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Combination of pembrolizumab and BCG treatment after endoscopic ablation of high-risk superficial upper urinary tract urothelial carcinoma in patients not candidates for radical nephroureterectomy: protocol for phase-II study

Introduction The treatment standard for high-risk upper urinary tract urothelial carcinoma (UUTUC) is radical nephroureterectomy. However, some patients may be unfit or unwilling, and in such patients the available alternatives are suboptimal. Therapies targeting the programmed death (PD) pathway have shown promise in urothelial carcinom (UC). We designed the current study to determine the safety and efficacy of administering MK-3475 (a monoclonal antibody targeting interaction between PD-1 and its ligand) in combination with bacillus Calmette-Guerin (BCG) in high-risk non-muscle invasive UUTUC patients. Methods This represents a single-centre phase-II efficacy study of MK-3475 therapy in combination with BCG for subjects, 18 years of age or older, with pathologically documented non-muscle invasive high-risk UUTUC unfit or unwilling to be treated with radical nephroureterectomy. Twenty subjects will be enrolled; patients will receive treatment with 200 mg of MK-3475 every 21 days, starting 2 weeks from the initial endoscopic resection and continuing for 6 weeks after the final dose of BCG. The primary objective is to determine the safety and efficacy of administering MK-3475 at a fixed dose of 200 mg every 3 weeks in conjunction with intrapelvic BCG. Secondary objectives include 19 week and the 3, 12 and 24-month post-treatment completion complete response and progression-free rate assessments. Ethics and dissemination The study has been approved by the Institutional Review Board of the Henry Ford Hospital. The results of this study will be published in a peer-reviewed journal and presented at a scientific conference. Trial registration number NCT03345134

Endoscopic treatment is an alternative to radical nephroureterectomy. Patients that are candidates for endoscopic management of high-risk disease have been extensively studied, and the eligibility criteria include lesions that are papillary and superficial, completely visualized and <2 cm in diameter with no computed tomographic evidence of parenchymal invasion (2). Further, in efforts to decrease recurrence, adjuvant BCG treatment has been utilized (3)(4)(5). Unfortunately, more than 50% of patients recur by 5-yr (2,3). Hence, identification of new agents active in this disease is warranted.
The blockade of immune checkpoint pathway involving Programmed Death 1 (PD-1) receptor and its ligands, CD274 (PD-L1) and CD273 (PD-L2), has been recently shown to be effective in cancer therapy including for UC (6,7). Furthermore, the treatment effect of PD-1 blockade may be exaggerated in UUTUC due the high prevalence of mismatch repair defects in these tumors (8,9). We hypothesize that a combination of Pembrolizumab and BCG following endoscopic treatment of high-risk UUTUC may be effective in improving outcomes of UUTUC after endoscopic treatment in patients unfit or unwilling to be treated with radical nephroureterectomy. The aim of this study is to determine the safety and response of administering MK-3475 (a monoclonal antibody designed to block the interaction between PD-1 and its ligand) in conjunction with BCG treatment in high-risk non-muscle invasive UUTUC patients. In this paper, we report the treatment protocol for the study. PDL-1 is widely expressed by hematopoietic, non-hematopoietic and tumor cells, whereas PDL-2 is expressed mainly by DCs and macrophages (10,11). Higher expression of PDL-1 in cancers is associated with poor survival as tumors can evade the immune system by stimulating PD-1 (12)(13)(14). expression in these tumors is also associated with high-grade disease. In addition, PDL-1 expression is found to be extremely abundant in the BCG-induced transitional epithelium granulomata in patients failing BCG treatment (15). Furthermore, as noted above, UUTUCs have aberrations in DNA mismatch repair genes that are detectable as microsatellite instability ([MSI]; in about 45%) (8). This is important because Pembrolizumab has been shown previously to have increased activity in tumors with mismatch repair deficiency. Le et al conducted a Phase-II study to evaluate the clinical activity of Pembrolizumab in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. The authors then found that the immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers (9). Based on these data, a combination of Pembrolizumab with BCG may be effective in preventing recurrence of UUTUC after endoscopic treatment.

Trial blinding
This is an open-label trial; therefore, the Sponsor, investigator and subject will know the treatment administered.

Study design
This is a single center Phase-II efficacy study of MK-3475 therapy used in combination with renal pelvis infused BCG treatment for subjects, 18 years or older, with pathologically documented high-risk superficial UUTUC unfit or unwilling to be treated with radical nephroureterectomy. Subjects will have confirmation of UUTUC non-invasive to the muscle. Approximately 30 subjects will be screened to treat 20 eligible subjects. Subjects will be enrolled to a single treatment group of a fixed dose of MK 3475 and BCG (Figure 1).

Trial schedule
Trial schedule (Figure 2) summarizes the trial procedures to be performed at each visit. It may be necessary to perform these procedures at unscheduled time points if deemed clinically necessary by the investigator. Furthermore, additional evaluations/testing may be deemed necessary by the sponsor for reasons related to subject safety. In some cases, such evaluation/testing may be potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional informed consent be obtained from the subject. In these cases, such evaluations/testing will be performed in accordance with those regulations.

Patient selection
Subjects, who meet required inclusion and exclusion criteria are eligible for enrollment into the study ( figure 3a and figure 3b).

