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9.2.1 storage and tracking of

All data associated with the patient samples is protected by using a secure database. All samples drawn at the NIH Clinical Center will be stored in the laboratories of the Urologic Oncology Branch as previously described. Serum samples will be stored in -80˚C freezers and PBMC samples will be stored in vapor phase liquid nitrogen at the NCI Frederick Central Repository in Frederick, MD. Samples drawn at the NIH Clinical Center will be transported by the Leidos couriers; samples from CINJ will be sent via Fed-Ex, as previously described. Samples will be tracked and managed by the Central Repository database. Leidos Biomedical Research, Inc. manages the NCI Frederick Central Repositories. NCI Frederick Central Repositories store, among other things, biological specimens in support of NIH clinical studies. All specimens are stored in secure, limited access facilities with sufficient security, back-up and emergency support capability and monitoring to ensure long-term integrity of the specimens for research. Sample data is stored in the BioSpecimen Inventory (BSI) System II. This inventory tracking system is used to manage the storage and retrieval of specimens as well as maintain specimen data. BSI is designed for controlled, concurrent access. It provides a real-time, multi-user environment for tracking millions of specimens. The system controls how and in what order database updates and searches are performed. This control prevents deadlocks and race conditions. For security, BSI has user password access, 3 types of user access levels, and 36 user permissions (levels of access) that can be set to control access to the system functions. BSI provides audit tracking for processes that are done to specimens including shipping, returning to inventory, aliquoting, thawing, additives, and other processes. BSI tracks the ancestry of specimens as they are aliquoted, as well as discrepancies and discrepancy resolution for specimens received by the repository. If a specimen goes out of the inventory, the system maintains data associated with the withdrawal request. Vials are labeled with a unique BSI ID which is printed in both eye-readable and bar-coded format. No patient-specific information is encoded in this ID. Investigators are granted view, input and withdrawal authority only for their specimens. They may not view specimen data or access specimens for which they have not been authorized.  
Access to specimen storage is confined to repository staff. Visitors to the repositories are escorted by repository staff at all times. 9.2.2 Protocol Completion/Sample Destruction Once primary research objectives for the protocol are achieved, intramural researchers can request access to remaining samples providing they have an IRB approved protocol and patient consent. Samples, and associated data, will be stored permanently unless the patient withdraws consent. The PI will report destroyed samples to the IRB if samples become unsalvageable or destroyed by environmental conditions (ex. Broken freezer or lack of dry ice in shipping container) or if a patient chooses to withdraw his/her consent. Samples will also be reported as lost if they are lost in transit or misplaced by a researcher.  
*Version Date: April 7, 2017*  
| Follow                                  | up10                                                                                                                                               | Xo                                     | Xo                                        |    |    |    |
|----------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------|-------------------------------------------|----|----|----|
| X                                |                                                                                                                                                    |                                        |                                           |    |    |    |
| 10 STUDY CALENDARS               | Evaluations below may be performed within ± 5 days of indicated time in order to accommodate patient schedules, holidays and  weather emergencies. | A) STUDY CALENDAR FOR BCG + PANVAC ARM | Baseline  (On study)  Week  0  Week17- 20 | X  | X  | X  |
| Week  15                         | X                                                                                                                                                  | X                                      |                                           |    |    |    |
| Week  11                         | X                                                                                                                                                  | X                                      |                                           |    |    |    |
| Week  8                          | X                                                                                                                                                  | X                                      |                                           |    |    |    |
| Week  7                          | X                                                                                                                                                  | X                                      | X                                         |    |    |    |
| Week  6                          | X                                                                                                                                                  | X                                      |                                           |    |    |    |
| Week  5                          | X                                                                                                                                                  | X                                      |                                           |    |    |    |
| Week  4                          | X                                                                                                                                                  | X                                      |                                           |    |    |    |
| Week  3                          | X                                                                                                                                                  | PANVAC-V1 X                            | PANVAC-F1 X                               | X  |    |    |
| X                                | X                                                                                                                                                  | X                                      | X                                         |    |    |    |
| NIH Advanced  Directives  Form11 | History and  Physical2                                                                                                                             | CBC with  differential3                | Chemistry3,4                              |    |    |    |
| BCG                              |                                                                                                                                                    |                                        |                                           |    |    |    |  
71  
| Follow-       | x.             | x   | X   | X   | X           | X          |    |    |    |
|---------------|----------------|-----|-----|-----|-------------|------------|----|----|----|
| Week17-       | x              | X   | x   | x   | X           | x          | X  | x  | X  |
|               | X              |     |     |     |             |            |    |    |    |
| II
X99M               | X              |     |     |     |             |            |    |    |    |
| 8
AM               | X              | X   |     |     |             |            |    |    |    |
| Week          | x              |     |     |     |             |            |    |    |    |
| 9
X93M               | X              |     |     |     |             |            |    |    |    |
| s
X93M               | X*             |     |     |     |             |            |    |    |    |
| X             |                |     |     |     |             |            |    |    |    |
| Week          | X              |     |     |     |             |            |    |    |    |
| Week
3               | X*             |     |     |     |             |            |    |    |    |
| X             | X              |     |     |     |             |            |    |    |    |
| Abbreviated Title: Vaccine and BCG for bladder ca
Version Date: February 13, 2018               | Week           | X   | X   |     |             |            |    |    |    |
| (On study)    | X              | X   | X   | X   | X           | X          | X  |    |    |
| PD (Research) | Jrine Cytology | Urine
(Research) 7     | Immunology
(Research blood) 8     | Serum Beta-
HCG 9     | Chest X-ray | CT urogram |    |    |    |
| Jrinalysis 5  |                |     |     |     |             |            |    |    |    |
| PT, PTT 3     | Biopsy         |     |     |     |             |            |    |    |    |  
Abbreviated Title: Vaccine and BCG for bladder ca
Version Date: February 13, 2018  
| Follow
up 10                                 | X             |                 |              |    |    |    |    |
|---------------------------------|---------------|-----------------|--------------|----|----|----|----|
| x                               | X             | Xº              | x            |    |    |    |    |
| Week17-
20                                 | X             | X               | X            | X  | X  | X  |    |
| Week
15                                 | X             |                 |              |    |    |    |    |
| ll
X93M                                 |               |                 |              |    |    |    |    |
| 8
XaoM                                 | X             | X               | x            |    |    |    |    |
| Week                            | x             | X               | X            |    |    |    |    |
| Week                            | X             | X               | X            |    |    |    |    |
| s
X93M                                 | x             | X               | x            |    |    |    |    |
| Week                            | x             | X               | x            |    |    |    |    |
| STUDY CALENDAR FOR BCG ONLY ARM | Week
3               | X               | X            | x  |    |    |    |
| Week                            |               |                 |              |    |    |    |    |
| On study)                       | X             | X               | X            | X  | X  | X  | X  |
| NIH Advanced
Directives
Form 11                                 | PD (Research) |                 |              |    |    |    |    |
| History and
Physical²                                 | CBC with
differential 3               | Chemistry 3 , 4 | Jrinalysis 5 |    |    |    |    |
| PT, PTT 3                       |               |                 |              |    |    |    |    |
| BCG                             |               |                 |              |    |    |    |    |  
73  
| Follow                                | up10   | 1 Applies only to treatment arm 2 (BCG + PANVACTM). Panvac administration continues as planned regardless of any delays in BCG  administration.   | *These time periods should correspond to BCG instillations #1 and #3, respectively. If a postponement of an instillation occurs, urine  collection will occur after instillations #1 and #3 irrespective of the trial week. On these weeks, urine will be collected twice: first  collection will be prior to BCG instillation and second collection of urine will be 2 hours after drainage of BCG.   | 2 Baseline: H & P and laboratory studies should be completed within 28 days of initiating treatment. This will include determination  of ECOG performance status.   | 3 Baseline laboratory studies should be completed within 28 days of initiating treatment.   |    |
|--------------------------------|--------|---------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------|----|
| X                              | Xo     | Xo                                                                                                                                                | X                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                     |                                                                                             |    |
| Week17- 20                     | X      | X                                                                                                                                                 | X                                                                                                                                                                                                                                                                                                                                                                                                      | X                                                                                                                                                                   | X                                                                                           | X  |
| Week  15  Week  11  Week  8    | X      |                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                     |                                                                                             |    |
| Week  7  Week  6  Week  5      | X*     |                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                     |                                                                                             |    |
| Week  4  Week  3               | X*     | X                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                     |                                                                                             |    |
| Week  0  (On study)  Baseline  | X      |                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                     |                                                                                             |    |
| Urine Cytology6 X              | X      | X                                                                                                                                                 | X                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                     |                                                                                             |    |
| Immunology  (Research  blood)8 | Serum BetaHCG9        | Chest X-ray                                                                                                                                       | CT urogram                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                     |                                                                                             |    |
| Urine  (Research)7             | Biopsy |                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                     |                                                                                             |    |  
4 Chemistry panel: Na+, K+, Cl-, CO2, glucose, BUN, creatinine, albumin, calcium, magnesium, phosphorus, alkaline phosphatase, ALT, AST, total bilirubin, pre-albumin, and LDH. 5 Urinalysis: If prior to an operative procedure, then will be obtained within 2-21 days of surgery and with an accompanying urine culture. If prior to BCG administration, a simple urinalysis will be performed in the hospital on the day of administration. ***Urine will be tested weekly prior to each BCG administration and the presence of gross hematuria or a urinary tract infection by urinalysis will result in holding that week's BCG dose as specified in the protocol.6 Urine cytology: will be obtained from voided urine within 2-21 days of operative procedures OR through instrumentation at the time of an operative procedure 7 Urine (research): Urine will be obtained for correlative immune studies checking for chemokine and cytokine levels 8 Blood will be obtained for immunologic/research assays. CTC analysis, plasma and sera will be isolated to look for CD4 antigenspecific responses, CD8 antigen-specific responses, and flow cytometry profiles of various immune cell types. 9 In females of child-bearing age, Beta-HCG to be done at baseline within 48 hours prior to receiving vaccinia 10 These patients will be followed every 3 months for 1 year after the completion of the initial 20 week study course with standard of care for non-muscle invasive high grade bladder cancer. This includes urinalysis, urine cytology, cystoscopy, possible bladder biopsy, and medical assessments. For patients unable to complete every 3 month follow-up at the NIH or CINJ after week 20, we will keep them on study and obtain their outside pathologic records and labs from their outside urologists who will perform these evaluations. 11 As indicated in section **14.3**, all subjects will be offered the opportunity to complete an NIH advanced directives form. This should be done preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly recommended, but is not required. Participating sites should adhere to appropriate procedures specific to their site. Xo = optional
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11 measurement of effect 11.1

