Lammers, et al DOI: 10.1200/JCO.2011.39.2936



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The following protocol information is provided solely to describe how the authors conducted the research underlying the published report associated with the following article: Intracutaneous and Intravesical Immunotherapy with Keyhole Limpet Hemocyanin is Inferior to Intravesical Mitomycin-C in Preventing Recurrences in Patients with Non-Muscle-Invasive Bladder Cancer: Results from a Prospective Randomized Phase III Trial Lammers, et al DOI: 10.1200/JCO.2011.39.2936 The information provided may not reflect the complete protocol or any previous amendments or modifications. As described in the Information for Contributors (http://jco.ascopubs.org/site/ifc/protocol.xhtml) only specific elements of the most recent version of the protocol are requested by JCO. The protocol information is not intended to replace good clinical judgment in selecting appropriate therapy and in determining drug doses, schedules, and dose modifications. The treating physician or other health care provider is responsible for determining the best treatment for the patient. ASCO and JCO assume no responsibility for any injury or damage to persons or property arising out of the use of these protocol materials or due to any errors or omissions. Individuals seeking additional information about the protocol are encouraged to consult with the corresponding author directly.

Intracutaneous and Intravesical Immunotherapy with Keyhole Limpet Hemocyanin is Not Equivalent to Intravesical Mitomycin-C in Preventing Recurrences in Patients with Non-Muscle-Invasive Bladder Cancer: Results from a Prospective Randomized Phase III Trial Redacted Protocol, Final version December 2002 Rianne J.M. Lammers a, Wim P.J. Witjes a,b, Maria H.C. Janzing-Pastors b, Christien T.M. Caris b, J. Alfred Witjes a a Radboud University Nijmegen Medical Centre, Department of Urology, Nijmegen, The Netherlands b CuraTrial SMO & Research, Arnhem, The Netherlands Author for correspondence Radboud University Nijmegen Medical Centre, Department of Urology Geert Grooteplein Zuid 10 (659) P.O. Box 9101 6500 HB Nijmegen The Netherlands Tel.: 0031 24 361 37 35 Fax.: 0031 24 354 10 31 E-mail: f.witjes@uro.umcn.nl

Participating centers St. Jansdal Hospital, Harderwijk, the Netherlands; Catharina Hospital, Eindhoven, the Netherlands; St. Elisabeth Hospital, Tilburg, the Netherlands; Canisius-Wilhelmina Hospital, Nijmegen, the Netherlands; Laurentius Hospital, Roermond, the Netherlands; Rijnstate Hospital, Arnhem, the Netherlands; Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherland; Rode Kruis Hospital, Den Haag, the Netherlands; Diaconessenhuis, Leiden, the Netherlands; VieCuri, Venlo, the Netherlands; Hospital Koningin Beatrix, Winterswijk, the Netherlands; TweeSteden Hospital, Tilburg, the Netherlands; Medisch Centrum Haaglanden, Den Haag, the Netherlands; Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; Martini Hospital, Groningen, the Netherlands; Bethesda Hospital, Hoogeveen, the Netherlands; Wilhelmina Hospital, Assen, the Netherlands; Isala Klinieken, Zwolle, the Netherlands. Selection of patients A total of 2 x 276 patients will be enrolled in each group: either Keyhole Limpet Hemocyanin (KLH) or Mitomycin-C (MMC). Inclusion criteria: One of the following three options: o Patients with solitary or multiple pta G2-3 tumor(s) or o Patients with solitary or multiple pt1 G1-3 tumor(s) or o Patients with multiple or recurrent ptag1 tumor(s) Tumor is pathologically confirmed transitional cell carcinoma of the urinary bladder (biopsy proven)

Complete transurethral resection of bladder tumor (TURBT) Patient is informed and has given written informed consent Exclusion criteria: Patients with a primary, solitary ptag1 tumor Patients with carcinoma in situ (CIS) at entry or papillary lesions stage T2 or higher Patients who received more than one intravesical MMC instillation within 2 years prior to inclusion Patients who are treated with intravesical therapy less than 6 months prior to entry this study Concurrent malignancy, except basal cell or squamous cell carcinoma of the skin Patients with a history of a cancer type other than transitional cell carcinoma of the urothelial tract, with a disease-free interval of less than 5 years (except basal cell or squamous cell carcinoma of the skin) WHO performance status higher than 2 Expected poor compliance such as difficulties in the follow-up related to other diseases or large distance between the patients home and the investigators center Uncontrollable urinary tract infection Previous systemic cancer therapy with immunotherapy or cytotoxic agents or radiotherapy less than 5 years prior to entry to the study Localization of transitional cell carcinoma in the prostatic urethra or in the upper urinary tract