Patient and public involvement
Patient and public were not involved in the design, recruitment and conduction of the present study. Results of the study will be disseminated to the study participants on request. Patient advisors were not used in the conduction of the this study.
Trial treatment dosage, formulation, preparation and frequency BCG: The rationale for the use of the indicated dose of BC is based upon FDA approved and commercially provided product. Dosing for the intraluminal treatment of carcinoma in situ and for the prophylaxis of recurrent papillary tumors consists of one vial of BCG suspended in 50 ml preservative-free saline. The preparation of the BCG suspension will be completed using aseptic technique and according to FDA approved labeling and use information. Patients may have treatment with more than one vial to ensure treatment of all tumor sites. BCG treatment will be administered beginning on day 1 of cycle 3 (Week 7) of MK-3475 after all procedures/assessments have been completed as detailed on the trial flowchart. BCG treatment will be repeated every 7 days at weeks 8, 9, 10, 11, and 12. BCG treatment may be administered up to 1 day before or after the scheduled date (at 7 days) due to administrative reasons. All trial treatments will be administered on an outpatient basis and according to institutional standards. BCG dose modification has been shown to reduce efficacy; therefore, no dose modifications will be attempted in this trial. The dose, frequency, route of administration and duration are summarized in (figure 4).
BCG treatment will be withheld for grade 2 hematuria, grade 1 fever, and drugrelated grade 4 hematologic toxicities, and all other non-hematological toxicity grade ≥3 Treatment will be postponed until resolution of febrile illness, urinary tract infection, or gross hematuria and adverse effect resolution to grade <1. In the event of a grade 1 or 2 immune related adverse event requiring corticosteroid treatment, BCG treatment may be delayed for up to 2 weeks with the expectation that steroid treatment will be discontinued prior to the next BCG treatment cycle. A delay of longer than 2 weeks will permanently discontinue the patient from BCG treatment within the study or permanent withdrawal of the patient from the study. The choice will be at the discretion of the treating investigator. Steroid treatment for grade 3 or higher immune mediated events will require discontinuation of BCG treatment and/or withdrawal from the study.   To determine safety and efficacy of administering MK-3475 at a fixed dose of 200 mg every three-weeks in conjunction with intrapelvic BCG in high-risk superficial UUTUC patients, who are unfit or unwilling to be treated with radical nephroureterectomy.

Secondary objective
To determine the 19 week and the 3, 12, and 24 month post-treatment completion complete response rate and progression-free rate with combined MK-3475 and intrapelvic BCG therapy for non-muscle invasive high-risk UUTUC for patients unfit or unwilling to be treated with radical nephroureterectomy.

Exploratory objectives
Three objectives: to examine pre-and post-treatment (MK-3475/BCG) renal pelvis biopsy specimens for PD-1, PD-L1, and CD3; to examine whole exome sequencing of subjects' germline DNA and formalin fixed, paraffin embedded tissue.
With analysis of the correlation of mutations to treatment outcomes; analysis of blood and urine specimens from enrollees for potential development of blood and urine assays for determination of response to treatment. The investigator or qualified designee will assess each subject to evaluate for potential new or worsening AEs as specified in the detailed trial schedule and more frequently, if clinically indicated. Adverse experiences will be graded and recorded throughout the study and during the follow-up period according to NCI CTCAE Version 4.0. Toxicities will be characterized in terms regarding seriousness, causality, toxicity grading, and action taken with regard to trial treatment. AEs will be recorded from the

Statistical Measures and Analysis
This protocol aims at assessing the activity of one treatment arm, BCG in combination with Pembrolizumab after endoscopic ablation in patients with UUTUC. We would like to detect an activity which is at least 25% greater than that of historical controls (50%) from case series of BCG only treatment. Sample size calculation for this protocol is based on the work by Kahn et al (2012). Using a one-stage design, 20 patients will be enrolled in this study. If 14 or more patients respond then the trial will stop. This design has 78.8% power to determine at the end whether the experimental arm is better than the historical control with a 1-sided type I error rate of 0.057. This design was selected because UUTUC is a rare disease, and we are willing to minimally sacrifice power to reach a sample size that could be achieved within the suggested study period and resources. Patient related data will be collected and transferred to an excel spreadsheet for statistical analysis. SAS software version 9.4 will be used for all data analysis. Descriptive statistics will be computed for all study variables. Continuous variables will be described with measures of central tendency (mean, median) and dispersion (range, standard deviation). Categorical For the secondary objective, subjects will be assessed at 19 weeks, 3 months, 12 months, and 24 months post treatment completion for complete response. The outcome endpoint will be complete response status at 19 weeks, 3 months, 12 months, and 24 months post treatment to measure complete response, recorded as yes or no and summarized as proportions and a percent.
For the exploratory objectives, Pearson or Spearman's rank correlation coefficient will be used to estimate correlations between expression level change and treatment outcome, as well as the correlations between mutation presence and treatment outcome.

ETHICS, CONFIDENTIALITY AND REGULATORY DETAILS
The Investigator and Sponsor of the study will adhere to all applicable data privacy and confidentiality laws and regulations (i.e. HIPAA). Those applicable include institutional, state, and Federal Law. It is the responsibility of the Investigator and Sponsor to ensure the subject data as well as sensitive study information is handled according to applicable guidelines and laws. Appropriate authorization and consent for use, disclosure, or transfer of protected health information must be obtained.
Subject names will not be recorded on the CRF. Only the subject number and subject's initials will be recorded, where permitted. If the subject's name appears on any other document (e.g., pathology report), it must be obliterated on the copy of the document as appropriate. Study data stored on a computer will be stored in accordance with local data protection laws. The subjects will be informed that representatives of the sponsor or its designee, IRB, and regulatory authorities may inspect their medical records to verify the information collected, and that all personal information made available for inspection will be handled in strictest confidence and in accordance with local data protection laws.
Based on the SPIRIT guidelines.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
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We designed the current study to determine the safety and efficacy of administering MK-3475 (a monoclonal antibody targeting interaction between PD-1 and its ligand) in combination with BCG in high-risk non-muscle invasive UUTUC patients.