All patients who receive at least one dose of protocol therapy will be evaluable for toxicity from the time of their first treatment with TICE BCG or rV-PANVAC™. All observed toxicities will be tabulated using CTCAE v4.1 and a previously published, vaccinia toxicity grading scale used with this vaccine 33 by the highest grade experienced for each patient and presented for each study arm.
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11.1.1.2 evaluable for objective response

Only those patients who have biopsy-proven disease present at baseline, have received at least one cycle of therapy (6 or fewer weekly intravesical doses of BCG), and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. In order to compare the secondary outcomes between study arms, all durations will be measured from the start of TICE BCG therapy (week 3). This is the start of therapy for the TICE BCG arm alone. BCG is not started at week 0 for two reasons: 1) to allow adequate healing from previous TURBT and 2) allow vaccinia priming vaccine to be given to combination group at week 0 with enough recovery time to possibly make the administration of BCG in week 3 safer in these patients. Furthermore, by following this schema, patients in each group will start BCG at the same time after study entry.
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11.1.1.3 recurrence-free survival

The recurrence-free survival duration will be measured from the start of TICE BCG therapy (week 3) until disease recurrence or death due to any cause in each arm. Recurrence is suspected and/or determined by urine cytology and/or cystoscopic exam and then confirmed pathologically after a TURBT. Positive cytology in the absence of pathologic confirmation is not considered to be a recurrence.
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11.1.1.4 progression-free survival

PFS is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first. The duration of progression-free survival is measured from the start of TICE BCG therapy (week 3) until progression or death due to any cause in each study arm. Progression is defined as upstaging from a lower stage to a higher stage (e.g., Ta to T1 or T1 to T2-4; or any N+ or M+ in these high grade tumors.
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11.1.1.5 time to tumor recurrence

The duration of time measured from the start of TICE BCG therapy (week 3) until recurrence is noted.
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12 data collection and reporting/regulatory

An enrollment log will be maintained in the regulatory binder/file which is the only location of personal identifiers with unique subject identification number. All data will be stored by a patient code identification number only, without personal identifiers, and linked to samples by that identification number in Labmatrix. Clinical Data will be entered into the NCI C3D database. Data will be collected using protocol-specific case report forms, and verified for accuracy and completeness. Data should be entered into the data base in a timely manner. Hard copies of data will be stored in locked secured areas and data will be entered onto a secured electronic data base. The following protocol-specific study forms will be complete and stored: eligibility checklist (developed by Central Registration Office, CRO). A copy of all serious AE forms will be kept in the research record. Data will be verified to ensure that:  
x Eligible patients are confirmed and eligibility checklist was completed. Consent form must be signed prior to registration with Central Registration.  
x Treatment is given according to protocol (dated notes about doses given, complications, and clinical outcomes).  
x Toxicity is assessed according to protocol (laboratory report slips, etc.) x Response is assessed according to protocol (X-ray, scan, lab reports, and date noted on clinical assessment, as appropriate).  
x Drug Accountability Records are kept for each patient.
All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Patients will be followed for adverse events for 30 days after removal from study treatment or until offstudy, whichever comes first.  
An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:
x Results in discontinuation from the study  
x Is associated with clinical signs or symptoms x Requires treatment or any other therapeutic intervention x Is associated with death or another serious adverse event, including hospitalization.
x Is judged by the Investigator to be of significant clinical impact  
x If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH Intramural Records Retention Schedule as applicable. Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, the IRB will be notified.
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12.2 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting until March 31, 2018. CTCAE version 5.0 will be utilized for AE reporting beginning April 1, 2018. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40).
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12.2.1 toxicity grading for vaccinia

Grade 1: Cutaneous reaction extending no more than 10 cm from the vaccination site (i.e., limited to the upper arm) Grade 2: Any autoinoculation syndrome that resolves without sequelae; Generalized vaccinia extending more than 10 cm from the vaccination site Grade 3: Any toxicity that is between grade 2 and 4 Grade 4: Autoinoculation syndrome (e.g. blindness); post vaccinia encephalitis; vaccinia gangrenosum; eczema gangrenosum; Stevens-Johnson syndrome
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12.3 data reporting

Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.
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12.3.1 method

This study will be monitored by the Clinical Data Update System (CDUS) Version 3.0. Cumulative protocol- and patient-specific CDUS data will be submitted electronically to CTEP on a quarterly basis, either by FTP burst of data or via the CDS web application. Reports are due January 31, April 30, July 31, and October 31. Instructions for submitting data using the CDUS can be found on the CTEP Web site
(http://ctep.cancer.gov/reporting/cdus.html).  
Note: If your study has been assigned to CDUS-Complete reporting, all adverse events (both routine and expedited) that have occurred on the study and meet the mandatory CDUS reporting guidelines must be reported via the monitoring method identified above. If your study has been assigned to CDUS-Abbreviated reporting, no adverse event reporting (routine or expedited) is required to be reported via CDUS
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12.3.2 responsibility for data submission

Participating study sites are responsible for submitting CDUS data and/or data forms to either the Coordinating Center or to the Lead Organization on the study quarterly. The date for submission to the Coordinating Center or to the Lead Organization will be set by them. CDUS does not accept data submissions from the participants on the study. When setting the dates, allow time for Coordinating Center compilation, Principal Investigator review, and timely submission to CTEP by the quarterly deadlines (see Section **12.3**). Either the Coordinating Center or the Lead Organization is responsible for compiling and submitting CDUS data to CTEP for all participants and for providing the data to the Principal Investigator for review.
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12.4 ctep multicenter guidelines

This protocol will adhere to the policies and requirements of the CTEP Multicenter Guidelines. The specific responsibilities of the Principal Investigator and the Coordinating Center (Study Coordinator) and the procedures for auditing are presented in Appendix B (Section 0).  
x The Principal Investigator/Coordinating Center is responsible for distributing all IND
Action Letters or Safety Reports received from CTEP to all participating institutions for submission to their individual IRBs for action as required.  
x Except in very unusual circumstances, each participating institution will order DCTDsupplied agents directly from CTEP. Agents may be ordered by a participating site only after the initial IRB approval for the site has been forwarded by the Coordinating Center to the CTEP PIO (PIO@ctep.nci.nih.gov) except for Group studies.
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12.5 collaborative agreements language

