A Multi-Institutional Phase 2 Study of Nivolumab or Nivolumab in Combination With Ipilimumab in Refractory Metastatic Squamous Cell Carcinoma of the Anal Canal
Phase: PHASE2
Conditions: Anal Canal Squamous Cell Carcinoma, Metastatic Anal Canal Carcinoma, Stage IV Anal Cancer AJCC v8
Sponsor: National Cancer Institute (NCI)

i 
 
SUMMARY OF CHANGES – PROTOCOL 9673 
 
For Protocol Amendment #21: This protocol is being amended to clarify the statistical analysis 
plan with regards to the timing of when the primary endpoint 
will be analyzed.  
 
Part A: A Multi-Institutional Phase 2 Study of Nivolumab in Refractory Metastatic 
Squamous Cell Carcinoma of the Anal Canal (Original Protocol) 
 
Part B: A Multi-Institutional Phase 2 Study of Nivolumab or Nivolumab in Combination 
with Ipilimumab in Refractory Metastatic Squamous Cell Carcinoma of the Anal Canal 
(Amendment) 
 
NCI Protocol #:  
 
9673 
Local Protocol #:  
 
NCI9673 
 
NCI Version Date:   
March 24, 2023 
Protocol Date:   
 
March 24, 2023 
 
The changes reflect revisions from v11.14.2022 to 03.24.2023 
 
 
# Section 
Page 
Change 
1. TITLE 
1 
OLD TEXT:  
Version Date: November 14, 2022 
 
NEW TEXT:  
Version Date: March 24, 2023 
 
RATIONALE:  Updated protocol header so document version date 
matches most recent resubmission date.  
2. TITLE 
3 
NEW TEXT:  
Amendment #21/Version Date: March 24, 2023 
 
RATIONALE:  Updated protocol title page with new amendment 
number and version date. 
3. 
14.6 
127 
NEW TEXT: The primary endpoint will be assessed when at least 8.2 
months of followup after the last patient has been reached or when the 
study has observed 87 progression events, whichever occurs sooner. 
 
RATIONALE:  Added text to clarify the timing of when the primary 
endpoint will be analyzed.  

NCI Protocol #9673 
Version Date: March 24, 2023 
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NCI Protocol #: 
9673 
 
Local Protocol #: 
NCI9673 
 
ClinicalTrials.gov Identifier:   NCT02314169 
 
TITLE:  PART A: A MULTI-INSTITUTIONAL PHASE 2 STUDY OF NIVOLUMAB IN 
REFRACTORY METASTATIC SQUAMOUS CELL CARCINOMA OF THE ANAL 
CANAL (ORIGINAL PROTOCOL) 
 
 
TITLE:  PART B: A MULTI-INSTITUTIONAL PHASE 2 STUDY OF NIVOLUMAB OR 
NIVOLUMAB IN COMBINATION WITH IPILIMUMAB IN REFRACTORY 
METASTATIC SQUAMOUS CELL CARCINOMA OF THE ANAL CANAL 
(AMENDMENT) 
 
 
 
Corresponding Organization: LAO-CT018 / Yale University Cancer Center LAO  
 
Principal Investigator: 
Cathy Eng, M.D., FACP 
Vanderbilt-Ingram Cancer Center 
2220 Pierce Avenue 
Preston Research Building, Suite 777 
Nashville, TN 37232 
Telephone: 615-936-8422 
Fax: 615-343-7602 
Cathy.eng@vumc.org 
 
Co-Principal Investigator:         Van Morris, M.D. 
 
1515 Holcombe Boulevard, Unit 426 
                                                      University of Texas – MD Anderson Cancer Center 
 
Houston, TX 77030 
 
Telephone: (713) 792-2828 
 
Fax: (713) 745-1163 
 
vkmorris@mdanderson.org 
 
 
Participating Organizations  
LAO-11030 / University Health Network Princess Margaret Cancer Center LAO 
LAO-CA043 / City of Hope Comprehensive Cancer Center LAO 
LAO-TX035 / University of Texas MD Anderson Cancer Center LAO 
LAO-MA036 / Dana-Farber - Harvard Cancer Center LAO 
LAO-MD017 / JHU Sidney Kimmel Comprehensive Cancer Center LAO 
LAO-MN026 / Mayo Clinic Cancer Center LAO 

NCI Protocol #9673 
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LAO-NC010 / Duke University - Duke Cancer Institute LAO 
LAO-NJ066 / Rutgers University - Cancer Institute of New Jersey LAO 
LAO-OH007 / Ohio State University Comprehensive Cancer Center LAO 
LAO-PA015 / University of Pittsburgh Cancer Institute LAO 
LAO-NCI/National Cancer Institute LAO 
EDDOP / Early Drug Development Opportunity Program 
CATCHUP / Creating Access to Targeted Cancer Therapy for Underserved Populations 
 
Non-Member Collaborators  
 
None 
 
Statistician:                                                                  Study Coordinator:        
Lianchun Xiao  
 