Methods:
This represents a single-center Phase-II efficacy study of MK-3475 therapy in combination with BCG for subjects, 18 years of age or older, with pathologically documented non-muscle invasive high-risk UUTUC unfit or unwilling to be treated with radical nephroureterectomy. Twenty subjects will be enrolled; patients will receive treatment with 200 mg of MK-3475 every 21 days, starting 2 weeks from the initial endoscopic resection and continuing for 6 weeks after the final dose of BCG. The primary objective is to determine the safety and efficacy of administering MK-3475 at a fixed dose of 200 mg every three-weeks in conjunction with intrapelvic BCG. Secondary objectives include 19 week and the 3, 12, and 24 month post-treatment completion complete response and progression-free rate assessments.

Ethics and dissemination:
The study has been approved by the Institutional Review Board of the Henry Ford Hospital. The results of this study will be published in a peerreviewed journal and presented at a scientific conference.

Strengths and limitations:
 One of the first clinical trials to assess the safety and efficacy of PD-1 inhibitors in combination with BCG for subjects with non-muscle invasive high-risk UUTUC after endoscopic ablation.
 Study provides extensive follow-up and multiple objective measures assessing the safety and efficacy of PD-1 inhibitors.
 This study is non-randomized and unblinded.  In the present study, accurate staging of non-muscle invasive UUTUC is solely dependent on endoscopic biopsy, which has been previously shown to result in understaging when compared to final pathology.  (3)(4)(5). Unfortunately, more than 50% of patients recur by 5-yr (2,3). Hence, identification of new agents active in this disease is warranted.
The blockade of immune checkpoint pathway involving Programmed Death 1 (PD-1) receptor and its ligands, CD274 (PD-L1) and CD273 (PD-L2), has been recently shown to be effective in cancer therapy including for UC (6,7). Furthermore, the treatment effect of PD-1 blockade may be exaggerated in UUTUC due the high prevalence of mismatch repair defects in these tumors (8,9). We hypothesize that a combination of Pembrolizumab and BCG following endoscopic treatment of high-risk UUTUC may be effective in improving outcomes of UUTUC after endoscopic treatment in patients unfit or unwilling to be treated with radical nephroureterectomy. The aim of this study is to determine the safety and response of administering MK-3475 (a monoclonal antibody designed to block the interaction between PD-1 and its ligand) in conjunction with BCG treatment in high-risk non-muscle invasive UUTUC patients. In this paper, we report the treatment protocol for the study.  PDL-1 expression, a marker for response to anti-PD-1 agents, is observed in 7% of pTa, 16% of pT1 and 45% of carcinoma in situ (CIS) urothelial tumors (15). PDL-1 objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers (9). Based on these data, a combination of Pembrolizumab with BCG may be effective in preventing recurrence of UUTUC after endoscopic treatment.

Trial blinding
This is an open-label trial; therefore, the Sponsor, investigator and subject will know the treatment administered.

Study design
This is a single center Phase-II efficacy study of MK-3475 therapy used in combination with renal pelvis infused BCG treatment for subjects, 18 years or older,  (Figure 1). Patients to undergo regular follow-up including applicable labs, in addition to post-treatment ureteroscopy with clinically indicated biopsy to assess recurrence. (Figure 2)

Trial schedule
Trial schedule (Figure 2) summarizes the trial procedures to be performed at each visit. It may be necessary to perform these procedures at unscheduled time points if deemed clinically necessary by the investigator. Furthermore, additional evaluations/testing may be deemed necessary by the sponsor for reasons related to subject safety. In some cases, such evaluation/testing may be potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional informed consent be obtained from the subject. In these cases, such evaluations/testing will be performed in accordance with those regulations.

Patient selection
Subjects, who meet required inclusion and exclusion criteria are eligible for enrollment into the study ( figure 3 and figure 4).

Patient and public involvement
Patient and public were not involved in the design, recruitment and conduction of the present study. Results of the study will be disseminated to the study participants on request. Patient advisors were not used in the conduction of the this study. days at weeks 8, 9, 10, 11, and 12. BCG treatment may be administered up to 1 day before or after the scheduled date (at 7 days) due to administrative reasons. All trial treatments will be administered on an outpatient basis and according to institutional standards. BCG dose modification has been shown to reduce efficacy; therefore, no dose modifications will be attempted in this trial. The dose, frequency, route of administration and duration are summarized in (figure 5). adverse effects including laboratory abnormalities and severe or life-threatening AEs.
Treatment will be postponed until resolution of febrile illness, urinary tract infection, or gross hematuria and adverse effect resolution to grade <1. In the event of a grade 1 or 2 immune related adverse event requiring corticosteroid treatment, BCG treatment may be delayed for up to 2 weeks with the expectation that steroid treatment will be discontinued prior to the next BCG treatment cycle. A delay of longer than 2 weeks will permanently discontinue the patient from BCG treatment within the study or permanent withdrawal of the patient from the study. The choice will be at the discretion of the treating investigator. Steroid treatment for grade 3 or higher immune mediated events will require discontinuation of BCG treatment and/or withdrawal from the study.    within the original study protocol and management of said risks and AE's is also noted within the original study protocol.

Primary objective
To determine safety and efficacy of administering MK-3475 at a fixed dose of 200 mg every three-weeks in conjunction with intrapelvic BCG in high-risk superficial UUTUC patients, who are unfit or unwilling to be treated with radical nephroureterectomy.