The PANVAC™-V (vaccinia) and PANVAC™-F (fowlpox) (hereinafter referred to as Agents) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI under a Collaborative Agreement (CRADA, CTA, CSA) between BNIT (Bavarian Nordic ImmunoTherapeutics) (hereinafter referred to as "Collaborator(s)") and the NCI. Therefore, the following obligations/guidelines, in addition to the provisions in the "Intellectual Property Option to Collaborator" (http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm) contained within the terms of award, apply to the use of the Agent(s) in this study: 1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can Agent(s) be transferred or licensed to any party not participating in the clinical study. Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall be maintained as such by the investigators. The protocol documents for studies utilizing Agents contain confidential information and should not be shared or distributed without the permission of the NCI. If a copy of this protocol is requested by a patient or patient's family member participating on the study, the individual should sign a confidentiality agreement. A suitable model agreement can be downloaded from: http://ctep.cancer.gov.  
2. For a clinical protocol where there is an investigational Agent used in combination with
(an)other Agent(s), each the subject of different Collaborative Agreements, the access to and use of data by each Collaborator shall be as follows (data pertaining to such combination use shall hereinafter be referred to as "Multi-Party Data"):
a) NCI will provide all Collaborators with prior written notice regarding the existence and nature of any agreements governing their collaboration with NCI, the design of the proposed combination protocol, and the existence of any obligations that would tend to restrict NCI's participation in the proposed combination protocol.  
b) Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial by any other Collaborator solely to the extent necessary to allow said other Collaborator to develop, obtain regulatory approval or commercialize its own Agent.  
c) Any Collaborator having the right to use the Multi-Party Data from these trials must agree in writing prior to the commencement of the trials that it will use the Multi-Party Data solely for development, regulatory approval, and commercialization of its own Agent.  
3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement will be made available to Collaborator(s), the NCI, and the FDA, as appropriate and unless additional disclosure is required by law or court order as described in the IP Option to Collaborator (http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm). Additionally, all Clinical Data and Results and Raw Data will be collected, used and disclosed consistent with all applicable federal statutes and regulations for the protection of human subjects, including, if applicable, the Standards for Privacy of Individually Identifiable Health Information set forth in 45 C.F.R. Part 164.  
4. When a Collaborator wishes to initiate a data request, the request should first be sent to the NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group studies, or PI for other studies) of Collaborator's wish to contact them.  
5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a DMC for this clinical trial.  
6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the Group office for Cooperative Group studies or by the principal investigator for nonCooperative Group studies for immediate delivery to Collaborator(s) for advisory review and comment prior to submission for publication. Collaborator(s) will have 30 days from the date of receipt for review. Collaborator shall have the right to request that publication be delayed for up to an additional 30 days in order to ensure that Collaborator's confidential and proprietary data, in addition to Collaborator(s)'s intellectual property rights, are protected. Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy review as soon as possible and preferably at least three (3) days prior to submission, but in any case, prior to presentation at the meeting or publication in the proceedings. Press releases and other media presentations must also be forwarded to CTEP prior to release.  
Copies of any manuscript, abstract and/or press release/ media presentation should be sent to:
Email: ncicteppubs@mail.nih.gov The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, manuscript or other form of public disclosure shall contain any of Collaborator's confidential/ proprietary information.
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12.5.1 material transfer agreement (mta)

A Material Transfer Agreement (MTA) is in process in order to receive specimens from participating sites for storage and future analysis.
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13 statistical considerations 13.1 primary

The study is to test the hypothesis that patients in the TICE BCG+PANVAC arm have better recurrence-free survival than patients in the TICE BCG alone arm. Patients to be accrued to the study are adults with high grade non-muscle invasive bladder cancer who failed at least 1 induction course of BCG. These patients comprise 3 subgroups, namely, BCG persistent with one failure of BCG induction course, BCG persistent with 2 or more failures of BCG induction courses, and BCG relapsing that can behave like BCG naïve. The ratio of the composition of the three subgroups is expected to be 1:5:1 with the majority of the patients being BCG persistent and having failed 2 or more BCG induction courses. In order to achieve the balance of treatment in each subgroup, stratified randomization will be implemented in this study. However, although the three subpopulations have different recurrence-free survival experience and may respond to treatment benefit differently, the study is powered to test the overall treatment benefit of BCG+PANVAC on the entire patient population. In the first subgroup of BCG persistent patients with one failure of BCG induction course, it is assumed that the 1-year recurrence-free survival rate is 37.5% 7,8 in the TICE BCG alone arm and 65% in the TICE BCG+PANVAC arm.**38,39** Under the assumption of exponential distribution for the recurrence-free survival in both arms, the difference in recurrence-free at 1-year corresponds to hazard ratio (HR) of 2.277. In the second subgroup of BCG persistent patients with 2 or more failures of BCG induction courses, the 1-year recurrence-free survival rate in the TICE BCG alone arm is expected to be 20%.40 Assume that the treatment benefit of TICE
BCG+PANVAC in the second subgroup is lower than in the first subgroup and HR is set at 2, which corresponds to a one-year recurrence-free survival of 44.7% in the TICE BCG+PANVAC
arm. In the third subgroup of BCG relapsing patients, it is expected that the 1-year recurrencefree survival rate is 48% and 65% in the TICE BCG alone and TICE BCG+PANVAC arm, respectively. This corresponds to HR=1.70. Patient accrual is targeted at one patient per month during the first 6 months and 2 patients per month afterwards, and follow-up period after completing accrual will be 12 months. The power calculation was based on one-sided log-rank test at 15% significance level. Sample size, power and duration of the study are presented in the table below. With a total number of 49 patients, the power is 84% (see table below). Under this scenario, the expected number of events (recurrence or death) is 40. In order to allow for the possibility of a small number of unevaluable patients, the accrual ceiling will be set at 54 patients.  
| Subgroup 1  1-year  recurrence-free  survival for BCG  persistent  patients who  failed 1 BCG  induction course   | Subgroup 2  1-year  recurrence-free  survival for BCG  persistent  patients who  failed 2 or more  BCG induction  courses   | Subgroup 3  1-year  recurrence-free  survival for BCG  naïve patients   | Total  sample  size                                   | Power   | Duration   |    |    |
|-------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------|-------------------------------------------------------|---------|------------|----|----|
| (%)                                                                                                               | of  accrual  (months)                                                                                                       | Expected  number of  events  (recurrence  or death)                     |                                                       |         |            |    |    |
| Treatment                                                                                                         | HR=2.277                                                                                                                    |                                                                         |                                                       |         |            |    |    |
| benefit                                                                                                           | 1-year RFS:  BCG alone:  37.5% BCG+PANVAC:  65%                                                                             | HR=2  1-year RFS:  BCG alone: 20%  BCG+PANVAC:  44.7%                   | HR=1.7  1-year RFS:  BCG alone: 48%  BCG+PANVAC:  65% | 49      | 84         | 28 | 40 |  
One interim analysis for futility will be performed according to the stopping procedure. 39 Specifically, the interim analysis will be performed when half of the expected events (20) have been observed. At that time, if the observed event rate of the TICE BCG+PANVAC arm is higher than that of the TICE BCG alone arm (i.e. the hazard ratio ≥ 1), we would consider terminating the trial and concluding that an advantage for the BCG+PANVAC regimen has not been established. For the purpose of protecting patient safety, we implement the following stopping rule for safety monitoring of patients who receive the combination therapy. If one patient experiences a grade 5 event, thought to be possibly or probably due to the combination therapy, the study will be stopped. For grade 3 or 4 event, the study will be stopped according to the following stopping rule. These adverse events are described in more detail in Section 7.  
| Number of patients   | No. of grade 3 or 4 serious adverse events   |
|----------------------|----------------------------------------------|
| 3-9                  | 3                                            |
| 10-18                | 4                                            |
| 19-26                | 5                                            |  
The operating characteristic of the stopping rule was assessed by simulations. Based on 10,000 simulations, the probability of stopping early for an excess of serious adverse event over the course of trial is 0.04 when the serious adverse event rate is at the anticipated level of 6% and 0.94 when the serious adverse event rate is 30%. This is deemed reasonable for protecting patient safety.
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13.2 secondary endpoints

Using Kaplan-Meier methods, we will estimate the probability distribution of progression-free survival and the time to recurrence. Results will be summarized by point estimates (12 months) with 95% CI. Exploratory analyses of these secondary objectives will compare the two treatment arms using the log rank test. Fisher's exact test will be used to compare frequency distributions of toxicity grades between the two arms.
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13.3 exploratory/correlative endpoints

