baseline characteristics (ITT population; N ¼ 12) (N ¼ 12) Age (y) Median (range)

71.0 (54–80)

Sex Male Female

11 (91.7) 1 (8.3)

Ethnicity Hispanic or Latino Non-Hispanic or non-Latino Not reported

0 12 (100.0) 0

Race American Indian or Alaskan native Asian Black or African American Native Hawaiian or other Pacific Islander White Other

0 0 1 (8.3) 0 10 (83.3) 1 (8.3)

Weight (kg) Median (range)

79.4 (56–109)

ECOG performance status Grade 0 Grade 1

11 (91.7) 1 (8.3)

Continuous variables listed as median (range) and categorical variables displayed as n (%). ECOG ¼ Eastern Cooperative Oncology Group; ITT ¼ intent-to-treat.

3. Results A total of 12 patients were screened, enrolled, and treated with TMX-101 between April 2013 and February 2014 and served as the intent-to-treat population. The cohort was predominantly male (92%) and white (83%), and the median age was 71 years (range: 54–80 years) (Table. 1). Disease characteristics of the study cohort are represented in Table 2. All patients had a history of CIS based on local pathologic evaluation. Median duration of CIS since diagnosis was 7.4 months (range: 0.4–109.5). Median number of previous transurethral resection of bladder tumors was 2 (range: 0–10). In total, 8 patients had received prior BCG, with 6 having received Z2 induction courses. Furthermore, 5 patients had received prior intravesical chemotherapy. The median number of prior instillations was 2 (range: 0–7). We observed statistically significant increases in the level of several urinary cytokines following intravesical treatment with TMX-101 including IL-6, IL-18, IL-1β, IL-1 receptor antagonist (IL-1ra), and vascular endothelial growth factor (VEGF) (Table 5). Changes in cytokine levels pretreatment and posttreatment were compared between those patients with a response (n ¼ 4) and those without a response (n ¼ 6). We observed that IL-18

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Table 2 Baseline disease characteristics of ITT population (N ¼ 12) Patient

Pathologic history

1-01 1-04

November 2012: CIS and high-grade Ta 1989: Unknown grade/stage April 2013: CIS May 2011: low-grade Ta December 2012: CIS August 2013: CIS May 2010: CIS

1-09 1-10 1-12 2-02 2-05 3-03 3-07 3-08 4-06 4-11

# Prior TURBTs

2008: CIS and high-grade Ta March 2011: CIS February 2013: CIS April 2011: CIS August 1996: low-grade Ta March 2007: CIS May 2011: high-grade Ta June 2014: CIS September 2004: CIS and high-grade Ta

Prior intravesical immunotherapy

2 4

BCG: 2012 BCG: 2011, 2012

6

BCG: 2011, 2013

1 1

BCG: 2010, 2011

10

2 8

Prior intravesical chemotherapy

Mitomycin: 2011, 2012 Valrubicin: 2012

BCG: 2008, 2009, 2012 BCG: 2011

Mitomycin: 2012

BCG: 2007, 2008, 2011 BCG þ interferon: 2013

Mitomycin: 2011 Mitomycin: 2008 Valrubicin: 2012 (2x 6-week courses)

2 3

BCG: 2004, 2005, 2006, 2010

ITT ¼ intent-to-treat; TURBT ¼ transurethral resection of bladder tumors.

demonstrated a significantly greater increase in patients with a response vs. those who did not experience a response (0.66 vs. 0.39, P ¼ 0.04) (Supplementary Table 1). All 12 patients enrolled received all 6 doses of TMX-101 per protocol. No patient required a dose reduction. Most patients were able to retain study drug for least 40 minutes, with most able to retain each dose for 1 hour by the second

dose. Overall, 92% (11/12) patients who received intravesical TMX-101 experienced a total of 51 AEs, of which 35 were deemed to be treatment related (Table 6). Treatment-related AEs included micturition urgency (6 patients; 50%), dysuria (4 patients; 33%), fatigue (3 patients; 25%), and hematuria (2 patients; 17%), among others. All but 1 AE was assessed by the investigator as

Table 3 Summary of histology and cytology findings and response to treatment by patient Patient

Evaluable?

