EUROPEAN UROLOGY 59 (2011)

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1 available at journal homepage: Bladder Cancer Sequential Intravesical Chemoimmunotherapy with Mitomycin C and Bacillus Calmette-Guérin and with Bacillus Calmette-Guérin Alone in Patients with Carcinoma in Situ of the Urinary Bladder: Results of an EORTC Genito-Urinary Group Randomized Phase 2 Trial (30993) Willem Oosterlinck a, *, Ziya Kirkali b, Richard Sylvester c, Fernando Calais da Silva d, Christer Busch e, Ferran Algaba f, Sandra Collette c, Aldo Bono g a Department of Urology, Ghent University Hospital, Ghent, Belgium b Department of Urology, Dokuz Eylul University School of Medicine, Izmir, Turkey c European Organization for Research and Treatment of Cancer Headquarters, Brussels, Belgium d Department of Urology, Centro Hospitalar de Lisboa Central, Lisbon, Portugal e University Hospital, Uppsala, Sweden f Fundacion Puigvert, Barcelona, Spain g Department of Urology, Ospedale Di Circolo E Fondazione Macchi, Varese, Italy Article info Abstract Article history: Accepted November 25, 2010 Published online ahead of print on December 7, 2010 Keywords: Bacillus Calmette-Guérin Carcinoma in situ Intravesical therapy Mitomycin C Non muscle-invasive bladder cancer Randomized clinical trial Urinary bladder Please visit europeanurology to read and answer questions on-line. The EU-ACME credits will then be attributed automatically. Background: Bacillus Calmette-Guérin (BCG) is the intravesical treatment of choice for carcinoma in situ (CIS). Objective: Our aim was to assess if sequential mitomycin C (MMC) plus BCG after transurethral resection (TUR) is worthy of further study in non muscle-invasive bladder cancer patients with CIS. Design, setting, and participants: In a noncomparative phase 2 study, 96 patients with primary/secondary/concurrent CIS of the urinary bladder were randomized to sequential MMC plus BCG or to BCG alone after TUR. Intervention: Patients received six weekly instillations of MMC followed by six weekly instillations of BCG or six weekly instillations of BCG, 3 wk rest, and three further weekly instillations of BCG. Complete responders received three weekly maintenance instillations at 6, 12, 18, 24, 30, and 36 mo in accordance with the initial randomization. Measurements: End points were complete response (CR) rate at the first control cystoscopy wk after start of treatment, disease-free interval, overall survival, and side effects. Results and limitations: Ninety-six patients were randomized, 48 to each treatment group. Ten patients were ineligible, and three did not start treatment. In all random- For the European Organization for Research and Treatment of Cancer (EORTC) Genito-Urinary Tract Cancer Group, Brussels, Belgium. * Corresponding author. Department of Urology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium. Tel ; Fax: address: willem.oosterlinck@ugent.be (W. Oosterlinck) /$ see back matter # 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 439 ized patients, CR rates on MMC plus BCG and BCG alone were 70.8% and 66.7%, respectively. In 83 eligible patients who started treatment, CR rates were 75.6% and 73.8%, respectively. Based on a median follow-up of 4.7 yr, 25 patients (52.1%) on MMC plus BCG and 22 patients (45.8%) on BCG alone were disease free. Twelve patients stopped treatment due to toxicity: three during induction (two MMC plus BCG, one BCG) and nine during maintenance (three MMC plus BCG, six BCG). Conclusions: In the treatment of patients with CIS, sequential chemoimmunotherapy with MMC plus BCG had acceptable toxicity. CR and disease-free rates were similar to those on BCG alone and to previous publications on sequential chemoimmunotherapy. Trial registration: This study was registered with the US National Cancer Institute clinical trials database (protocol ID: EORTC-30993). search/viewclinicaltrials.aspx?cdrid=68869&version=healthprofessional& protocolsearchid= # 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved. 1. Introduction Bacillus Calmette-Guérin (BCG) is the intravesical treatment of