13.3.1 To characterize the expression of MUC1 and CEA in bladder tumor specimens pre-
(week 0) and the change in expression post-vaccination (on tissue obtained between weeks 17-20) for patients treated with TICE BCG+PANVAC™.  
13.3.1.1 MUC1 expression will be evaluated using IHC. A positive result is defined if >5% of tumor cells reacted with any intensity. Intensity will be arbitrarily scored on a 4 point scale: 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). Potential antibody will be clone Ma695 dilution 1:100 (Novocastra, Newcastle upon Tyne, England) but has to be analytically validated using appropriate positive and negative controls prior to use.22 13.3.1.2 CEA expression in FFPE pre- and post-treatment tissues will be evaluated by IHC. A
positive result is defined when the cytoplasmic staining is equal in intensity to glycocalyceal staining. Tumors will be scored as 0 (no signal), 1+ (signal in <25% sampled tumor area), 2+ (26-75% of tumor cells), and 3+ (>75% of tumor cells). The potential antibody will be CD66e (clone 12-140-10; Novocastra, Newcastle upon-Tyne, England; dilution 1:500; Ventana NexES immunostainer; protease digestion).  
13.3.1.3 **Statistical Analysis**: For both MUC1 and CEA expression the distributions of IHC
staining intensity at baseline and post-vaccination as well as the change in score will be summarized with descriptive statistics. The results will be summarized by calculating the proportion and 95% confidence interval of positive staining tissue samples at
baseline and the distribution of pre and post-vaccination combinations (--, -+, +-, ++) characterizing the change with treatment. Results will be presented separately for each treatment arm. The distributions for the two treatment arms will be compared using a chi square test.  
13.3.2 To explore the CD4 Antigen-Specific response to MUC1 and CEA 13.3.2.1 The CD4 antigen-specific response will be assessed by culturing CD4+ T cells obtained from PBMCs pre- and post-treatment with irradiated APCs in the presence of MUC1 and CEA peptides/proteins. The IFN-γ levels will be measured by ELISA. Flu protein (positive control) and myoglobulin (negative control) will serve as controls.  
13.3.2.2 **Statistical Analysis:** Descriptive statistics will be calculated to summarize the CD4 antigen-specific response to MUC1 and CEA determined pre-treatment as well as the change post-vaccination. The same methods for analysis for each treatment arm and the comparison of arms as described for evaluation of the immune response to the vaccines will be applied.
13.3.3 To explore the CD8-mediated immune response to MUC1 and CEA 13.3.3.1 Assess CD8 T cell response by ELISPOT assay in pre- and post-treatment PBMCs using CEA and MUC1-specific T lymphocytes. We will be using an overlapping peptide library that will allow for the ability to test ~65% of HLA groups for MUC1 and ~80% of HLA groups for CEA
13.3.3.2 **Statistical Analysis:** A positive CD8 T cell response is defined as ≥ 2-fold increase post treatment compared with the pretreatment value in IFN-γ-secretion. Descriptive statistics will be calculated to summarize the CD8 T cell response as continuous variables to MUC1 and CEA at both time points for each study arm. In addition, the change from pre- to post-treatment will be summarized by the proportion of patients achieving a positive response. The proportion with 95% confidence intervals will be presented for each treatment arm along with additional descriptive statistics (e.g. mean change, the mean percent change). Fisher's exact test will be performed to compare the positive CD8 T cell response between the two treatment arms.
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13.4 reporting and exclusions 13.4.1

All patients will be evaluable for toxicity from the time of their first treatment with rV-PANVAC™ and rF-PANVAC™.
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13.4.2 evaluation of response

All patients included in the study must be assessed for response to treatment, even if there are major protocol treatment deviations or if they are ineligible. All of the patients who met the eligibility criteria (with the possible exception of those who received no study medication) should be included in the main analysis of the response rate. All conclusions should be based on all eligible patients. Subanalyses may then be performed on the basis of a subset of patients, excluding those for whom major protocol deviations have been identified (*e.g.*, early death due to other reasons, early discontinuation of treatment, major protocol deviations, etc.). However, these subanalyses may not serve as the basis for drawing conclusions concerning treatment efficacy, and the reasons for excluding patients from the analysis should be clearly reported. The 95% confidence intervals should also be provided.
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14 human subjects protections 14.1

Subjects from all racial/ethnic groups and both genders are eligible for this study if they meet the eligibility criteria. To date, there is no information that suggests that differences in drug metabolism, immune response or disease response would be expected in one group compared with another. Efforts will be made to extend accrual to a representative population, but in this preliminary study, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic and/or ineffective treatments on one hand and the need to explore gender and ethnic aspects of clinical research on the other hand. If differences in outcome that correlate with ethnic identity are noted, accrual may be expanded or a follow-up study may be written to investigate those differences more fully.
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14.1.2 justification for exclusions

Due to impaired cellular immunity, HIV patients are at an increased risk of serious side effects from vaccinations with infectious agents and are excluded. This is based on recommendations from the CDC and FDA. In addition, pregnant women are also excluded due to potentially increased risks of serious side effects from vaccinations with infectious agents.
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14.2 participation of children

Individuals under the age of 18 will not be eligible for participation in this study based on the fact that patients under 18 are unlikely to have this disease and there are unknown toxicities in pediatric patients.
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14.3 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol. However re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section **14.5**), will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team for evaluation. For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in MEC Policy 87-4 for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of attorney, will be followed. Participating sites should adhere to appropriate procedures specific to their site.
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14.4 evaluation of benefits and

BCG is a standard of care treatment and will be used in this study. The biggest risk of using this agent after failing an initial induction course of BCG is progression of disease to muscle-invasive disease. However, this occurs after multiple, failed BCG induction treatments and occurs more often after 2 years of such therapy40. In addition, patients will be accurately staged for disease with imaging, TURBT, and in some cases, repeat TURBT. They will be followed quite rigorously throughout the study and thereafter with a planned biopsy between weeks 17-20 to ensure that no progression of disease has occurred during the treatment period. Patients who progress will promptly undergo radical cystectomy. Radical cystectomy and pelvic lymphadenectomy is also considered a standard of care treatment for high grade BCG refractory NMIBC. However, since enrollment into this study requires patients to refuse radical surgery or not be medically fit for surgery, we believe the risk of proceeding with another induction course of BCG is outweighed by its benefit. Other risks of the use of BCG include local bladder symptoms such as blood in the urine, urinary frequency, urgency of urination, dysuria (pain), and bladder spasms which are often self-limiting and usually resolve within a week by withholding BCG and administering standard of care medications (e.g. anti-inflammatories, anti-spasmodics, hydration, etc.). Finally, a rare potential risk of BCG administration is development of "BCG-osis" which is a form of bacteremia/urosepsis or systemic BCG infection that occurs with BCG administration on very rare occasions. In this event, patients will be taken off the study and treated with anti-microbials as per recommendations by infectious disease consultants. The addition of PANVAC™ is unknown but given the relative safety of both agents and different routes of administration, we presume that the risk is minimal.
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14.4.1 alternative approaches or treatments

Patients will be consented verbally and in writing regarding the risks and benefits of this trial, the treatment requirements, and alternative approaches to entering on this trial.  
14.4.2 Procedure for Protecting Against or Minimizing any Potential Risks All care will be taken to minimize side effects, but they can be unpredictable in nature and severity. This study may involve risks to patients, which are currently unforeseeable. All patients will have blood tests, examinations and CT scans of the chest/abdomen/pelvis as described in the monitoring schedule as described in Appendix C (Section **16.3**). Patients will also be required to have a local physician to provide long-term care and to monitor for complications. If patients suffer any physical injury as a result of the participation in this study, immediate medical treatment is available at the Warren Grant Magnuson Clinical Center, Bethesda, Maryland. Although no compensation is available, any injury will be evaluated and treated in keeping with the benefits or care to which patients are entitled under applicable regulations. 14.4.3 Provisions for Monitoring Data Collection to Ensure Safety of Subjects As information is gathered from this trial, clinical results will be shared with patients. Laboratory and clinical data will be frequently gathered and any new significant finding(s) found during the course of the research, which may affect a patient's willingness to participate further, will be explained. Confidentiality of information concerning participants will be maintained, including in all publications and presentations resulting from this study. Names of participants or material identifying participants will not be released without permission, except as such release is required by law. Records at the Clinical Center are maintained according to current legal requirements, and are made available for review by Cancer Therapy Evaluation Program, the Food and Drug Administration, or other authorized users, as stated under the guidelines established by the Federal Privacy Act.
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14.5 risks/benefits analysis

