Pembrolizumab and aMVAC Chemotherapy as Neoadjuvant Therapy in Non-Urothelial Histology Muscle-Invasive Bladder Cancer: A Pilot Trial
Phase: PHASE2
Conditions: Stage II Bladder Cancer AJCC v8, Stage IIIA Bladder Cancer AJCC v8, Muscle Invasive Bladder Carcinoma
Sponsor: University of Washington

Neoadjuvant aMVAC+Pembrolizumab – non-UC MIBC Protocol 
Consortium Study No.: RG1006206
Version 8: 20-DEC-2023
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SPONSOR: Petros Grivas 
TITLE: Pembrolizumab and aMVAC chemotherapy as neoadjuvant therapy in non-
urothelial histology muscle-invasive bladder cancer: a pilot trial
INSTITUTIONAL PROTOCOL NUMBER: RG1006206
IND NUMBER:
Principal Investigator: Petros Grivas, MD, PhD
Sub-Investigators: 
Heather Cheng, MD, PhD
John Gore, MD
Andrew Hsieh, MD, PhD
Meredith Hullar, PhD
Ali Raza Khaki, MD
Hung-Ming Lam, PhD
John Lee, MD, PhD
Daniel Lin, MD
Zhengwei Mao, MD, MT, ASCP, NCA
Robert Bruce Montgomery, MD
Sarah Psutka, MD
Mary Redman, PhD
George Schade, MD
Michael Schweizer, MD
Funda Vakar-Lopez, MD
Jonathan Wright, MD
Todd Yezefski, MD
Evan Yu, MD
Fred Hutch IRB
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Version 8: 20-DEC-2023
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TABLE OF CONTENTS
1.0
TRIAL SUMMARY ....................................................................................................8
2.0
BACKGROUND & RATIONALE ............................................................................8
2.1
Background ..............................................................................................................8
2.1.1
Pharmaceutical and Therapeutic Background .......................................................8
2.1.2
Preclinical and Clinical Trial Data.........................................................................9
2.2
Rationale...................................................................................................................9
2.2.1
Rationale for the Trial and Selected Population ....................................................9
2.2.2
Justification for Dose...........................................................................................12
2.2.3
Rationale for Endpoints .......................................................................................13
3.0
TRIAL DESIGN ........................................................................................................14
3.1
Trial Design............................................................................................................14
3.2
Trial Diagram ........................................................................................................15
4.0
OBJECTIVE(S) & HYPOTHESIS(ES) ..................................................................16
4.1
Primary Objective(s) & Hypothesis(es)...............................................................16
4.2
Secondary Objective(s)..........................................................................................16
4.3
Exploratory Objective...........................................................................................16
5.0
METHODOLOGY ....................................................................................................17
5.1
Study Population....................................................................................................17
5.1.1
Participant Inclusion Criteria...............................................................................17
5.1.2
Participant Exclusion Criteria..............................................................................19
5.1.3
Lifestyle Restrictions...........................................................................................21
5.1.4
Pregnancy.............................................................................................................21
5.1.5
Use in Nursing Women........................................................................................21
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5.2
Enrollment..............................................................................................................22
5.3
Trial Treatments....................................................................................................22
4.3.1 Timing of Dose Administration..............................................................................22
5.3.2
Dose modification and toxicity management for immune-related AEs associated 
with pembrolizumab ............................................................................................23
5.3.3
Dose Modifications for aMVAC chemotherapy (recommendations)..................28
5.4
Concomitant Medications/Vaccinations (allowed & prohibited)......................29
5.4.1
Acceptable Concomitant Medications.................................................................30
5.4.2
Prohibited Concomitant Medications ..................................................................30
5.4.3
Rescue Medications & Supportive Care..............................................................31
5.5
Participant Withdrawal/Discontinuation Criteria .............................................31
5.6
Participant Replacement Strategy .......................................................................32
5.7
Clinical Criteria for Early Trial Termination ....................................................32
6.0
TRIAL FLOW CHART............................................................................................33
6.1
Study Flow Chart...................................................................................................33
7.0
TRIAL PROCEDURES ............................................................................................36
7.1
Trial Procedures ....................................................................................................36
7.1.1
Administrative Procedures...................................................................................36
7.1.2
Clinical Procedures/Assessments ........................................................................38
7.1.3
Laboratory Procedures/Assessments ...................................................................45
7.1.4
Other Procedures..................................................................................................47
7.1.5
Visit Requirements ..............................................................................................47
7.2
Assessing and Recording Adverse Events ...........................................................48
7.2.1
Definition of an Overdose for This Protocol and Reporting of Overdose to Merck
.............................................................................................................................48
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7.2.2
Reporting of Pregnancy and Lactation to Merck.................................................48
7.2.3
Adverse Event......................................................................................................49
7.2.4
Serious Adverse Events .......................................................................................49
7.2.5
Unexpected Adverse Event..................................................................................50
7.2.6
Monitoring and Recording Adverse Events.........................................................50
7.2.7
Grading Adverse Event Severity .........................................................................51
7.2.8
Attribution of an Adverse Event..........................................................................51
7.2.9
Adverse Event Recording Period.........................................................................51
7.2.10 Adverse Event Reporting Requirements..............................................................52
7.2.11 FDA Reporting Requirements .............................................................................52
8.0
STATISTICAL CONSIDERATIONS.....................................................................56
9.0
DATA AND SAFETY MONITORING PLAN .......................................................56
9.1
Safety of pembrolizumab and chemotherapy .....................................................56
9.2
Monitoring Plan.....................................................................................................57
10.0
LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL 
SUPPLIES ..................................................................................................................58
10.1
Investigational Product .........................................................................................58
10.2
Packaging and Labeling Information..................................................................58
10.3
Clinical Supplies Disclosure..................................................................................58
10.4
Storage and Handling Requirements...................................................................58
10.5
Returns and Reconciliation...................................................................................59
10.6
Methotrexate ..........................................................................................................59
10.6.1 Other Names ........................................................................................................59
10.6.2 Classification .......................................................................................................59
10.6.3 Mode of Action....................................................................................................59
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10.6.4 Description...........................................................................................................59
10.6.5 Preparation and Administration...........................................................................59
10.6.6 Storage and Stability............................................................................................59
10.6.7 Drug Interactions .................................................................................................59
10.6.8 Agent Availability................................................................................................60
10.6.9 Agent Ordering ....................................................................................................60
10.6.10
Agent Accountability....................................................................................60
10.6.11
Side Effects...................................................................................................60
10.7
Vinblastine..............................................................................................................60
10.7.1 Other Names ........................................................................................................60
10.7.2 Classification .......................................................................................................60
10.7.3 Mode of Action....................................................................................................60
10.7.4 Description...........................................................................................................60
10.7.5 Preparation and Administration...........................................................................60
10.7.6 Storage and Stability............................................................................................60
10.7.7 Drug Interactions .................................................................................................61
10.7.8 Agent Availability................................................................................................61
10.7.9 Agent Ordering ....................................................................................................61
10.7.10
Agent Accountability....................................................................................61
10.7.11
Side Effects...................................................................................................61
10.8
Doxorubicin............................................................................................................61
10.8.1 Classification .......................................................................................................61
10.8.2 Mode of Action....................................................................................................61
10.8.3 Description...........................................................................................................61
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10.8.4 Preparation and Administration...........................................................................61
10.8.5 Storage and Stability............................................................................................61
10.8.6 Drug Interactions .................................................................................................62
10.8.7 Agent Availability................................................................................................62
10.8.8 Agent Ordering ....................................................................................................62
10.8.9 Agent Accountability...........................................................................................62
10.8.10
Side Effects...................................................................................................62
10.9
Cisplatin..................................................................................................................62
10.9.1 Other Names ........................................................................................................62
10.9.2 Classification .......................................................................................................62
10.9.3 Mode of Action....................................................................................................62
10.9.4 Description...........................................................................................................62
10.9.5 Preparation and Administration...........................................................................62
10.9.6 Storage and Stability............................................................................................62
10.9.7 Drug Interactions .................................................................................................63
10.9.8 Agent Availability................................................................................................63
10.9.9 Agent Ordering ....................................................................................................63
10.9.10
Agent Accountability....................................................................................63
10.9.11
Side Effects...................................................................................................63
11.0
INVESTIGATOR OBLIGATIONS.........................................................................63
12.0
ADMINISTRATIVE AND REGULATORY DETAILS .......................................63
12.1
Pre-Study Documentation.....................................................................................63
12.2
Study Site Training................................................................................................63
12.3
Documentation.......................................................................................................64
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12.4
Access to Source Data............................................................................................64
12.5
Data Collection.......................................................................................................64
12.6
Protocol Interpretation and Compliance ............................................................64
12.7
Study Monitoring and Data Collection................................................................65
12.8
Disclosure of Data/Publication .............................................................................65
12.9
Ethical Considerations ..........................................................................................65
12.10 Informed Consent..................................................................................................66
12.11 Institutional Review Board/Ethics Committee ...................................................66
12.12 Subject Privacy ......................................................................................................67
13.0
STOPPING THE STUDY.........................................................................................67
14.0
REFERENCES ..........................................................................................................68
15.0
APPENDICES............................................................................................................73
Appendix 1: ECOG Performance Status.........................................................................73
Appendix 2: Contraceptive Guidance and Pregnancy Testing .....................................74
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1.0  TRIAL SUMMARY
Abbreviated Title
Neoadjuvant aMVAC and Pembro for non-UC MIBC
Trial Phase
Pilot trial
Clinical Indication
Neoadjuvant chemoimmunotherapy
Trial Type
Pilot trial
Type of control
None
Route of administration
IV
Trial Blinding
None
Treatment Groups 
Single-arm of patients with new diagnosis of non-UC MIBC
Number of trial participants
14-17
Estimated enrollment period
17-26 months
Estimated duration of trial
2-3 years
Duration of Participation
6 weeks of neoadjuvant treatment (up to 2 years follow-up)
Estimated average length of 
treatment per patient
6 weeks
2.0 BACKGROUND & RATIONALE
2.1
Background
Pembrolizumab is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) 
with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting 
its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 
(PD-L2). Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor 
blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety profile and is 
approved as an intravenous (IV) immunotherapy for advanced malignancies, including two 
indications in advanced urothelial cancer. It is FDA-approved in combination with platinum-
based chemotherapy in metastatic non-small cell lung cancer (NSCLC), while it has been 
evaluated in combination with cisplatin-based chemotherapy in patients with localized muscle 
invasive bladder cancer (MIBC).1,2 Moreover, the combination of pembrolizumab with cisplatin-
based chemotherapy is being investigated in advanced urothelial cancer (Keynote 361 trial that 
has completed accrual). For more details on specific indications refer to the Investigator 
Brochure (IB).
2.1.1
Pharmaceutical and Therapeutic Background
The importance of intact immune surveillance function in controlling outgrowth of neoplastic 
transformations has been known for decades [Disis, 2010].3  Accumulating evidence shows a 
correlation between tumor-infiltrating lymphocytes in cancer tissue and favorable prognosis in 
various malignancies. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector 
T-cells/FoxP3+ regulatory T-cells (T-regs) correlates with improved prognosis and long-term 
survival in solid malignancies, such as ovarian, colorectal, pancreatic, hepatocellular, renal cell, 
urothelial carcinoma, and melanoma. Tumor-infiltrating lymphocytes can be expanded ex vivo 
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and reinfused, inducing durable objective tumor responses in cancers, such as melanoma [Dudley 
et al., 2005; Hunder et al., 2008].4,5 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune 
control. The normal function of PD-1, expressed on the cell surface of activated T-cells under 
healthy conditions, is to down-modulate unwanted or excessive immune responses, including 
autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an immunoglobulin (Ig) 
superfamily member related to cluster of differentiation 28 (CD28) and cytotoxic T-lymphocyte-
associated protein 4 (CTLA-4) that has been shown to negatively regulate antigen receptor 
signaling upon engagement of its ligands (PD-L1 and/or PD-L2) [Greenwald et al., 2005; 
Okazaki et al., 2001].6,7 
The structure of murine PD-1 has been resolved [Zhang et al., 2004].8 PD-1 and its family 
members are type I transmembrane glycoproteins containing an Ig-variable–type (IgV-type) 
domain responsible for ligand binding and a cytoplasmic tail responsible for the binding of 
signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs, 
an immunoreceptor tyrosine-based inhibition motif, and an immunoreceptor tyrosine-based 
switch motif. Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases, SHP-1 and 
SHP-2, to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to 
the de-phosphorylation of effector molecules, such as CD3 zeta (CD3ζ), protein kinase C-theta 
(PKCθ), and zeta-chain-associated protein kinase (ZAP70), which are involved in the CD3 T-cell 
signaling cascade [Okazaki et al., 2001; Chemnitz et al., 2004; Sheppard et al., 2004; and Riley, 
2009].7,9–11  The mechanism by which PD-1 down-modulates T-cell responses is similar to, but 
distinct from, that of CTLA-4, because both molecules regulate an overlapping set of signaling 
proteins [Parry et al., 2005; Francisco, 2010].12,13 Consequently, the PD-1/PD-L1 pathway is a 
very attractive target for therapeutic intervention, including as neoadjuvant treatment of MIBC, a 
malignancy with remarkable responses to (and benefit from) immunotherapeutic strategies. 
2.1.2
Preclinical and Clinical Trial Data
Refer to the Investigator Brochure for several preclinical and clinical datasets with pembrolizumab 
across various malignancies, including MIBC and advanced urothelial carcinoma.
2.2
Rationale
2.2.1
Rationale for the Trial and Selected Population
2.2.1.1
Bladder Cancer – Disease Overview
Bladder cancer (BC) is one of the most common malignancies in the United States (US) with an 
estimated 80,470 new cases and 17,670 deaths in 2019 (https://www.cancer.org/cancer/bladder-
cancer/about/key-statistics.html).14 It is the 4th most common cancer in men and there are >500,000 
BC patients alive in the US alone; it accounts for about 5% of all new cancers in the US. Almost 
a quarter of BC patients present with MIBC; among the ~65% of BC patients presenting with non-
muscle-invasive bladder cancer (NMIBC), up to 15-20% may progress to MIBC every year.15 
Radical cystectomy (RC) is the standard of care for MIBC, however it results in a 5-year 
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recurrence-free survival rate of 68% (only 35% for those with lymph node involvement). This is 
most probably due to micro-metastases present at the time of surgery considering the high rate of 
distant recurrence.16,17 Systemic cisplatin-based combination chemotherapy prior to RC results in 
high pathologic complete response (pCR) rates (30-35%) and clinically meaningful increase in 
overall survival (OS) compared to local therapy alone, and has become the standard of care for 
cisplatin-eligible patients with MIBC.18-20 
2.2.1.2
Variant Histology Bladder Cancer Background
Notably, most MIBC cases consist of urothelial carcinoma (UC), while the presence of squamous 
cell or glandular features admixed with a predominant UC histology pattern does not appear to 
compromise pCR rates with neoadjuvant cisplatin-based combination chemotherapy.21 However, 
the data is not that clear with other histologic variants, and also when non-UC variants represent 
the predominant histology of MIBC. Non-UC BC variants account for 5-10% of all BC cases and 
are comprised of a variety of histologic types, including squamous cell carcinoma, 
adenocarcinoma, small cell carcinoma, sarcomatoid, micropapillary, plasmacytoid, nested, among 
others.22,23 These non-UC variants may not respond as well to cisplatin-based chemotherapy alone 
and represent a major unmet clinical need.
2.2.1.3
Rationale for Immunotherapy in MIBC
The role of immunotherapy in BC is well established. Intra-vesical administration of Bacillus 
Calmette-Guerin (BCG), a bovine mycoplasma-derived agent that lowers bladder tumor 
recurrence by inciting a robust immunologic reaction in the bladder microenvironment, is standard 
of care for high grade NMIBC. The evident clinical benefit with intravesical BCG immunotherapy 
is accompanied by the established benefit from systemic immune checkpoint inhibitors, like 
pembrolizumab in advanced UC.24 The presence of CD8+ tumor-infiltrating lymphocytes (TILs) 
has been associated with longer disease-free and overall survival in BC.25,26 Aberrant expression 
of the immune checkpoint programmed death-ligand 1 (PD-L1) has been shown in UC, implying 
that tumor-associated PD-L1 can play a regulatory role in anti-tumor immunity and be a relevant 
therapeutic target.27 PD-L1 expression was associated with high tumor grade, tumor infiltration by 
mononuclear cells, stage progression and attenuated response to BCG immunotherapy by 
neutralizing T cells.28 Less is known about the impact of immunotherapy in non-UC MIBC.
2.2.1.4
Rationale for pembrolizumab plus chemotherapy in MIBC
Breakthrough clinical data have been noted with agents targeting cytotoxic T-programmed death-1 
(PD-1) and PD-L1. PD-1 expression in TILs has been observed in almost all MIBC patients 
undergoing cystectomy with nearly all TILs expressing CD8; 76% of patients with PD-L1-positive 
tumors had moderate or marked TILs PD-1 expression. PD-L1 expression in bladder tumors and 
TILs PD-1 expression were significantly associated with advanced pathologic stage, and PD-L1 
expression independently predicted all-cause mortality after cystectomy. In a phase I expanded 
cohort of post-platinum advanced UC, anti-PD-L1 single agent therapy induced very high response 
rate in tumors with high PD-L1 immunostaining, including rapid and durable responses with an 
excellent safety profile.29,30 A phase II trial with single agent anti-PDL1 confirmed durable activity 
and good tolerability in this patient population; the level of PD-L1 expression on immune cells, 
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The Cancer Genome Atlas (TCGA) subtype and mutational load correlated with response.31 
Significant anti-tumor activity and favorable tolerability was also noted with two anti-PD1 agents, 
pembrolizumab and nivolumab, and two other anti-PD-L1 agents, durvalumab and avelumab.32–36 
Pembrolizumab, atezolizumab, nivolumab, durvalumab and avelumab are FDA-approved for 
platinum-resistant advanced UC. Pembrolizumab demonstrated a significant OS benefit and better 
tolerability over salvage chemotherapy in a large phase III trial of platinum-resistant advanced UC 
(with level I evidence in this setting).34 Pembrolizumab and atezolizumab are also FDA-approved 
as first line therapy in patients with advanced UC with PD-L1 positive tumors (based on 
companion diagnostic assay) or patients who cannot tolerate any platinum (cisplatin or 
carboplatin) based on notable established activity in large phase II trials.37,38 Two large phase III 
randomized trials (Keynote361 and IMvigor130) in the first line treatment setting of advanced UC 
are investigating the role of the combination of chemotherapy plus anti-PD-1/PD-L1 agents; both 
trials have completed accrual and results are anticipated soon. As mentioned before, the 
combination of platinum-based chemotherapy plus pembrolizumab has been FDA-approved in 
metastatic NSCLC, further supporting the safety and feasibility for the combination. 
Moreover, recent data showed significant efficacy of anti-PD-1/PD-L1 agents as neoadjuvant 
immunotherapy in MIBC. The single arm phase II PURE-01 trial enrolled patients with T2-
3bN0M0 MIBC regardless of cisplatin eligibility and met its primary endpoint (based on pCR 
rate).39 Three doses of pembrolizumab led to pCR (pT0 in intention-to-treat population) in 42% of 
patients, while most patients with pCR had PD-L1 combined positive score ≥ 10. These results are 
very encouraging as the pCR with single-agent pembrolizumab appears comparable to 38% pCR 
with neoadjuvant conventional-dose MVAC, though it warrants investigation in larger randomized 
trials. Furthermore, in the single-arm phase II ABACUS trial, two doses of neoadjuvant 
atezolizumab led to pCR 29% in cisplatin-unfit patients with T2-4N0M0 MIBC.40 pCR included 
pT0 (24%) and pTcis (6%); primary endpoint was pCR in ≥20% patients. PD-L1 positive status 
(≥5% in TILs based on SP142 assay) was noted in almost half of the patients and pCR rates were 
38% in PD-L1-positive and 27% in PD-L1-negative tumors. Both studies confirmed the safety of 
neoadjuvant immunotherapy. Data from the NABUCCO trial further supports the safety of 
neoadjuvant immunotherapy. In this trial, neoadjuvant combination immune checkpoint inhibitor 
blockade with nivolumab and anti-CTLA-4 ipilimumab was shown to be safe among the first 24 
patients enrolled with 23 of 24 proceeding to cystectomy on schedule. NCT02365766 trial is a 
phase Ib/II study, currently evaluating the combination of neoadjuvant chemotherapy plus 
pembrolizumab in cisplatin-eligible (cohort I) and ineligible (cohort II) patients with T2-4aN0M0 
MIBC.41 In cohort I, therapy consisted of pembrolizumab plus cisplatin-based chemotherapy, 
while in cohort II of pembrolizumab plus gemcitabine. Available data from 40 evaluable patients 
from cohort I demonstrated feasibility and safety of the combination, with 60% (95%CI: 42-74%) 
pathologic non-muscle invasive rate, which did not correlate with PD-L1 expression score. Similar 
studies with immune checkpoint inhibitors combined with chemotherapy have to be performed in 
non-UC bladder cancer; this approach represents a major critical unmet need with potential 
registration pathway in an “orphan disease”.
2.2.2
Justification for Dose 
The planned dose of pembrolizumab for this study is 200 mg every 3 weeks (Q3W).  Based on 
the totality of data generated in the pembrolizumab development program, 200 mg Q3W is the 
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appropriate dose of pembrolizumab for adults across all indications and regardless of tumor type.  
As outlined below, this dose is justified by:

Clinical data from 8 randomized studies in melanoma and NSCLC indications 
demonstrating flat dose- and exposure-efficacy relationships from 2 mg/kg Q3W to 10 
mg/kg every 2 weeks (Q2W), representing an approximate 5- to 7.5-fold exposure range 
(refer to IB, Section 5.2.2)

Population PK analysis showing that both fixed dosing and weight-based dosing provides 
similar control of PK variability with considerable overlap in the distributions of exposures, 
supporting suitability of 200 mg Q3W

Clinical data showing meaningful improvement in benefit-risk including OS at 200 mg 
Q3W across multiple indications, and

Pharmacology data showing full target saturation in both systemic circulation (inferred 
from pharmacokinetic [PK] data) and tumor (inferred from physiologically-based PK 
[PBPK] analysis) at 200 mg Q3W
2.2.3
Rationale for Endpoints
2.2.3.1
Efficacy Endpoints
Pathologic complete response (pCR) has been shown in a meta-analysis to be associated 
with improved recurrence-free survival (RFS) and OS and can be evaluated in a timely 
fashion at the time of standard of care RC, which is very common in neoadjuvant trials.42
2.2.3.2
Biomarker Research
Density of CD8+ TILs has been found to correlate with OS in UC.25,26 An older study 
assessed the prognostic value of TILs in a cohort of 514 patients with UC over a period of 
9 years. In advanced stage tumors (T3-T4) increased TIL density was related to less 
aggressive behavior, whereas in a multivariate analysis, dense TILs were a highly 
significant factor of favorable prognosis and allowed to separate UC into prognostic groups 
based on TIL density. A study by Sharma et al. in 2007 included 69 patients with UC who 
underwent cystectomy. Patients were separated into those with high vs low density of TILs 
in cystectomy specimens. Patients with higher pathologic stage (pT2-4) who had increased 
TILs density had longer disease-free survival and overall survival, compared to patients 
with the same stage who had lower TIL density.26 A study by Faraj et al., evaluated 56 
tissue microarrays from cystectomy specimens from a single institution.43 Patient samples 
were divided into high and low CD8+ T cell density categories. High CD8+ T cell density 
was associated with longer overall and disease-specific survival even when adjusted for 
demographic and clinicopathologic parameters. Another study showed that CD8+ to Treg 
TIL density in the pre-treatment tissues predicted response to neoadjuvant cisplatin-based 
chemotherapy.44 Taken together, data suggests that CD8+ TIL density is associated with 
better outcomes in MIBC and should be further evaluated as a surrogate biomarker of 
clinical benefit. There is a plethora of additional putative tumor tissue and blood based 
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biomarkers that have shown correlation with response to immunotherapy and/or 
chemotherapy in several studies.24 Such biomarkers need further investigation in 
neoadjuvant MIBC clinical trials in order to facilitate biomarker discovery and validation.
2.2.3.3
Exploratory Microbiome Research
Not all patients experience favorable response to immunotherapy or chemotherapy and 
many may not respond and/or experience adverse events.45 Understanding how exactly 
patient and tumor characteristics impact immunotherapy response is limited, but existing 
data suggests that the gut microbiota appear intimately involved with immune regulation 
and response to immunotherapy.46 A primary role for the gut microbiota is protecting the 
host from invading pathogens and participating in immune surveillance. The gut 
microbiota are involved in T cell expansion and activation, and are further responsible for 
induction by antigen exposure of Foxp3+ Treg cells, important in maintaining immune 
tolerance. Specific to the gut, CD103+CD11b+ dendritic cells (DCs) produce TGF-β, 
which induces Treg expansion, which are then absorbed into systemic circulation.47 
Similarly, Tregs can be induced by Bacteroides fragilis and several Clostridia species. 
Mice that have a microbiome lacking Bifidobacterium have reduced intra-tumoral DCs and 
poor responses to anti-PD1 therapy, and germ-free or antibiotic-treated mice fail to mount 
an appropriate immune response to tumor development.48 A number of investigations in 
humans have assessed the role of specific microbiota in response to immunotherapy. In a 
study by Routy et al., patients with renal cell carcinoma or NSCLC receiving antibiotic 
treatment within two months of anti-PD-1 therapy had reduced efficacy.49 Several bacterial 
species were associated with improved response in this study, including Akkermansia 
muciniphila, Alistipes indistinctus, and Enterococcus hirae (combined with Akkermansia), 
and patients with memory T cell response targeting these bacteria have been shown to have 
longer progression-free survival. Other human studies have identified Bifidobacteria spp, 
Collinsella aerofaciens, and Enterococcus faecium to be associated with improved 
response.50 On the contrary, several species were negatively associated with response, 
including Blautia obeum and Roseburia intestinalis. Translocation of bacterial products 
can contribute to stimulation of immune response and to systemic inflammatory 
environment, while anti-PD-1 agents have been shown to promote loss of gut barrier 
integrity thus facilitating this translocation.51 These findings raise interesting questions 
about the contribution of the gut microbiota to the delicate balance between immune 
activation and suppression, regarding clinical response to therapy. In fact, in addition to an 
initial effect of the microbiome on immune surveillance, Mao et al. reported that the 
microbial community could itself be influenced by the innate and adaptive immune 
responses, thus potentially modifying both the composition and function of the bacterial 
community members.52 This evidence provides support for prospective collection of stool 
samples before and after initiation of therapy for a more comprehensive assessment of the 
role of microbial dynamics on treatment response. We will use the 16S rRNA gene analysis 
to target changes in diversity and specific bacteria as well as the total microbiome in 
efficacy of this treatment.
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3.0 TRIAL DESIGN
3.1
Trial Design
Single center, single arm, unblinded study of neoadjuvant accelerated MVAC plus 
pembrolizumab for patients with muscle invasive bladder cancer with pure or predominant 
non-urothelial histology. Primary endpoint is pathologic complete response. Diagram below.
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Radical 
Cystectomy
Maximal 
TURBT
Endpoints

Pathologic 
complete response 
rate

Toxicity

Tumor infiltrating 
lymphocyte 
density

Two-year 
recurrence-free 
survival

Translational 
studies with tissue, 
blood, urine, stool
aMVAC x 4
weeks 0, 2, 4, 6
Key Eligibility

Cisplatin-eligible 

Muscle Invasive 
Bladder Cancer 
(Clinical T2-T4a, 
N0-N1, M0)

Candidates for 
radical cystectomy

Predominant or 
pure non-urothelial 
carcinoma 
histology (pure 
small cell 
excluded)

No active 
autoimmune 
disease
Pembrolizumab x 3 
weeks 0, 3, 6
3.2
Trial Diagram