choice according to American and European guidelines [1,2] for carcinoma in situ (CIS). Complete response (CR) rates of 65 70% can be expected on BCG alone. With mitomycin C (MMC), a CR of about 50% is obtained [3]. It seems logical to expect a higher response rate with the sequential use of both treatments. At the moment of designing this trial, laboratory and clinical data supported this idea. An in vitro study using combination therapy with bladder cancer cells showed more pronounced antitumoral activity than chemotherapy or BCG alone [4]. Three small clinical studies [5 7] demonstrated low recurrence rates in non muscle-invasive bladder cancer (NMIBC) with the combination of chemoimmunoprophylaxis. The objective of this study was to assess in a phase 2 trial if sequential MMC plus BCG after transurethral resection (TUR) is worthy of further study in CIS. 2. Material and methods 2.1. Study design The main eligibility criteria were primary, concurrent, or recurrent biopsy-proven CIS, no previous upper urinary tract tumors, no pretreatment with BCG, and no intravesical treatment with chemotherapeutic agents within 3 mo prior to TUR. All papillary lesions were resected with a deep biopsy to allow correct staging. Targeted biopsies of all suspicious areas, and in the case of suspicious cytology only, random biopsies from normal-looking mucosa (left and right bladder wall, dome, both ureteric orifices, posterior wall, and prostatic urethra) were taken. Neither fluorescence TUR nor re-tur was standard when this study was designed. After pathology reports showing CIS and absence of muscle involvement, patients were randomized in the trial. Patients were stratified for institution and type of CIS: primary (no previous or concurrent carcinomas of the bladder), secondary (detected during follow-up of NMIBC), or concurrent (in the presence of NMIBC). After TUR, patients were randomized to one of the following induction cycles: (1) weekly MMC 40 mg in 50 ml saline for 6 wk, followed by six weekly BCG (TICE 5 10*8 CFU) instillations, or (2) six weekly BCG (TICE 5 10*8 CFU in 50 ml saline) instillations, followed by 3 wk of rest and then by 3 wk of BCG. The first instillation was administered within d after TUR. Complete responders after induction received three weekly maintenance instillations at 6, 12, 18, 24, 30, and 36 mo in accordance with the initial randomization (Fig. 1). The maintenance treatment in group 1 was one instillation of MMC followed by two BCG instillations weekly. The first evaluation of CR was done wk after the start of therapy by cytology; TUR and biopsies were performed either of suspected lesions or at random. Negative TUR/biopsies and cytology were required for a CR. Positive cytology, even with negative biopsies, was considered persistent disease. The appearance of a papillary tumor T2 or greater was classified as progression to muscle-invasive disease. Further follow-up was adjusted according to endoscopic and pathologic findings. CRs were treated with maintenance therapy and followed by cystoscopy and cytology every 3 mo for 3 yr, every 6 mo for 2 yr, and yearly thereafter. In the case of Ta or T1 G1 2 tumors with no CIS, tumors were resected and a further course of therapy according to the initial randomization was given. Persistent CIS, pt1g3 tumors, second recurrence of ptat1 G1 2 tumors, recurrence after completion of maintenance treatment, positive cytology, or progression to muscleinvasive disease were considered treatment failures and went off protocol Statistical considerations The primary end point was CR after the first induction course (or after the second induction if needed). In this noncomparative randomized phase 2 study, a Fleming one-stage design [8] was applied independently to each treatment group with the aim of excluding a CR rate 60% with a type I error rate of 10%. To have 95% power to reject this hypothesis under the alternative that the true CR rate was 80%, 45 eligible patients should be Fig. 1 Treatment schedule. BCG = bacillus Calmette-Guérin; MMC = mitomycin C; TUR = transurethral resection.