The patients we will be enrolling have high grade NMIBC that has already failed the standard of care therapy, BCG induction. These patients are on the verge of developing muscle-invasive disease which requires potentially morbid surgery and change in quality of life. Therefore, they are very interested in strategies to preserve the bladder. Repeat induction BCG therapy is a standard of care and is being given to all patients in this study. The addition of PANVAC™ may potentially improve upon the poor RFS seen with BCG alone and hopefully this study will bear that out. Alternative treatments besides surgery include other intravesical agents that have not shown to be that effective. Whether the vaccine will have any clinical effect upon improving the outcome with BCG is unknown, therefore, benefit cannot be promised nor can the chance of benefit be accurately predicted. Patients' participation in this study is voluntary and refusal will not result in penalty or loss of benefit to which patient is otherwise entitled. Participation may be discontinued at any time without penalty and the patient can ask questions.
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14.6 consent process and documentation

The investigational nature and objectives of this trial, the procedures involved, and their attendant risks and discomforts, potential benefits, and potential alternative therapies will be explained to the patient and a signed informed consent document obtained. All listed associate investigators are permitted to obtain informed consent. The informed consent process will be documented on a progress note by the consenting investigator and a copy of the informed consent document and note will be kept in the subject's research record.
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14.6.1 telephone consent

The informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. A witness to the subject's signature will sign and date the consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with the date the consent was obtained via telephone. A fully executed copy will be returned via mail for the subject's records. The informed consent process will be documented on a progress note by the consenting investigator and a copy of the informed consent document and note will be kept in the subject's research record. 14.6.2 Informed consent of non-English speaking subjects If there is an unexpected enrollment of a research participant for whom there is no translated extant IRB approved consent document, the principal investigator and/or those authorized to obtain informed consent will use the Short Form Oral Consent Process as described in MAS Policy M77-2, OHSRP SOP 12, 45 CFR 46.117 (b) (2), (*If a study with an IND or IDE, also cite* 21 CFR 50.27 (b) (2)). The summary that will be used is the English version of the extant IRB approved consent document. Signed copies of both the English version of the consent and the translated short form will be given to the subject or their legally authorized representative and the signed original will be filed in the medical record. Unless the PI is fluent in the prospective subject's language, an interpreter will be present to facilitate the conversation (using either the long translated form or the short form). Preferably someone who is independent of the subject (i.e., not a family member) will assist in presenting information and obtaining consent. Whenever possible, interpreters will be provided copies of the relevant consent documents well before the consent conversation with the subject (24 to 48 hours if possible). We request prospective IRB approval of the use of the short form process for non-English speaking subjects and will notify the IRB at the time of continuing review of the frequency of the use of the Short Form.
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15 references

1. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin 62:10-29, 2012 2. Donat SM: Evaluation and follow-up strategies for superficial bladder cancer. Urol Clin North Am 30:765-76, 2003 3. Herr HW, Morales A: History of bacillus Calmette-Guerin and bladder cancer: an immunotherapy success story. J Urol 179:53-6, 2008 4. Crawford ED: Intravesical therapy for superficial cancer: need for more options. J Clin Oncol 20:3185-6, 2002 5. Morales A: Long-term results and complications of intracavitary bacillus Calmette-Guerin therapy for bladder cancer. J Urol 132:457-9, 1984 6. Di Lorenzo G, Perdona S, Damiano R, et al: Gemcitabine versus bacille Calmette-Guerin after initial bacille Calmette-Guerin failure in non-muscle-invasive bladder cancer: a multicenter prospective randomized trial. Cancer 116:1893-900, 2010 7. Kavoussi LR, Torrence RJ, Gillen DP, et al: Results of 6 weekly intravesical bacillus Calmette-Guerin instillations on the treatment of superficial bladder tumors. J Urol 139:935-40, 1988 8. O'Donnell MA, Boehle A: Treatment options for BCG failures. World J Urol 24:481-7, 2006 9. Shabsigh A, Korets R, Vora KC, et al: Defining early morbidity of radical cystectomy for patients with bladder cancer using a standardized reporting methodology. Eur Urol 55:164-74, 2009 10. Stein JP, Lieskovsky G, Cote R, et al: Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. J Clin Oncol 19:666-75, 2001 11. Joudi FN, Smith BJ, O'Donnell MA, et al: Contemporary management of superficial bladder cancer in the United States: a pattern of care analysis. Urology 62:1083-8, 2003 12. Lightfoot AJ, Rosevear HM, O'Donnell MA: Recognition and treatment of BCG failure in bladder cancer. ScientificWorldJournal 11:602-13, 2011 13. Ratliff TL, Gillen D, Catalona WJ: Requirement of a thymus dependent immune response for BCG-mediated antitumor activity. J Urol 137:155-8, 1987 14. Ratliff TL, Ritchey JK, Yuan JJ, et al: T-cell subsets required for intravesical BCG immunotherapy for bladder cancer. J Urol 150:1018-23, 1993 15. Luo Y, Knudson MJ: Mycobacterium bovis bacillus Calmette-Guerininduced macrophage cytotoxicity against bladder cancer cells. Clin Dev Immunol 2010:357591, 2010  
16. Prescott S, James K, Hargreave TB, et al: Intravesical Evans strain BCG therapy: quantitative immunohistochemical analysis of the immune response within the bladder wall. J Urol 147:1636-42, 1992 17. Hodge JW, Grosenbach DW, Aarts WM, et al: Vaccine therapy of established tumors in the absence of autoimmunity. Clin Cancer Res 9:1837-49, 2003 18. Madan RA, Arlen PM, Gulley JL: PANVAC-VF: poxviral-based vaccine therapy targeting CEA and MUC1 in carcinoma. Expert Opin Biol Ther 7:543-54, 2007 19. Kufe DW: Mucins in cancer: function, prognosis and therapy. Nat Rev Cancer 9:874-85, 2009 20. Raina D, Kosugi M, Ahmad R, et al: Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells. Mol Cancer Ther 10:806-16, 2011 21. Walsh MD, Hohn BG, Thong W, et al: Mucin expression by transitional cell carcinomas of the bladder. Br J Urol 73:256-62, 1994 22. Lau SK, Weiss LM, Chu PG: Differential expression of MUC1, MUC2, and MUC5AC in carcinomas of various sites: an immunohistochemical study. Am J Clin Pathol 122:61-9, 2004 23. Goldenberg DM, Sharkey RM, Primus FJ: Immunocytochemical detection of carcinoembryonic antigen in conventional histopathology specimens. Cancer 42:1546-53, 1978 24. Cardillo MR, Castagna G, Memeo L, et al: Epidermal growth factor receptor, MUC-1 and MUC-2 in bladder cancer. J Exp Clin Cancer Res 19:225-33, 2000 25. Hodge JW, Grosenbach DW, Rad AN, et al: Enhancing the potency of peptide-pulsed antigen presenting cells by vector-driven hyperexpression of a triad of costimulatory molecules. Vaccine 19:3552-67, 2001 26. Gulley JL, Arlen PM, Tsang KY, et al: Pilot study of vaccination with recombinant CEA-MUC-1-TRICOM poxviral-based vaccines in patients with metastatic carcinoma. Clin Cancer Res 14:3060-9, 2008 27. Mohebtash M, Tsang KY, Madan RA, et al: A pilot study of MUC1/CEA/TRICOM poxviral-based vaccine in patients with metastatic breast and ovarian cancer.  
Clin Cancer Res 17:7164-73, 2011 28. C. Heery NI, R.A. Madan, M. Mohebtash, P. Arlen, M. Bilusic, J. Kim, N.K. Singh, S McMahon, S. Hodge, S.M. Steinberg, J. Schlom, J. Hodge, J.L. Gulley: A phase 2 randomized trial of docetaxel alone or in combination with therapeutic cancer vaccine, CEA-, MUC-1-TRICOM. Presented at the ESMO 2012, Vienna, Austria, 2012 29. M. Morse DN, J. Marshall, C. R. Garrett, D. Z. Chang, M. Aklilu, T. S. Crocenzi, D. J. Cole, S. Dessureault, A. Hobeika, T. Osada, B. M. Clary, S. D. Hsu, G. Devi, A. Bulusu, R. Annechiarico, V. Chadaram, T. M. Clay, H. K. Lyerly: Survival rates among patients vaccinated following resection of colorectal cancer metastases in a phase II randomized study compared with contemporary controls. Presented at the 2011 ASCO Annual Meeting, 2011  
30. Kantoff PW, Schuetz TJ, Blumenstein BA, et al: Overall survival analysis of a phase II randomized controlled trial of a Poxviral-based PSA-targeted immunotherapy in metastatic castration-resistant prostate cancer. J Clin Oncol 28:1099-105, 2010 31. Martinez-Pineiro JA, Flores N, Isorna S, et al: Long-term follow-up of a randomized prospective trial comparing a standard 81 mg dose of intravesical bacille CalmetteGuerin with a reduced dose of 27 mg in superficial bladder cancer. BJU Int 89:671-80, 2002 32. Ojea A, Nogueira JL, Solsona E, et al: A multicentre, randomised prospective trial comparing three intravesical adjuvant therapies for intermediate-risk superficial bladder cancer: low-dose bacillus Calmette-Guerin (27 mg) versus very low-dose bacillus Calmette-Guerin (13.5 mg) versus mitomycin C. Eur Urol 52:1398-406, 2007 33. Gulley J, Chen AP, Dahut W, et al: Phase I study of a vaccine using recombinant vaccinia virus expressing PSA (rV-PSA) in patients with metastatic androgenindependent prostate cancer. Prostate 53:109-17, 2002 34. Lamm DL: Efficacy and safety of bacille Calmette-Guerin immunotherapy in superficial bladder cancer. Clin Infect Dis 31 Suppl 3:S86-90, 2000 35. Vaccinia (smallpox) vaccine. Recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR Recomm Rep 40:1-10, 1991 36. Theodorescu D, Wittke S, Ross MM, et al: Discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis. Lancet Oncol 7:230-40, 2006 37. Ludwig AT, Moore JM, Luo Y, et al: Tumor necrosis factor-related apoptosis-inducing ligand: a novel mechanism for Bacillus Calmette-Guerin-induced antitumor activity. Cancer Res 64:3386-90, 2004 38. Mitropoulos DN: Novel insights into the mechanism of action of intravesical immunomodulators. In Vivo 19:611-21, 2005 39. Wieand S, Schroeder G, O'Fallon JR: Stopping when the experimental regimen does not appear to help. Stat Med 13:1453-8, 1994 40. Herr HW, Sogani PC: Does early cystectomy improve the survival of patients with high risk superficial bladder tumors? J Urol 166:1296-9, 2001
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appendices 16.1 appendix a performance