Secondary objective
To determine the 19 week and the 3, 12, and 24 month post-treatment completion complete response rate and progression-free rate with combined MK-3475 and intrapelvic BCG therapy for non-muscle invasive high-risk UUTUC for patients unfit or unwilling to be treated with radical nephroureterectomy.

Exploratory objectives
Three objectives: to examine pre-and post-treatment (MK-3475/BCG) renal pelvis biopsy specimens for PD-1, PD-L1, and CD3; to examine whole exome sequencing of subjects' germline DNA and formalin fixed, paraffin embedded tissue.
With analysis of the correlation of mutations to treatment outcomes; analysis of blood and urine specimens from enrollees for potential development of blood and urine assays for determination of response to treatment.

Adverse effects
The investigator or qualified designee will assess each subject to evaluate for potential new or worsening AEs as specified in the detailed trial schedule and more frequently, if clinically indicated. Adverse experiences will be graded and recorded throughout the study and during the follow-up period according to NCI CTCAE Version 4.0. Toxicities will be characterized in terms regarding seriousness, causality, toxicity grading, and action taken with regard to trial treatment. AEs will be recorded from the

Statistical Measures and Analysis
This protocol aims at assessing the activity of one treatment arm, BCG in combination with Pembrolizumab after endoscopic ablation in patients with UUTUC. We Sample size calculation for this protocol is based on the work by Kahn et al (2012). Using a one-stage design, 20 patients will be enrolled in this study. If 14 or more patients respond then the trial will stop. This design has 78.8% power to determine at the end whether the experimental arm is better than the historical control with a 1-sided type I error rate of 0.057. This design was selected because UUTUC is a rare disease, and we are willing to minimally sacrifice power to reach a sample size that could be achieved within the suggested study period and resources. Patient related data will be collected and transferred to an excel spreadsheet for statistical analysis. SAS software version 9.4 will be used for all data analysis. Descriptive statistics will be computed for all study variables. Continuous variables will be described with measures of central tendency (mean, median) and dispersion (range, standard deviation). Categorical  For the secondary objective, subjects will be assessed at 19 weeks, 3 months, 12 months, and 24 months post treatment completion for complete response. The outcome endpoint will be complete response status at 19 weeks, 3 months, 12 months, and 24 months post treatment to measure complete response, recorded as yes or no and summarized as proportions and a percent.
For the exploratory objectives, Pearson or Spearman's rank correlation coefficient will be used to estimate correlations between expression level change and treatment outcome, as well as the correlations between mutation presence and treatment outcome.

ETHICS, CONFIDENTIALITY AND REGULATORY DETAILS
The Investigator and Sponsor of the study will adhere to all applicable data privacy and confidentiality laws and regulations (i.e. HIPAA). Those applicable include institutional, state, and Federal Law. It is the responsibility of the Investigator and Sponsor to ensure the subject data as well as sensitive study information is handled according to applicable guidelines and laws. Appropriate authorization and consent for use, disclosure, or transfer of protected health information must be obtained.
Subject names will not be recorded on the CRF. Only the subject number and subject's initials will be recorded, where permitted. If the subject's name appears on any other document (e.g., pathology report), it must be obliterated on the copy of the document as appropriate. Study data stored on a computer will be stored in accordance with local data protection laws. The subjects will be informed that representatives of the sponsor or its designee, IRB, and regulatory authorities may inspect their medical records to verify the information collected, and that all personal information made available for inspection will be handled in strictest confidence and in accordance with local data protection laws.
Qualified staff of the Sponsor will monitor the study according to a pre-arranged monitoring plan. Monitoring of the study will include: Protocol deviations will be identified and recorded on a deviation log.  The combination of BCG therapy has been shown to decrease recurrence, yet despite its efficacy, recurrence remains relatively high.
The design of the present study will allow the researchers to discern the safety

Figure 1
a Ablation may be performed in an antegrade or retrograde fashion. b Six weekly doses of BCG in total. c Patients with suspicious lesions, or positive cytology, will be evaluated with renal pelvis biopsy under general anesthesia. will occur to monitor for fever and symptoms of systemic infection. Subjects will each be given an oral thermometer and a diary to record oral temperature twice daily (morning and evening) and to record any symptoms they may be having. The diary will be utilized Days 1-7 for each of the 6 BCG cycles (Weeks 7-12). h Trans-urethral Resections: All TUBR will occur as part of SOC and as clinically indicated. For the purposes of entry into this study, the screening window for endoscopic ablation with repeat biopsy will be within 90 days of Day 1. i Adverse Events will be collected from time of Main study informed consent through 30 days post last study drug infusion/treatment. j Physical Exam: Full physical exams are required at screening and at the 30 day follow up. All other exams may be symptom directed. k Performance status: ECOG performance status scale should be used. l ECG, Chest x-ray, and Pulmonary Function Tests will be completed prior to Day 1 Cycle 1 and will only be repeated as clinically indicated. m PT/INR and aPTT will be completed prior to all resections/ possibly biopsies as standard of care. All other monitoring of PT/INR and aPTT will be completed as clinically indicated (i.e. if on Coumadin). n Ureteroscopy will be performed as standard of care but will be considered measures for efficacy. Biopsy will be performed as clinically indicated. o Specimen Collection/Correlative studies: Whole blood, serum, and urine will be collected at time of screening consent/ main study consent (only 1 collection at either time point) as well as each visit through Week 19 repeat ureteroscopy (12 visits). Saliva will be collected only at baseline. Tumor tissue will only be collected at baseline and at Week 19 biopsy if a biopsy is clinically indicated at the time of ureteroscopy. Specimen collection is requested at the 3, 6, 9, 12, 18, and 24 month follow up visits if subject is being treated locally at the site. Tissue will only be collected if a biopsy is clinically indicated at time of ureteroscopy. p Chest X-rays performed in the 90 days window prior to the screening visit are acceptable. q CBC, CMP, and correlative labs do not need to be repeated on Day 1 Cycle 1 if they were completed within 10 day prior to Day 1 Cycle 1 during screening.