1-01

Yes

1-04a

No

1-09

Yes

1-10

Yes

1-12

Yes

2-02

Yes

2-05

Yes

3-03

Yes

3-07

Yes

3-08a

No

4-06

Yes

4-11

Yes

a

Timepoint

Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up Screening Follow-up

Histology

Cytology

Response

Local read

Central read

Local read

Central read

Positive Negative Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Negative Positive Positive Positive Positive Positive Negative Positive Positive

Positive Negative Negative Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Negative Positive Positive Negative Negative Equivocal Negative Positive Positive

Negative Negative Negative Negative Positive Positive Negative Positive Positive Positive Negative Negative Positive Negative Negative Negative Negative Negative Positive Positive Negative Negative Positive Positive

Equivocal Positive Equivocal Negative Positive Positive Positive Positive Positive Positive Equivocal Positive Positive Positive Positive Equivocal Negative Equivocal Negative Negative Positive Negative Positive Equivocal

Excluded from evaluable population based on a finding of no CIS on screening evaluation.

Nonresponder Not evaluable Nonresponder Nonresponder Nonresponder Nonresponder Nonresponder Responder Nonresponder Not evaluable Responder Nonresponder

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Table 4 Summary of follow-up data through 6 mo after the last TMX-101 dose in responders (N ¼ 2) Patient

Timepoint a

Alternative systemic treatment of bladder cancer within 6 mo after last dose?

Comments

6-mo cystoscopy, biopsy, and cytology negative. 6-mo cystoscopy negative.

6 wk after last dose

6-mo follow-up period

Histology

Cytology

Histology

Cytology

3-03

Negative

Equivocal

Negative

Negative

No

4-06

Negative

Negative

Not done

Not done

BCG

a

Based on central pathologic review.

grade 1 or 2 in intensity. A patient experienced a grade 3 (severe) AE (urinary tract infection), which also was the only serious AE reported during the study. All AEs were managed with oral medical therapy. No patients required hospitalization. Based on a central pathologic review of baseline biopsy specimens, 2 patients in the intent-to-treat population were deemed not to have CIS, and as such these 2 patients were excluded from the evaluable population. Of the 10 remaining evaluable patients, 2 patients were free of recurrence at the 6-week examination based on the central cytologic and pathologic examinations and were considered treatment responders (Table 3). In these 2 patients, one received no additional treatment, whereas the other received BCG. Both remained free of disease at 6 months (Table 4). Review of clinical laboratory results revealed that mean changes from baseline were small and not clinically meaningful, with no apparent trend in hematologic, clinical chemistry including liver function tests, or urinalysis parameters. No meaningful changes were noted in vitals’ signs or temperature. Any treatment-related electrocardiography abnormalities observed were considered clinically insignificant.

4. Discussion In this phase 2 prospective multicenter study of patients with CIS bladder cancer, 6 weekly instillations of intravesical TMX-101 0.4% were safe and well tolerated. No

patients discontinued treatment or required dose reduction. Although most patients had AEs, all except 1 were grade 1 or 2, and most events were limited to the genitourinary system, with micturition urgency, dysuria, and hematuria being most common. In total, 2 patients demonstrated clinical responses to treatment. Significant increases in urinary levels of IL-6, IL-18, IL-1β, IL-1ra, and VEGF were seen following treatment, consistent with the agent’s mechanism of action. The safety findings herein largely parallel those of the phase 1 dose-escalation trial of TMX101, in which no serious AEs occurred, there was no doselimiting toxicity, and although AEs were common, all were grade 1 or 2. The safety profile of TMX-101 is hence comparable to that of other intravesical chemotherapeutic agents that have been evaluated as alternatives to BCG, including gemcitabine [16], valrubicin [8], and docetaxel [17]. Both TMX-101 and BCG bind TLRs within the membrane of endosomes and lysosomes within host immune and epithelial cells, leading to activation of the intracellular adapter protein MyD88 and up-regulation of NF-kB and MAP-kinases that result in the production of proinflammatory molecules, such as IL-8, IL-6, and IL-1b [18]. BCG, however, is less specific and acts via stimulation of multiple TLRs, including TLR2, TLR4, and TLR9 [19]. TMX-101, in contrast, is believed to act via targeted agonism of TLR7, and although this ultimately leads to up-regulation of a cascade of intracellular proteins similar to that of BCG, it specifically for TLR7 may provide for fewer adverse effects when compared with the somewhat less-specific immunoactivation seen with BCG. Furthermore, TMX-101 may retain

Table 5 Mean difference between pretreatment and posttreatment cytokine values (ITT population; N ¼ 12) Parameter

Mean pretreatment to posttreatment Δ

P value

Interleukin-6 (IL-6) Interleukin-8 (IL-8) Interleukin-18 (IL-18) Macrophage inflammatory protein-1 beta (MIP-1β) Monocyte chemotactic protein-1 (MCP-1) Interleukin-1 beta (IL-1β) Interleukin-1 receptor antagonist (IL-1ra) Vascular endothelial growth factor (VEGF)

0.36 0.19 0.50 0.10 0.02 0.44 1.36 0.15

0.0021 0.1343 o0.0001 0.2186 0.7752 0.0167 o0.0001 0.0437

Bolded values represent statistically significant changes. ITT ¼ intent-to-treat.