| ECOG Performance Status Scale   |                                                                                                                                                                |
|---------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade                           | Descriptions                                                                                                                                                   |
| 0                               | Normal activity. Fully active, able to carry on all pre-disease  performance without restriction. Symptoms, but ambulatory. Restricted in physically strenuous |
| 1                               | activity, but ambulatory and able to carry out work of a light or  sedentary nature (e.g., light housework, office work).  In bed <50% of the time. Ambulatory and capable of all selfcare, but unable to carry out any work activities. Up and about                                                                                                                                                                |
| 2                               | more than 50% of waking hours.                                                                                                                                 |
| 3                               | In bed >50% of the time. Capable of only limited self-care,  confined to bed or chair more than 50% of waking hours.                                           |
| 4                               | 100% bedridden. Completely disabled. Cannot carry on any selfcare. Totally confined to bed or chair.                                                                                                                                                                |
| 5                               | Dead.                                                                                                                                                          |
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16.2 appendix b ctep multicenter

If an institution wishes to collaborate with other participating institutions in performing a CTEP sponsored research protocol, then the following guidelines must be followed.
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responsibility of the protocol chair

x The Protocol Chair will be the single liaison with the CTEP Protocol and Information Office (PIO). The Protocol Chair is responsible for the coordination, development, submission, and approval of the protocol as well as its subsequent amendments. The protocol must not be rewritten or modified by anyone other than the Protocol Chair. There will be only one version of the protocol, and each participating institution will use that document. The Protocol Chair is responsible for assuring that all participating institutions are using the correct version of the protocol.  
x The Protocol Chair is responsible for the overall conduct of the study at all participating institutions and for monitoring its progress. All reporting requirements to CTEP are the responsibility of the Protocol Chair.  
x The Protocol Chair is responsible for the timely review of Adverse Events (AE) to assure safety of the patients.  
x The Protocol Chair will be responsible for the review of and timely submission of data for study analysis.
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responsibilities of the coordinating center

x Each participating institution will have an appropriate assurance on file with the Office for Human Research Protection (OHRP), NIH. The Coordinating Center is responsible for assuring that each participating institution has an OHRP assurance and must maintain copies of IRB approvals from each participating site.  
x Prior to the activation of the protocol at each participating institution, an OHRP form 310
(documentation of IRB approval) must be submitted to the CTEP PIO.  
x The Coordinating Center is responsible for central patient registration. The Coordinating Center is responsible for assuring that IRB approval has been obtained at each participating site prior to the first patient registration from that site.  
x The Coordinating Center is responsible for the preparation of all submitted data for review by the Protocol Chair.  
x The Coordinating Center will maintain documentation of AE reports. There are two options for AE reporting: (1) participating institutions may report directly to CTEP with a copy to the Coordinating Center, or (2) participating institutions report to the Coordinating Center who in turn report to CTEP. The Coordinating Center will submit AE reports to the Protocol Chair for timely review.  
x Audits may be accomplished in one of two ways: (1) source documents and research records for selected patients are brought from participating sites to the Coordinating Center for audit, or (2) selected patient records may be audited on-site at participating sites. If the NCI chooses to have an audit at the Coordinating Center, then the Coordinating Center is responsible for having all source documents, research records, all IRB approval documents, NCI Drug Accountability Record forms, patient registration lists, response assessments scans, x-rays, etc. available for the audit.
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inclusion of multicenter guidelines in

x The protocol must include the following minimum information:
¾ The title page must include the name and address of each participating institution and the name, telephone number and e-mail address of the responsible investigator at each participating institution.  
¾ The Coordinating Center must be designated on the title page. ¾ Central registration of patients is required. The procedures for registration must be stated in the protocol.  
¾ Data collection forms should be of a common format. Sample forms should be submitted with the protocol. The frequency and timing of data submission forms to the Coordinating Center should be stated.  
¾ Describe how AEs will be reported from the participating institutions, either directly to CTEP or through the Coordinating Center.  
¾ Describe how Safety Reports and Action Letters from CTEP will be distributed to participating institutions.
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agent ordering

x Except in very unusual circumstances, each participating institution will order DCTDsupplied investigational agents directly from CTEP. Investigational agents may be ordered by a participating site only after the initial IRB approval for the site has been forwarded by the Coordinating Center to the CTEP PIO.
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16.3 appendix c panvac [recombinant

1. What vaccination site reactions can you expect? 2. How should you care for the vaccination site? 3. Are there any activities I should avoid? 4. What about contact with other people? 5. Who do I contact when I have a question?
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1. what vaccination site reactions

A typical reaction in a patient who has been previously vaccinated with vaccinia includes the appearance of a small swelling in 2-3 days that may enlarge to 1-2 inches across, a small blister or pustule after 5-7 days, and healing with little scarring within 2-3 weeks. Some patients may have very little skin reaction. Often the leg that received the vaccine may become swollen. Swollen or tender lymph nodes ("glands") in the groin may be felt. A fever to 100-101qF may occur on the second or third day. You may notice that you feel tired for 3 or 4 days. The vaccination site may itch for about 2 weeks while the scab is forming. You can take acetaminophen ("Tylenol") if you have any aches or fever but should avoid aspirin. If fever continues for more than a day or two, you should call to speak to the clinic nurse or the research nurse. In patients who have never received vaccinia or in some who received it a very long time ago, a red swelling is followed by a blisters on day 5 to 6 and then formation of a pustule (or "boil") 1-2 inches in diameter on day 9 to 11. A large area of redness may surround this area. A crusted scab usually forms by the second week and falls off by the third week leaving a scar roughly 1/2 inch in diameter. Fever and malaise (the "blahs") may occur during the blister and pustular phases. Swollen and tender lymph nodes may persist for months. Many of the local toxicities described (e.g., pustule and scab formation) are typical of reactions seen when vaccinia is administered via scarification or intradermal administration. These reactions may be seen, but are usually not seen when administered via subcutaneous injection.
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2. how should you care