 
               Thomas Leonard-Martin 
University of Texas –                                                     Vanderbilt-Ingram Cancer Center 
 MD Anderson Cancer Center                                        Clinical Trials Shared Resource 
1515 Holcombe Boulevard, Unit 1411                          3322 West End, Suite 1000                                                
Houston, TX 77030                                                        Nashville, TN 37203 
Telephone: (713) 745-5984                                            Telephone: 615-936-6726   
lxiao@mdanderson.org                                                   thomas.leonard-martin@vumc.org 
                                                                                         Fax: 615-936-5850 
 
 
           
 
Responsible Research Nurse: 
        Responsible Data Manager:  
Jennifer Teller                                                                 Bo Whitson 
Vanderbilt-Ingram Cancer Center                                Vanderbilt-Ingram Cancer Center 
Clinical Trials Shared Resource                                   Clinical Trials Shared Resource 
2220 Pierce Avenue, 491 PRB                                     3322 West End, Suite 1000 
Nashville, TN 37232                                                     Nashville, TN 37203 
Telephone: 615-875-9327                                             Telephone: 615-875-6515  
jennifer.l.teller@vumc.org 
                                 bo.k.whitson@vumc.org 
 
 
 
 
NCI-Supplied Agent:   
Nivolumab (BMS-936558, MDX-1106, and ONO-4538) (NSC #748726) 
Ipilimumab (MDX-010, NSC #732442) 
 
 
IND #:  IND 
 
IND Sponsor:  DCTD, NCI 
ClinicalTrials.gov Registration:  NCT02314169 
 
 

NCI Protocol #9673 
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Protocol Type / Version # / Version Date:  Original/ Version Date:  October 30, 2014   
                      
Amendment # 1/Version Date: December 31, 2014 
Amendment # 2/Version Date: January 27, 2015 
Amendment # 3/Version Date: March 23, 2015 
Amendment # 4/Version Date: March 2, 2016 
Amendment # 5/Version Date: April 12, 2016 
Amendment # 6/Version Date: February 22, 2017 
Amendment # 7/Version Date: November 17, 2017 (CTEP Disapproved) 
Amendment # 8/Version Date: December 19, 2017(CIRB approved pending modification) 
Amendment # 9/Version Date: February 28, 2017 (CIRB Approved, CTEP Disapproved) 
Amendment #10/Version Date: May 7, 2018 
Amendment #11/Version Date: August 16, 2018 
Amendment #12/Version Date: April 23, 2019 
Amendment #13/Version Date: May 24, 2019 
Ammendment 14/Version Date: June 25, 2019 
Amendment #15/Version Date: November 19, 2019 
Amendent #16/Version Date: July 15, 2020 
Amendment #17/Version Date: August 27, 2020 
Amendment #18/Version Date: February 3, 2021 
Amendment #19/Version Date: March 18, 2021 
Amendment #20/Version Date: November 14, 2022 
Amendment #21/Version Date: March 24, 2023 
 
 

NCI Protocol #9673 
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PART A:  SCHEMA 
A Multi-Institutional Phase 2 Study of Nivolumab in Refractory Metastatic Squamous Cell 
Carcinoma of the Anal Canal (Original Protocol) 
 
 
 
 
PART B:  SCHEMA  
A Multi-Institutional Phase 2 Study of Nivolumab or Nivolumab in Combination with 
Ipilimumab in Refractory Metastatic Squamous Cell Carcinoma of the Anal Canal 
(Amendment) 
 
100 patients 
with metastatic  
squamous cell 
carcinoma of the 
anal canal 
Nivolumab 
Nivolumab + ipilimumab 
1:1  
randomization 

TABLE OF CONTENTS 
 
TITLE:  Part A: A Multi-Institutional Phase 2 Study of Nivolumab in Refractory 
Metastatic Squamous Cell Carcinoma of the Anal Canal (Original 
Protocol) ......................................................................................................1 
TITLE:  Part B: A Multi-Institutional Phase 2 Study of Nivolumab or Nivolumab in 
Combination with Ipilimumab in Refractory Metastatic Squamous 
Cell Carcinoma of the Anal Canal (Amendment) ...................................1 
Part A:  SCHEMA ......................................................................................................................4 
Part B:  SCHEMA ......................................................................................................................4 
TABLE OF CONTENTS ..............................................................................................................5 
1. 
OBJECTIVES ............................................................................................8 
1.1 
Primary Objectives ...................................................................................................8 
1.2 
Secondary Objectives ...............................................................................................8 
1.3 
Exploratory Objectives ............................................................................................8 
2. 
BACKGROUND ........................................................................................9 
2.1 
Squamous Cell Carcinoma of the Anal Canal .........................................................9 
2.2 
Rationale (Parts A+B) ............................................................................................27 
2.3 
Rationale for modified dosing for nivolumab and ipilimumab (Part B) ................30 
2.4 
Correlative Studies Background (Part A) ..............................................................32 
2.5 
Correlative Studies Background (Part B) ..............................................................35 
3. 
PATIENT SELECTION .........................................................................36 
3.1 
Eligibility Criteria (Parts A+B) ..............................................................................36 
3.2 
Exclusion Criteria (Parts A+B) ..............................................................................40 
3.3 
Inclusion of Women and Minorities ......................................................................41 
4. 
REGISTRATION PROCEDURES ........................................................41 
4.1 
Investigator and Research Associate Registration with CTEP ..............................41 
4.2 
Site Registration .....................................................................................................42 
4.3 
Patient Registration ................................................................................................44 
4.4 
General Guidelines .................................................................................................46 
5. 
TREATMENT PLAN ..............................................................................46 
5.1 
Treatment with nivolumab (Parts A+B) ................................................................46 
5.2 
Nivolumab Administration ....................................................................................46 
5.3 
General Concomitant Medication and Supportive Care Guidelines (Parts 
A+B) ....................................................................................................................47 
5.4 
Duration of Therapy (Parts A+B) ..........................................................................47 
5.5 
Duration of Follow Up ...........................................................................................48 
5.6 
Criteria for Removal from Study (Parts A+B) .......................................................48 