Figure 3
a All pathology specimens used for enrollment are required to be trad by the research site pathology laboratory. b Patient are to have provided tissue from an archival tissue sample or newly obtained core or excisional biopsy of a tumor lesion. Tissue must be obtained from the most recent upper urinary tract biopsy. However, in patients with small tumors or carcinoma in situ of the upper urinary tract, and considering the difficulty of obtaining sufficient tissue for testing beyond pathologic diagnosis, not providing this tissue is not going to exclude the patient from the study. c If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. , and urogram are allowed to ascertain the superficial nature of the disease when indicated, but not required. If urogram protocol is not available or contrast allergy/poor renal function precludes such imaging, then non-contrast CT or MRI of the abdomen/pelvis within 90 days of study entry will suffice.) b Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, localized prostate cancer with no recurrence after curative surgery or radiation, or in situ cervical cancer that has undergone potentially curative therapy. Lower urinary tract transitional cell carcinoma is also allowable on study as high-risk transitional cell carcinoma is commonly multifocal, and intraluminal BCG therapy is also used for treatment of lower urinary tract lesions in a manner similar to that of UUTUC c Use of disease modifying agents, corticosteroids or immunosuppressive drugs. Replacement therapy (eg. thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment. d This includes anything that may confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator. e Subjects will not be specifically tested for the study; however, subjects that are tested within 28 days of beginning study or while on study and test positive with the PPD test before treatment should have active tuberculosis ruled out before therapy begins for their superficial renal pelvis cancer Figure 5 a The MK-3475 dosing interval may be increased due to toxicity as is described in the Dose Modification and Adverse Effects section. BCG treatment may be interrupted or delayed as per the Dose Modification and Adverse Effects section. b BCG: One vial of TICE  BCG suspended in 50 ml preservative-free saline. BCG is to be given through a urinary catheter into the renal pelvis either retrograde or antegrade through a nephrostomy tube. BCG treatment will occur once per week for 6 weeks. BCG treatment will commence on the 3rd cycle of treatment with MK-3475, Week 7. BCG is a FDA approved treatment and will be supplied commercially through the study site pharmacy. The preparation of the BCG suspension will be completed using aseptic technique and according to FDA approved labeling and use information. Patients may have treatment with more than one vial to ensure treatment of all tumor sites. Additional lesions in patients with multi-focal disease will be treated per intuitional standard and directed by the treating urologist.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
Your article may not currently address all the items on the checklist. Please modify your text to include the missing information. If you are certain that an item does not apply, please write "n/a" and provide a short explanation.
Upload your completed checklist as an extra file when you submit to a journal.
In your methods section, say that you used the SPIRIT reporting guidelines, and cite them as: Author notes 1. n/a in original protocol which has been submitted with the manuscript 2. n/a in original protocol which has been submitted with the manuscript 3. 10,11, as well as original study protocol 4. 20, extensive details within original study protocol 5. n/a, detailed within original study protocol 6. 11,12,19,20 7. n/a, this is a non-randomized, un-blinded clinical trial, both researchers and patients are aware of involvement and intervention within the study Therapies targeting the Programmed Death (PD) pathway have shown promise in UC.
We designed the current study to determine the safety and efficacy of administering MK-3475 (a monoclonal antibody targeting interaction between PD-1 and its ligand) in combination with BCG in high-risk non-muscle invasive UUTUC patients.

Ethics and dissemination:
The study has been approved by the Institutional Review Board of the Henry Ford Hospital. The results of this study will be published in a peerreviewed journal and presented at a scientific conference.

Strengths and limitations:
 One of the first clinical trials to assess the safety and efficacy of PD-1 inhibitors in combination with BCG for subjects with non-muscle invasive high-risk UUTUC after endoscopic ablation.
 Study provides extensive follow-up and multiple objective measures assessing the safety and efficacy of PD-1 inhibitors.
 This study is non-randomized and unblinded with Long-term enrollment and frequent clinical obligations may result in failure in the retention of subjects.
 Small sample size given rarity of disease and patient eligibility criteria.
 In the present study, accurate staging of non-muscle invasive UUTUC is solely dependent on endoscopic biopsy, which has been previously shown to result in understaging when compared to final pathology. The blockade of immune checkpoint pathway involving Programmed Death 1 (PD-1) receptor and its ligands, CD274 (PD-L1) and CD273 (PD-L2), has been recently shown to be effective in cancer therapy including for UC (6, 7). Furthermore, the treatment effect of PD-1 blockade may be exaggerated in UUTUC due the high prevalence of mismatch repair defects in these tumors (8,9). We hypothesize that a combination of Pembrolizumab and BCG following endoscopic treatment of high-risk UUTUC may be effective in improving outcomes of UUTUC after endoscopic treatment  PDL-1 expression, a marker for response to anti-PD-1 agents, is observed in 7% of pTa, 16% of pT1 and 45% of carcinoma in situ (CIS) urothelial tumors (15). PDL-1 objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers (9). Based on these data, a combination of Pembrolizumab with BCG may be effective in preventing recurrence of UUTUC after endoscopic treatment.

Trial blinding
This is an open-label trial; therefore, the Sponsor, investigator and subject will know the treatment administered.