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Table 6 Summary of adverse events and stratified by system organ classa (safety population; N ¼ 12) All patients (N ¼ 12) Events, n

Patients, n (%)

Summary All AEs All treatment-related AEs 4Grade 1 treatment-related AEs Serious treatment-related AEs AEs leading to drug discontinuation

51 35 24 1 0

11 (91.7) 9 (75.0) 8 (66.7) 1 (8.3) 0

Renal and urinary disorders Dysuria Micturition urgency Hematuria Urinary retention

24 13 6 4 1

9 4 6 2 1

(75.0) (33.3) (50.0) (16.7) ( 8.3)

General disorders and administration site conditions Fatigue

8

3 (25.0)

8

3 (25.0)

Infections and infestations Urinary tract infection

1 1

1 (8.3) 1 (8.3)

Reproductive system and breast disorders Penis disorder

1 1

1 (8.3) 1 (8.3)

Skin and Subcutaneous tissue disorders Rash

1 1

1 (8.3) 1 (8.3)

a

symptoms (cystitis and hematuria) in up to 50% of patients and systemic symptoms (fever and malaise) in 30% of patients [22,23], along with the potential for systemic infection are potential deterrents to its use and may explain why a proportion of potential candidates for BCG therapy never receive it [24]. Better-tolerated immunotherapeutic treatments may encourage more widespread use. Immunotherapy represents a rational therapeutic strategy for NMIBC for several reasons. First, immunotherapy has historically proven most effective for tumors that harbor high mutational burdens, such as melanoma, lung cancer, and bladder cancers [25]. Second, immunotherapy has already demonstrated significant efficacy in the metastatic and advanced space [26]. Finally, intravesical immunotherapy may ultimately prove more effective than chemotherapy, because traditional chemotherapeutic agents, such as mitomycin and gemcitabine, work through interference with s-phase DNA replication [27,28]. Given that at any time only a subset of malignant cells would be undergoing DNA replication, the efficacy of these agents is likely limited by their dwell time—typically 60 minutes—within the bladder. Combination therapy is also under investigation, and several clinical trials are evaluating novel immunotherapeutic agents in combination with BCG, including systemic anti–programmed cell death ligand-1 (NCT02792192), systemic anti–cytotoxic T-lymphocyte antigen-4 (NCT01838200 and NCT00880854), and intravesical anti–programmed cell death-1 (NCT02808143).

As defined by the Medical Dictionary for Regulatory Activities.

5. Conclusions efficacy in the BCG refractory or resistant space as it signals to similar downstream products through a separate pathway. We observed significant increases in urinary levels of IL-6, IL-18, IL-1β, IL-1ra, and VEGF following treatment with TMX-101, all of which are known to be involved in the response of the urothelium to BCG [20]. IL-6 is secreted in response to internalization of BCG within the urothelium and acts as a neutrophil chemoattractant. IL-18 stimulates macrophage cytotoxicity against bladder cancer cells and induces interferon-gamma production in T cells and natural killer cells. Both IL-1β and IL-1ra are known inducible cytokines in response to BCG. Of note, in the present study, responders had a significant increase in urinary IL-18 levels when compared with nonresponders (0.66 vs. 0.39, P ¼ 0.04). Investigators have previously demonstrated that increases in IL-18 can predict freedom from disease in patients treated with BCG [21]. These observations suggest a potential role for IL-18 as a predictive biomarker for identifying patients who are at high risk of failing intravesical immunotherapy and in whom early cystectomy may be considered. The excellent safety profile of TMX-101 demonstrated herein is important in light of the tolerability and safety concerns associated with BCG therapy. The known AEs profile of BCG therapy, which includes both localized

In this phase 2 pilot study, intravesical TMX-101 was used to treat 12 patients with CIS bladder cancer. The study drug was well tolerated, and no patients required dose reduction or treatment discontinuation. Treatment-related AEs were common and mild, with the majority limited to the genitourinary tract. Clinical activity was suggested by the increase in posttreatment urinary cytokines, and the clinical responses observed in several patients. Intravesical TMX-101 is a promising, safe, and novel immunotherapeutic agent for CIS bladder cancer that warrants further prospective evaluation. Acknowledgments The investigators would like to acknowledge the contributions of Dr. Subodh M. Lele, M.D., who served as the single central pathologist and assessed all pathologic specimens in the trial. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. urolonc.2016.09.006.

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