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5.7 
Criteria to Resume Treatment (Parts A+B) ...........................................................49 
5.8 
Treatment of Nivolumab-Related Infusion Reactions (Parts A+B) .......................50 
5.9 
Ipilimumab Administration (Part B) ......................................................................52 
5.10  Prohibited and Restricted Therapies ......................................................................61 
5.11 
Treatment Beyond Progression ..............................................................................62 
5.12 
Discontinuation of Study Therapy .........................................................................62 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ....................................63 
6.1 
Dosing Modifications.............................................................................................63 
6.2 
Dosing Delays ........................................................................................................63 
7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS
....................................................................................................................68 
7.1 
Comprehensive Adverse Events and Potential Risks List (CAEPR) .....................68 
7.2 
Adverse Event Characteristics ...............................................................................77 
7.3 
Expedited Adverse Event Reporting ....................................................................778 
7.4 
Routine Adverse Event Reporting .......................................................................799 
7.5 
Secondary Malignancy .........................................................................................790 
7.6 
Second Malignancy ................................................................................................80 
7.7 
Safety Monitoring Plan ..........................................................................................80 
8. 
PHARMACEUTICAL INFORMATION ............................................801 
8.1 
CTEP IND Agent .................................................................................................801 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES (Part A 
Only, see section 10 for part b).................................................................86 
9.1 
Integral Laboratory or Imaging Studies .................................................................86 
9.2 
Integrated Correlative Studies ................................................................................86 
9.3 
Exploratory Correlative Studies .............................................................................86 
9.4 
Immunohistochemical (IHC) Staining and Analysis for PD-L1, PD-1, 
CD8+,  and CD4+ Tumor-Infiltrating Lymphocytes (Parts A and B) ......................92 
9.5 
Specimen Submission Summary............................................................................92 
9.6 
Special Studies - Analysis for the Presence of HPV ..............................................95 
9.7 
Optional Banking of Residual Tissue and Blood Samples for Future 
Research .................................................................................................................96 
10. 
Part B – Correlative and blood work: ....................................................96 
10.1 
Integral Laboratory or Imaging Studies .................................................................96 
10.2 
Integrated Correlative Studies ................................................................................96 
10.3 
Exploratory Correlative Studies .............................................................................96 
10.4 
Specimen Submission Summary..........................................................................104 
10.5 
Optional Banking of Residual Tissue and Blood Samples for Future 
Research ...............................................................................................................106 
11. 
STUDY CALENDAR ............................................................................107 
11.1 
Part A: ..................................................................................................................107 
11.2   Part B: ..................................................................................................................109 

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12. 
MEASUREMENT OF EFFECT ..........................................................113 
12.1 
Antitumor Effect – Solid Tumors ........................................................................113 
12.2 
Other Response Parameters .................................................................................119 
13. 
Study Oversight and DATA REPORTING / REGULATORY 
REQUIREMENTS .................................................................................119 
13.1 
Study Oversight ...................................................................................................119 
13.2 
Data Reporting .....................................................................................................120 
13.3 
CTEP Multicenter Guidelines ..............................................................................122 
13.4 
Collaborative Agreements Language ...................................................................122 
14. 
STATISTICAL CONSIDERATIONS .................................................124 
14.1 
Study Design/Endpoints (Part A).........................................................................124 
14.2 
Sample Size/Accrual Rate ....................................................................................125 
14.3 
Stratification Factors ............................................................................................125 
14.4 
Analysis of Secondary Endpoints ........................................................................125 
14.5 
Analyses of Exploratory Endpoints: ....................................................................126 
14.6 
Study Design/Endpoints (Part B) .........................................................................126 
14.7 
Reporting and Exclusions ....................................................................................128 
15. 
Study Status Updates and Study Closure ............................................129 
15.1 
Definitions of Study Status Changes ...................................................................129 
15.2 
Responsibility for Filing Protocol Status Update Forms .....................................130 
REFERENCES 
..................................................................................................................131 
APPENDIX A 
PERFORMANCE STATUS CRITERIA ............................................139 
APPENDIX B 
CTEP MULTICENTER GUIDELINES FOR NON-ETCTN TRIALS
..................................................................................................................140 
APPENDIX C 
BIOASSAY TEMPLATES ...................................................................141 
APPENDIX D 
MANAGEMENT ALGORITHMS FOR ENDOCRINOPATHY, 
GASTROINTESTINAL, HEPATIC, NEUROLOGICAL, 
PULMONARY, RENAL, AND SKIN ADVERSE EVENTS ............142 
APPENDIX E  ANALYSES OF SAMPLES FOR EXPLORATORY STUDIES ................150 
Appendix F ParticiPant Wallet Card .......................................................................................151 
 