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4.0 OBJECTIVE(S) & HYPOTHESIS(ES)
4.1
Primary Objective(s) & Hypothesis(es)
(1) Objective: To evaluate the antitumor efficacy of neoadjuvant aMVAC and pembrolizumab as 
measured by the rate of patients achieving pCR (defined as pT0N0) at radical cystectomy (RC) 
in pure or predominant non-UC histologic variants. 
Hypothesis: We hypothesize that neoadjuvant aMVAC and pembrolizumab will lead to a high 
rate of pCR comparable to data in urothelial cancer.
4.2
Secondary Objective(s)
(1) Objective: To assess the frequency and severity of toxicity according to Common 
Terminology Criteria for Adverse Events version 5.0 (CTCAE v5.0) in patients treated with 
aMVAC and pembrolizumab
(2) Objective: To evaluate the feasibility of neoadjuvant aMVAC and pembrolizumab as 
measured by the rate of patients able to receive RC within 10 weeks from completion of study 
therapy.
(3) Objective: To assess the recurrence-free survival (RFS), defined as time from trial enrollment 
to recurrence or death at the two-year time point in patients treated with aMVAC and 
pembrolizumab
(4) Objective: To assess the absolute and percentage (%) change in CD8+ TIL density at RC 
compared to pre-treatment TURBT
4.3
Exploratory Objective
(1)
Objective: To describe relationship between pre- and post- treatment TILs, prior BCG 
exposure, pathologic partial and complete response (defined by pathologic staging <pT2N0 
and pT0N0, respectively) as well as two-year RFS.
(2)
Objective: To describe relationship between PD-L1 by IHC, tumor mutational burden, 
mutation signatures, neo-epitope burden, intrinsic molecular subtypes (basal vs. luminal), 
homologous recombination deficiency, loss of heterozygosity, DNA damage response gene 
alterations, pathologic partial and complete response as well as two-year RFS.
(3)
Objective: To describe relationship between tumor-associated macrophages, myeloid 
derived suppressor cell (MDSC) subsets, combined “immune-score” of distribution and 
density of infiltrative CD3+/CD8+ cells, pathologic partial and complete response as well as 
two-year RFS.
(4)
Objective: To describe relationship between pre- and post-treatment peripheral blood T-cell 
subsets, CD4+/CD8+, CD4+/FOXP3+ ratios, monocytes and plasma cytokine multiplex 
panels, pathologic partial and complete response as well as two-year RFS.
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(5)
Objective: To describe relationship between myeloid gene signature, methylome 
sequencing, T cell clonality & diversity in tissue & blood, pathologic partial and complete 
response as well as two-year RFS.
(6)
Objective: To describe relationship between pre-treatment microbiome and microbiome 
change, pathologic partial and complete response as well as two-year RFS. 
5.0 METHODOLOGY
5.1
Study Population
5.1.1
Participant Inclusion Criteria
Participants are eligible to be included in the study only if all the following criteria apply:
1. Participants must be at least 18 years of age on the day of signing informed consent 
2. Participants must have histologically confirmed diagnosis of muscle invasive bladder 
cancer (cT2-T4a, N0-N1, M0 clinical stage per American Joint Commission on Cancer 
[AJCC]). Clinical node-positive (N1) patients are eligible provided the lymph nodes (LNs) 
are confined to the true pelvis and are within the planned surgical LN dissection template.
3. Histology must be either pure or predominant non-urothelial histology (noted on any 
TURBT)
4. Participants must be deemed eligible for cisplatin-based chemotherapy, radical cystectomy 
(RC) and pelvic lymph node dissection (PLND) by urologist and medical oncologist. 
5. Patients must agree to undergo curative intent surgery. 
6. TURBT that showed muscularis propria invasion should be within 12 weeks prior to 
beginning study therapy. Patients must have available tumor tissue from either initial or 
repeat TURBT, prior to starting study therapy. Archival and/or fresh tumor tissue sample 
of a tumor lesion (TURBT specimen) should be provided and must contain muscle invasive 
component, at least ≥T2 tumor. Formalin-fixed, paraffin embedded (FFPE) tissue blocks 
are preferred to slides. If submitting unstained cut slides, newly cut slides should be 
submitted to the testing laboratory, preferably within 14 days from the date slides are cut 
if possible. Patient must be willing to provide tumor tissue for research. Research samples 
will not be used for any studies unrelated to this trial.
7. Must have clinical non-metastatic bladder cancer (M0) determined by cross-sectional CT 
CAP or MRI imaging
8. A male participant must agree to use a contraception as detailed in Appendix 2 of this 
protocol during the treatment period and for at least 180 days after the last dose of study 
treatment and refrain from donating sperm during this period.
9. A female participant is eligible to participate if she is not pregnant (see Appendix 2), not 
breastfeeding, and at least one of the following conditions applies: 
a.) Not a woman of childbearing potential (WOCBP) as defined in Appendix 2 
OR 
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b.) A WOCBP who agrees to follow the contraceptive guidance in Appendix 2 during the 
treatment period and for at least 180 days after the last dose of study treatment.
10. The participant (or legally acceptable representative if applicable) provides written 
informed consent for the trial.  
11. Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. 
Evaluation is to be performed within 7 days prior to the date of enrolment.  
12. Have adequate organ function as defined in the following table (Table 1).  Specimens must 
be collected within 10 days prior to the start of study treatment.
Table 1. Adequate Organ Function Laboratory Values
System
Laboratory Value
Hematological
Absolute neutrophil count (ANC)
≥1500/µL
Platelets
≥100 000/µL
Hemoglobin
≥9.0 g/dL or ≥5.6 mmol/La
Renal
Serum Creatinine
Measured or calculatedb creatinine clearance
(GFR can be used in place of creatinine 
clearance; 24-hour urine collection can be used 
for more accurate estimate as needed)
≤1.5x ULN OR calculated creatinine clearance 
(GFR can be used in place of creatinine or 
creatinine clearance) ≥50ml/min
Hepatic
Total bilirubin
≤1.5 ×ULN OR direct bilirubin ≤ULN for 
participants 
with 
total 
bilirubin 
levels 
>1.5 × ULN
AST (SGOT) and ALT (SGPT)
≤2.5 × ULN 
Coagulation
International normalized ratio (INR) OR 
prothrombin time (PT)
Activated partial thromboplastin time (aPTT)
≤1.5 × ULN unless participant is receiving 
anticoagulant therapy, as long as PT or aPTT is 
within therapeutic range of intended use of 
anticoagulants
ALT (SGPT)=alanine aminotransferase (serum glutamic pyruvic transaminase); AST (SGOT)=aspartate 
aminotransferase (serum glutamic oxaloacetic transaminase); GFR=glomerular filtration rate; ULN=upper 
limit of normal.
a Criteria must be met without erythropoietin dependency and without packed red blood cell (pRBC) 
transfusion within last 2 weeks. 
b Creatinine clearance (CrCl) should be calculated per institutional standard.
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5.1.2
Participant Exclusion Criteria
Participants are excluded from the study if any of the following criteria apply:
1. A WOCBP who has a positive urine pregnancy test within 72 hours prior to enrolment 
(see Appendix 2).  If the urine test is positive or cannot be confirmed as negative, a serum 
pregnancy test will be required.
2. Patients with pure small cell histology will be excluded. Mixed histology including 
partial neuroendocrine small cell features will be permitted.
3. Patients considered to be medically unfit for accelerated (dose dense) MVAC 
chemotherapy, TURBT or RC (per Investigator discretion) will be excluded.
4. Has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or with an 
agent directed to another stimulatory or co-inhibitory T-cell receptor (e.g., CTLA-4, 
OX-40, CD137).
5. Has received prior systemic anti-cancer therapy including investigational agents within 
4 weeks. Intravesical therapies are allowed without specified treatment interval. 
Note: Participants must have recovered from all AEs due to previous systemic therapies to 
≤Grade 1 or baseline. If participant had major surgery, they must have recovered 
adequately from the toxicity and/or complications from the intervention prior to starting 
study treatment.
6. Has received prior radiotherapy within 2 weeks of start of study treatment. Participants 
must have recovered from all radiation-related toxicities, not require corticosteroids, and 
should not have active radiation pneumonitis.
7. Has received a live vaccine within 30 days prior to the first dose of study drug. Examples 
of live vaccines include, but are not limited to, the following: measles, mumps, rubella, a 
version of varicella/zoster (chicken pox), yellow fever, rabies, Bacillus Calmette–Guérin 
(BCG), and typhoid vaccine. Seasonal influenza vaccines for injection are generally 
killed virus vaccines and are allowed; however, intranasal influenza vaccines 
(eg, FluMist®) are live attenuated vaccines and are not allowed.
8. Is currently participating in or has participated in a study of an investigational agent or 
has used an investigational device within 4 weeks prior to the first dose of study 
treatment.
Note: This table includes eligibility-defining laboratory value requirements for treatment; laboratory value 
requirements should be adapted according to local regulations and guidelines for the administration of 
specific chemotherapies.
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Note:  Participants who have entered the follow-up phase of an investigational study may 
participate as long as it has been 4 weeks after the last dose of the previous 
investigational agent.
9. Has diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy (in 
dosing > 10 mg daily of prednisone dose equivalent) or any other form of 
immunosuppressive therapy within 7 days prior to the first dose of study drug.
10. Has known additional malignancy that is progressing or has required active systemic 
treatment within the past 2 years. Note:  Participants with basal cell carcinoma or squamous 
cell carcinoma of the skin, or any carcinoma in situ that have undergone potentially curative 
therapy are not excluded. Low/intermediate risk prostate cancer with prior potentially 
curative therapy, or no intent of future systemic therapy and/or radiation is allowed. Non-
invasive (Tis, Ta) upper urinary tract (renal pelvis/ureter) is allowed. Urethra cancer with 
prior curative intent therapy with no active recurrence is also allowed regardless of time 
elapsed. 
11. Has known locally advanced (unresectable) or metastatic cancer on baseline radiographic 
imaging (CT or MRI) obtained within 28 days prior to study registration.
12. Has severe hypersensitivity (≥Grade 3) to pembrolizumab and/or any of its excipients.
13. Has active autoimmune disease that has required systemic treatment in the past 2 years (i.e. 
with use of disease modifying agents, corticosteroids or immunosuppressive drugs). 
Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement 
therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic 
treatment and is allowed. Note: Patients with active well controlled type 1 diabetes 
mellitus, vitiligo, Graves’ disease, Hashimoto disease, eczema, lichen simplex chronicus, 
or psoriasis, not requiring systemic immunosuppression within the past 2 years are not 
excluded.
14. Has history of (non-infectious) pneumonitis that required steroids or has current 
pneumonitis.
15. Has an active infection requiring systemic therapy.
16. Has known history of Human Immunodeficiency Virus (HIV). Note: no HIV testing is 
required. 
17. Has known history of active Hepatitis B (defined as Hepatitis B surface antigen [HBsAg] 
detected) or known active Hepatitis C virus (defined as HCV RNA [qualitative] detected) 
infection. Note: no testing for Hepatitis B and Hepatitis C is required. 
18. Has known history of active TB (Bacillus Tuberculosis). Note: no testing is required 
unless it is clinically indicated 
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19. Has history or current evidence of any condition, therapy, or laboratory abnormality that 
might confound the results of the study, interfere with the subject’s participation for the 
full duration of the study, or is not in the best interest of the subject to participate, in the 
opinion of the treating investigator.  
20. Has known psychiatric or substance abuse disorders that would interfere with cooperation 
with the requirements of the trial.
21. Has had allogeneic solid visceral organ transplant.
5.1.3
Lifestyle Restrictions
5.1.3.1
Meals and Dietary Restrictions
Participants should maintain a normal diet unless modifications are required to manage an AE such 
as diarrhea, nausea or vomiting.
5.1.3.2
Contraception
Pembrolizumab may have adverse effects on a fetus in utero.  Refer to Appendix 2 for approved 
methods of contraception.
For this study, male participants will be considered to be of non-reproductive potential if they 
have azoospermia (whether due to having had vasectomy or due to an underlying medical 
condition).  
5.1.4
Pregnancy
If a participant inadvertently becomes pregnant while on treatment with pembrolizumab, the 
participant will be immediately discontinued from study treatment.  The site will contact the 
participant at least monthly and document the participant’s status until the pregnancy has been 
completed or terminated. The outcome of the pregnancy will be reported to Merck within 2 
working days if the outcome is a serious adverse experience (e.g., death, abortion, congenital 
anomaly, or other disabling or life-threatening complication to the mother or newborn).  The 
study Investigator will make every effort to obtain permission to follow the outcome of the 
pregnancy and report the condition of the fetus or newborn to Merck. If a male participant 
impregnates his female partner, the study personnel at the site must be informed immediately and 
the pregnancy must be reported to Merck and followed as described in Section 7.2.2.
5.1.5
Use in Nursing Women
It is unknown whether pembrolizumab is excreted in human milk. Since many drugs are excreted 
in human milk, and because of the potential for serious adverse reactions in the nursing infant, 
participants who are breast-feeding are not eligible for enrollment.
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5.2
Enrollment
Enrollment will be defined as the date participant starts treatment. Prior to this, while patient is 
being considered for the trial will be the screening period.
5.3
Trial Treatments
The treatment to be used in this trial is outlined below in Table 2
Table 2.  Trial Treatment
Drug
Dose/Potency
Dose 
Frequency
Route of 
Administration
Regimen/Treatment 
Period
Use
Pembrolizumab
200 mg
Q3W
IV infusion
Day 1 of weeks 0, 
3 and 6 of 
NAC***
Experimental
Methotrexate
30 mg/m2
Q2W
IV push
Day 1 of weeks 0, 2, 
4 and 6 in NAC. 
Administered first.
SOC
Vinblastine
3mg/m2
Q2W
IV infusion
Day 1 of weeks 0, 2, 
4 and 6 in NAC. 
Administered second.
SOC
Doxorubicin
30mg/m2
Q2W
IV push
Day 1 of weeks 0, 2, 
4 and 6 in NAC. 
Administered third.
SOC
Cisplatin
70mg/m2*
Q2W
IV infusion
Day 1 of weeks 0, 2, 
4 and 6 in NAC. 
Administered last.
SOC
Pegfilgrastim 
or Filgrastim** 
Standard 
dose per 
manufacturer 
Q2W
Subcutaneous 
injection
Day 1 or 2 of weeks 
0, 2, 4 and 6 in NAC. 
Administered after 
chemotherapy
SOC
Trial treatment should begin on the day of enrollment.
*Cisplatin dose may be split as 35 mg/m2 on days 1 and 2 (day 3 or day 4 is allowed for the second 
split dose), based on investigator’s discretion
** Granulocyte colony-stimulating factor (G-CSF): Either On-Pro or any biosimilar to (a) 
Pegfilgrastim or to (b) On-Pro or to (c) Filgrastim is allowed (G-CSF is given as a standard best 
supportive care to aMVAC chemotherapy)
***On days when pembrolizumab and aMVAC will be administered (week 0 and 6), 
pembrolizumab will be administered first (before  aMVAC)
5.3.1 Timing of Dose Administration
Trial treatment should be administered on Day 1 of each cycle after all procedures/assessments 
have been completed as detailed on the Trial Flow Chart (Section 6.0). Trial treatment may be 
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administered up to 4 days before or after the scheduled Day 1 of each cycle due to administrative 
and logistical reasons.
Pembrolizumab 200 mg will be administered as 30-minute IV infusion every 3 weeks. Sites should 
make every effort to target infusion timing to be as close to 30 minutes as possible. However, given 
the variability of infusion pumps from site to site, a window of +/-10 minutes is permitted (i.e., 
infusion time is 30 minutes: -10 min/+10 min).
The Investigator’s Brochure contains specific instructions for the preparation of the 
pembrolizumab infusion fluid and administration of infusion solution.
Accelerated (a)MVAC will be administered as sequential chemotherapy every 2 weeks. 
Medications will be administered per pharmacy protocol. G-CSF should ideally be given 
subcutaneously on day 1 or 2 in the form of either pegfilgrastim or filgrastim (or On-Pro or 
biosimilar) after the completion of the chemotherapy infusion.
On dates when aMVAC will be co-administered with pembrolizumab (week 0 and 6), aMVAC 
should be given after pembrolizumab.
5.3.2
Dose modification and toxicity management for immune-related AEs associated 
with pembrolizumab
AEs associated with pembrolizumab exposure , including coadministration with additional 
compounds, may represent an immune-related response. These irAEs may occur shortly after the 
first dose or several months after the last dose of pembrolizumab/combination treatment and may 
affect more than one body system simultaneously. Therefore, early recognition and initiation of 
treatment is critical to reduce complications. Based on existing clinical study data, most irAEs 
were reversible and could be managed with interruptions of pembrolizumab/combination 
treatment, administration of corticosteroids and/or other supportive care. For suspected irAEs, 
ensure adequate evaluation to confirm etiology or exclude other causes. Additional procedures or 
tests such as bronchoscopy, endoscopy, skin biopsy may be included as part of the evaluation. 
Dose modification and toxicity management guidelines for irAEs associated with 
pembrolizumab/combination treatment are provided in Table 3.
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Table 3. Dose Modification and Toxicity Management Guidelines for Immune-related 
AEs Associated with Pembrolizumab monotherapy and IO Combinations 
General instructions:
1.
Severe and life-threatening irAEs should be treated with IV corticosteroids followed by oral steroids. Other 
immunosuppressive treatment should begin if the irAEs are not controlled by corticosteroids. 
2.
Study Intervention must be permanently discontinued if the irAE does not resolve to ≤ Grade 1 and the 
corticosteroid dose is not ≤10 mg/day within 12 weeks of the last study intervention treatment. 
3.
The corticosteroid taper should begin when the irAE is ≤ Grade 1 and continue at least 4 weeks.
4.
If study intervention has been withheld, study intervention may resume after the irAE decreased to ≤ Grade 1 
and corticosteroid dose is tapered to ≤10 mg/day (within 12 weeks of last dose).
irAEs
Toxicity grade 
(CTCAE V5.0)
Action with 
pembrolizumab
Corticosteroid and/or 
other therapies 
Monitoring and follow-
up 
Grade 2
Withhold 
Pneumonitis 
Recurrent 
Grade 2, 
Grade 3 or 4 
Permanently 
discontinue
Administer 
corticosteroids (initial 
dose of 1 - 2 mg/kg 
prednisone or 
equivalent) followed 
by taper
Add prophylactic 
antibiotics for 
opportunistic 
infections
Monitor participants 
for signs and symptoms 
of pneumonitis
Evaluate participants 
with suspected 
pneumonitis with 
radiographic imaging 
and initiate 
corticosteroid treatment
Grade 2 or 3
Withhold
Diarrhea / Colitis 
Recurrent Grade 3 
or Grade 4
Permanently 
discontinue
Administer 
corticosteroids (initial 
dose of 1 - 2 mg/kg 
prednisone or 
equivalent) followed 
by taper
Monitor participants 
for signs and symptoms 
of enterocolitis (ie, 
diarrhea, abdominal 
pain, blood or mucus in 
stool with or without 
fever) and of bowel 
perforation (ie, 
peritoneal signs and 
ileus)
Participants with 
≥Grade 2 diarrhea 
suspecting colitis 
should consider GI 
consultation and 
performing endoscopy 
to rule out colitis
Participants with 
diarrhea/colitis should 
be advised to drink 
liberal quantities of 
clear fluids. If 
sufficient oral fluid 
intake is not feasible, 
fluid and electrolytes 
should be substituted 
via IV infusion
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Grade 2 a
Withhold
Administer 
corticosteroids (initial 
dose of 0.5 - 1 mg/kg 
prednisone or 
equivalent) followed 
by taper
AST or ALT 
elevation or 
Increased Bilirubin
Grade 3 b or 4 c
Permanently 
discontinue
Administer 
corticosteroids (initial 
dose of 1 - 2 mg/kg 
prednisone or 
equivalent) followed 
by taper
Monitor with liver 
function tests (consider 
weekly or more 
frequently until liver 
enzyme value returned 
to baseline or is stable)
Type 1 diabetes 
mellitus (T1DM) or 
Hyperglycemia
New onset T1DM 
or  Grade 3 or 4 
hyperglycemia 
associated with 
evidence of -cell 
failure
Withhold d
Initiate insulin 
replacement therapy 
for participants with 
T1DM
Administer anti-
hyperglycemic in 
participants with 
hyperglycemia
Monitor participants 
for hyperglycemia or 
other signs and 
symptoms of diabetes
Grade 2
Withhold
Hypophysitis
Grade 3 or 4
Withhold or 
permanently 
discontinue d
Administer 
corticosteroids and 
initiate hormonal 
replacements as 
clinically indicated
Monitor for signs and 
symptoms of 
hypophysitis (including 
hypopituitarism and 
adrenal insufficiency)
Grade 2
Continue
Hyperthyroidism 
Grade 3 or 4
Withhold or 
permanently 
discontinue d
Treat with 
nonselective beta-
blockers (eg, 
propranolol) or 
thionamides as 
appropriate
Monitor for signs and 
symptoms of thyroid 
disorders
Hypothyroidism
Grade 2, 3, 4
Continue
Initiate thyroid 
replacement 
hormones (eg, 
levothyroxine or 
liothyronine) per 
standard of care
Monitor for signs and 
symptoms of thyroid 
disorders
Grade 2
Withhold
Nephritis:
grading according to 
increased creatinine 
or acute kidney injury
Grade 3 or 4
Permanently 
discontinue
Administer 
corticosteroids 
(prednisone 
1 to 2 mg/kg or 
equivalent) followed 
by taper
Monitor changes of 
renal function
Grade 2
Withhold
Neurological 
Toxicities
Grade 3 or 4
Permanently 
discontinue
Based on severity of 
AE administer 
corticosteroids
Ensure adequate
evaluation to confirm 
etiology and/or exclude 
other causes
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Grade 1 or 2
Withhold
Myocarditis
Grade 3 or 4
Permanently 
discontinue
Based on severity of 
AE administer 
corticosteroids
Ensure adequate 
evaluation to confirm 
etiology and/or exclude 
other causes
Suspected SJS, 
TEN, or DRESS
Withhold
Exfoliative 
Dermatologic 
Conditions
Confirmed SJS, 
TEN, or DRESS
Permanently 
discontinue
Based on severity of 
AE administer 
corticosteroids
Ensure adequate 
evaluation to confirm 
etiology or exclude 
other causes
Persistent Grade 2
Withhold
Grade 3
Withhold or 
discontinue 
based on the 
event e 
All Other immune-
related AEs
Recurrent Grade 3 
or Grade 4 
Permanently 
discontinue
Based on severity of 
AE administer 
corticosteroids
Ensure adequate 
evaluation to confirm 
etiology or exclude 
other causes
AE(s)=adverse event(s); ALT= alanine aminotransferase; AST=aspartate aminotransferase; CTCAE=Common 
Terminology Criteria for Adverse Events; DRESS=Drug Rash with Eosinophilia and Systemic Symptom; 
GI=gastrointestinal; IO=immuno-oncology; ir=immune related; IV=intravenous; SJS=Stevens-Johnson Syndrome; 
T1DM=type 1 diabetes mellitus; TEN=Toxic Epidermal Necrolysis; ULN=upper limit of normal.
Note: Non-irAE will be managed as appropriate, following clinical practice recommendations.
a AST/ALT: >3.0 to 5.0 x ULN if baseline normal; >3.0 to 5.0 x baseline, if baseline abnormal; 
bilirubin:>1.5 to 3.0 x ULN if baseline normal; >1.5 to 3.0 x baseline if baseline abnormal
b AST/ALT: >5.0 to 20.0 x ULN, if baseline normal; >5.0 to 20.0 x baseline, if baseline abnormal; 
bilirubin:>3.0 to 10.0 x ULN if baseline normal; >3.0 to 10.0 x baseline if baseline abnormal
c AST/ALT: >20.0 x ULN, if baseline normal; >20.0 x baseline, if baseline abnormal; 
bilirubin: >10.0 x ULN if baseline normal; >10.0 x baseline if baseline abnormal
d The decision to withhold or permanently discontinue pembrolizumab is at the discretion of the Investigator or 
treating physician. If control achieved or ≤ Grade 2, pembrolizumab may be resumed.
e Events that require discontinuation include but are not limited to: encephalitis and other clinically important 
irAEs (eg, vasculitis and sclerosing cholangitis).
Dose Modification and Toxicity Management of Infusion Reactions Related to 
Pembrolizumab 
Pembrolizumab may cause severe or life-threatening infusion-reactions including severe 
hypersensitivity or anaphylaxis. Signs and symptoms usually develop during or shortly after 
drug infusion and generally resolve completely within 24 hours of completion of infusion. 
Dose modification and toxicity management guidelines on pembrolizumab associated infusion 
reaction are provided in Table 4.
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Table 4. Pembrolizumab Infusion Reaction Dose Modification and Treatment 
Guidelines
NCI CTCAE Grade
Treatment
Premedication at 
Subsequent Dosing
Grade 1
Mild reaction; infusion 
interruption not indicated; 
intervention not indicated
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator
None
Grade 2
Requires therapy or infusion 
interruption but responds 
promptly to symptomatic 
treatment (eg, antihistamines, 
NSAIDs, narcotics, IV fluids); 
prophylactic medications 
indicated for ≤24 hrs
Stop Infusion
Additional appropriate medical therapy may 
include but is not limited to:
IV fluids
Antihistamines
NSAIDs
Acetaminophen
Narcotics
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator.
If symptoms resolve within 1 hour of 
stopping drug infusion, the infusion may be 
restarted at 50% of the original infusion rate 
(eg, from 100 mL/hr to 50 mL/hr). Otherwise 
dosing will be held until symptoms resolve 
and the participant should be premedicated 
for the next scheduled dose.
Participants who develop Grade 2 toxicity 
despite adequate premedication should be 
permanently discontinued from further study 
drug intervention.
Participant may be 
premedicated 1.5 h (±30 
minutes) prior to infusion 
of pembrolizumab with:
Diphenhydramine 50 mg 
PO (or equivalent dose of 
antihistamine).
Acetaminophen 500-1000 
mg PO (or equivalent dose 
of analgesic).
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NCI CTCAE Grade
Treatment
Premedication at 
Subsequent Dosing
Grades 3 or 4
Grade 3:
Prolonged (ie, not rapidly 
responsive to symptomatic 
medication and/or brief 
interruption of infusion); 
recurrence of symptoms 
following initial improvement; 
hospitalization indicated for 
other clinical sequelae (eg, 
renal impairment, pulmonary 
infiltrates)
Grade 4:
Life-threatening; pressor or 
ventilatory support indicated
Stop Infusion.
Additional appropriate medical therapy may 
include but is not limited to:
Epinephrine**
IV fluids
Antihistamines
NSAIDs
Acetaminophen
Narcotics
Oxygen
Pressors
Corticosteroids
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator.
Hospitalization may be indicated.
**In cases of anaphylaxis, epinephrine 
should be used immediately.
Participant is permanently discontinued from 
further study drug intervention.
No subsequent dosing
Appropriate resuscitation equipment should be available at the bedside and a physician readily available 
during the period of drug administration.
For further information, please refer to the Common Terminology Criteria for Adverse Events v5.0 
(CTCAE) at http://ctep.cancer.gov
Other Allowed Dose Interruption for Pembrolizumab
Pembrolizumab may be interrupted for situations other than treatment-related AEs, such as 
medical / surgical events or logistical reasons not related to study therapy. Participants should 
be placed back on study therapy within 3 weeks of the scheduled interruption. The reason for 
interruption should be documented in the participant's study record.
5.3.3
Dose Modifications for aMVAC chemotherapy (recommendations only; actual 
decisions for chemotherapy will be based on the best clinical judgment and 
institutional guidelines, if applicable)
5.3.3.1
Hematological Toxicity
Grade 1 and Grade 2 toxicities will be accepted without dose interruption or dose 
modification. However, for grade 2 neutropenia on day 1 of any chemotherapy cycle 
(absolute neutrophil count 1-1.5x109/L), dose reduction to 75% dose of methotrexate, 
vinblastine and doxorubicin is recommended.
If a patient experiences Grade 3 hematological toxicity on day 1 of any chemotherapy 
cycle, study chemotherapy will be held until toxicity has resolved or ≤ Grade 1. For 
grade 3 neutropenia on day 1 of any chemotherapy cycle (absolute neutrophil count 
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1-1.5x109/L) that resolves to ≤ Grade 1 within 4 weeks, dose reduction to 66% dose 
of methotrexate, vinblastine and doxorubicin is recommended. If the Grade 3 toxicity 
does not resolve to ≤ Grade 1 within 4 weeks, all study chemotherapy may be 
permanently discontinued. Pembrolizumab may continue in that case as per 
physician discretion based on attribution.
If the patient experiences Grade 4 treatment related hematologic toxicity, all study 
chemotherapy may be permanently discontinued regardless of the time to resolution 
≤ Grade 1. Pembrolizumab may continue in that case as per physician discretion based 
on attribution.
5.3.3.2
Renal Dysfunction
Recommended dosing based on CrCl of cisplatin as listed below.
CrCl (mL/min)
Dose
≥60 mL/min
100%
45-59 mL/min
Cisplatin: Split dose 
over 2 days with 
hydration
Methotrexate: 75%
< 45 mL/min*
Hold
               * Note CrCl<50 ml/min is exclusion criteria for enrollment
5.3.3.3
Other non-hematological toxicity
If the patient develops any chemotherapy-related non-hematological toxicity, dose interruption 
or discontinuation should be carefully managed by the treating physician, ideally with 
discussion with trial principal investigator, based on the best clinical judgment and local 
institutional guidelines, if applicable. Best possible determination of adverse event grade and 
attribution to each agent(s) should be pursued along with proper management and best 
supportive care in each case. Considering that the trial will be open in one site, this is feasible.
Chemotherapy may be interrupted for situations other than treatment-related AEs, such as 
medical / surgical events or logistical reasons not related to study therapy. Participants should 
be placed back on study therapy within 4 weeks of the scheduled interruption. The reason for 
interruption should be documented in the participant's study record.
5.4
Concomitant Medications/Vaccinations (allowed & prohibited)
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed 
during the ongoing trial.  If there is a clinical indication for one of these or other medications 
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or vaccinations specifically prohibited during the trial, discontinuation from trial therapy or 
vaccination may be required.  The final decision on any supportive therapy or vaccination rests 
with the Investigator and/or the participant's treating physician. 
5.4.1
Acceptable Concomitant Medications
All treatments that the Investigator considers necessary for a participant’s welfare may be 
administered at the discretion of the Investigator in keeping with the community standards of 
medical care. All concomitant medication will be recorded on the case report form (CRF) 
including all prescription, over-the-counter (OTC), herbal supplements, and IV medications 
and fluids.  If changes occur during the trial period, documentation of drug dosage, frequency, 
route, and date may also be included on the CRF.
All concomitant medications received within 28 days before the first dose of trial treatment 
and 30 days after the last dose of trial treatment should be recorded. Concomitant medications 
administered after 30 days after the last dose of trial treatment should be recorded only for 
SAEs and ECIs as defined in Section 7.2.
5.4.2
Prohibited Concomitant Medications
Participants are prohibited from receiving the following therapies during the Screening and 
Treatment Phase of this trial:

Antineoplastic systemic chemotherapy or biological therapy 

Immunotherapy not specified in this protocol

Chemotherapy not specified in this protocol

Investigational agents other than pembrolizumab

Radiation therapy 

Live vaccines within 30 days prior to the first dose of study treatment and while 
participating in the study.  Examples of live vaccines include, but are not limited to, the 
following: measles, mumps, rubella, a version of varicella/zoster, yellow fever, rabies, 
BCG, and typhoid vaccine. Seasonal influenza vaccines for injection are generally 
killed virus vaccines and are allowed; however, intranasal influenza vaccines (e.g., 
FluMist®) are live attenuated vaccines and are not allowed. 

Systemic glucocorticoids (> 10 mg of prednisone equivalent daily dose) for any 
purpose other than to modulate symptoms from an event of clinical interest of suspected 
immunologic etiology. The use of physiologic doses of corticosteroids are allowed. 
Standard use of steroids as premedication for cisplatin infusion is allowed with dose 
and duration at investigator discretion.
Participants who, in the assessment by the investigator, require the persistent use of any of the 
aforementioned treatments for clinical management should be removed from the study. All 
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treatments that the Investigator considers necessary for a participant’s welfare may be 
administered at the discretion of the Investigator in keeping with the community standards of 
medical care.  There are no prohibited therapies during the Post-Treatment Follow-up Phase.
5.4.3
Rescue Medications & Supportive Care
Participants should receive appropriate supportive care measures as deemed necessary by the 
treating investigator. Suggested supportive care measures for the management of AEs with 
potential immunologic etiology are outlined along with the dose modification guidelines in 
Section 5.2.2, [Table 3].  Where appropriate, these guidelines include the use of oral or IV 
treatment with corticosteroids, as well as additional immunosuppressive agents, esp. if 
symptoms do not improve with administration of corticosteroids. Note that several courses of 
steroid tapering may be necessary as symptoms may worsen when the steroid dose is 
decreased.  For each disorder, attempts should be made to rule out other causes, such as 
metastatic disease or any infection, which might require additional supportive care. The 
treatment guidelines are intended to be applied when the Investigator determines the events 
to be related to pembrolizumab. 
Note: If after the evaluation of the event, it is determined not to be related to pembrolizumab, 
the Investigator does not need to follow the treatment guidance.  Refer to [Table 3] in Section 
5.2.2 for guidelines regarding dose modification and supportive care. 
It may be necessary to perform conditional procedures, such as bronchoscopy, endoscopy, or 
skin photography, etc., as part of evaluation of the event. 
5.5
Participant Withdrawal/Discontinuation Criteria
Participants may discontinue study treatment at any time for any reason or be dropped from 
the study treatment at the discretion of the Investigator should any untoward effect occur. In 
addition, a participant may be discontinued from study treatment by the Investigator if study 
treatment is inappropriate, the trial plan is violated, or for administrative and/or other safety 
reasons. Specific details regarding procedures to be performed at study treatment 
discontinuation are provided in Section 7.1.4 – Other Procedures.
A participant must be discontinued from study treatment for any of the following reasons:

The participant or participant’s legally acceptable representative requests to 
discontinue study treatment

Confirmed radiographic disease progression outlined in Section 7.1.2.6

Any progression of malignancy, or any occurrence of another malignancy that 
requires active treatment

Unacceptable adverse events as described in Section 5.2.2.
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
The participant has a medical condition or personal circumstance which, in the 
opinion of the investigator, placed the participant at unnecessary risk from continued 
administration of study treatment.

The participant has a confirmed positive serum pregnancy test or breastfeeds

Noncompliance with study treatment or procedure requirements

The participant is lost to follow-up or died

Administrative/logistical reasons
5.6
Participant Replacement Strategy
A patient will be replaced if following TURBT and registration into the study, they do not 
meet any of the following criteria:

Begin neoadjuvant study therapy within 12 weeks of first TURBT that showed 
muscularis propria invasion

Receive at least one study treatment dose (accelerated MVAC or pembrolizumab)

Predominant or pure non-UC histology in any TURBT meeting criteria outlined in 
inclusion and exclusion criteria (Section 5.1.1 and 5.1.2).
If a patient withdraws informed consent any time before radical cystectomy (not due to therapy 
related toxicity or cancer progression), they can be replaced and will not be considered 
evaluable for efficacy assessment.
5.7
Clinical Criteria for Early Trial Termination
Early trial termination will be the result of the criteria specified below:
1. Quality or quantity of data recording is inaccurate or incomplete
2. Poor adherence to protocol and regulatory requirements
3. Incidence or severity of adverse drug reaction in this or other studies indicates a potential 
health hazard to participants
4. Plans to modify or discontinue the development of the study drug
In the event of Merck decision to no longer supply study drug, ample notification will be 
provided so that appropriate adjustments to participant treatment can be made.
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6.0 TRIAL FLOW CHART
6.1
Study Flow Chart
Trial Period
Treatment Cycles*
aMVAC
Treatment Cycle/Title:
 
Screening 
P1
P2 
P3
1
2
3
4
Surgery7
End of 
Treatment
/Discontin
uation9
Survival 
Follow-
Up10
Scheduling Window (Days):
-28 to -1
±4
± 4
± 4
± 4
± 4
± 4
± 4
At time of 
pre-op for 
RC
  1-month 
(+/- 1wk) 
post-op of 
RC 
or 
after 
discontinu
ation14 
SOC 
Informed Consent1
X
Inclusion/Exclusion Criteria
X
Demographics and Medical History
X
Prior and Concomitant Medication Review
X
X
X
X
X
X
X
X
X
X
Pulse Oximetry first and then trial 
Treatment Administration
X
X
X
X
X
X
X
Post-study anticancer therapy status
X
X
X
Survival Status
X
X
X
X
X
X
X
X
X
X
Review Adverse Events12
X
X
X
X
X
X
X
X
X
Full Physical Examination
X
X
X
X
X
X
X
X
X
X
Vital Signs and Weight
X
X
X
X
X
X
X
X
X
X
ECOG Performance Status
X
X
X
X
X
X
X
X
X
X
Pregnancy Test: Urine or Serum -HCG16
X
PT/INR and aPTT
X
X
CBC with Differential
X
X
X
X
X
X
X
X
X
X
Comprehensive Serum Chemistry Panel
X
X
X
X
X
X
X
X
X
X
Urinalysis and EKG13
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Trial Period
Treatment Cycles*
aMVAC
Treatment Cycle/Title:
 