Pregnancy, or lactation, or females in the reproductive ages who refuse to take adequate contraceptive measures Patients with congenital or acquired immune deficiency syndromes, leukemia, transplant recipients and patients with known positive HIV serology Known hypersensitivity to MMC Previous (with in 1 month preceding the start of the trial) or current treatment with an investigational drug Scheme and treatment plan Arm KLH Screening and informed consent Baseline: TURBT and pathology results within 1 week after TURBT Pre-immunization: week 1-2 after TURBT After randomization for treatment with KLH, patients will start initial pre-immunization directly after pathology results and written informed consent is obtained. Patients will receive an intracutaneous (ic.) injection of 1 mg Immucothel, solved in 0.5mL water, into the left forearm (in case of left-handed, into the right forearm). The ic. injection is repeated up to 2 times at interval of 2-7 days until a delayed type hypersensitivity (DTH) response is obtained. In case a DTH is not obtained after 3 ic. injections, an ic. injection is repeated at week 6 of the intravesical instillation therapy period. A DTH response is a palpable infiltrate in the skin with a diameter of at least 3-5mm that contains T-lymphocytes, macrophages and neutrophilic granulocytes, and is comparable to the skin reaction that is obtained after tuberculin vaccination.

Intravesical therapy: week 1/2 month 12 after TURBT Intravesical instillations are begun independent of the pre-immunization, within 2 weeks after TURBT. During the first 6 weeks, 2x10mg (20mg) Immucothel in 2x10mL solvent once a week (at week 1,2,3,4,5,6,) will be installed, followed by 2x10mg (20mg) Immucothel in 2x10mL solvent once a month for 1 year (at month 3,4,5,6,7,8,9,10,11 and 12). In case a DTH is not obtained after 3 ic. injections, another ic. injection is repeated at week 6 of the intravesical instillation therapy period. Post-therapy follow-up After the intravesical therapy, post-therapy follow-up (cystoscopy and urinary cytology) will be done every 3 months in the first year, every 4 months during the 2th and 3 rd year, every 6 months thereafter. All visible lesion seen at cystoscopy must be biopsied. Therefore it is not allowed to coagulate any suspicious lesions during cystoscopy without having taken biopsies. If cytology is positive and no lesions are visible at cystoscopy, selected biopsies should be done because CIS can be expected. Arm MMC Screening and informed consent Baseline: TURBT and pathology results within 1 week after TURBT Pre-immunization: week 1-2 after TURBT In the MMC-arm patients will not be pre-immunized

Intravesical therapy: week 1/2 month 12 after TURBT Intravesical instillations will start within 2 weeks after TURBT. Patients will receive a total of 11 intravesical instillations of 40mg MMC in 50mL saline (at week 1,2,3,4 and month 2,3,4,5,6,9 and 12). Post-therapy follow-up In the MMC-arm the post-therapy follow-up is the same as in the KLH-arm. Rules for dose modification In case an instillation needs to be delayed or the dose needs to be modified due to any adverse events, this is at the discretion of the investigator. Any deviation from the required dose or schedule, as well as the reason for the deviation should be documented carefully in the case report form by the investigator. Measurement of treatment effect Disease-free survival: the time between the transurethral resection of the tumor till the time of first recurrence. Recurrence: positive histological examination of biopsy Recurrence rate: the number of cystoscopies at which the recurrences are observed, divided by the total number of months of patient follow-up (multiplied x 100) Adverse event: any undesirable clinical occurrence in a subject, whether it is considered to be drug related or not. An adverse event is classified by the investigator as mild, moderate or severe

Serious adverse event: any untoward medical occurrence that at any dose results in death, is life-treating, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability / incapacity, or is a congenital anomaly / birth defect. Reasons for early cessation of trial therapy Off study criteria: Withdrawal of informed consent by the patient When the investigator considers it in the best interest of the patient that he/she will be withdrawn Second recurrence during treatment period First recurrence after completion of treatment 5 years disease-free survival Occurrence of CIS Occurrence of urothelial carcinoma of the upper tract, or in the prostatic urethra Occurrence of distant metastases In case treatment is stopped due to unacceptable toxicity the follow-up schedule for assessment of recurrences still has to be followed, so patient is not officially off study. The study may be terminated prematurely in case of the occurrence of severe, unexpected advent, necessitating a review of the product s safety profile. Also unforeseen events or new information relating to the product, making it unlikely that the objectives of the study or clinical program will be met, may warrant discontinuing the study prematurely.