3 440 randomized to each treatment arm. The combination MMC plus BCG would be rejected if 31 CRs were observed. Secondary end points were the duration of the disease-free interval, duration of CR and type of recurrence in patients with CR, duration of survival, and side effects. For the disease-free interval and overall survival, the starting point was the date of randomization. Patients who could not be assessed for response were considered to have recurred at time zero. The duration of CR was calculated as the difference between the date of first recurrence after CR and the date of first assessment of CR. The disease-free interval and the duration of CR were estimated using cumulative incidence functions taking death as a competing risk. The duration of survival was estimated using the Kaplan-Meier technique. The randomization was not done for the purpose of making a treatment comparison but to provide a simultaneous screening of the two treatments. Thus no formal treatment comparisons were made, and no p values are given for any of the end points. 3. Results Between June 2001 and February 2005, 96 patients were randomized by 16 institutions, 48 to each arm (intent-totreat population). Central pathology review was carried out in 73 of the 96 patients. CIS was not pathologically confirmed in nine patients (five on MMC plus BCG and four on BCG alone). These nine patients and a patient with previous upper tract tumors were ineligible (Fig. 2). Two additional patients did not start the treatment, and for one patient no data were available. Eighty-three eligible patients started the treatment, 41 on MMC plus BCG and 42 on BCG alone. Table 1 displays the demographic and disease characteristics. The median age was 69 yr, 86.5% were male, 93.8% had a World Health Organization performance status prognostic score of 0, 33.3% had primary CIS, 8.3% had secondary CIS, 47.9% had concurrent CIS, and 55.2% had papillary lesions. Only four patients were pretreated with MMC. Table 1 Patient and disease characteristics 3.1. Induction treatment (n = 48) (n = 48) (n = 96) Age, yr Median (range) 68 (42 83) 70 (55 83) 69 (42 83) Gender, n(%) Male 44 (91.7) 39 (81.3) 83 (86.5) Female 4 (8.3) 8 (16.7) 12 (12.5) Missing 0 (0.0) 1 (2.1) 1 (1.0) WHO PS, n(%) PS 0 48 (100.0) 42 (87.5) 90 (93.8) PS 1 0 (0.0) 5 (10.4) 5 (5.2) Missing 0 (0.0) 1 (2.1) 1 (1.0) Type of CIS *,n(%) No CIS 5 (10.4) 4 (8.3) 9 (9.4) Primary 15 (31.3) 17 (35.4) 32 (33.3) Secondary 4 (8.3) 4 (8.3) 8 (8.3) Concurrent 24 (50.0) 22 (45.8) 46 (47.9) Missing 0 (0.0) 1 (2.1) 1 (1.0) Papillary lesions *, n (%) No 21 (43.8) 21 (43.8) 42 (43.8) Yes 27 (56.3) 26 (54.2) 53 (55.2) pta pt ptx Missing 0 (0.0) 1 (2.1) 1 (1.0) BCG = bacillus Calmette-Guérin; CIS = carcinoma in situ; MMC = mitomycin C; PS = performance status; WHO = World Health Organization. * Type of CIS/papillary lesions according to local pathologist unless review pathologist could not confirm. Three patients did not start treatment (Fig. 2). Thus 47 patients on MMC plus BCG and 46 patients on BCG alone started instillations. The median duration of the first induction course was 77 d (Table 2). Forty-two patients on Pa ents with CIS of the urinary bladder randomly assigned a er TUR (n=96) Allocated to MMC + BCG (n=48) Allocated to BCG alone (n=48) Insufficient data (n=1) Ineligible pa ents (n=6): - CIS not confirmed: 5 - History of upper tract tumors: 1 Treatment not started (n=1): -Refusal: 1 Ineligible pa ents (n=4): - CIS not confirmed: 4 (one did not start treatment) Treatment not started (n=1): -Refusal: 1 Eligible pa ents who started the treatment (n=41) Eligible pa ents who started the treatment (n=42) Fig. 2 Consolidated Standards of Reporting Trials (CONSORT) diagram. BCG = bacillus Calmette-Guérin; CIS = carcinoma in situ; MMC = mitomycin C; TUR = transurethral resection.