Screening 
P1
P2 
P3
1
2
3
4
Surgery7
End of 
Treatment
/Discontin
uation9
Survival 
Follow-
Up10
Scheduling Window (Days):
-28 to -1
±4
± 4
± 4
± 4
± 4
± 4
± 4
At time of 
pre-op for 
RC
  1-month 
(+/- 1wk) 
post-op of 
RC 
or 
after 
discontinu
ation14 
SOC 
T3, free T4, TSH, cortisol
X
X
X
X
Hepatitis B & C only if clinically indicated2
X
Tumor Imaging3
X
X8
X15
X
Archival and/or Fresh Tissue Collection4
X
X
Correlative Studies: Blood Collection5
X
X
X
X
X11
Correlative Studies: Urine Collection5
X
X
X
X
X11
Correlative Studies: Stool Collection6
X
X
X11
*Scheduled Visits: +/-4 day window is allowed for scheduled study therapy, required tests and/or visits except as otherwise noted. Delay due to holidays, weekends, bad 
weather or other unforeseen circumstances will be permitted up to +/-7 days from target date. Study procedures, e.g. history, physical exam, pulse oximetry, blood, urine, 
stool collection, at each time point should be performed prior to study therapy administration. (P=pembrolizumab)
1
Cystoscopy with TURBT showing muscularis propria invasion should be performed within 12 weeks (84 days) of starting study therapy (C1D1 of treatment). 
Patients will complete all other screening studies (i.e. labs, imaging, history, exam, etc.) within 28 days of starting study therapy.
2
Hepatitis B surface antigen (HBsAg) and Hepatitis C (HCV) testing is not required unless clinically indicated (as standard of care). These tests may be 
repeated during the course of the study, if clinically indicated (as standard of care).
3
For abdomen/pelvis, CT with IV contrast is the preferred method (IV contrast is not required for CT chest). MRI abdomen/pelvis (preferably with gadolinium 
if possible) can be performed, if CT with IV contrast cannot be obtained. CT abdomen/pelvis without IV contrast is allowed if IV contrast cannot be given 
and MRI cannot be performed. Follow-up scans will be done as clinically indicated based on pathologic stage at time of RC (as standard of care).
     4     TURBT and RC tumor tissue (any number of block or slides are OK for eligibility). 
Time points (up to three): TURBT, radical cystectomy, and at the time of recurrence (if possible)
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-Fresh frozen OCT (and extra fresh if possible): block/tissue to be collected and stored after FFPE (Stored samples will not be used for any studies unrelated 
to this trial.)
5
Research blood & urine, samples shall be obtained at time points noted below. (Stored samples will not be used for any studies unrelated to this trial.)
Before first & second pembrolizumab dose, pre-radical cystectomy, post-op visit, and at the time of recurrence (if possible) - up to five time points
           -Peripheral blood: two 10 mL purple top tubes, two 5 mL purple top tubes, and one 10 mL Streck DNA tube
-Urine: At least 30 mL in standard urine cup
6
Research stool samples shall be obtained before first & second pembrolizumab dose, and at the time of recurrence (if possible) - up to three time points 
(Stored samples will not be used for any studies unrelated to this trial.)
7
Radical cystectomy (RC) with bilateral (standard or extended) pelvic lymph node dissection to be performed as soon as deemed safe, and ideally within 10 
weeks, after the last neoadjuvant infusion (RC is standard of care).
8
CT (or MRI as noted above) after final dose of study therapy and before surgery (standard of care)
9
If patient is removed from treatment for reason(s) other than progression, follow with regular tumor assessments as per standard of care until progression or 
start of new treatment. Adverse events should be reviewed at that post-RC safety visit, while phone follow-up may also be done as clinically needed to 
review potential subsequent adverse events; if those are felt to be at least possibly related to study therapy, they should be recorded as such. 
10
Patients will be followed clinically and radiographically for at least 2 years post-cystectomy or when study-wide follow-up ends as per standard of care. 
First surveillance scan will be approximately 3 months after RC and then will continue every 3-6 months for 2 years as per treating physician and standard 
practice. Date of diagnosis for progression, first subsequent therapy and survival shall be reported. Phone follow-up may also be done for patients unable or 
unwilling to return for follow-up evaluations.
11
At time of recurrence, if feasible
12. All AEs up until 30 days post study intervention and SAEs up until 90 days post study treatment or 30 days if participant starts new anticancer therapy, 
whichever is earlier, should be collected
13
Urinalysis and EKG to be done during treatment if clinically indicated
14
For patients who are discontinued while on treatment, scheduling of end of treatment visit will be at earliest convenience per provider discretion.
15 Tumor imaging is only necessary for those participants who discontinue study treatment per 7.1.2.5.1. For patients who complete study treatment and RC, 
tumor imaging should be per SOC survival follow-up.
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7.0 TRIAL PROCEDURES
7.1
Trial Procedures
The Trial Flow Chart - Section 6.0 summarizes the trial procedures to be performed at each visit. 
Individual trial procedures are described in detail below. 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 Investigator.
7.1.1
Administrative Procedures
7.1.1.1
Informed Consent
The Investigator must obtain documented consent from each potential participant prior to 
participating in a clinical trial.
7.1.1.1.1 General Informed Consent
Consent must be documented by the participant’s dated signature or by the participant’s legally 
acceptable representative’s dated signature on a consent form along with the dated signature of the 
person conducting the consent discussion. 
A copy of the signed and dated consent form should be given to the participant before participation 
in the trial.
The initial informed consent form, any subsequent revised written informed consent form and any 
written information provided to the participant must receive the IRB/ERC’s approval/favorable 
opinion in advance of use. The participant or his/her legally acceptable representative should be 
informed in a timely manner if new information becomes available that may be relevant to the 
participant’s willingness to continue participation in the trial. The communication of this 
information will be provided and documented via a revised consent form or addendum to the 
original consent form that captures the participant’s dated signature or by the participant’s legally 
acceptable representative’s dated signature. Specifics about a trial and the trial population will be 
added to the consent form template at the protocol level.  The informed consent will adhere to 
IRB/ERC requirements, applicable laws and regulations requirements.
7.1.1.2
Inclusion/Exclusion Criteria
All inclusion and exclusion criteria will be reviewed by the Investigator or qualified designee to 
ensure that the participant qualifies for the trial. 
7.1.1.3
Medical History
A medical history will be obtained by the Investigator or qualified designee. Medical history will 
include all active conditions, and any condition diagnosed within the prior 10 years that are 
considered to be clinically significant by the Investigator. Details regarding the disease for which 
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the participant has enrolled in this study will be recorded separately and not listed as medical 
history.  
7.1.1.4
Prior and Concomitant Medications Review
7.1.1.4.1 Prior Medications
The Investigator or qualified designee will review prior medication use, including any protocol-
specified washout requirement, and record prior medication taken by the participant within 28 days 
before starting the trial.  Treatment for the disease for which the participant has enrolled in this 
study will be recorded separately and not listed as a prior medication. 
7.1.1.4.2 Concomitant Medications
The Investigator or qualified designee will record medication, if any, taken by the participant 
during the trial. All medications related to reportable SAEs and ECIs should be recorded as defined 
in Section 7.2.
7.1.1.5
Disease Details and Treatments
7.1.1.5.1 Disease Details
The Investigator or qualified designee will obtain prior and current details regarding disease status. 
7.1.1.5.2 Prior Treatment Details
The Investigator or qualified designee will review all prior cancer treatments including systemic 
treatments, radiation and surgeries.
7.1.1.5.3 Subsequent Anti-Cancer Therapy Status
The Investigator or qualified designee will review all new anti-neoplastic therapy initiated after 
the last dose of trial treatment. If a participant initiates a new anti-cancer therapy after the last dose 
of trial treatment, the safety follow-up visit should ideally occur before the first dose of the new 
therapy. Once new anti-cancer therapy has been initiated the participant will move into survival 
follow-up. 
7.1.1.6
Assignment of Screening Number
Patients will be assigned a screening number after they sign informed consent.
7.1.1.7
Assignment of Enrollment Number 
Patients will be enrolled locally into the study based on local procedures based on eligibility 
criteria signed off by sub-investigator.
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7.1.1.8
Trial Compliance (Medication/Diet/Activity/Other) 
All study procedures should be followed per protocol. 
7.1.2
Clinical Procedures/Assessments
7.1.2.1
Adverse Event (AE) Monitoring
The Investigator or qualified designee will assess each participant to evaluate for potential new 
or worsening AEs as specified in the Trial Flow Chart and more frequently if clinically indicated. 
AEs will be graded and recorded throughout the study and during the follow-up period according 
to NCI CTCAE Version 5.0 (The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for 
adverse event reporting; 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm#ctc_50). 
Toxicities will be characterized in terms regarding seriousness, causality, grading, and action taken 
with regard to trial treatment. Please refer to section 7.2 for detailed information regarding the 
assessment and recording of AEs.  
7.1.2.2
Full Physical Exam
The Investigator or qualified designee will perform a complete physical exam during the screening 
period. Clinically significant abnormal findings should be recorded as medical history. A full 
physical exam should be performed during screening, 
7.1.2.3
Vital Signs
The Investigator or qualified designee will take vital signs at screening, prior to the administration 
of each dose of trial treatment and at treatment discontinuation as specified in the Trial Flow Chart 
(Section 6.0). Vital signs should include temperature, pulse, respiratory rate, weight and blood 
pressure. Height will be measured at screening only.
7.1.2.4
Eastern Cooperative Oncology Group (ECOG) Performance Scale
The Investigator or qualified designee will assess ECOG status (see Appendix 1) at screening, 
prior to the administration of each dose of trial treatment and discontinuation of trial treatment as 
specified in the Trial Flow Chart.  
7.1.2.5
Tumor Imaging and Assessment of Disease
Tumor imaging is strongly preferred to be acquired by computed tomography (CT) with IV 
contrast. For the abdomen / pelvis, magnetic resonance imaging (MRI) may be used when CT 
with IV contrast is contraindicated, or when local practice mandates it. CT abdomen / pelvis 
without IV contrast is acceptable if IV contrast is contraindicated and MRI cannot be done. MRI 
is the strongly preferred modality for imaging the brain (as clinically indicated). The same 
imaging technique regarding modality, ideally the same scanner, and the use of contrast should 
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ideally be used in a participant throughout the study to optimize the reproducibility of the 
assessment of existing and new tumor burden and improve the accuracy of the assessment of 
response or progression based on imaging.
7.1.2.6
Tumor Imaging
Initial tumor imaging at screening must be performed within 28 days prior to the date of 
enrolment. The site study team must review screening images to confirm the participant does not 
have metastatic disease. 
Tumor Imaging During the Study
The first on-study imaging assessment should be performed at the completion of neoadjuvant 
chemoimmunotherapy and prior to radical cystectomy. Subsequent tumor imaging should be 
performed post-operatively based on pathologic stage at time of radical cystectomy. Tumor 
imaging intervals will be based on NCCN guidelines for given pathologic stage. 
End of Treatment and Follow-up Tumor Imaging
In participants who discontinue study treatment prior to trial completion, tumor imaging should 
ideally be performed within 4 weeks or at the earliest convenience per provider discretion. If 
previous imaging was obtained within 4 weeks prior to the date of discontinuation, then imaging 
at treatment discontinuation is not mandatory.
In participants who discontinue study treatment without documented disease progression, every 
effort should be made to continue monitoring their disease status by tumor imaging using ideally 
the same imaging schedule used while on treatment to monitor disease status until the start of a 
new anticancer treatment, disease progression, pregnancy, death, withdrawal of consent, or the 
end of the study, whichever occurs first.
7.1.2.7
Tumor Tissue Collection and Correlative Studies Blood Sampling
7.1.2.7.1 Tumor Tissue Assessments

Pre-treatment TURBT (archived tissue may be used)

Radical Cystectomy

At the time of recurrence, if feasible
7.1.2.7.2 Tissue Banking
TURBT specimens will be banked locally at UWMC GU Research Lab (Dr. Ming Lam). Stored 
samples will not be used for any studies unrelated to this trial.
Intraoperative RC specimens will be collected directly from the operating room. Tissue necessary 
for standard of care pathology evaluation will be sent to the UWMC pathology department and 
additional specimens will be sent directly to UWMC lab. 
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Tissue sorting priority will be the following:
(1) Pathology/Clinical Evaluation
(2) Formalin-fixed paraffin-embedded blocks
(3) Fresh and fresh frozen tissue
7.1.2.7.3 Biospecimen and Data Repository 
Biospecimen banks are collections of stored human biological materials, assembled either 
through blood, urine, or surgical acquisition procedures, and which are linked to a database of 
de-identified patient information that can be associated with each sample. Patient information 
will be stored in RedCap. RedCap requires username, password and a 2-factor authentication.  
All personnel who have access to RedCap are HIPAA trained. All tissue, peripheral blood, 
urine, and stool samples collected will be labeled with a unique numeric identifier that will be 
coded for patient privacy protection before releasing specimens for processing. All de-
identified specimens will be processed and stored at the UWMC GU Research Lab (Dr. Ming 
Lam). The data and samples stored in this repository may be used by researches and will be 
used for research purposes only and will not be sold. 
7.1.2.7.4 Tumor Infiltrating CD8+ T-Cell Density
Post-treatment tumor infiltrating CD8+ T-cell density is a secondary endpoint in the trial. In 
pre- and post-treatment tumor tissue specimens CD8+ T-cell density will be examined by IHC 
performed on whole tissue histology sections. CD8+ T cell density will be assessed using a 
fluorescent tryamide-based immunohistochemistry (mIHC) approach to identify CD8 and 
tumor (pancytokeratin AE1/AE3). The assays will be performed on representative slides from 
formalin-fixed paraffin-embedded pre-treatment TURBT and post-treatment cystectomy tissue 
in the CLIA-regulated Integrated Clinical Trials Pathology Lab (ICTPL) at the Fred 
Hutchinson Cancer Center. Fluorescent whole-slide scanning will also be performed on the 
Aperio Versa (Leica). Digital images will be exported into Halo (Indica Labs) for quantitative 
image analysis of CD8+ T cell counts, CD8+ T cell density (CD8+/mm2) as well as CD8+ T 
cell density at (within 50 microns) of the tumor-stroma interface.  
7.1.2.7.5 Multiplex Immunohistochemistry mIHC on tumor microarrays (TMAs)
For additional exploratory analysis, tissue microarrays (TMAs) of pre- and post-treatment 
tumor tissues may be constructed using available tumor FFPE blocks. The TMAs will be 
constructed using three 1.0 mm diameter tumor core tissues containing at least 100 malignant 
cells representing the greatest extent of infiltration of lymphocytes. Also, a minimum of 20 (30 
preferred) unstained 5-micron thick sections from both the pre- and post-treatment tumor 
samples will be cut into slides for additional correlative investigations. A 7-color mIHC will 
be performed using spectrally-resolvable fluorescent tryamides, followed by imaging and 
spectral deconvolution using the Vectra 3.0 slide scanner (Perkin-Elmer) as previously 
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described.  Downstream analysis will be performed using Halo (Indica Labs).  The mIHC panel 
will include the following analytes: (1) PD-1, (2) PD-L1, (3) CD8 (4) CD4 (5) CD66b (6) 
macrophage cocktail (CD68+CD163).  
7.1.2.7.6 Additional Analysis
Other analysis that may be performed depending on resources, tissue quality and availability 
may include the following:
Additional IHC Analyses
As tissue availability permits, additional IHC and immunofluorescence (IF) staining is planned 
to further characterize functional immune phenotypes and targets of interest for future drug 
development. Utilizing the TMAs constructed from the pre- and post-treatment archived tumor 
specimens, additional mIHC stains planned, but not limited to the following: FOXP3, Ki-67, 
CTLA-4, Hsp27, PTEN, OX40, LAG3, TIM-3, KIR2DL, CSF1R, 4-1BB, IDO, GITR, TIGIT, 
CD27, CD73, CD40L, pSTAT1, RBP-J, CMAF, LGR4, LGR5, LGR6, SLAMF7, etc. Exact 
list of IHC biomarkers may be modified according to new target identification and emerging 
translational science. Immunoscore and immune reports may also be evaluated.
Immune Gene Expression Analysis
Baseline and post-treatment tumor cells will be isolated and enriched from patient tumor slides 
by macro-dissection. RNA will be extracted and assessed for quality control utilizing standard 
manufacturer RNA extraction kits and per manufacturer instructions. Extracted RNA from 
each sample passing quality control will be analyzed for the expression of immune mediating 
genes by standard quantitative PCR. 
Nanostring
Nanostring-based transcriptional analysis will be performed on serial sections from the same 
FFPE specimens utilized for CD8+ T cell density measurements (see above). In brief, RNA 
will be extracted from unstained FFPE samples from microscope slides and transcripts of 
immunological interest will be quantified using the established panels from Nanostring using 
nCounter’s Advance analyses technology. Up to 30 transcripts of particular interest for bladder 
cancer biology will be simultaneously measured using a custom “add-in” probe set. 
T-Cell Receptor Repertoire Analysis
Pre- and post-treatment tumor cells will be isolated and enriched from patient pre- and post-
treatment tumor slides by macro-dissection. DNA will be extracted and assessed for quality 
control utilizing standard manufacturer DNA extraction kits and per manufacturer instructions. 
Extracted DNA from each sample will be analyzed for T-cell and B-cell receptor mutations by 
next generation sequencing platform, statistical and bioinformatics analysis.
RNASeq
RNA from frozen pre- and post- treatment tissues will be isolated by the UW GU research 
laboratory and generated into RNASeq libraries through the Fred Hutch Genomics Core. 
Sequencing alignment and analysis will be conducted by the Hsieh Lab using standard 
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platforms including EdgeR and DESeq2.
Exome Sequencing
Baseline tumor, post-treatment tumor, and germline DNA will be extracted from patient tissues 
(punches from FFPE or fresh frozen tissues). Extracted DNA from each sample will be 
analyzed for somatic mutations, copy number alterations, and mutational load estimate. 
Additional DNA based investigations on tumor DNA may be performed as tumor DNA 
availability and emerging analysis platforms permit.
7.1.2.8
Correlative Studies: Mandatory Peripheral Blood Samples and Analysis
7.1.2.8.1 Peripheral Blood Assessments

Before First and Second Neoadjuvant Pembrolizumab Dose
- Two 10 mL and two 5mL purple top tubes
- One 10 mL Streck DNA tube 

Pre-RC
- Two 10 mL and two 5mL purple top tubes
- One 10 mL Streck DNA tube 

Safety Follow Up Visit (after RC)
- Two 10 mL and two 5mL  purple top tubes
- One 10 mL Streck DNA tube 

At time of recurrence, if feasible
- Two 10 mL and two 5mL  purple top tubes
- One 10 mL Streck DNA tube 
7.1.2.8.2 Peripheral Blood Lymphocyte Subsets
T-cell subset proportions (%CD4+ T-cells, %CD8+ T-cells, %Treg T-cells, %Myeloid Derived 
Suppressor Cells, %Natural Killer cells, etc.) will be determined by flow cytometry analysis.
7.1.2.8.3 ctDNA Analysis
Cell-free ctDNA analysis will be performed on Streck DNA tube samples at time points of 
collection. Germline DNA can be extracted from “buffy coat”.
7.1.2.8.4 Cytokine Analysis
The functional effects of study therapy will be analyzed by assessment of pre- and post-
treatment plasma cytokine markers of interest (e.g. IFN-α, TGF-β, IL-10, IL-4, IL-5, IL-13, 
IFN-γ, etc.). The exact list of cytokines analyzed will be selected based on assay methodology 
optimization and validation testing. It is expected that a panel of a few biomarkers will be 
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analyzed to distinguish between major immune effector pathways. Additional exploratory 
analysis of the relationship between circulating free tumor DNA/RNA, micro-RNA, and other 
novel platforms will be pursued as resources and specimen availability permit.
7.1.2.9
Correlative Studies: Mandatory Urine Samples and Analysis
7.1.2.9.1 Urine Assessment and Sample Collection

Before First and Second Neoadjuvant Pembrolizumab Dose

Pre-RC

Safety Follow Up Visit (after RC)

At time of recurrence, if feasible
Urine should be collected in standard analysis cup and 30mL aliquoted into two 15mL falcon 
tubes.
7.1.2.9.2 Banking for Future Research: Urine Biomarker Analysis
Urine samples will be banked and stored for future correlative work related to this study. Future 
analyses of urinary cytokine changes, urine cell pellet profiles, T-cell receptor repertoire, gene 
methylation and mutation analysis are proposed as resources and specimen availability permit. 
The exact list of urine biomarkers may be modified according to new target identification and 
emerging translational science. Stored samples will not be used for any studies unrelated to 
this trial.
7.1.2.10 Correlative Studies: Stool Sample Collection
7.1.2.10.1 Stool Assessment and Sample Collection
Before First and Second Neoadjuvant Pembrolizumab Dose
At time of recurrence, if feasible
Two stool samples per time point should be collected in standard analysis tubes, one 
tube with 5 mL RNA later and one tube without preservative. Samples should be stored 
at -80 oC until analysis. Plan to record the stool collection date.
7.1.2.10.2 Banking for Future Research
Stool samples will be banked and stored. Future analyses will include microbiome diversity, 
metagenomic, and meta-transcriptomic evaluation and bacterial culturing depending on 
availability of resources. Stored samples will not be used for any studies unrelated to this trial.
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7.1.2.11 Research Samples Processing
All tissue, peripheral blood and urine samples collected will be labeled with a unique numeric 
identifier that will be coded for patient privacy protection. All specimens will be processed and 
stored at the GU Research Lab (Dr. Ming Lam). Stored samples will not be used for any studies 
unrelated to this trial.
7.1.2.11.1 Tissue Block or Slide Samples
Tissue will be formalin-fixed and paraffin embedded (FFPE). Five-μm sections will be cut and 
put on slides for H&E and IHC analysis. For frozen tissue processing (if 1 FFPE has been 
fulfilled), a cryomold will be prefilled with OCT halfway, then the tissue will be placed in the 
middle of the OCT followed by filling the rest of the cryomold with OCT. The cryomold will 
be immediately put into an isopentane bath on dry ice for freezing. The frozen OCT-embedded 
tissue will be stored in -80°C. Stored samples will not be used for any studies unrelated to this 
trial.
7.1.2.11.2 Peripheral Blood Samples
Peripheral blood should be collected as noted above.
Two 5ml purple top will be sent to Zhengwei (Jenny) Mao's lab for peripheral blood 
lymphocytes analysis.
Two 10ml purple top and 1 Streck DNA tube will be sent to the GU Research Lab (Dr. Ming 
Lam) for processing. 
Purple Top Tubes Processing for Plasma and Buffy Coat
**Process samples ideally within 120 minutes post-collection (record the 
blood draw time)**

Gently mix each blood sample by inversion 10 times (do not shake).