Objectives and statistical methods Primary study objective The primary study objective is to compare the effect of KLH intracutaneous + intravesical treatment with MMC intravesical treatment in a group of patients with intermediate and high risk for recurrence on duration of disease-free survival. Secondary study objective The secondary study objectives are to compare the effect of KLH intracutaneous + intravesical treatment with MMC intravesical treatment in a group of patients with intermediate and high risk for recurrence on: Recurrence rate, including the number of recurrent tumors Rate of progression to a higher stage (T2 or higher) of the disease Incidence and severity of side-effects Another secondary study objective is to compare, within the groups of patients randomized for KLH treatment, the group of patients with a positive DTH response with the group of patients with a negative DTH response for the effects on: Duration of disease-free survival Recurrence rate, including the number of recurrent tumors Rate of progression to a higher stage (T2 or higher) of the disease Incidence and severity of side-effects Statistical methods Justification of patient number

Primary endpoint for sample size calculation is median time to first recurrence. In order to establish therapeutic equivalence, the true ratio of median time to first recurrence (t KLH / t MMC ) must not be lower than 0.75 (e.g. 1.31 / 1.75 years). Taking this ratio as the lower margin of the one-sided equivalence range, 251 evaluable patients must be entered in each treatment group to reject the hypothesis of inferiority at error rates of α/2 = 0.025 and β = 0.20. With this sample size calculation, the required number of patients will have to be entered within 3 years and the additional follow-up after the end of recruitment will be 2 years. To account for a drop out percentage of 10% we will need 552 patients (276 patients per treatment arm) for randomization in this study. Statistical analyses Statistical analyses will be performed by CuraTrial SMO & Research. Patient characteristics, demographic and baseline measurements will be summarized in order to provide a characterization of the patient population. Descriptive statistics, e.g. mean, standard deviation, median, range, frequency distributions as appropriate will be presented for each randomization group: MMC versus KLH, as well as within the KLH treated group, the group of patients with a positive DTH response versus the group of patients with a negative DTH response. The safety analysis will be performed when all patients have finished the treatment phase (after 4 years). The incidence and severity of all adverse events will be calculated per treatment group: MMC versus KLH, as well as within the KLH treated group, the group of patients with a positive DTH response versus the group of patients with a negative DTH response.

The efficacy analysis will be performed when the last included patient has a follow-up of 2 years. The efficacy analysis will be performed including all patients who are randomized and treated with at least on KLH or MMC dosage, and who have a regularly end of the study. This could be a recurrence during the study, as well as the completed instillation schedule, and follow-up in case the patient does not develop a recurrence. Efficacy variables will be analyzed per treatment group: MMC versus KLH, as well as within the KLH treated group, the group of patients with a positive DTH response versus the group of patients with a negative DTH response. Efficacy variables to be analyzed are duration of disease-free survival, recurrence rate including the number of recurrent tumors, rate of progression to a higher stage (T2 or higher) of the disease. Duration of disease-free interval will be estimated by means of the Kaplan-Meier method and comparison between treatment groups, and DTH positive versus DTH negative groups will be done by means of the log-rank test. A Cox proportional hazards model will be applied to correct for treatment effects and assess influence of prognostic and confounding factors. Observed sample percentages will be compared between treatment groups and DTH positive versus DTH negatives groups by means of a Chi-square test. The analyses of other outcome measurements will be analyses in a exploratory way with methods appropriate for the type of the variable. Both the safety data and the efficacy data will be summarized by appropriate descriptive statistics. For each patient entering the reason for discontinuation (e.g. patients discussion, urologists decision, lack of efficacy, adverse events) should be clarified. The description of the reason for discontinuation will be performed. All tests of efficacy hypotheses based on a between-treatment comparisons will be performed at the 2.5% level of

significance, and will be one-sided. Statistical tests based on within-treatment comparisons will be performed at the 5% level of significance and will be two-sided. No adjustments will be made to nominal significance levels to account for multiple comparisons made on the same data.