 
 

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1. OBJECTIVES 
 
1.1 Primary Objectives  
 
To evaluate overall response rate (ORR) with nivolumab in patients with previously treated 
metastatic squamous cell carcinoma (SCCA) of the anal canal (Part A). 
 
To determine an improvement in Progression-Free Survival (PFS) when nivolumab is combined 
with ipilimumab vs. nivolumab alone in patients with previously treated metastatic SCCA (Part 
B).   
 
1.2 Secondary Objectives  
 
Part A:  
 
1.2.1 
To evaluate progression-free survival (PFS) of nivolumab in 
patients with previously treated metastatic SCCA of the anal canal. 
 
1.2.2 
To evaluate overall survival (OS) in patients with previously 
treated metastatic SCCA of the anal canal treated with nivolumab. 
 
1.2.3 
To evaluate the grade 3 and 4 toxicity rate in patients with 
previously treated metastatic SCCA of the anal canal when treated with 
nivolumab. 
 
Part B:  
 
1.2.4 
To evaluate the overall response rate (ORR) of nivolumab plus or minus ipilimumab in 
patients with previously treated metastatic SCCA of the anal canal. 
 
 
1.2.5 To evaluate overall survival (OS) in patients with previously treated metastatic SCCA of 
the anal canal treated with nivolumab plus or minus ipilimumab. 
 
1.2.6 
To evaluate the grade 3 and 4 toxicity rate in patients with 
previously treated metastatic SCCA of the anal canal when treated with 
nivolumab plus or minus ipilimumab. 
 
1.3 Exploratory Objectives  
 
Part A: 

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1.3.1 
To evaluate ORR, PFS, and OS based on expression of PD-L1, 
PD-1, peritumoral CD8+ tumor infiltrating lymphoctyes (TILs), 
peritumoral CD4+ TILs, and regulatory T cells as analyzed from tumor 
biopsies in previously treated patients with metastatic SCCA of the anal 
canal when treated with nivolumab.  
1.3.2 
To evaluate radiographic responses according to relative 
changes in proportions of anti-HPV specific CD8+ and CD4+ TILs and 
regulatory T cells in patients with previously treated metastatic SCCA of 
the anal canal following treatment with nivolumab, analyzed from serial 
peripheral blood samples. 
 
Part B:  
 
1.3.3 To evaluate ORR, PFS, and OS based on expression of PD-L1, PD-1, peritumoral CD8+ 
tumor infiltrating lymphoctyes (TILs), peritumoral CD4+ TILs, and regulatory T cells as 
analyzed from tumor biopsies in previously treated patients with metastatic SCCA of the 
anal canal when treated with nivolumab plus or minus ipilimumab.  
1.3.4 To evaluate radiographic responses according to relative changes in proportions of anti-
HPV specific CD8+ and CD4+ TILs and regulatory T cells in patients with previously 
treated metastatic SCCA of the anal canal following treatment with nivolumab plus or 
minus ipilimumab. 
 
2. BACKGROUND 
 
2.1 Squamous Cell Carcinoma of the Anal Canal 
 
Squamous cell carcinoma (SCCA) of the anal canal accounts for an estimated 2% of all 
gastrointestinal malignancies in the US1.  However, the annual incidence has risen steadily over 
the past two decades, with more than 8200 new cases expected in the United States in 20172,3.  
Initially considered an orphan malignancy, the standard treatment paradigm of concurrent 
chemoradiation has largely remained unchanged in the US for greater than 3 decades4,5.  
Approximately 25% of patients initially treated with chemoradiation will develop locally 
advanced disease and/or distant metastases6-9, and an additional 10% of patients will be 
diagnosed with stage IV disease at initial presentation10.  There is currently no standard treatment 
approach for patients diagnosed with metastatic SCCA of the anal canal.  Historically, 
chemotherapy regimens utilized in the treatment of these patients have largely been extrapolated 
from data in more common metastatic squamous cell carcinomas11-15 (e.g., head and neck cancer 
and cervical cancer).  A phase II international study has recently been initiated [InterAACT: C 
Eng (US Lead PI)] and is globally supported by the IRCI/NCI/EORTC/ECOG to identify the 
best chemotherapy backbone in treatment-naïve (HIV+ and HIV-) patients with SCCA of the 
anal canal).  To that end, large academic institutions serve as major referral sources for this 
disease given that outside providers are limited by the paucity of treatment options available to 
this population of patients.   
 