Place tubes immediately on wet ice for five minutes

Centrifuge at 1500 RPM for 15 minutes at 4°C. 
After centrifugation, the plasma layer will be at the top half of the tube and the 
nucleated cells (WBC) will be in a whitish layer called the “buffy coat”, just under 
the plasma and above the red blood cells.
Plasma Preparation

Using a transfer pipette for each tube take the top two-thirds of the plasma and 
transfer plasma into a 15-mL conical centrifuge tube, be careful not to disturb the 
buffy coat layer in each purple top tube (NOTE: see below for buffy coat 
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processing instructions). 

Transfer equal amounts of plasma from each tube into two labeled polypropylene 
tubes for cryopreservation.

Store the two aliquots of plasma samples from each tube in a freezer at ≤ -70°C or 
colder. DO NOT ALLOW SAMPLES TO THAW.

Stored samples will not be used for any studies unrelated to this trial.
Buffy Coat Preparation

From each purple top tube remove and aliquot the “buffy coat”; be careful not to 
disturb the layer of red blood cells.

Store the aliquot of cells from each tube in one labeled polypropylene tube for 
cryopreservation.

Store the samples in the freezer at ≤ -70°C or colder. DO NOT ALLOW SAMPLES 
TO THAW.

Stored samples will not be used for any studies unrelated to this trial.
Streck Tube Processing
Fill tube completely. IMMEDIATELY mix the blood sample by gentle inversion 8-10 
times (do not shake). One inversion is a complete turn of the wrist (180 degrees and 
back). Store at ambient temperature (15-30°C). Stored samples will not be used for any 
studies unrelated to this trial.
7.1.2.11.3 Urine Sample Processing 
Sites should collect urine samples as noted in Section 7.1.2.9.1

Centrifuge the two 15 mL urine samples for 10 minutes at 3500 RPM.

The supernatant from each tube should be transferred into three 5 mL labeled 
polypropylene tube for cryopreservation.

The cell pellet from each tube should be re-suspended in 1 mL phosphate-buffered 
saline (PBS) and individually transferred into one labeled polypropylene tube. 
Centrifuge polypropylene tube at 3500 RPM for 10 minutes then aspirate the 
supernatant. The remaining cell pellet will be cryopreserved.

Store the samples in the freezer at ≤ -70°C or colder. DO NOT ALLOW 
SAMPLES TO THAW. 

Stored samples will not be used for any studies unrelated to this trial.
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7.1.3
Laboratory Procedures/Assessments
Details regarding specific laboratory procedures/assessments to be performed in this trial are 
provided below. Laboratory tests for hematology, chemistry, urinalysis, and others are 
specified in Table 5.  
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Table 5. Laboratory Tests
Hematology
Chemistry*
Urinalysis
Other
Hematocrit
Albumin
Blood
Serum β-human chorionic gonadotropin†
Hemoglobin
Alkaline phosphatase
Glucose
  (β-hCG)†
Platelet count
Alanine aminotransferase (ALT)
Protein
PT (INR)
WBC (total and differential)
Aspartate aminotransferase (AST)
Specific gravity 
aPTT
Red Blood Cell Count
Blood Urea Nitrogen & Creatinine
Microscopic exam (If abnormal) 
Total thriiodothyronine (T3)
Absolute Neutrophil Count
Carbon Dioxide ‡
results are noted
Free tyroxine (T4)
  (CO2 or bicarbonate)
Urine pregnancy test †
Thyroid stimulating hormone (TSH)
Calcium
Cortisol
Chloride
Blood/urine/stool for correlative studies
Glucose
 
Potassium  
 
Sodium
 
Total Bilirubin
 
Direct Bilirubin (If total bilirubin is 
elevated above the upper limit of 
normal)
Total protein
† Perform on women of childbearing potential only. If urine pregnancy results cannot be confirmed as negative, a serum pregnancy test will be required.  
‡ If considered standard of care in the site region.
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Laboratory tests for screening should be performed within 10 days prior to the first dose of 
treatment. After Cycle 1, pre-dose laboratory procedures can be conducted up to 72 hours prior 
to dosing. Results must be reviewed by the Investigator or qualified designee and found to be 
acceptable prior to each dose of trial treatment. 
7.1.4
Other Procedures
7.1.4.1
Withdrawal/Discontinuation
When a participant discontinues/withdraws prior to trial completion, all applicable activities 
scheduled for the final trial visit should be performed at the End of Treatment visit (which 
should be scheduled at earliest convenience per provider discretion, ideally within 4 weeks of 
discontinuation, if possible). Any adverse events which are present at the time of 
discontinuation/withdrawal should be followed in accordance with the safety requirements 
outlined in Section 7.2 - Assessing and Recording Adverse Events.  
7.1.5
Visit Requirements
Visit requirements are outlined in Section 6.0 - Trial Flow Chart.  Specific procedure-related 
details are provided above in Section 7.1 - Trial Procedures.
7.1.5.1
Screening
Screening is performed after attaining informed consent and within 12 weeks of diagnosis of 
MIBC by TURBT.
7.1.5.1.1 Screening Period
Screening should be completed as soon as possible and no later than 28 days from screening 
initiation.
7.1.5.2
Treatment Period
Treatment period is a 6-week neoadjuvant treatment after diagnosis of MIBC by TURBT and 
prior to RC. Treatment schedule will include aMVAC chemotherapy every 2 weeks (weeks 0, 
2, 4, and 6) and pembrolizumab every 3 weeks (weeks 0, 3 and 6).
7.1.5.3
Post-Treatment Visits
Post treatment visits will follow standard of care guidelines per pathologic stage at time of RC.
7.1.5.4
Safety Follow-Up Visit
The mandatory Safety Follow-Up Visit should be conducted after RC or before the initiation 
of a new anti-cancer treatment, whichever comes first. All AEs that occur prior to the Safety 
Follow-Up Visit should be recorded. Participants with an AE of Grade > 1 will be followed 
until the resolution of the AE to Grade 0-1 or until the beginning of a new anti-cancer therapy, 
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whichever occurs first. Study treatment-related SAEs that occur after the last dose of treatment 
or before initiation of a new anti-cancer treatment should also be recorded, if known.
7.1.5.5
Follow-up Visits
Participants who discontinue study treatment for a reason other than disease progression will 
move into the Follow-Up Phase and should be assessed per standard of care.  Every effort 
should be made to collect information regarding disease status until the start of new anti-cancer 
therapy, disease progression, death, end of the study.  
7.1.5.6
Survival Follow-up
Participants who experience confirmed disease progression or start a new anticancer therapy, 
will move into the Survival Follow-Up Phase and should be contacted by telephone if needed 
to assess for survival status until death, withdrawal of consent, or the end of the trial, whichever 
occurs first. 
7.2
Assessing and Recording Adverse Events
7.2.1
Definition of an Overdose for This Protocol and Reporting of Overdose to Merck
For purposes of this study, an overdose of pembrolizumab will be defined as any dose of 1,000 
mg or greater (≥5 times the indicated dose). No specific information is available on the 
treatment of overdose of pembrolizumab. In the event of overdose, the participant should be 
observed closely for signs of toxicity. Appropriate supportive treatment should be provided if 
clinically indicated.
If an adverse event(s) is associated with (“results from”) the overdose of a Merck product, the 
adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria are 
met.
If a dose of Merck’s product meeting the protocol definition of overdose is taken without any 
associated clinical symptoms or abnormal laboratory results, the overdose is reported as a non-
serious Event of Clinical Interest (ECI), using the terminology “accidental or intentional 
overdose without adverse effect.”
All reports of overdose with and without an adverse event must be reported within 2 working 
days hours to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215-661-6229)
7.2.2
Reporting of Pregnancy and Lactation to Merck
Although pregnancy and infant exposure during breast feeding are not considered adverse 
events, it is the responsibility of investigators or their designees to report any pregnancy or 
lactation in a participant (spontaneously reported to them) that occurs during the study.
Pregnancies and infant exposures during breastfeeding that occur after study therapy starts but 
before treatment allocation/randomization must be reported by the Investigator if they cause 
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the participant to be excluded from the trial or are the result of a protocol-specified 
intervention, including but not limited to washout or discontinuation of usual therapy, diet, 
placebo treatment or a procedure.  
Pregnancies and infant exposures during breastfeeding that occur from the time of treatment 
allocation through 7 months following cessation of the product, or 30 days following cessation 
of treatment if the participant initiates new anticancer therapy, whichever is earlier, must be 
reported by the investigator. All reported pregnancies must be followed to the 
completion/termination of the pregnancy. Pregnancy outcomes of spontaneous abortion, 
missed abortion, benign hydatidiform mole, blighted ovum, fetal death, intrauterine death, 
miscarriage and stillbirth must be reported as serious events (Important Medical Events). If 
the pregnancy continues to term, the outcome (health of infant) must also be reported.
Such events must be reported within 2 working days to Merck Global Safety. (Attn: Worldwide 
Product Safety; FAX: 215-661-6229)
7.2.3
Adverse Event
According to ICH guidelines (Federal Register. 1997; 62(90):25691-25709) and 21 CFR 
312.32, IND Safety Reports, and ICH E2A, Definitions and Standards for Expedited 
Reporting, an adverse event is defined as follows:
An adverse event is any untoward medical occurrence in a clinical investigation subject 
administered a medicinal product and which does not necessarily have a causal relationship 
with this treatment. An AE can therefore be any unfavorable and unintended sign (including 
an abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal product, whether or not considered related to the medicinal product.
Abnormal laboratory values for laboratory parameters specified in the study should not be 
recorded as an adverse event unless an intervention is required (repeat testing to confirm the 
abnormality is not considered intervention), the laboratory abnormality results in a serious 
adverse event or the adverse event results in study termination or interruption/discontinuation 
of study treatment.
Medical conditions present at screening (i.e., before the study treatment is administered) are 
not adverse events and should not be recorded on adverse event pages of the CRFs.  These 
medical conditions should be adequately documented on the subject chart.  However, medical 
conditions present at baseline that worsen in intensity or frequency during the treatment or 
post-treatment periods should be reported and recorded as adverse events.
7.2.4
Serious Adverse Events
An adverse event should be classified as an SAE if it meets one of the following criteria
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Fatal
Adverse event results in death.
Life threatening: 
The adverse events placed the subject at immediate risk of death. This 
classification did not apply to an adverse event that hypothetically might 
cause death if it were more severe. 
Hospitalization: 
It required or prolonged inpatient hospitalization. Hospitalizations for 
elective medical or surgical procedures or treatments planned before 
enrollment in the treatment plan or routine check-ups are not SAEs by 
this criterion. Admission to a palliative unit or hospice care facility is not 
considered to be a hospitalization. 
Disabling/incapacitating 
Resulted in a substantial and permanent disruption of the subject’s ability 
to carry out normal life functions. 
Congenital anomaly or birth 
defect: 
An adverse outcome in a child or fetus of a subject exposed to the 
molecule or treatment plan regimen before conception or during 
pregnancy. 
Medically significant: 
The adverse event did not meet any of the above criteria but could have 
jeopardized the subject and might have required medical or surgical 
intervention to prevent one of the outcomes listed above. 
7.2.5
Unexpected Adverse Event
An unexpected adverse event is defined as an event that has a nature or severity, or frequency 
that is not consistent with the applicable Investigator Brochure, or the prior medical condition 
of the subject or other treatment given to the subject.  “Unexpected,” as used in this definition, 
refers to an adverse drug experience that has not been previously observed and reported in 
preclinical or clinical studies rather than an experience that has not been anticipated based on 
the pharmacological properties of the study drug.
7.2.6
Monitoring and Recording Adverse Events
All AEs will be assessed by the Investigator or qualified designee and recorded in the CRFs. 
The Investigator should attempt to establish a diagnosis of the event on the basis of signs, 
symptoms and/or other clinical information. In such cases, the diagnosis should be documented 
as the adverse event and/or serious adverse event and not described as the individual signs or 
symptoms. The following information should be recorded:

Description of the adverse event using concise medical terminology

Description as to whether or not the adverse event is serious, noting all criteria that 
apply 

The start date (date of adverse event onset)

The stop date (date of adverse event resolution)

The severity (grade) of the adverse event 

A description of the potential relatedness of the adverse event to study drug, a study 
procedure, or other causality 
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
The action taken due to the adverse event

The outcome of the adverse event
Subjects will be followed for safety per iRAE clinical guidelines and standard clinical 
procedure if they terminate early or experience a non-serious AE considered to be possibly or 
definitely related to study treatment.
7.2.7
Grading Adverse Event Severity
All AEs will be graded in severity according to the NCI Common Terminology Criteria for 
Adverse Events (CTCAE) Version 5.0 (Table 6). If a CTCAE criterion does not exist, the 
Investigator should use the grade or adjectives: Grade 1 (mild), Grade 2 (moderate), Grade 3 
(severe), Grade 4 (life-threatening), or Grade 5 (fatal) to describe the maximum intensity of 
the adverse event. 
7.2.8
Attribution of an Adverse Event
Association or relatedness to the study agent will be assessed by the Investigator as follows:

Definite: The event follows a reasonable temporal sequence from exposure to the 
investigational agent, has been previously described in association with the 
investigational agent, and cannot reasonably be attributed to other factors such as the 
subject’s clinical state, other therapeutic interventions or concomitant medications; 
AND the event disappears or improves with withdrawal of the investigational agent 
and/or re-appears on re-exposure (e.g., in the event of an infusion reaction).

Probable: The event follows a reasonable temporal sequence from exposure to the 
investigational agent and has been previously been described in association with the 
investigational agent OR cannot reasonably be attributed to other factors such as the 
subject’s clinical state, other therapeutic interventions or concomitant medications.

Possible: The event follows a reasonable temporal sequence from exposure to the 
investigational agent but could be attributable to other factors such as the subject’s 
clinical state, other therapeutic interventions or concomitant medications.

Unlikely: Toxicity is doubtfully related to the investigational agent(s).  The event may 
be attributable to other factors such as the subject’s clinical state, other therapeutic 
interventions or concomitant medications.
Unrelated: The event is clearly related to other factors such as the subject’s clinical 
state, other therapeutic interventions or concomitant medications.
For general AE assessment, an AE is considered related if it is assessed as definitely, probably, 
or possibly related; unrelated if it is assessed as unlikely related or unrelated. 
7.2.9
Adverse Event Recording Period
AEs will be monitored and recorded in study-specific case report forms (CRFs) from the time 
of first exposure to an investigational product in this study (i.e., the start of the first \ infusion). 
AEs with an onset date prior to the first exposure to an investigational product will not be 
recorded. 
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7.2.10 Adverse Event Reporting Requirements 
7.2.10.1 Reporting to Merck
The Sponsor-Investigator or designee must report events to Merck (the financial sponsor of 
the study) as outlined in the contract. 
For the time period beginning at study therapy initiation through the safety follow up visit 
following cessation of treatment, or 30 days following cessation of treatment if the participant 
initiates new anticancer therapy, whichever is earlier, any serious adverse event, or follow up 
to a serious adverse event, including death due to any cause whether or not related to the Merck 
product, must be reported within 2 working days to Merck Global Safety.
Additionally, any serious adverse event, considered by an Investigator who is a qualified 
physician to be related to Merck product that is brought to the attention of the investigator at 
any time following consent through the end of the specified safety follow-up period specified 
in the paragraph above, or at any time outside of the time period specified in the previous 
paragraph also must be reported when feasible to Merck Global Safety.
All participants with serious adverse events must be followed up for outcome.
SAE reports and any other relevant safety information are to be forwarded to Merck Global Safety 
facsimile number:  +1-215-661-6229.
A copy of all IND Safety Reports and Annual Progress Reports is submitted as required by 
FDA., The Sponsor-Investigator will submit a copy of these reports to Merck & Co., Inc. (Attn: 
Worldwide Product Safety; FAX 215-661-6229) at the time of submission to FDA.
7.2.10.2 Reporting to IRB
The Sponsor-Investigator or designee must report events to the Fred Hutch IRB in accordance 
with the policies of the IRB.
7.2.11 FDA Reporting Requirements
All Adverse Events will be reported by the Sponsor-Investigator to regulatory 
authorities, IRB/IECs and investigators in accordance with all applicable global laws 
and regulations.
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Table 6. Evaluating Adverse Events
An Investigator who is a qualified physician, will evaluate all adverse events as to CTCAE v5.0. 
V5.0 CTCAE 
Grading
Grade 1
Mild; asymptomatic or mid symptoms; clinical or diagnostic observations only; intervention not indicated.
Grade 2
Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate instrumental ADL.
Grade 3
Severe or medically significant but not immediately life-threatening; hospitalization or prolongation or hospitalization indicated; disabling; 
limiting self-care ADL.
Grade 4
Life threatening consequences; urgent intervention indicated.
Grade 5
Death related to AE
Seriousness
A serious adverse event is any adverse event occurring at any dose or during any use of Merck product that:
†Results in death; or
†Is life threatening; or places the participant, in the view of the investigator, at immediate risk of death from the event as it occurred (Note: This does not include an 
adverse event that, had it occurred in a more severe form, might have caused death.); or
†Results in a persistent or significant disability/incapacity (substantial disruption of one’s ability to conduct normal life functions); or
†Results in or prolongs an existing inpatient hospitalization (hospitalization is defined as an inpatient admission, regardless of length of stay, even if the 
hospitalization is a precautionary measure for continued observation. (Note: Hospitalization for an elective procedure to treat a pre-existing condition that has not 
worsened is not a serious adverse event.  A pre-existing condition is a clinical condition that is diagnosed prior to the use of a Merck product and is documented in the 
patient’s medical history.); or
†Is a congenital anomaly/birth defect (in offspring of participant taking the product regardless of time to diagnosis);or
Is a new cancer (that is not a condition of the study) (although not serious per ICH definition, is reportable within 24 hours and to Merck within 2 working days to 
meet certain local requirements); or
Is an overdose (whether accidental or intentional).  Any adverse event associated with an overdose is considered a serious adverse event for collection purposes. An 
overdose that is not associated with an adverse event is considered a non-serious event of clinical interest and must be reported to Merck within 2 working days.