Live vaccinia virus is in skin cells at the vaccination site during the 1-2 weeks until healing has occurred. Maximal viral "shedding" from the vaccination site occurs from days 4-14, but can continue until the scab falls off from the skin. After that there is no vaccinia virus in your body. You can spread the virus to other parts of your body or to other people by touching the vaccination site and then touching your eyes, mouth, a cut or some other break in the skin.You do not pass vaccinia virus by coughing or sneezing or by sharing food or cups and dishes. In general, frequent careful hand washing by you and by any persons in physical contact with you is the best way to prevent transmission of virus. You should also use two types of barriers over your vaccination site at all times until the scab is gone. These barriers are (1) the bandage and (2) clothing (pants or elbow length sleeves depending on the site of vaccination). These barriers should remain in place until the scab has fallen off. For dressing care you will have a bag with some no-stick "First-Aid" or "Telfa" pads, disposable gloves, and zip-lock plastic bags. If you run out of supplies between visits, you can use a dry sterile bandage (gauze or "First-Aid" or "Telfa") from the drug store. The no-stick pad ("First-Aid" or "Telfa" pad) dressing should be worn until the site has healed. If it remains clean and dry and is not coming off, you do not need to change it. If the dressing gets wet either from drainage from the vaccination or from water when you are showering or if it starts coming off, you should remove it and put on a clean bandage. Wear the gloves when handling the old dressings. Put the old dressing and the gloves in the zip-lock bag, then wash your hands, put on the new bandage, and wash your hands again. You do not need to wear gloves for the new bandage. You do not need to wash the vaccination site, but while the dressing is off, you may wash it lightly with a cloth, soap, and water. If you do wash, blot the site dry with a towel (don't rub), then put the wash cloth and the towel in the laundry. Do not let the shower run on the unbandaged site because live virus could be washed onto other areas on your body. Do not put any steroid cream, medicated creams, or other ointments on the vaccination site. Before you throw away the zip-lock bag with the old dressing and gloves in it, pour a little bleach (about a quarter cup) in the bag to help kill any virus.  
Wash your hands after each step.
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3. are there any activities

You should not go swimming until the vaccination site has healed and you no longer need to wear a bandage on it. If you wear contact lenses, have removable dentures, have a colostomy or any other "open" area on your body that needs daily care, always wash your hands very well before handling your contact lenses, dentures, dressings, etc. Take care of all of these procedures before changing your vaccination dressing.
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4. what about contact with

Remember, frequent careful hand washing by you and by any persons with whom you have physical contact is the best way to prevent transmission of virus. During the time you need to wear a bandage (for 7-14 days after vaccination) there are several kinds of people with whom you should avoid close contact. "Close contact" means that you sleep in the same bed with the person, give the person baths, and/or touch their bare skin to change their clothes (or diapers), apply ointments, or change their bandages. The individuals you should avoid include children < 3years of age; pregnant women or nursing women; individuals with eczema, history of eczema or other skin conditions such as active cases of extensive psoriasis, severe rashes, generalized itching, infections, burns, chicken pox, or skin trauma; and/or immune suppressed individuals such as individuals with leukemia or lymphoma, with AIDS, or those receiving immunosuppressive treatment (for example, after organ transplant).
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5. who do i contact

If you have any questions at any time, please call. A nurse or a physician is available 24 hours a day by telephone. To speak with your main doctor or with a clinic nurse, call the Urology Clinic between 8 AM and 4:30 PM Monday to Friday. To speak with the research nurses, call the research nurse office during the day; during nights, weekends, and sometimes during the day, when the research office is empty, you may leave a message for the research nurse on the answering machine. You can call Dr. Piyush Agarwal (the principal investigator) any time during weekday hours. In an emergency on weekends, evenings, or holidays, you can always get in touch with the on-call urologic oncology doctor (fellow) (listed below). This doctor can be contacted by calling the NIH Clinical Care Center operator, who will page the on call doctor. The on call doctor will then call you back. If you have to go to an emergency room near your home, go to the hospital first, and then have the doctors there call the NIH for more information.
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phone numbers

Urology Outpatient Clinic (3rd floor) (301) 496-5484  
| Sonia Bellfield, RN   | (240) 760-6118   |
|-----------------------|------------------|  
Rebecca Dolan, CRNP (301)827-1007  
Piyush Agarwal, MD (240) 760-6242*  
*after clinic hours the NCI Urology Oncology physician on call through NIH page operator (301) 496-1211  
| 16.4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | APPENDIX D: NIH PROBLEM REPORT FORM                                                                |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------|
| NCI Protocol #:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Protocol Title:  Report version: (select one) ____Initial Report  ____Revised Report ____Follow-up |
| Site Principal Investigator:  Date of problem:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Location of problem: (e.g., patient's home,  doctor's office )                                     |
| Who identified the problem? (provide role (not name of person): nurse, investigator, monitor,  etc…) Brief Description of Subject (if  applicable)  (Do NOT include personal  identifiers) Sex: ___ Male ___ Female Age:   ___ Not applicable (more than subject is involved) Diagnosis under study:  Name the problem: (select all that apply)  [ ] Adverse drug reaction  [ ] Abnormal lab value  [ ] Death  [ ] Cardiac Arrest/ code  [ ] Anaphylaxis  [ ] Sepsis/Infection  [ ] Blood product reaction  [ ] Unanticipated surgery/procedure  [ ] Change in status (e.g. increased level of care required)  [ ] Allergy (non-medication)  [ ] Fall  [ ] Injury/Accident (not fall)  [ ] Specimen collection issue  [ ] Informed consent issue  [ ] Ineligible for enrollment |                                                                                                    |  
Detailed Description of the problem: (Include any relevant treatment, outcomes or pertinent history): *Is this problem unexpected? *(see the definition of unexpected in the protocol))* __YES __NO Please explain: *Is this problem related or possibly related to participation in the research? __YES __NO Please explain: *Does the problem suggest the research places subjects or others at a greater risk of harm than was previously known or recognized? __YES __NO Please explain: Is this problem? (*select all that apply*) [ ] An Unanticipated Problem* that is: [ ] Serious [ ] Not Serious [ ] A Protocol Deviation that is: [ ] Serious [ ] Not Serious [ ] Non-compliance *Note if the 3 criteria starred above are answered, "YES", then this event is also a UP. Is the problem also *(select one)* [ ] AE [ ] Non-AE Have similar problems occurred on this protocol at your site? __YES __NO If "Yes", how many? ____ Please describe:
Describe what steps you have already taken as a result of this problem:
In addition to the NCI IRB, this problem is also being reported to: (*select all that apply*)
[ ] Local IRB
[ ] Study Sponsor [ ] Manufacturer : ____________________ [ ] Institutional Biosafety Committee [ ] Data Safety Monitoring Board [ ] Other: ____________________________________ [ ] None of the above, not applicable INVESTIGATOR'S SIGNATURE: DATE:
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16.5 appendix e specimen collection

The following protocols are used in the Agarwal Laboratory and efforts will be made at collaborating centers to collect bio-specimens in a similar fashion. When feasible and when an appropriate materials transfer agreement is in place specimens used for correlative endpoints can be transferred from collaborating centers to the NIH for storage and future analysis. Until then, specimens will be stored at the collaborating sites under the following conditions.
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urine protocol

A. Urine will be collected at room temperature from patients through voiding or instrumentation and transported to laboratory staff on ice. Collection will be 30-100 mL of urine in a sterile urine container.  
B. Time points for collection  
| BCG/PANVAC arm   | BCG arm                                                                                                                                                                                                                                                 |    |                                                                                                                                                                                                                                                        |
|------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| x                | Week 0 - collect before vaccine  administration                                                                                                                                                                                                         | x  | Week 3 - Two samples:  o Pre-BCG urine prior to BCG  instillation  o Post-BCG urine after BCG is drained  from bladder. This sample is the first  voided urine obtained after the BCG  instillation is completed and all of the  BCG is first drained. |
| x                | Week 3 - Two samples:  o Pre-BCG urine prior to BCG  instillation  o Post-BCG urine after BCG is  drained from bladder. This sample  is the first voided urine obtained  after the BCG instillation is  completed and all of the BCG is first  drained. | x  | Week 5 - Two samples:  o Pre-BCG urine prior to BCG  instillation  o Post-BCG urine after BCG is drained  from bladder. This sample is the first  voided urine obtained after the BCG  instillation is completed and all of the  BCG is first drained. |
| x                | Week 5 - Two samples:  o Pre-BCG urine prior to BCG  instillation  o Post-BCG urine after BCG is  drained from bladder. This sample  is the first voided urine obtained  after the BCG instillation is  completed and all of the BCG is first  drained. | x  | Week 17-20 - collect urine prior to  bladder biopsy.                                                                                                                                                                                                   |
| x                | Week 17-20 - collect urine prior to  bladder biopsy.                                                                                                                                                                                                    |    |                                                                                                                                                                                                                                                        |
| C.               | Centrifuge urine at 1200xg (2491 rpm for 20 minutes using our 5810R Eppendorf                                                                                                                                                                           |    |                                                                                                                                                                                                                                                        |  
C. Centrifuge urine at 1200xg (2491 rpm for 20 minutes using our 5810R Eppendorf  
Centrifuge) at 4°C in 50 mL conical tube.  
D. Filter the supernatant (cell-free urine) with .45 μm filter to remove large debris. Save cell  
  
pellet.  
E. Store filtered urine as cell-free, filtered urine in 6 vials (1.8 mL cryovial each) at -80°C
without any cryopreservative.  
F. Resuspend urine cell pellet in 1 mL of 5% DMSO (95% MEM media) and transfer to 1.8 mL
cryovial tube.  
G. Spin this cryovial at 1200xg for 20 minutes at 4°C and leave cell pellet packed. H. Do slow freeze of cell pellet with Mr. Frosty and then store at -80. I. Schematic: Urine will be processed at: Piyush K. Agarwal, MD c/o Julian Custer or Quentin Li Urologic Oncology Branch 10 Center Drive, 1W-5888 Bethesda, MD 20892 For patients at CINJ, research samples consisting of urine (week 0, week 3, week 5 and week 17-20) will be obtained and processed as above and then shipped via Fed-Ex to the above address.
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blood protocol