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2.1.1 
Nivolumab (Parts A and B) 
 
Nivolumab (BMS-936558, MDX-1106, and ONO-4538) is a fully human monoclonal 
immunoglobulin G4 (IgG4) antibody (HuMAb) that is specific for human programmed death-1 
(PD-1, cluster of differentiation 279 [CD279]) cell surface membrane receptor16.  PD-1 is a 
negative regulatory molecule that is expressed transiently following T-cell activation and on 
chronically stimulated T cells characterized by an “exhausted” phenotype.  Nivolumab binds to 
cynomolgus monkey PD-1 but not mouse, rat, or rabbit molecules.  Clinical activity of 
nivolumab has been observed in patients with melanoma, non-small cell lung cancer (NSCLC), 
and renal cell carcinoma (RCC).   The combination of nivolumab and ipilimumab (anti-cytotoxic 
T lymphocyte associated antigen-4 [anti-CTLA-4]) in a phase II trial showed enhanced clinical 
activity relative to other agent alone, with an acceptable safety profile, in melanoma patients17.   
 
The clinical use of monoclonal antibodies to T-cell inhibitory receptors has provided 
transformative information on the nature of the immune system and cancer.  An emerging picture 
suggests that endogenous immune responses can mediate effective tumor regression and/or 
improved survival even in patients with large volume tumors resistant to other forms of therapy.  
Some of the unique features of this type of therapy, based largely on experience in advanced 
melanoma, include:  improved overall survival (OS) with or without radiographic responses or 
improved progression-free survival (PFS); responses that may be delayed or occur after 
radiographic disease progression; combinations of immune modulators with enhanced or novel 
activities (in the example of ipilimumab and nivolumab); and toxicity that is almost exclusively 
immune or inflammatory in nature.  It is not yet clear what factors determine responses and 
which components of the immune system are needed for this to occur.  It seems likely that both 
memory helper and effector cells would be needed to sustain long-term responses. Increasing 
emphasis has been placed on understanding the relationships of the tumor, cellular infiltrate, and 
immunologic milieu surrounding each tumor.   
 
PD-1, a 55-kDa type 1 transmembrane protein, is a member of the CD28 family of T-cell co-
stimulatory receptors that include Ig super family member CD28, CTLA-4, inducible co-
stimulator (ICOS), and B and T lymphocyte attenuator (BTLA)16.  PD-1 is transiently but highly 
expressed on activated T cells functioning to limit immune effectors at the site of activation.  
Chronic stimulation may prevent the re-methylation of the PD-1 gene leading to continuous 
expression and characterizes a state of “exhausted” T cells that lose function and proliferative 
capacity while enhancing a suppressive tumor microenvironment.  PD-1 may act together with 
other T-cell modulating molecules, including CTLA-4, TIM-3, lymphocyte-activation gene 3 
(LAG-3) as well as indoleamine-pyrrole 2, 3-dioxygenase 1 (IDO-1), cytokines, and 
transforming growth factor beta (TGF-beta).   
 
Two ligands specific for PD-1 have been identified:  PD-ligand 1 (PD-L1, also known as B7-H1 
or CD274, expressed on tumor, antigen-presenting cells [APCs], and dendritic cells [DCs]) and 
PD-L2 (also known as B7-DC or CD273, expressed on endothelial cells).  The interaction of PD-
1 with PD-L1 and PD-L2 results in negative regulatory stimuli that down-modulate the activated 
T-cell immune response through SHP-1 phosphatase.   
 
PD-1 knockout mice develop strain-specific lupus-like glomerulonephritis (C57BL/6) and 

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cardiomyopathy (BALB/c).  In transplantable tumor models that expressed PD-1 and LAG-3 on 
tumor-infiltrating CD4+ and CD8+ T cells dual anti-LAG-3/anti-PD-1 antibody treatment cured 
most mice of established tumors that were largely resistant to single antibody treatment18.  
Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual 
knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, 
leading to eventual lethality. 
 
PD-L1 expression is found on a number of tumors, and is associated with poor prognoses based 
on OS in many tumors, including melanoma19, renal20-22, esophageal23, gastric24, ovarian25, 
pancreatic26, lung27, and other cancers16. 
 
The PD-1/PD-L1 axis plays a role in human infections, particularly in hepatitis C virus (HCV) 
and human immunodeficiency virus (HIV).  In these cases, high expression levels of PD-1 were 
found in viral-specific CD8+ T cells that also display a non-responsive or exhausted phenotype.  
Non-responsive PD-1-high T cells were observed in simian immunodeficiency virus (SIV) 
infection in rhesus macaques.  Treatment of SIV-infected macaques with an anti-PD-1 mAb (3 
mg/kg x4) resulted in decreased viral loads and increased survival along with expanded T cells 
with increased T-cell functionality.   
 