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Other important medical events that may not result in death, not be life threatening, or not require hospitalization may be considered a serious adverse event when, 
based upon appropriate medical judgment, the event may jeopardize the participant and may require medical or surgical intervention to prevent one of the outcomes 
listed previously (designated above by a †).
Duration
Record the start and stop dates of the adverse event. If less than 1 day, indicate the appropriate length of time and units
Action taken 
Did the adverse event cause Merck product to be discontinued?
Relationship to 
Merck Product 
Did Merck product cause the adverse event? The determination of the likelihood that Merck product caused the adverse event will be provided by an Investigator who 
is a qualified physician. The investigator’s signed/dated initials on the source document or worksheet that supports the causality noted on the AE form, ensures that a 
medically qualified assessment of causality was done. This initialed document must be retained for the required regulatory time frame. The criteria below are intended 
as reference guidelines to assist the Investigator in assessing the likelihood of a relationship between the test drug and the adverse event based upon the available 
information.
The following components are to be used to assess the relationship between Merck product and the AE; the greater the correlation with the components and their 
respective elements (in number and/or intensity), the more likely Merck product caused the adverse event (AE):
Exposure
Is there evidence that the participant was actually exposed to Merck product such as: reliable history, acceptable compliance assessment (pill count, 
diary, etc.), expected pharmacologic effect, or measurement of drug/metabolite in bodily specimen?
Time Course
Did 
the 
AE 
follow 
in 
a 
reasonable 
temporal 
sequence 
from 
administration 
of 
Merck 
product? 
Is the time of onset of the AE compatible with a drug-induced effect (applies to trials with investigational medicinal product)?
Likely Cause
Is the AE not reasonably explained by another etiology such as underlying disease, other drug(s)/vaccine(s), or other host or environmental factors

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Relationship
The following components are to be used to assess the relationship between the test drug and the AE: (continued)
to Merck 
Product
(continued) 
Dechallenge
Was Merck product discontinued or dose/exposure/frequency reduced?
If yes, did the AE resolve or improve?
If yes, this is a positive dechallenge. If no, this is a negative dechallenge.
(Note: This criterion is not applicable if: (1) the AE resulted in death or permanent disability; (2) the AE resolved/improved despite continuation of 
the product; or (3) the trial is a single-dose drug trial); or (4) Merck’s product(s) is/are only used one time.)
Rechallenge
Was the participant re-exposed to Merck product in this study?
If yes, did the AE recur or worsen?
If yes, this is a positive rechallenge. If no, this is a negative rechallenge.
(Note: This criterion is not applicable if: (1) the initial AE resulted in death or permanent disability, or (2) the trial is a single-dose drug trial); or (3) 
Merck product(s) is/are used only one time).
NOTE: IF A RECHALLENGE IS PLANNED FOR AN ADVERSE EVENT WHICH WAS SERIOUS AND WHICH MAY HAVE BEEN 
CAUSED BY MERCK PRODUCT, OR IF REEXPOSURE TO MERCK PRODUCT POSES ADDITIONAL POTENTIAL SIGNIFICANT RISK 
TO THE PARTICIPANT, THEN THE RECHALLENGE MUST BE APPROVED IN ADVANCE BY THE SPONSOR-INVESTIGATOR AS PER 
DOSE MODIFICATION GUIDELINES IN THE PROTOCOL.
Consistency 
with Trial 
Treatment 
Profile
Is the clinical/pathological presentation of the AE consistent with previous knowledge regarding Merck product or drug class pharmacology or 
toxicology?
The assessment of relationship will be reported on the case report forms /worksheets by an Investigator who is a qualified physician according to his/her best clinical judgment, including 
consideration of the above elements.
Record one of the following
Use the following scale of criteria as guidance (not all criteria must be present to be indicative of Merck product relationship).
Yes, there is a reasonable 
possibility of Merck product 
relationship.
There is evidence of exposure to Merck product.  The temporal sequence of the AE onset relative to the administration of Merck product is reasonable.  
The AE is more likely explained by Merck product than by another cause.
No, there is not a reasonable 
possibility of Merck product 
relationship
Participant did not receive the Merck product OR temporal sequence of the AE onset relative to administration of Merck product is not reasonable 
OR the AE is more likely explained by another cause than the Merck product.  (Also entered for a participant with overdose without an associated 
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8.0 STATISTICAL CONSIDERATIONS 
The primary objective is to evaluate the pCR rate at RC with neoadjuvant aMVAC and 
pembolizumab in non-UC predominant histologic variants. A pCR rate of 8% or less would be 
considered of insufficient activity based on historical data for TURBT alone. A design with 
91% exact power to rule out an 8% pCR rate at the 1-sided 4% exact level, if the true pCR rate 
is 36% would require 17 patients. The observation of at least 4 patients with pCR (23.5% pCR 
rate) would be considered evidence to rule out a pCR rate of 8%. The calculations are based 
on the one-arm binomial calculator at www.swogstat.org.  
The anticipated duration of accrual is 17-26 months (1 patient every 30-45 days) given low 
prevalence (10-25%) of non-UC predominant histologic variants. However, if after the study 
has been active for 18 months and 8 or fewer patients have been enrolled, the accrual goal may 
be modified to target a total of 14 patients. In this case, the design would have 96% exact power 
to rule out an 8% pCR rate at the 1-sided 10% exact level, if the true pCR rate is 40%. The 
observation of at least 3 patients with pCR (21.4% pCR rate) would be considered evidence to 
rule out a pCR rate of 8%.
Secondary objectives include an evaluation of the frequency and severity of toxicity of the 
regimen, evaluation of recurrence-free survival at 2 years and TIL density at RC. With 17 
patients, binary proportions can be estimated to within 24% with 95% confidence. Any toxicity 
with true prevalence of 10% or greater is likely to be observed with an 83% chance. The 
feasibility of the regimen will be evaluated by the percentage of patients able to undergo RC 
within 10 weeks from end of study therapy. This proportion along with 95% confidence 
intervals will be evaluated. The relationship (or lack thereof) between any delay of RC beyond 
10 weeks and study therapy will be described. 
Distributions of time-to-event outcomes will be estimated using the method of Kaplan-Meier.  
The rates at specified time points will use these estimates and the associated 95% confidence 
intervals. Continuous outcomes, such as TIL density and PD-L1 expression levels will be 
summarized by means, medians, and quantiles. Changes in continuous outcomes (such as CD8, 
TILs) will be evaluated as both absolute and percentage change. Descriptive statistics will also 
be used when needed. 
9.0 DATA AND SAFETY MONITORING PLAN
9.1
Safety of pembrolizumab and chemotherapy
Multiple trials are presently underway (including neoadjuvant trials) investigating the 
combination of platinum-based chemotherapy and anti-PD-1/PD-L1 agents in MIBC. 
Two large phase III randomized trials (Keynote361 and IMvigor130) in the first line treatment 
setting of advanced UC are investigating the role of the combination of chemotherapy plus 
anti-PD-1/PD-L1 agents; both trials have completed accrual and results are anticipated soon. 
Preliminary results from IMvigor 130 showed that the combination of platinum-based 
chemotherapy was safe for patient with aUC. The combination of platinum-based 
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chemotherapy plus pembrolizumab has also been FDA-approved in metastatic NSCLC, further 
supporting the safety and feasibility for the combination. 
Moreover, recent data showed significant efficacy of anti-PD-1/PD-L1 agents as neoadjuvant 
immunotherapy in MIBC. The single arm phase II PURE-01 trial enrolled patients with T2-
3bN0M0 MIBC regardless of cisplatin eligibility and met its primary endpoint (based on pCR 
rate).39 Three doses of pembrolizumab led to pCR (pT0 in intention-to-treat population) in 42% 
of patients, while most patients with pCR had PD-L1 combined positive score ≥ 10. These 
results are very encouraging as the pCR with single-agent pembrolizumab appears comparable 
to 38% pCR with neoadjuvant conventional-dose MVAC, though it warrants investigation in 
larger randomized trials. Furthermore, in the single-arm phase II ABACUS trial, two doses of 
neoadjuvant atezolizumab led to pCR 29% in cisplatin-unfit patients with T2-4N0M0 MIBC.40 
pCR included pT0 (24%) and pTcis (6%); primary endpoint was pCR in ≥20% patients. PD-
L1 positive status (≥5% in TILs based on SP142 assay) was noted in almost half of the patients 
and pCR rates were 38% in PD-L1-positive and 27% in PD-L1-negative tumors. Both studies 
confirmed the safety of neoadjuvant immunotherapy. Data from the NABUCCO trial further 
supports the safety of neoadjuvant immunotherapy. In this trial, neoadjuvant combination 
immune checkpoint inhibitor blockade with nivolumab and anti-CTLA-4 ipilimumab was 
shown to be safe among the first 24 patients enrolled with 23 of 24 proceeding to cystectomy 
on schedule; pathologic complete response was 46% in this trial. Finally, NCT02365766 trial 
is a phase Ib/II study, currently evaluating the combination of neoadjuvant chemotherapy plus 
pembrolizumab in cisplatin-eligible (cohort I) and ineligible (cohort II) patients with T2-
4aN0M0 MIBC.41 In cohort I, therapy consisted of pembrolizumab plus cisplatin-based 
chemotherapy, while in cohort II of pembrolizumab plus gemcitabine. Available data from 40 
evaluable patients from cohort I demonstrated feasibility and safety of the combination, with 
10% (4/40) of patients not proceeding to RC but only one of which due to toxicity. 
9.2
Monitoring Plan
Institutional support of trial monitoring will be in accordance with the Fred Hutchinson Cancer 
Center/University of Washington Cancer Consortium Institutional Data and Safety Monitoring 
Plan. Under the provisions of this plan, Fred Hutch Clinical Research Support (CRS) 
coordinates data and compliance monitoring conducted by consultants, contract research 
organizations, or Fred Hutch employees unaffiliated with the conduct of the 
study. Independent monitoring visits occur at specified intervals determined by the assessed 
risk level of the study and the findings of previous visits per the institutional DSMP. 
In addition, protocols are reviewed at least annually and as needed by the Consortium Data 
and Safety Monitoring Committee (DSMC), Fred Hutch Scientific Review Committee (SRC) 
and the Fred Hutch/University of Washington Cancer Consortium Institutional Review Board 
(IRB).  The review committees evaluate accrual, adverse events, stopping rules, and adherence 
to the applicable data and safety monitoring plan for studies actively enrolling or treating 
subjects.  The IRB reviews the study progress and safety information to assess continued 
acceptability of the risk-benefit ratio for human subjects.  Approval of committees as 
applicable is necessary to continue the study.
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Due to the very small sample size and existing safety data on the combination of chemotherapy 
and anti-PD1 agents, no interim stopping rules were included in the statistical considerations. 
However, the experienced research team, statistician and Sponsor-Investigator will monitor 
closely the trial, which will comply with the standard guidelines set forth by these regulatory 
committees and other institutional, state and federal guidelines.
10.0 LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL 
SUPPLIES
10.1 Investigational Product 
The Sponsor-Investigator and designees shall take responsibility for and shall take all steps to 
maintain appropriate records and ensure appropriate supply, storage, handling, distribution and 
usage of investigational product in accordance with the protocol and any applicable laws and 
regulations. Pembrolizumab will be provided by Merck as summarized in Table 7.
Table 7. Product Descriptions
Product Name & Potency
Dosage Form
Pembrolizumab 100 mg/4mL
Solution for Injection
10.2 Packaging and Labeling Information
Supplies will be labeled in accordance with regulatory requirements.
10.3 Clinical Supplies Disclosure
This trial is open-label; therefore, the participant, the trial site personnel, the Sponsor-
Investigator and/or designees are not blinded to treatment. Drug identity (name, strength) is 
included in the label text; random code/disclosure envelopes or lists are not provided.
10.4 Storage and Handling Requirements
Clinical supplies must be stored in a secure, limited-access location under the storage 
conditions specified on the label.  
Receipt and dispensing of trial medication must be recorded by an authorized person at the 
trial site.
Clinical supplies may not be used for any purpose other than that stated in the protocol.
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10.5 Returns and Reconciliation
The Sponsor-Investigator and designees is responsible for keeping accurate records of the 
clinical supplies received from Merck or designee, the amount dispensed to and returned by 
the participants and the amount remaining at the conclusion of the trial. 
Upon completion or termination of the study, all unused and/or partially used investigational 
product will be destroyed at the site per institutional policy. It is the Investigator’s 
responsibility to arrange for disposal of all empty containers, provided that procedures for 
proper disposal have been established according to applicable federal, state, local and 
institutional guidelines and procedures, and provided that appropriate records of disposal are 
kept.
10.6 Methotrexate
10.6.1 Other Names
Additional name is amethopterin. 
10.6.2 Classification 
Antimetabolite
10.6.3 Mode of Action
Methotrexate acts as an antimetabolite to interfere with DNA synthesis, repair and 
cellular replication. Methotrexate inhibits dihydrofolic acid reductase which acts to 
reduce dihydrofolate to tetrahydrofolate. Tetrahydrofolate is an essential metabolite 
used for synthesis of purine nucleotides and thymidylate.
10.6.4 Preparation and Administration
Methotrexate should be prepared and administered per pharmacy institutional policy 
(standard of care). 
10.6.5 Storage and Stability
Clinical supplies must be stored in a secure, limited-access location. Methotrexate vials 
should be stored at controlled room temperature of 25°C with excursions permitted to 
15°C to 30°C and should be protected from light. 
The administration of methotrexate must be completed within 24 hours of preparation. 
If not used immediately, the infusion solution may be stored at room temperature (15°-
30°C, 59°-86°F) for up to 24 hours. 
10.6.6 Drug Interactions
See Package Insert for full summary of drug interactions. Notably, no interactions 
reported with vinblastine, doxorubicin, cisplatin, GCSF or pembrolizumab.
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10.6.7 Agent Availability
Methotrexate supply will be managed per institutional pharmacy policy (standard of 
care). 
10.6.8 Agent Ordering
Pharmacy will be responsible for ordering per institutional policy (standard of care).
10.6.9  Agent Accountability
Methotrexate will be stored and accessed per pharmacy institutional policy (standard 
of care).
10.6.10 Side Effects
See Package Insert for full summary of side effects.
10.7 Vinblastine
10.7.1 Other Names
Additional names include vinblastine sulfate and vincaleukoblastine. 
10.7.2 Classification 
Plant alkaloid
10.7.3 Mode of Action
Vinblastine inhibits microtubule formation in the mitotic spindle, resulting in an arrest 
of dividing cells at the metaphase stage. 
10.7.4 Preparation and Administration
Vinblastine should be prepared and administered per pharmacy institutional policy 
(standard of care). 
Vinblastine is a vesicant so care with administration to minimize perivenous 
infiltrations is imperative. Leakage into surrounding tissue during intravenous 
administration of vinblastine sulfate may cause considerable irritation. If extravasation 
occurs, the injection should be discontinued immediately, and any remaining portion of 
the dose should then be introduced into another vein. 
10.7.5 Storage and Stability
Clinical supplies must be stored in a secure, limited-access location. Vinblastine vials 
should be stored refrigerated at 2° to 8°C (36° to 46°F) to assure extended stability. 
The administration of vinblastine infusion must be completed within 24 hours of 
preparation. If not used immediately, the infusion solution may be stored at room 
temperature (20°-25°C, 68°-77°F) for up to 24 hours. 
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10.7.6 Drug Interactions
See Package Insert for full summary of drug interactions. Notably, no interactions 
reported with methotrexate, doxorubicin, cisplatin, GCSF or pembrolizumab.
10.7.7 Agent Availability
Vinblastine supply will be managed per institutional pharmacy policy (standard of 
care). 
10.7.8 Agent Ordering
Pharmacy will be responsible for ordering per institutional policy (standard of care).
10.7.9  Agent Accountability
Vinblastine will be stored and accessed per pharmacy institutional policy (standard of 
care).
10.7.10 Side Effects
See Package Insert for full summary of side effects.
10.8 Doxorubicin
10.8.1 Classification 
Anthracycline antibiotic
10.8.2 Mode of Action
Doxorubicin binds to nucleic acids by specific intercalation of the planar anthracycline 
nucleus with the DNA double helix. 
10.8.3 Preparation and Administration
Doxorubicin should be prepared and administered per pharmacy institutional policy 
(standard of care). 
Doxorubicin is a vesicant so care should be taken with administration to reduce the 
chance of perivenous infiltration. If any signs or symptoms of extravasation have 
occurred, the injection should be terminated and restarted in another vein.
10.8.4 Storage and Stability
Clinical supplies must be stored in a secure, limited-access location. Doxorubicin vials 
should be stored refrigerated at 2° to 8°C (36° to 46°F) and should be protected from 
light. 
The administration of doxorubicin must be completed within 24 hours of preparation. 
If not used immediately, the infusion solution may be stored at room temperature (20°-
25°C, 68°-77°F) for up to 24 hours. Opened vials can be stored in a hood for up to 6 
hours at room temperature (20°-25°C, 68°-77°F). 
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10.8.5 Drug Interactions
See Package Insert for full summary of drug interactions. Notably, no interactions 
reported with methotrexate, vinblastine, cisplatin, GCSF or pembrolizumab.
10.8.6 Agent Availability
Doxorubicin supply will be managed per institutional pharmacy policy (standard of 
care). 
10.8.7 Agent Ordering
Pharmacy will be responsible for ordering per institutional policy (standard of care).
10.8.8 Agent Accountability
Doxorubicin will be stored and accessed per pharmacy institutional policy (standard of 
care).
10.8.9  Side Effects
See Package Insert for full summary of side effects.
10.9 Cisplatin
10.9.1 Other Names
Additional names include CDDP.
10.9.2 Classification 
Platinum analog
10.9.3 Mode of Action
Chlorine atoms of cisplatin are subject to chemical displacement by nucleophiles, such 
as water or sulfhydryl groups causing direct damage to DNA.
 