4 441 Table 2 Induction treatment (first course) MMC plus BCG BCG alone (n = 47) (n = 46) Duration of the induction, d Median (range) 77 (7 92) 77 (35 98) No. of BCG instillations, n(%) 0 3 (6.4) 0 (0.0) 6 44 (93.6) 3 (6.5) 8 0 (0.0) 2 (4.3) 9 0 (0.0) 41 (89.1) No. of MMC instillations, n(%) 0 0 (0.0) 46 (100.0) (10.6) 0 (0.0) 6 42 (89.4) 0 (0.0) Stop of induction due to side effects, n(%) No stop due to side effects 45 (95.7) 45 (97.8) Stop due to local side effects 0 (0.0) 1 (2.2) Stop due to systemic side effects 1 (2.1) 0 (0.0) Stop due to local and systemic side effects 1 (2.1) 0 (0.0) Three patients (one on MMC plus BCG and two on BCG alone) did not start the induction course; one patient in the BCG-alone arm had two cycles of induction without side effects during the two treatments courses. MMC plus BCG received the full treatment (six instillations of MMC followed by six instillations of BCG), and 41 patients on BCG alone received all nine instillations. One patient on the BCG-alone arm had two cycles of induction treatment. The most frequent local side effect was chemical cystitis (16.1%), dysuria (23.7%), and frequency (25.9%) with no tendency to be more frequent or severe on MMC plus BCG. General fatigue (n = 9) and fever (n = 9) were the most frequent systemic side effects in both groups. One patient developed BCG sepsis. During the induction course, two patients on MMC plus BCG stopped treatment due to side effects, one for allergic reaction and the other for fever (grade 3), incontinence (grade 1), and chemical cystitis. One patient on BCG alone stopped because of a penile skin eruption Tumor response after induction Table 3 lists CR at first cystoscopy. In the intent-to-treat population of all randomized patients, the CR rates on MMC plus BCG and BCG alone were 70.8% and 66.7%, respectively. In the 83 eligible patients who started treatment, the CR rates on MMC plus BCG and BCG alone were 75.6% and 73.8%, respectively (Table 4) Maintenance therapy Three patients in CR at the end of induction did not start maintenance therapy. Maintenance treatment in patients achieving a CR is displayed in Table 5. The frequency of side effects (local and/or systemic) remained stable during maintenance. Eight patients (three on MMC plus BCG and five on BCG alone) stopped maintenance due to side effects. In addition, seven patients (one on MMC plus BCG and six on BCG alone) of the 33 patients who did not achieve a CR started maintenance treatment (not presented in Table 5), one of whom stopped because of side effects. Table 3 Response to induction treatment in the intent-to-treat * population (n = 48) (n = 48) (n = 96) Response to induction, n(%) CR 28 (58.3) 34 (70.8) 26 (54.2) 32 (66.7) 54 (56.3) 66 (68.8) CR but not cytology confirmed 6 (12.5) 6 (12.5) 12 (12.5) No change 9 (18.6) 9 (18.6) 18 (18.8) Progressive disease 1 (2.1) 1 (2.1) 2 (2.1) Not assessable for other reason 4 (8.3) 6 (12.5) 10 (10.4) BCG = bacillus Calmette-Guérin; CR = complete response; MMC = mitomycin C. * Intent-to-treat: all patients. Table 4 Response to induction treatment in the per-protocol * population (n = 41) (n = 42) (n = 83) Response to induction, n(%) CR 25 (61.0) 31 (75.6) 26 (61.9) 31 (73.8) 51 (61.4) 62 (74.7) CR but not cytology confirmed 6 (14.6) 5 (11.9) 11 (13.3) No change 7 (17.1) 7 (16.7) 14 (16.9) Progressive disease 1 (2.4) 1 (2.4) 2 (2.4) Not assessable for other reason 2 (4.9) 3 (7.1) 5 (6.0) BCG = bacillus Calmette-Guérin; CR = complete response; MMC = mitomycin C. * Per-protocol population: all eligible patients who started the induction treatment.