Study design
This is a single center Phase-II efficacy study of MK-3475 therapy used in combination with renal pelvis infused BCG treatment for subjects, 18 years or older,  (Figure 1). Patients to undergo regular follow-up including applicable labs, in addition to post-treatment ureteroscopy with clinically indicated biopsy to assess recurrence. (Figure 2)

Trial schedule
Trial schedule (Figure 2) summarizes the trial procedures to be performed at each visit. It may be necessary to perform these procedures at unscheduled time points if deemed clinically necessary by the investigator. Furthermore, additional evaluations/testing may be deemed necessary by the sponsor for reasons related to subject safety. In some cases, such evaluation/testing may be potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional informed consent be obtained from the subject. In these cases, such evaluations/testing will be performed in accordance with those regulations.

Patient selection
Subjects, who meet required inclusion and exclusion criteria are eligible for enrollment into the study ( figure 3 and figure 4).

Patient and public involvement
Patient and public were not involved in the design, recruitment and conduction of the present study. Results of the study will be disseminated to the study participants on request. Patient advisors were not used in the conduction of the this study.  Treatment will be postponed until resolution of febrile illness, urinary tract infection, or gross hematuria and adverse effect resolution to grade <1. In the event of a grade 1 or 2 immune related adverse event requiring corticosteroid treatment, BCG treatment may be delayed for up to 2 weeks with the expectation that steroid treatment will be discontinued prior to the next BCG treatment cycle. A delay of longer than 2 weeks will permanently discontinue the patient from BCG treatment within the study or permanent withdrawal of the patient from the study. The choice will be at the discretion of the treating investigator. Steroid treatment for grade 3 or higher immune mediated events will require discontinuation of BCG treatment and/or withdrawal from the study.    within the original study protocol and management of said risks and AE's is also noted within the original study protocol.

Primary objective
To determine safety and efficacy of administering MK-3475 at a fixed dose of 200 mg every three-weeks in conjunction with intrapelvic BCG in high-risk superficial UUTUC patients, who are unfit or unwilling to be treated with radical nephroureterectomy.

Secondary objective
To determine the 19 week and the 3, 12, and 24 month post-treatment completion complete response rate and progression-free rate with combined MK-3475 and intrapelvic BCG therapy for non-muscle invasive high-risk UUTUC for patients unfit or unwilling to be treated with radical nephroureterectomy.

Exploratory objectives
Three objectives: to examine pre-and post-treatment (MK-3475/BCG) renal pelvis biopsy specimens for PD-1, PD-L1, and CD3; to examine whole exome sequencing of subjects' germline DNA and formalin fixed, paraffin embedded tissue.
With analysis of the correlation of mutations to treatment outcomes; analysis of blood and urine specimens from enrollees for potential development of blood and urine assays for determination of response to treatment.

Adverse effects
The investigator or qualified designee will assess each subject to evaluate for potential new or worsening AEs as specified in the detailed trial schedule and more frequently, if clinically indicated. Adverse experiences will be graded and recorded throughout the study and during the follow-up period according to NCI CTCAE Version 4.0. Toxicities will be characterized in terms regarding seriousness, causality, toxicity grading, and action taken with regard to trial treatment. AEs will be recorded from the

Statistical Measures and Analysis
This protocol aims at assessing the activity of one treatment arm, BCG in combination with Pembrolizumab after endoscopic ablation in patients with UUTUC. We For the secondary objective, subjects will be assessed at 19 weeks, 3 months, 12 months, and 24 months post treatment completion for complete response. The outcome endpoint will be complete response status at 19 weeks, 3 months, 12 months, and 24 months post treatment to measure complete response, recorded as yes or no and summarized as proportions and a percent.
For the exploratory objectives, Pearson or Spearman's rank correlation coefficient will be used to estimate correlations between expression level change and treatment outcome, as well as the correlations between mutation presence and treatment outcome.

ETHICS, CONFIDENTIALITY AND REGULATORY DETAILS
The Investigator and Sponsor of the study will adhere to all applicable data privacy and confidentiality laws and regulations (i.e. HIPAA). Those applicable include institutional, state, and Federal Law. It is the responsibility of the Investigator and Sponsor to ensure the subject data as well as sensitive study information is handled according to applicable guidelines and laws. Appropriate authorization and consent for use, disclosure, or transfer of protected health information must be obtained.
Subject names will not be recorded on the CRF. Only the subject number and subject's initials will be recorded, where permitted. If the subject's name appears on any other document (e.g., pathology report), it must be obliterated on the copy of the document as appropriate. Study data stored on a computer will be stored in accordance with local data protection laws. The subjects will be informed that representatives of the sponsor or its designee, IRB, and regulatory authorities may inspect their medical records to verify the information collected, and that all personal information made available for inspection will be handled in strictest confidence and in accordance with local data protection laws.
Qualified staff of the Sponsor will monitor the study according to a pre-arranged monitoring plan. Monitoring of the study will include:     (Weeks 7-12) will occur to monitor for fever and symptoms of systemic infection. Subjects will each be given an oral thermometer and a diary to record oral temperature twice daily (morning and evening) and to record any symptoms they may be having. The diary will be utilized Days 1-7 for each of the 6 BCG cycles (Weeks 7-12). h Trans-urethral Resections: All TUBR will occur as part of SOC and as clinically indicated. For the purposes of entry into this study, the screening window for endoscopic ablation with repeat biopsy will be within 90 days of Day 1. i Adverse Events will be collected from time of Main study informed consent through 30 days post last study drug infusion/treatment. j Physical Exam: Full physical exams are required at screening and at the 30 day follow up. All other exams may be symptom directed. k Performance status: ECOG performance status scale should be used. l ECG, Chest x-ray, and Pulmonary Function Tests will be completed prior to Day 1 Cycle 1 and will only be repeated as clinically indicated. m PT/INR and aPTT will be completed prior to all resections/ possibly biopsies as standard of care. All other monitoring of PT/INR and aPTT will be completed as clinically indicated (i.e. if on Coumadin). n Ureteroscopy will be performed as standard of care but will be considered measures for efficacy. Biopsy will be performed as clinically indicated. o Specimen Collection/Correlative studies: Whole blood, serum, and urine will be collected at time of screening consent/ main study consent (only 1 collection at either time point) as well as each visit through Week 19 repeat ureteroscopy (12 visits). Saliva will be collected only at baseline. Tumor tissue will only be collected at baseline and at