A. Blood will be collected at various time points before any treatments and in various types of tubes and processed as below.  
B. Time points for collection  
| BCG/PANVAC arm   | BCG arm                                                                                                                                                                      |    |                                                                                                                                                                              |
|------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| x                | Week 0 or at randomization - 1 purple  top tube (contains EDTA), total  volume 10 mL                                                                                         |    |                                                                                                                                                                              |
| x                | Week 0 - 1 purple top tube (contains  EDTA), total volume 10 mL; 6 green top  tubes (contains sodium heparin), total  volume 60 mL; 2 red top tubes, total  volume 20 mL     | x  | Week 3 - 6 green top tubes (contains  sodium heparin), total volume 60 mL;  2 red top tubes, total volume 20 mL                                                              |
| x                | Week 3 - 6 green top tubes (contains  sodium heparin), total volume 60 mL; 2  red top tubes, total volume 20 mL                                                              | x  | Week 8 - 6 green top tubes (contains  sodium heparin), total volume 60 mL;  2 red top tubes, total volume 20 mL                                                              |
| x                | Week 8 - 6 green top tubes (contains  sodium heparin), total volume 60 mL; 2  red top tubes, total volume 20 mL                                                              | x  | Week 17-20 - 1 purple top tube  (contains EDTA), total volume 10 mL;  6 green top tubes (contains sodium  heparin), total volume 60 mL; 2 red  top tubes, total volume 20 mL |
| x                | Week 11 - 6 green top tubes (contains  sodium heparin), total volume 60 mL; 2  red top tubes, total volume 20 mL                                                             |    |                                                                                                                                                                              |
| x                | Week 15 - 6 green top tubes (contains  sodium heparin), total volume 60 mL; 2  red top tubes, total volume 20 mL                                                             |    |                                                                                                                                                                              |
| x                | Week 17-20 - 1 purple top tube (contains  EDTA), total volume 10 mL; 6 green top  tubes (contains sodium heparin), total  volume 60 mL; 2 red top tubes, total  volume 20 mL |    |                                                                                                                                                                              |  
C. Red Top tubes and Green Top Tubes will be processed as follows:
Clinical Services Program (CSP) NCI Frederick Cancer Research and Development Center PO Box B Frederick MD 21702 301-846-1000 On days samples are drawn, Jen Bangh at CSP should be notified (phone: [301] 846-5893; fax [301] 846-6222). She will arrange same day courier delivery of the specimens drawn at NIH. For samples drawn at outside sites, please refer to the Research Sample Shipment section below. Once a patient's treatment schedule has been determined, Caroline Jochems should be notified at jochemscm@mail.nih.gov for planning purposes.  
D. Purple Top Tubes will be processed in Dr. Agarwal's Lab as follows:
a. Centrifuge blood at 4,000 rpm for 10-20 minutes using our 5810R Eppendorf Centrifuge at 4°C.  
b. Transfer plasma to 1.8 mL cryovial tubes. Store the tubes with plasma at -80C. c. Collect buffy coat layer (white blood cells) using individually packaged 3 mL transfer pipets.  
d. Extract DNA from buffy coat layer (white blood cells) with Promega MAXWELL 16 instrument using the Maxwell Blood DNA Purification Kit as follows:
i. Remove the seal from each cartridge.  
ii. Place plunger in well \#7 and sample (white blood cells) in well \#1.  
iii. Open the door. Press "Run/Stop" to extend platform.  
iv. Transfer cartridges to platform. Add elution tubes and 400 L elution buffer to each tube.  
v. Close the door. Press "Run" to perform purification for 50 min.  
vi. Run completed. Verify that plungers are clear.  
vii. Transfer elution tubes to magnetic elution rack and transfer DNA samples to storage tubes.  
viii. Label and store the tubes at -80C.  
e. For patients at CINJ, research samples consisting of 2 purple top tubes (week 0 and week 17-20) will be drawn, processed as above, and then shipped via Fed-Ex to Dr. Agarwal's lab.
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tissue protocol

Tissue will be collected for diagnostic and therapeutic purposes at the beginning of the trial and in weeks 17-20 of the trial following BCG or BCG/PANVAC therapy. Additional tissue that is safely obtained and not needed for pathologic diagnosis can be placed in a sterile container and processed in the following manner:
i. Remove samples to be frozen from the container with sterile forceps and gently dab on sterile gauze to remove excess fluid.  
ii. Once dry, place sample on a piece of tin foil above dry ice to freeze samples iii. Once frozen, move the samples to empty labeled vial.  
iv. If there are enough specimens in the sample to freeze in Tissue Tek O.C.T.  
Compound (OCT), then place one sample on plastic OCT plate instead of tin foil v. Add OCT medium to the OCT plate. Then place the plate on top of dry ice with the plastic side down (the OCT medium and embedded tissue should be facing you).  
vi. If tissue culture is going to be performed on sample, leave at least one piece of tissue in the original container (without freezing it) and deliver this sample to the tissue culture lab.  
vii. Once labeled and placed on dry ice, store samples in a -80 freezer.
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research sample shipment

For samples drawn at outside sites to be sent to the NCI Frederick Repository, please FED-EX Overnight to the shipping address below. PBMCs are to be sent on the day of draw; serum is to be frozen and batch-shipped at intervals. USE NIH/NCI/LTIB FedEx Account Number: 281312103
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1. materials

Pack blood vials in the shipping kit provided to the site by the NCI research team. Contents of the kit: EXAKT-PAK for Vials Category B D-pak MD8702V06 (Accommodates 6 vials) Includes Inner pack (Ambient) and Insulated Cooler With 2 cool packs per cooler, part \#CP1003, slightly cooled (refrigerated, not frozen)
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2. address

Leidos Biomedical Research Attn: Bill Kopp/Theresa Burks 1050 Boyles Street Bldg. 469/Room 121 Frederick, MD 21702 Phone 301-846-5125, or 301-846-1707 x Please notify the Frederick laboratory when specimens are being shipped. Please email Frederick prior to shipping to notify the lab.  
x Emails should be sent to the following individuals:
Bill Kopp, koppw@mail.nih.gov Theresa Burks, burkst@mail.nih.gov Caroline Jochems, jochemscm@mail.nih.gov Piyush Agarwal, piyush.agarwal@nih.gov James Gulley gulleyj@mail.nih.gov  
x Additionally, NO specimens should be shipped on Fridays or the day before a Federal holiday.
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3. labeling of blood samples

Please label research tubes with a coding mechanism. List the patient's enrollment number first (will be provided by the Central Registration Office at time of registration), followed by the patient's initials, and lastly followed by the enrollment site. Example: 01-ABC-CINJ
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4. supply for shipping kits

SHIPPING PAKS to be ordered by the NCI and shipped to participating cancer center. Please contact the NCI team for requesting additional shipping kits.  
EXAKT Technologies, Inc. Home office: 7002 N. Broadway Extension Oklahoma City, OK 73116-9006 405-848-5800
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16.6 appendix f sample shipping

| Ship From:            | Ship To:          |
|-----------------------|-------------------|
| Address:              | Attn:             |
|                       | Shipping Manifest |
| Phone:  Contact Name: | Phone:            |
| Clinical Site:        | E-mail:           |
| Shipping Date:        | Carrier:          |  
| Clinical Protocol: CTEP 9539   |                         |             |                 |          |
|--------------------------------|-------------------------|-------------|-----------------|----------|
| In Package                     | Unique Patient ID*      | Description | Number of items | Comments |
|                                | Total number of items = |             |                 |          |