5 Recurrence After a median follow-up of 4.7 yr and a maximum of 6.5 yr, 25 patients (52.1%) on MMC plus BCG and 22 patients (45.8%) on BCG alone are disease free. The primary reason for recurrence was no CR in 20 patients (10 in each arm) (Table 6). The main type of first recurrence after CR was a superficial recurrence in nine patients on MMC plus BCG and seven patients on BCG alone. Ten patients were not assessable and considered treatment failures at baseline; 48.6% of patients (95% confidence interval [CI], ) on MMC plus BCG versus 56.4% (95% CI, ) on BCG alone had recurred at 5 yr (Fig. 3). Table 5 Maintenance treatment in complete responders MMC plus BCG BCG alone (n = 33) (n = 30) Duration of the maintenance, mo Median (range) 29.0 ( ) 25.3 ( ) No. of courses, n(%) (27.3) 7 (23.3) (24.2) 11 (36.7) 6 15 (45.5) 12 (40.0) >6 1 (3.0) 0 (0.0) Stop of maintenance due to side effects, n(%) No stop due to side effects 30 (90.9) 25 (83.3) Stop due to local side effects 1 (3.3) 3 (10.0) Stop due to systemic side effects 1 (3.3) 1 (3.3) Stop due to local and 0 (0.0) 1 (3.3) systemic side effects Unknown 1 (3.3) 0 (0.0) Three patients (one on MMC plus BCG and two on BCG alone) did not start the maintenance after achieving complete response. Table 6 Type of first recurrence 3.5. Disease evolution and overall survival Table 7 presents the patients disease status. Globally, 13 patients (13.5%) had a cystectomy, 7 (7.3%) progressed to muscle-invasive disease, 6 (6.3%) developed distant metastases, and 18 (18.8%) died, 6 (6.3%) due to bladder cancer (all on BCG). The 5-yr overall survival rate was 82.7% (95% CI, ) on the MMC plus BCG arm versus 77.8% (95% CI, ) on the BCG-alone arm (Fig. 4). Among 66 patients in CR after the induction, 26.5% (95% CI, ) in the MMC plus BCG arm versus 28.1% (95% CI, 12.2% 44.1) in the BCG-alone arm had a recurrence (CIS, Ta, T1, T2) at 5 yr (considering cystectomy as a competing risk) (Fig. 5). 4. Discussion (n = 48) (n = 48) (n = 96) Disease free, n (%) 25 (52.1) 22 (45.8) 47 (49.0) Recurrence, n (%) 23 (47.9) 26 (54.2) 49 (51.0) No CR Superficial recurrence after CR Muscle invasive after CR Distant metastasis after CR Not assessable BCG = bacillus Calmette-Guérin; CR = complete response; MMC = mitomycin C. CIS has a high risk of progression. Cystectomy is often necessary during follow-up due to the persistence of CIS or Treatment failure (%) (years) O N Number of patients at risk: Treatment MMC + BCG BCG alone Fig. 3 Time to recurrence.

6 443 Table 7 Disease status (n = 48) (n = 48) (n = 96) Cystectomy, n(%) 8 (16.7) 5 (10.4) 13 (13.5) Progression to muscle 2 (4.2) 5 (10.4) 7 (7.3) invasive, n(%) Extravesical tumor, n(%) 0 (0.0) 4 (8.3) 4 (4.2) Distant metastasis, n(%) 1 (2.1) 5 (10.4) 6 (6.3) Second cancer, n(%) 2 (4.2) 3 (6.3) 5 (5.2) Survival status, n(%) Alive 41 (85.4) 37 (77.1) 78 (81.3) Dead, cause: 7 (14.6) 11 (22.9) 18 (18.8) Progression of disease Second primary Associated chronic disease Intercurrent death not due to bladder cancer Other Unknown progression [3]. Intravesical BCG is the standard of care according to the American Urological Association and the European Association of Urology. The CR rate for CIS is about 55% after one cycle of six weekly BCG instillations. An additional 3- to 6-wk course of BCG further increases the CR rate to approximately 75 80%. Maintenance prophylaxis has been proven to be superior to a single course of BCG, but the optimal duration of maintenance is not yet defined [9]. BCG reduces the odds of progression by 35% compared with intravesical chemotherapy or different immunotherapy [10]. Intravesical chemotherapy has been shown to have activity against CIS but to a lesser extent than BCG. The disease-free interval is also shorter after intravesical chemotherapy as compared with BCG [9]. Twelve randomized trials including 845 patients with CIS compared BCG with different chemotherapy regimens [3]. There was a 68% CR rate on BCG and a 49% CR rate on chemotherapy. In the CRs, after a median follow-up of 3.75 yr, 68% of the patients treated with BCG remained disease free as compared with 47% after chemotherapy. The overall disease-free rates were 51% and 27% on BCG and chemotherapy, respectively. It was logical to explore if treatment with sequential chemoimmunotherapy with two different antitumor mechanisms of action might be more active than BCG alone. At the time this protocol started, the data referenced here supported the idea that the combination of intravesical chemotherapy and BCG could improve the CR rate and the disease-free interval. In vitro studies support the hypothesis that the association of chemotherapy and BCG may have a more pronounced antitumoral activity than chemotherapy