FIGURE LEGENDS
Week 19 biopsy if a biopsy is clinically indicated at the time of ureteroscopy. Specimen collection is requested at the 3, 6, 9, 12, 18, and 24 month follow up visits if subject is being treated locally at the site. Tissue will only be collected if a biopsy is clinically indicated at time of ureteroscopy. p Chest X-rays performed in the 90 days window prior to the screening visit are acceptable. q CBC, CMP, and correlative labs do not need to be repeated on Day 1 Cycle 1 if they were completed within 10 day prior to Day 1 Cycle 1 during screening. Figure 3 a All pathology specimens used for enrollment are required to be trad by the research site pathology laboratory. b Patient are to have provided tissue from an archival tissue sample or newly obtained core or excisional biopsy of a tumor lesion. Tissue must be obtained from the most recent upper urinary tract biopsy. However, in patients with small tumors or carcinoma in situ of the upper urinary tract, and considering the difficulty of obtaining sufficient tissue for testing beyond pathologic diagnosis, not providing this tissue is not going to exclude the patient from the study. c If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. , and urogram are allowed to ascertain the superficial nature of the disease when indicated, but not required. If urogram protocol is not available or contrast allergy/poor renal function precludes such imaging, then non-contrast CT or MRI of the abdomen/pelvis within 90 days of study entry will suffice.) b Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, localized prostate cancer with no recurrence after curative surgery or radiation, or in situ cervical cancer that has undergone potentially curative therapy. Lower urinary tract transitional cell carcinoma is also allowable on study as high-risk transitional cell carcinoma is commonly multifocal, and intraluminal BCG therapy is also used for treatment of lower urinary tract lesions in a manner similar to that of UUTUC c Use of disease modifying agents, corticosteroids or immunosuppressive drugs. Replacement therapy (eg. thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment. d This includes anything that may confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator. e Subjects will not be specifically tested for the study; however, subjects that are tested within 28 days of beginning study or while on study and test positive with the PPD test before treatment should have active tuberculosis ruled out before therapy begins for their superficial renal pelvis cancer Figure 5 a The MK-3475 dosing interval may be increased due to toxicity as is described in the Dose Modification and Adverse Effects section. BCG treatment may be interrupted or delayed as per the Dose Modification and Adverse Effects section. b BCG: One vial of TICE  BCG suspended in 50 ml preservative-free saline. BCG is to be given through a urinary catheter into the renal pelvis either retrograde or antegrade through a nephrostomy tube. BCG treatment will occur once per week for 6 weeks. BCG treatment will commence on the 3rd cycle of treatment with MK-3475, Week 7. BCG is a FDA approved treatment and will be supplied commercially through the study site pharmacy. The preparation of the BCG suspension will be completed using aseptic technique and according to FDA approved labeling and use information. Patients may have treatment with more than one vial to ensure treatment of all tumor sites. Additional lesions in patients with multi-focal disease will be treated per intuitional standard and directed by the treating urologist.      1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y Reporting checklist for protocol of a clinical trial.
Based on the SPIRIT guidelines.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
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13. 12,13, extensive detailing of management of adverse events within the original study protocol.

Ethics and dissemination:
The study has been approved by the Institutional Review Board of the Henry Ford Hospital. The results of this study will be published in a peerreviewed journal and presented at a scientific conference.

Strengths and limitations:
 One of the first clinical trials to assess the safety and efficacy of PD-1 inhibitors in combination with BCG for subjects with non-muscle invasive high-risk UUTUC after endoscopic ablation.
 Study provides extensive follow-up and multiple objective measures assessing the safety and efficacy of PD-1 inhibitors.
 This study is non-randomized and unblinded with Long-term enrollment and frequent clinical obligations may result in failure in the retention of subjects.
 Small sample size given rarity of disease and patient eligibility criteria.
 In the present study, accurate staging of non-muscle invasive UUTUC is solely dependent on endoscopic biopsy, which has been previously shown to result in understaging when compared to final pathology.  (3)(4)(5). Unfortunately, more than 50% of patients recur by 5-yr (2,3). Hence, identification of new agents active in this disease is warranted.
The blockade of immune checkpoint pathway involving Programmed Death 1 (PD-1) receptor and its ligands, CD274 (PD-L1) and CD273 (PD-L2), has been recently shown to be effective in cancer therapy including for UC (6,7). Furthermore, the treatment effect of PD-1 blockade may be exaggerated in UUTUC due the high prevalence of mismatch repair defects in these tumors (8,9). We hypothesize that a combination of Pembrolizumab and BCG following endoscopic treatment of high-risk UUTUC may be effective in improving outcomes of UUTUC after endoscopic treatment in patients unfit or unwilling to be treated with radical nephroureterectomy. The aim of this study is to determine the safety and response of administering MK-3475 (a monoclonal antibody designed to block the interaction between PD-1 and its ligand) in conjunction with BCG treatment in high-risk non-muscle invasive UUTUC patients. In this paper, we report the treatment protocol for the study.   objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers (9). Based on these data, a combination of Pembrolizumab with BCG may be effective in preventing recurrence of UUTUC after endoscopic treatment.