10.9.4 Preparation and Administration
Cisplatin should be prepared and administered per pharmacy institutional policy 
(standard of care). 
10.9.5 Storage and Stability
Clinical supplies must be stored in a secure, limited-access location. Cisplatin vials 
should be stored at room temperature of 25°C (77°F). 
The administration of cisplatin must be completed within 24 hours of preparation. If not 
used immediately, the infusion solution may be stored at room temperature (20°-25°C, 
68°-77°F) for up to 24 hours. 
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10.9.6 Drug Interactions
See Package Insert for full summary of side effects. Notably, no side effects reported 
with vinblastine, doxorubicin, cisplatin, G-CSF or pembrolizumab.
10.9.7 Agent Availability
Cisplatin supply will be managed per institutional pharmacy policy (standard of care). 
10.9.8 Agent Ordering
Pharmacy will be responsible for ordering per institutional policy (standard of care).
10.9.9  Agent Accountability
Cisplatin will be stored and accessed per pharmacy institutional policy (standard of 
care).
10.9.10 Side Effects
See Package Insert for full summary of side effect.
11.0 INVESTIGATOR OBLIGATIONS
The Sponsor-Investigator is responsible for the conduct of the clinical trial at the site and is 
responsible for personally overseeing the treatment of all study subjects. The Sponsor-
Investigator must assure that all study site personnel, including Sub-Investigators and other 
study staff members, adhere to the study protocol and to all applicable regulations and 
guidelines regarding clinical trials both during and after study completion.
All subjects are informed of the nature of the program, its possible hazards, and their right to 
withdraw at any time, and each subject signs a form indicating their consent to participate prior 
to receiving any study-related procedures.
12.0 ADMINISTRATIVE AND REGULATORY DETAILS
12.1 Pre-Study Documentation
The following documentation required by the FDA must be received by Merck, or its designee, 
prior to initiation of the trial: FDA Form 1572; curricula vitae of the Sponsor-Investigator and 
all Sub-Investigators; signed Protocol Agreement; copy of the correspondence from the 
IRB/EC indicating approval of the protocol and Informed Consent Forms, signed by the 
IRB/EC chairperson or designee; an IRB/EC membership list containing the names and 
occupations of the IRB/EC members; copy of the Informed Consent Forms that were reviewed 
and approved by the IRB/EC.
12.2 Study Site Training
Before initiation of the study, the Sponsor-Investigator, or its designated representatives will 
review and discuss the following items with the Sub-Investigators and clinic staff: the protocol, 
study procedures, record keeping and administrative requirements, drug accountability, AE 
reporting, Good Clinical Practice guidelines, CRF/electronic case report form (eCRF) 
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completion guidelines, monitoring requirements, and the ability of the site to satisfactorily 
complete the protocol. Additional documents with instructions for study compliance and 
CRF/eCRF completion will be provided.
12.3 Documentation
The documentation of clinical data must be stored by the Sponsor-Investigator according to 
legal requirements. The Sponsor-Investigator and study staff have responsibility for 
maintaining a comprehensive and centralized filing system containing all study-related 
documentation. These files must be suitable for inspection by Merck, the FDA, and/or other 
applicable regulatory agencies/competent authorities at any time, and should consist of the 
following elements: subject files (complete medical records, laboratory data, supporting source 
documentation, and the Informed Consent); study files (the protocol with all amendments, 
copies of all pre-study documentation, and all correspondence between the Competent 
Authorities, IRB/EC, site, and Sponsor); and drug accountability files, containing a complete 
account of the receipt and disposition of the study drug.
12.4 Access to Source Data
The Sponsor-Investigator will permit monitoring to be conducted by consultants, contract 
research organizations, or Fred Hutch employees unaffiliated with the conduct of the study 
to determine that protocol adherence and data recording are satisfactory. The CRF/eCRF and 
related source documents will be reviewed in detail by the Sponsor-Investigator 
representative at each site visit. Only original source documents are acceptable for review. 
This review includes inspection of data acquired as a requirement for participation in this 
study and other medical records as required to confirm information contained in the 
CRF/eCRF, such as past history, secondary diagnoses, and concomitant medications. Other 
study records, such as correspondence with Merck, the Sponsor-Investigator, the Competent 
Authorities, IRB/EC, and screening and drug accountability logs will also be inspected. All 
source data and study records must also be available for inspection by representatives of the 
FDA or other regulatory agencies.
12.5 Data Collection
eCRFs must be completed and submitted for each subject enrolled in the study. Any changes 
or corrections made to the CRF/eCRF must be subsequently reviewed and signed by the PI. 
All data fields in the CRF/eCRF must be completed to avoid queries. Study data were collected 
and managed using Research Electronic Data Capture (REDCap)53 electronic data capture 
tools hosted at the Institute of Translational Health Sciences. REDCap is a secure web-based 
designed to support data capture of research studies.
12.6 Protocol Interpretation and Compliance
The procedures defined in the protocol are carefully reviewed by the Sponsor-Investigator, 
Sub-Investigators, and clinic staff prior to the time of study initiation to ensure accurate 
representation and implementation. Protocol amendments, if any, are reviewed and 
implemented promptly following IRB/EC and relevant Competent Authorities approval. The 
Sponsor-Investigator is responsible for submitting protocol amendments to the FDA as 
described in 21 CFR § 312.30 (Protocol Amendments) and other regulatory agencies 
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according to national, state or local requirements. The Sponsor-Investigator, or designee, is 
always available to answer protocol- or subject-related questions.
12.7 Study Monitoring and Data Collection
A representative from Fred Hutch Clinical Research Support (consultant, contract research 
organizations, or Fred Hutch unaffiliated employee) will visit the study center periodically to 
monitor adherence to the protocol, applicable FDA regulations and/or other regulatory 
agencies national, state or local requirements, and the maintenance of adequate and accurate 
clinical records. eCRFs are reviewed to ensure that key safety and efficacy data are collected 
and recorded as specified by the protocol. The Fred Hutch Clinical Research Support 
monitoring designee is permitted to access subject medical records, laboratory data and other 
source documentation as needed to appropriately monitor the trial.
12.8 Disclosure of Data/Publication
Individual subject medical information obtained as a result of this study is considered 
confidential and disclosure to third parties other than those noted below is prohibited. Such 
medical information may be given to the subject’s personal physician or to other appropriate 
medical personnel responsible for the subject’s welfare. Data generated as a result of this study 
are to be available for inspection on request by the FDA or other regulatory agencies, Merck 
or its designee, and by the IRB/EC.
It is anticipated that the final results of this study will be submitted to a peer-reviewed scientific 
journal. Authorship on such a paper will be acknowledged with customary scientific practice. 
As such, without the expressed permission of Merck, only clinical Study data relating the Study 
as a whole will be published. If permission is granted by Merck for publication of ancillary 
data, prior to submission for publication of any manuscript or presentation of any poster, 
presentation, abstract or other written or oral material that describes the results of Study, 
Institution and/or Sponsor-Investigator shall provide Merck at least 60 days (or as otherwise 
specified in the sites executed Clinical Trial Agreement) to review any such materials. Such 
materials shall not divulge any of Merck’s Confidential Information, and Institution and/or 
Sponsor-Investigator shall promptly remove any Confidential Information as requested by 
Merck. If requested by Merck, the Sponsor-Investigator and Institution shall delay the 
submission of any publication or presentation up to 60 days from the date of Merck’s request 
for such a delay. In addition, Merck has the right to require that any publication or presentation 
concerning the Study will acknowledge Merck’s support.
12.9 Ethical Considerations
The Sponsor-Investigator agrees to conduct this study in accordance with applicable United 
States FDA clinical trial regulations and guidelines, applicable United States FDA clinical trial 
regulations and guidelines, the ICH (E6) GCP guidelines, the European Union Directive 
2001/20/EC for clinical trials conducted in the European Union, the IRB/EC and local legal 
requirements and with the Declaration of Helsinki (1989). The Sponsor-Investigator will 
conduct all aspects of this study in accordance with all national, state, and local laws of the 
applicable regulatory agencies.
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12.10 Informed Consent
The Sponsor-Investigator assumes the responsibility of obtaining written Informed Consent 
for each subject or the subject’s legally authorized representative before any study-specific 
procedures are performed.
Subjects meeting the criteria set forth in the protocol will be offered the opportunity to 
participate in the study. To avoid introduction of bias, the Sponsor-Investigator must exercise 
no selectivity with regard to offering eligible subjects the opportunity to participate in the 
study. Subjects or parents/legal guardians of all candidate subjects will receive a 
comprehensive explanation of the proposed treatment, including the nature of the therapy, 
alternative therapies available, any known previously experienced adverse reactions, the 
investigational status of the study drug, and other factors that are part of obtaining a proper 
Informed Consent. Subjects will be given the opportunity to ask questions concerning the 
study, and adequate time to consider their decision to or not to participate.
Informed Consent will be documented by the use of a written Consent Form that includes all 
the elements required by FDA regulations and ICH guidelines. The Sponsor-Investigator or 
designee will review the informed consent prior to submission to the IRB/EC. The form is to 
be signed and dated by the subject or subject's legally authorized representative and by the 
person who administers the consent process. A copy of the signed form will be given to the 
person who signed it, the original signed Consent Form will be filed with the subject’s medical 
records, and copy maintained with the subject’s study records. The date and time of time of 
the Informed Consent must be recorded in the source documents.
If an amendment to the protocol changes the subject participation schedule in scope or activity, 
or increases the potential risk to the subject, the Informed Consent Form must be amended. 
Any amended Informed Consent must be reviewed by the Sponsor-Investigator or designee 
and approved by the IRB/EC prior to use. The revised Informed Consent Form must be used 
to obtain re-consent from any subjects currently enrolled in the study if the subject is affected 
by the amendment and must be used to document consent from any new subjects enrolled after 
the approval date of the amendment.
12.11 Institutional Review Board/Ethics Committee
The Sponsor-Investigator will assure that an appropriately constituted IRB/EC that complies 
with the requirements of 21 CFR Section 56 or written assurance of compliance with ICH (E6) 
guidelines will be responsible for the initial and continuing review and approval of the clinical 
study. Before initiation of the study, the Sponsor-Investigator or designee will forward copies 
of the protocol and Consent Form to be used for the study to the IRB/EC for its review and 
approval. A photocopy of the IRB/EC notification of approval must be forwarded to Merck 
before any investigational supplies will be shipped to the Sponsor-Investigator.
The Sponsor-Investigator or designee will also assure that all changes in the research activity 
and all unanticipated problems involving risks to human subjects or others will be reported 
promptly to the IRB/EC, and that no changes will be made to the protocol without prior Merck 
and IRB/EC approval, except where necessary to eliminate apparent immediate hazards to 
human subjects.
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Copies of all study-related correspondence between the Sponsor-Investigator and the IRB/EC 
must be provided to Merck by the Sponsor-Investigator or designee. The Sponsor-Investigator 
or designee must promptly notify the IRB/EC of any SAE occurring at the site and of any 
safety reports that meet the reporting criteria of the IRB of record (e.g., IND Safety Reports) 
received from Merck, and must copy Merck on that correspondence.
The Sponsor-Investigator or designee will be responsible for submitting periodic progress 
reports to the IRB/EC at intervals appropriate to the degree of subject risk involved in the 
study, but not less than once per year and at the completion or termination of the study.
12.12 Subject Privacy
Merck, the Sponsor-Investigator, and designees affirm and uphold the principle of the subject's 
right to privacy. Merck, the Sponsor-Investigator, and designees shall comply with applicable 
national and local privacy laws.
To verify compliance with this protocol, the Sponsor-Investigator will permit monitoring to be 
conducted by consultants, contract research organizations, or Fred Hutch employees 
unaffiliated with the conduct of the study to monitor the study as frequently as the DSMC 
deems necessary to review the subject's original medical records. Should access to such 
medical records require a waiver or authorization separate from the statement of Informed 
Consent, the Investigator will obtain such permission in writing from the subject before the 
subject is entered into the study.
13.0 STOPPING THE STUDY 
The Sponsor-Investigator may decide to stop the study at any point, for any reason. The 
following reasons will lead to premature termination of the trial:

New convincing information leading to unfavorable risk-benefit assessment of IP, 
including occurrence of significant toxicity associated with IP;

Sponsor-Investigator’s decision that continuation of the trial is unjustifiable for 
medical or ethical reasons;

Discontinuation of development of IP.
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15.0 APPENDICES
Appendix 1: ECOG Performance Status
Grade
Description
0
Normal activity. Fully active, able to carry on all pre-disease 
performance without restriction.
1
Symptoms, but ambulatory. Restricted in physically strenuous 
activity, but ambulatory and able to carry out work of a light or 
sedentary nature (e.g., light housework, office work).
2
In bed <50% of the time. Ambulatory and capable of all self-care, but 
unable to carry out any work activities. Up and about 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 self-care. 
Totally confined to bed or chair.
5
Dead.
* As published in Am. J. Clin. Oncol.:  Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., 
McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology 
Group. Am J Clin Oncol 5:649-655, 1982. The Eastern Cooperative Oncology Group, Robert Comis 
M.D., Group Chair.
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Appendix 2: Contraceptive Guidance and Pregnancy Testing
Woman of Childbearing Potential (WOCBP)
A woman is considered fertile following menarche and until becoming post-menopausal 
unless permanently sterile (see below) 
Women in the following categories are not considered WOCBP:
●
Premenarchal
●
Premenopausal female with 1 of the following:
○
Documented hysterectomy
○
Documented bilateral salpingectomy
○
Documented bilateral oophorectomy
Note:  Documentation can come from the site personnel’s review of the participant’s 
medical records, medical examination, or medical history interview.
●
Postmenopausal female
○
A postmenopausal state is defined as no menses for 12 months without an alternative 
medical cause. 
▪
A high follicle stimulating hormone (FSH) level in the postmenopausal range may 
be used to confirm a postmenopausal state in women not using hormonal 
contraception or hormonal replacement therapy (HRT). However, in the absence 
of 12 months of amenorrhea, confirmation with two FSH measurements in the 
postmenopausal range is required. 
○
Females on HRT and whose menopausal status is in doubt will be required to use one of 
the non-hormonal highly effective contraception methods if they wish to continue their 
HRT during the study. Otherwise, they must discontinue HRT to allow confirmation of 
postmenopausal status before study enrollment.
Contraception Requirements
Male Participants:
Male participants with female partners of childbearing potential are eligible to participate if 
they agree to one of the following during the protocol defined time frame in section X: 
●
Be abstinent from penile-vaginal intercourse as their usual and preferred lifestyle 
(abstinent on a long term and persistent basis) and agree to remain abstinent
●
Use a male condom plus partner use of a contraceptive method with a failure rate of <1% 
per year as described in Table 8 when having penile-vaginal intercourse with a woman of 
childbearing potential who is not currently pregnant.
○
Note: Men with a pregnant or breastfeeding partner must agree to remain abstinent 
from penile-vaginal intercourse or use a male condom during each episode of penile 
penetration.
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Female Participants:
Female participants of childbearing potential are eligible to participate if they agree to use a 
highly effective method of contraception that has a low user dependency consistently and 
correctly as described in Table 8 during the protocol-defined time frame in the protocol.  
Table 8. Highly Effective Contraceptive Methods That Have Low User Dependency
Highly Effective Methods That Have Low User Dependency 
Failure rate of <1% per year when used consistently and correctly.
●
Progestogen- only contraceptive implant a, b
●
Intrauterine hormone-releasing system (IUS) b
●
Intrauterine device (IUD)
●
Bilateral tubal occlusion
●
Vasectomized partner 
A vasectomized partner is a highly effective contraception method provided that the partner is the 
sole male sexual partner of the WOCBP and the absence of sperm has been confirmed. If not, an 
additional highly effective method of contraception should be used. 
●
Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining from 
heterosexual intercourse during the entire period of risk associated with the study treatment. The 
reliability of sexual abstinence needs to be evaluated in relation to the duration of the study and the 
preferred and usual lifestyle of the participant. 
WOCBP should only be included after a negative highly sensitive urine or serum pregnancy 
test. Pregnancy testing will be done whenever an expected menstrual cycle is missed or when 
pregnancy is otherwise suspected.
Fred Hutch IRB
Approved 
3/13/2024