or BCG alone [4].In a preliminary study in 14 patients, epirubicin plus BCG instilled together weekly for 6 wk and then monthly for 6 mo was able to reduce the recurrence rate to 11% [5]. Side effects were heavy and delayed the instillations in half of the patients. In another study with chemoimmunoprophylaxis, the recurrence rate was only 3%, but side effects were frequent [6]. Intravesical chemoimmunoprophylaxis was found to be highly effective in a small series of T1 G3 tumors [7]. Combination therapy with MMC and BCG proved to be effective in two series of patients with CIS [11,12], but it was compared with MMC and not with BCG alone. There were also studies suggesting no benefit of the sequential treatment. In a marker lesion study of the European Organization for Research and Treatment of Cancer Genito-Urinary Group, the CR rate was 50% and thus not higher than expected after BCG alone [13]. In a Finnbladder Group study, alternating MMC and BCG was not superior to Overall survival (%) (years) O N Number of patients at risk: Treatment MMC + BCG BCG alone Fig. 4 Duration of survival.

7 Recurrence (%) (years) O N Number of patients at risk : Treatment MMC + BCG BCG alone Fig. 5 Time to recurrence in complete responders. MMC alone in the prophylaxis of recurrent Ta-T1 bladder tumors [14]. Because a possible synergistic effect between these two treatments could be expected, the present study was undertaken to assess the activity of sequential MMC plus BCG and BCG alone in patients with CIS. However, similar complete response rates were observed, and there was no suggestion of an advantage for the sequential treatment. Clinical results of other randomized trials have been published [15,16]. They have come to the same conclusion as in our study. A CR rate of about 75% is initially obtained with intravesical therapy, and approximately 55 75% of complete responders remain disease free for 5 yr. A few trials have compared BCG plus chemotherapy to BCG alone. The Nordic trial [17] compared alternating MMC and BCG instillations with BCG alone in 304 patients with CIS. No difference in CR rate was seen between the two treatment regimens, but based on a median follow-up of 56 mo, there was a significantly longer disease-free interval in the BCG arm. Fifty-five percent were disease free on BCG alone, compared with 45% on the combination therapy. Thus alternating monthly MMC and BCG was not superior to BCG alone [17]. It can be postulated that giving BCG instillations every 2 mo in the alternating arm is suboptimal for producing an adequate response. This is also supported by the results of a Finnbladder Group study [18] in which monthly BCG was more effective in reducing recurrences in highly recurrent Ta-T1 tumors than when BCG was given every 2 mo alternating with interferon, with 5-yr diseasefree rates of 54% and 20%, respectively. It can be postulated that the intensity of BCG therapy plays an important role. In recent years, three prospective randomized studies comparing BCG alone versus its combination with chemotherapy have appeared [19 21]. They included high-risk NMIBC in general and not specifically CIS. In the study by Cai et al. [19], one immediate instillation with epirubicin was included. This resulted in a 57.5% recurrence-free rate versus 50.6% in the BCG-alone group. An immediate instillation after TUR is clearly different from the treatment schedule in our study. In another study [20], MMC was used with electromotive application. Whether the large difference in recurrence-free rate (58% for BCG alone and 91% for the MMC-electromotive group) was due to the electromotive application cannot be deduced from the data. The third study [21] included 56 recurrent Ta-T1 tumors; grade was not mentioned. The treatment schedule included an immediate MMC instillation after TUR followed by a 4 weekly course of MMC. As in the Finnbladder Group study, patients received BCG monthly for 1 yr. In the BCG arm, an immediate instillation was not given. The time to first recurrence was significantly longer in the first group. It can be expected that the early instillation at least partially reduced the recurrence rate, but monthly BCG can also be responsible for the better outcome, confirming the Finnbladder report. Whether CIS behaves different from papillary tumors also remains an open question. 5. Conclusions The present study and data from the literature do not support the use of sequential intravesical chemotherapy and BCG for the treatment of CIS. The side effects were not more than with BCG alone. Author contributions: Willem Oosterlinck 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: Oosterlinck, Kirkali, Sylvester, da Silva, Busch, Algaba, Bono.