2.1.1.1 
Nonclinical Development of Nivolumab (Parts A and B) 
 
In intravenous (IV) repeat-dose toxicology studies in cynomolgus monkeys, nivolumab alone 
was well tolerated16.  Combination studies have highlighted the potential for toxicity when 
combined with ipilimumab, MDX-1408, and BMS-986016.  Nivolumab bound specifically to 
PD-1 (and not to related members of the CD28 family such as CD28, ICOS, CTLA-4, and 
BTLA) with a Kd = 3.06 nM.  A surrogate rat anti-mouse PD-1 antibody (4H2) was derived and 
expressed as chimeric IgG1 murine antibody.  Antitumor activity was seen for several tumor 
models, including colon carcinoma and fibrosarcoma. 
 
2.1.1.2 
Clinical Development of Nivolumab (Parts A and B) 
 
Nivolumab is being evaluated as monotherapy and in combination with cytotoxic chemotherapy, 
other immunotherapy (such as ipilimumab), anti-angiogenesis therapy, and targeted therapies in 
completed and ongoing BMS-sponsored clinical trials in NSCLC, melanoma, RCC, 
hepatocellular carcinoma (HCC), gastrointestinal (GI) malignancies including microsatellite 
instability (MSI) in colorectal cancer, and triple-negative breast cancer (TNBC) with an 
expanding group of indications16.  In addition, two investigator-sponsored trials (ISTs) of 
nivolumab in combination with a peptide vaccine in melanoma are being conducted in the 
adjuvant setting and advanced disease.  
 
Seven nivolumab studies were conducted in Japan, including six studies in advanced solid 
tumors and recurrent or unresectable stage III/IV melanoma sponsored by Ono Pharmaceuticals 
Co. Ltd., and one IST in recurrent or advanced platinum-refractory ovarian cancer.   
 
2.1.1.2.1. Pharmacokinetics (Parts A and B) 
 

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Pharmacokinetics (PK) of nivolumab was linear in the range of 0.3 to 10 mg/kg, with dose-
proportional increases in maximum serum concentration (Cmax) and area under the 
concentration-time curve from time zero to infinity (AUC0-∞), with low to moderate inter-subject 
variability observed at each dose level16.  Clearance of nivolumab is independent of dose in the 
dose range (0.1 to 10 mg/kg) and tumor types studied.  Body weight normalized dosing showed 
approximately constant trough concentrations over a wide range of body weights.  The mean 
terminal elimination half-life of BMS-936558 is 17 to 25 days consistent with the half-life of 
endogenous IgG4.   
 
2.1.1.2.2. Efficacy (Parts A and B) 
 
In a phase 1 (1, 3, and 10 mg/kg nivolumab doses) dose-escalation study the 3 mg/kg dose was 
chosen for expanded cohorts. Among 236 patients, objective responses (ORs) (complete or 
partial responses [CR or PR]) were seen in NSCLC, melanoma, and RCC.  ORs were observed 
at all doses28.  Median OS was 16.8 months across doses and 20.3 months at the 3 mg/kg dose.  
Median OS across all dose cohorts was 9.2 months and 9.6 months for squamous and non-
squamous NSCLC, respectively29.  In the RCC cohort, median duration of response was 12.9 
months for both doses with 5 of the 10 responses lasting ≥1 year30.   
 
In an advanced melanoma phase 1 study, nivolumab and ipilimumab were administered IV every 
3 weeks for 4 doses followed by nivolumab alone every 3 weeks for 4 doses (concurrent 
regimen) 17.  The combined treatment was subsequently administered every 12 weeks for up to 8 
doses.  In a sequenced regimen, patients previously treated with ipilimumab received nivolumab 
every 2 weeks for up to 48 doses.  In the concurrent regimen (53 patients), 53% of patients had 
an OR at doses 1 mg/kg nivolumab and 3 mg/kg ipilimumab, with tumor reduction of 80% or 
more (modified World Health Organization [mWHO] criteria).  In the sequenced-regimen (33 
patients), the objective response rate (ORR) was 20%. 
 
In a phase 1 study of nivolumab plus platinum-based doublet chemotherapy (PT-doublet) in 
chemotherapy-naïve NSCLC patients, 43 patients were treated with nivolumab + PT-doublet31.  
No dose-limiting toxicities (DLTs) were reported and total/confirmed ORRs were 43/33%, 
40/33%, and 31/31% in nivolumab/gemcitabine/cisplatin, nivolumab/pemetrexed/cisplatin, and 
nivolumab/carboplatin/paclitaxel arms, respectively. 
 
2.1.1.2.3. Toxicology (Parts A and B) 
 
A maximum tolerated dose (MTD) of nivolumab was not defined32.  Serious adverse events 
(SAEs) occurred in 32 of 296 patients (11%) similar to the immune-related inflammatory events 
seen with ipilimumab:  pneumonitis, vitiligo, colitis, hepatitis, hypophysitis, and thyroiditis (with 
noted pulmonary toxicity resulting in 3 deaths. Renal failure, symptomatic pancreatic and DM, 
neurologic events, and vasculitis have also been reported.).   In combination with ipilimumab in 
the concurrent-regimen group17, grade 3 or 4 treatment-related events were noted in 53% of 
patients.  Skin rash represents the majority of these events.  
 