Trial blinding
This is an open-label trial; therefore, the Sponsor, investigator and subject will know the treatment administered.

Study design
This is a single center Phase-II efficacy study of MK-3475 therapy used in combination with renal pelvis infused BCG treatment for subjects, 18 years or older,  (Figure 1). Patients to undergo regular follow-up including applicable labs, in addition to post-treatment ureteroscopy with clinically indicated biopsy to assess recurrence. (Figure 2)

Trial schedule
Trial schedule (Figure 2) summarizes the trial procedures to be performed at each visit. It may be necessary to perform these procedures at unscheduled time points if deemed clinically necessary by the investigator. Furthermore, additional evaluations/testing may be deemed necessary by the sponsor for reasons related to subject safety. In some cases, such evaluation/testing may be potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional informed consent be obtained from the subject. In these cases, such evaluations/testing will be performed in accordance with those regulations.

Patient selection
Subjects, who meet required inclusion and exclusion criteria are eligible for enrollment into the study ( figure 3 and figure 4).   Treatment will be postponed until resolution of febrile illness, urinary tract infection, or gross hematuria and adverse effect resolution to grade <1. In the event of a grade 1 or 2 immune related adverse event requiring corticosteroid treatment, BCG treatment may be delayed for up to 2 weeks with the expectation that steroid treatment will be discontinued prior to the next BCG treatment cycle. A delay of longer than 2 weeks will permanently discontinue the patient from BCG treatment within the study or permanent withdrawal of the patient from the study. The choice will be at the discretion of the treating investigator. Steroid treatment for grade 3 or higher immune mediated events will require discontinuation of BCG treatment and/or withdrawal from the study.

Primary objective
To determine safety and efficacy of administering MK-3475 at a fixed dose of 200 mg every three-weeks in conjunction with intrapelvic BCG in high-risk superficial UUTUC patients, who are unfit or unwilling to be treated with radical nephroureterectomy.

Secondary objective
To determine the 19 week and the 3, 12, and 24 month post-treatment completion complete response rate and progression-free rate with combined MK-3475 and intrapelvic BCG therapy for non-muscle invasive high-risk UUTUC for patients unfit or unwilling to be treated with radical nephroureterectomy.

Exploratory objectives
Three objectives: to examine pre-and post-treatment (MK-3475/BCG) renal pelvis biopsy specimens for PD-1, PD-L1, and CD3; to examine whole exome sequencing of subjects' germline DNA and formalin fixed, paraffin embedded tissue.
With analysis of the correlation of mutations to treatment outcomes; analysis of blood and urine specimens from enrollees for potential development of blood and urine assays for determination of response to treatment.

Adverse effects
The investigator or qualified designee will assess each subject to evaluate for potential new or worsening AEs as specified in the detailed trial schedule and more frequently, if clinically indicated. Adverse experiences will be graded and recorded throughout the study and during the follow-up period according to NCI CTCAE Version 4.0. Toxicities will be characterized in terms regarding seriousness, causality, toxicity grading, and action taken with regard to trial treatment. AEs will be recorded from the

ETHICS, CONFIDENTIALITY AND REGULATORY DETAILS
The Investigator and Sponsor of the study will adhere to all applicable data privacy and confidentiality laws and regulations (i.e. HIPAA). Those applicable include institutional, state, and Federal Law. It is the responsibility of the Investigator and Sponsor to ensure the subject data as well as sensitive study information is handled according to applicable guidelines and laws. Appropriate authorization and consent for use, disclosure, or transfer of protected health information must be obtained.
Subject names will not be recorded on the CRF. Only the subject number and subject's initials will be recorded, where permitted. If the subject's name appears on any other document (e.g., pathology report), it must be obliterated on the copy of the document as appropriate. Study data stored on a computer will be stored in accordance with local data protection laws. The subjects will be informed that representatives of the sponsor or its designee, IRB, and regulatory authorities may inspect their medical records to verify the information collected, and that all personal information made available for inspection will be handled in strictest confidence and in accordance with local data protection laws.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
Your article may not currently address all the items on the checklist. Please modify your text to include the missing information. If you are certain that an item does not apply, please write "n/a" and provide a short explanation.
Upload your completed checklist as an extra file when you submit to a journal.
In your methods section, say that you used the SPIRIT reporting guidelines, and cite them as: Chan Biological specimens #33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in the current trial and for future use in ancillary studies, if applicable 12,21 Author notes 1. n/a in original protocol which has been submitted with the manuscript 2. n/a in original protocol which has been submitted with the manuscript 3. 10,11, as well as original study protocol 4. 20, extensive details within original study protocol 5. n/a, detailed within original study protocol 6. 11,12,19,20 7. n/a, this is a non-randomized, un-blinded clinical trial, both researchers and patients are aware of involvement and intervention within the study  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 F o r p e e r r e v i e w o n l y 8. n/a, this is a non-randomized, un-blinded clinical trial, both researchers and patients are aware of involvement and intervention within the study 9. n/a, this is a non-randomized, un-blinded clinical trial, both researchers and patients are aware of involvement and intervention within the study 10. n/a, this is a non-randomized, un-blinded clinical trial, both researchers and patients are aware of involvement and intervention within the study 11. n/a, this is a non-randomized, un-blinded clinical trial, both researchers and patients are aware of involvement and intervention within the study 12. 15,16, extensive detailing within the original study protocol.
13. 12,13, extensive detailing of management of adverse events within the original study protocol.
14. n/a, within original study protocol.
15. 21, specific mention of use of data for current studies and subsequent studies within original consent form.