8 445 Acquisition of data: Oosterlinck, Kirkali, Sylvester, da Silva, Busch, Algaba, Colette, Bono; EORTC-GU group. Analysis and interpretation of data: Oosterlinck, Sylvester, Colette. Drafting of the manuscript: Oosterlinck, Sylvester, Colette. Critical revision of the manuscript for important intellectual content: Busch, Bono, Kirkali. Statistical analysis: Sylvester, Colette. Obtaining funding: Oosterlinck, Kirkali, Sylvester, da Silva, Busch, Algaba, Colette, Bono; EORTC-GU group. Administrative, technical, or material support: None. Supervision: None. 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: None. Funding/Support and role of the sponsor: This paper was supported by grants number 5U10 CA through 5U10 CA from the NationalCancerInstitute (Bethesda, MD, USA) andby Fonds Cancer (FOCA) from Belgium. Its content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Cancer Institute. This study was also supported by educational grants from Kyowa Hakko Ltd (now Kyowa Hakko Kirin UK Ltd) and Organon Teknika, now part of Merck. Appendix A. Participating Centers and Investigators W. Oosterlinck, Universiteit Gent, Gent, Belgium; Z. Kirkali, Dokuz Eylul University School of Medicine, Izmir, Turkey; A.V. Bono, Ospedale Di Circolo E Fondazione Macchi, Varese, Italy; F. Calais Da Silva, Hospital Do Desterro, Lisbon, Portugal; P. Gontero, Università Di Torino, Torino, Italy; G.O.N. Oosterhof, I. Van De Beek, Academisch Ziekenhuis Maastricht, Maastricht, The Netherlands; Th.M. De Reijke, Academisch Medisch Centrum, Amsterdam, The Netherlands; I. Billiet, AZ Groeninghe Campus St. Maarten, Kortrijk, Belgium; K. Vekemans, Virga Jesse Hospital, Hasselt, Belgium; F. Keuppens, D. Michielsen, Universitair Zienkenhuis Brussel, Brussels, Belgium; W. Kirkels, Erasmus MC, Rotterdam, The Netherlands; P. Carpentier, Onze Lieve Vrow Ziekenhuis, Aalst, Belgium; C. Selli, Azienda Ospedaliera Universitaria Pisana Ospedale St. Chiara, Pisa, Italy; J.A. Witjes, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; H. Kynaston, University of Wales College of Medicine, Cardiff, United Kingdom; R. Persad, University Hospitals Bristol NHS Foundation Trust Bristol Royal Infirmary, Bristol, United Kingdom. References [1] Hall MC, Chang SS, Dalbagni G, et al. Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1and Tis): 2007 update. J Urol 2007;178: [2] Babjuk M, Oosterlinck W, Sylvester R, Kaasinen E, Böhle A, Palou-Redorta J. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder. Eur Urol 2008;54: [3] van der Meijden APM, Sylvester R, Oosterlinck W, et al. EAU guidelines on the diagnosis and treatment of urothelial carcinoma in situ. Eur Urol 2005;48: [4] Rajala P, Kaasinen E, Rintala E, et al. Cytostatic effect of different strains of Bacillus Calmette-Guerin on human bladder cancer cells in vitro alone and in combination with mitomycin C and interferonalpha. Urol Res 1992;20: [5] Erol A, Ozgur S, Basar M, Cetin S. Trial with bacillus Calmette-Guerin and epirubicin combination in the prophylaxis of superficial bladder cancer. Urol Int 1994;52: [6] Uekado Y, Hirano A, Shinka T, Ohkawa T. The effects of intravesical chemoimmunotherapy with epirubicin and bacillus Calmette-Guerin for prophylaxis of recurrence of superficial bladder cancer: a preliminary report. Cancer Chemother Pharmacol 1994;35(Suppl):S65 8. [7] Bono AV, Lovisolo JA, Saredi