2.1.1.2.4. Pharmacodynamics/Biomarkers (Parts A and B) 
 

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Tumor-cell expression (melanoma) of PD-L1 was characterized in combination with ipilimumab 
with the use of IHC staining and pharmacodynamics changes in the peripheral-blood absolute 
lymphocyte count17.  With PD-L1 positivity defined as expression in at least 5% of tumor cells, 
biopsy specimens from 21 of 56 patients (38%) were PD-L1–positive.  Among patients treated 
with the concurrent regimen of nivolumab and ipilimumab, ORs were observed in patients with 
either PD-L1–positive tumor samples (6 of 13 patients) or PD-L1–negative tumor samples (9 of 
22).  In the sequenced regimen cohorts, a higher number of overall responses was seen among 
patients with PD-L1–positive tumor samples (4 of 8 patients) than among patients with PD-L1–
negative tumor samples (1 of 13) suggesting the possibility that these tumors have higher 
response rates to the combination. The relationship between PDL-1 expression and responses 
may not be present in patients treated with the combination.  Tissue expression of PDL-2, 
interferon-γ (IFN- γ), IDO, and T cell CD8+ are of current interest.  Until more reliable data 
based on standardized procedures for tissue collection and assays are available, PD-L1 status 
cannot be used to select patients for treatment at this time.   
 
2.1.2 Ipilimumab (Part B) 
 
Ipilimumab (MDX-010, MDX-CTLA4, BMS-734016) is being developed by CTEP as an 
anticancer agent in collaboration with Bristol-Myers-Squibb (BMS).  On March 25, 2011, the 
FDA approved ipilimumab injection (YERVOY, BMS) for the treatment of unresectable or 
metastatic melanoma.  Ipilimumab is a human IgG1κ monoclonal antibody (mAb); it is specific 
for human cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152) expressed on 
activated T cells.  Ipilimumab is now produced and formulated from transfected Chinese hamster 
ovary (CHO) cells.   
 
CTLA-4 is a negative regulator of T-cell responses following T-cell stimulation33,34.  CTLA-4 
knockout mice suffer from a fatal lymphoproliferative disorder, supporting the idea that CTLA-4 
functions as a negative regulator of T-cell responses in vivo35-37.  Disrupting CTLA-4 interaction 
with its ligands B7-1 (CD80) and B7-2 (CD86), which are expressed on antigen-presenting cells 
(APCs), with ipilimumab, augments immune responses (Investigator Brochure, 2011).  In vivo 
blockade of CTLA-4, utilizing anti-CTLA-4 mAb, induced regression of established tumors and 
enhanced antitumor immune responses in several murine tumor models.  Blockade of CTLA-4-
mediated signals is effective in inducing rejection of immunogenic cancers in mice.  These 
findings suggest that CTLA-4 blockade, alone or in combination with antigenic stimulation and 
other immune modulating agents can induce a potent antitumor response.   
 
2.1.2.1  Pharmacology of Ipilimumab (Part B) 
 
In vitro studies were performed with ipilimumab to demonstrate that it is specific for CTLA-4, 
actively inhibits CTLA-4 interactions with B7.1 and B7.2, does not show any cross-reactivity 
with human B7.1 or B7.2 negative cell lines, and stains the appropriate cells without non-specific 
cross-reactivity in normal human tissues.  Ipilimumab does cross-react with CTLA-4 in non-
human primates including cynomolgus monkeys.  Blockade of CTLA-4/B7 interactions 
enhanced T-cell responses to CD3 / CD28, peptide antigens, or superantigens in mice38-41.  
CTLA-4 knockout mice appear to have spontaneously activated T cells evident at approximately 
1 week after birth, followed by rampant lymphoproliferation and lymphadenopathy.  These mice 

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die at approximately 3 weeks of age, either as a result of polyclonal T-cell expansion and tissue 
destruction or as a result of toxic shock resulting from lymphokine production.  Genetically 
engineered mice heterozygous for CTLA-4 (CTLA-4+/-), appeared healthy and gave birth to 
healthy CTLA-4+/- heterozygous offspring.  Mated CTLA-4+/- heterozygous mice also 
produced offspring deficient in CTLA-4 (homozygous negative, CTLA-4-/-).  Since thymocyte 
differentiation and selection proceed normally in CTLA-4-deficient mice, the rampant T-cell 
expansion that occurs in the mice indicates that CTLA-4 plays a critical role in down-regulating 
post- thymic T-cell responses in the periphery following stimulation of naïve, memory, and 
effector T cells41.   
 