G. Transurethral resection and sequential chemo-immunoprophylaxis in primary T1G3 bladder cancer. Eur Urol 2000;37: [8] Fleming TR. One-sample multiple testing procedure for phase II clinical trials. Biometrics 1982;38: [9] Gontero P, Bohle A, Malmstrom PU, et al. The role of bacillus Calmette-Guérin in the treatment of non muscle-invasive bladder cancer. Eur Urol 2010;57: [10] Sylvester RJ, van der Meijden AP, Lamm DL. Intravesical bacillus Calmette-Guerin reduces the risk of progression in patients with superficial bladder cancer: a meta-analysis of the published results of randomized clinical trials. J Urol 2002;168: [11] Rintala E, Jauhiainen K, Rajala P, Ruutu M, Kaasinen E, Alfthan O. Alternating mitomycin C and bacillus Calmette-Guerin instillation therapy for carcinoma in situ of the bladder. The Finnbladder Group. J Urol 1995;154: [12] Witjes JA, Caris CT, Mungan NA, Debruyne FM, Witjes WP. Results of a randomized phase III trial of sequential intravesical therapy with mitomycin C and bacillus Calmette-Guerin versus mitomycin C alone in patients with superficial bladder cancer. J Urol 1998;160: [13] Van der Meijden APM, Hall RR, Macaluso MP, Pawinsky A, Sylvester R, Van Glabbeke M. Marker tumour response to the sequential combination of intravesical therapy with mitomycin-c and BCG-RIVM in multiple superficial bladder tumours: Report from the European Organisation for Research and Treatment on Cancer-Genitourinary Group (EORTC 30897). Eur Urol 1996;29: [14] Rintala E, Jauhiainen K, Kaasinen E, Nurmi M, Alfthan O, Finnbladder Group. Alternating mitomycin C and bacillus Calmette-Guerin instillation prophylaxis for recurrent papillary (stages Ta to T1) superficial bladder cancer. J Urol 1996;156:56 9, discussion [15] Jakse G, Hall R, Bono A, et al. Intravesical BCG in patients with carcinoma in situ of the urinary bladder: long-term results of EORTC GU Group phase II protocol Eur Urol 2001;40: [16] de Reijke TM, Kurth KH, Sylvester RJ, et al. Bacillus Calmette-Guerin versus epirubicin for primary, secondary or concurrent carcinoma in situ of the bladder: results of a European Organization for the Research and Treatment of Cancer Genito-Urinary Group Phase III Trial (30906). J Urol 2005;173: [17] Kaasinen E, Wijkström H, Malmström PU, et al. Alternating mitomycin C and BCG instillations versus BCG alone in treatment of carcinoma in situ of the urinary bladder: a Nordic study. Eur Urol 2003;43: [18] Kaasinen E, Rintalla E, Hellström P, et al. Factors explaining recurrence in patients undergoing chemoimmunotherapy regimens for frequently recurring superficial bladder carcinoma. Eur Urol 2002; 42: [19] Cai T, Nesi G, Tinacci G, et al. Can early single dose instillation of epirubicin improve bacillus Calmette-Guerin efficacy in patients with non-muscle invasive high risk bladder cancer? Results from a

9 446 prospective, randomized, double-blind controlled study. J Urol 2008;180: [20] Di Stasi SM, Giannantoni A, Giurioli A, et al. Sequential BCG and electromotive mitomycin versus BCG alone for high-risk superficial bladder cancer: a randomised controlled trial. Lancet Oncol 2006;7: [21] El Mohsen MA, Shelbaia A, El Ghobashy S, et al. Sequential chemoimmunotherapy using Mitomycin followed by bacillus Calmette- Guérin versus single agent immunotherapy (BCG) for recurrent superficial bladder tumors. UroToday Int J 2010;3. doi: / uij