2.1.2.2  Pharmacokinetics (Part B) 
 
The pharmacokinetics (PK) of ipilimumab was studied in 499 patients with unresectable or 
metastatic melanoma who received doses of 0.3, 3, or 10 mg/kg administered once every 3 
weeks (q3w) for four doses.  Peak concentration (Cmax), trough concentration (Cmin), and area 
under the curve (AUC) of ipilimumab were found to be dose proportional within the dose range 
examined.  Upon repeated dosing of ipilimumab administered q3w, ipilimumab clearance was 
found to be time-invariant, and minimal systemic accumulation was observed as evident by an 
accumulation index of 1.5-fold or less.  Ipilimumab steady-state concentration was reached by 
the third dose.  The following mean (percent coefficient of variation) parameters were generated 
through population PK analysis: terminal half-life of 14.7 days (30.1%); systemic clearance (CL) 
of 15.3 mL/h (38.5%); and volume of distribution at steady-state (Vss) of 7.21 L (10.5%).  The 
mean (±SD) ipilimumab Cmin achieved at steady-state with the 3-mg/kg regimen was 21.8 
mcg/mL (±11.2). 
 
Specific Populations: Cross-study analyses were performed on data from patients with a variety 
of conditions, including 420 patients with melanoma who received single or multiple infusions of 
ipilimumab at doses of 0.3, 3, or 10 mg/kg.  The effects of various covariates on ipilimumab PK 
were assessed in population PK analyses. 
 
Ipilimumab CL increased with increasing body weight; however, no dose adjustment of 
ipilimumab is required for body weight after administration on a mg/kg basis. 
 
The following factors had no clinically meaningful effect on the CL of ipilimumab: age (range 
26 to 86 years), gender, concomitant use of budesonide, performance status, HLA-A2*0201 
status, positive anti-ipilimumab antibody status, prior use of systemic anticancer therapy, or 
baseline lactate dehydrogenase (LDH) levels.  The effect of race was not examined as there were 
insufficient numbers of patients in non-Caucasian ethnic groups. 
 
Renal Impairment: Creatinine clearance at baseline did not have a clinically important effect on 
ipilimumab PK in patients with calculated creatinine clearance values of 29 mL/min or greater. 
 
Hepatic Impairment: Baseline AST, total bilirubin, and ALT levels did not have a clinically 
important effect on ipilimumab PK in patients with various degrees of hepatic impairment. 
 
2.1.2.3  Clinical Pharmacodynamics (Part B) 

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In clinical studies, ipilimumab increased absolute lymphocytes counts (ALC) in peripheral blood 
(Investigator Brochure, 2011).  However, CD4+/CD8+ ratio did not appear to be affected.  
Across three phase 2 studies in 463 subjects with advanced melanoma, ipilimumab increased 
ALC in a dose-dependent manner, with the largest increase observed at 10 mg/kg dose.  ALC 
continued to increase over time during the induction treatment at least until week 12 at the 3 
mg/kg and 10 mg/kg dose, but not at the 0.3 mg/kg dose.  The slope of ALC increase also 
suggested the 10 mg/kg dose is more biologically active than the 3.0 mg/kg or 0.3 mg/kg dose.   
 
2.1.2.4  Mechanism of Action (Part B) 
 
The proposed mechanism of action for ipilimumab is T-cell potentiating through interference of 
the interaction of CTLA-4 with B7 (CD80 or CD86) molecules on APCs, with subsequent 
blockade of the inhibitory function of CTLA-4 (Investigator Brochure, 2011).  Ipilimumab 
impacts tumor cells indirectly, and measurable clinical effects emerge after the immunological 
effects.  Tumor infiltration with lymphocytes and the associated inflammation is likely the 
cornerstone of the effect of ipilimumab and can manifest in various patterns of clinical activity 
leading to tumor control.  These immunologic responses may take time to develop and so tumor 
responses may be delayed and tumor progression may occur during the initial period followed by 
responses.  In some cases, tumor response based on tumor infiltration with immune cells may be 
preceded by an apparent increase in initial tumor volume and/or the appearance of new lesions, 
which may be taken for tumor progression on radiological evaluations. Delayed responses 
following increasing tumor size or appearance of new lesions have been seen in approximately 
10-20% of patients with metastatic melanoma.  For patients who are not experiencing rapid 
clinical deterioration, allowing sufficient time to observe responses including disease 
stabilization or confirmation of progression is recommended; as discussed in the section “Overall 
Risk/Benefit Assessment” may allow better assessment of clinical activity and avoid 
unnecessarily initiating additional therapies in subjects who might be benefitting from treatment.  
Immune-related (ir) response criteria were developed based on these observations in patients 
with melanoma to systematically categorize novel patterns of clinical activity and are currently 
being prospectively evaluated in clinical studies. 
 
2.1.2.5  Nonclinical Toxicology (Part B) 
 
Please note relevant toxicity for single agent ipilimumab has been almost completely derived 
from clinical studies.  
 
In a study using cynomolgus macaques, anti-melanocyte responses were observed in animals 
given up to four doses of 10 mg/kg ipilimumab after receiving a melanoma cell vaccine42.  
Depigmentation has been observed in other nonclinical immunotherapy studies that involve 
treatment with melanoma peptides43-48.  The symptoms in animals appear to resemble vitiligo 
observed in clinical immunotherapy trials of melanoma patients and may be an unavoidable 
consequence of treatment49. 
 
Additional repeat-dose toxicity studies conducted using cynomolgus macaques demonstrated that 
the IV administration of ≤30 mg/kg every 3 days for three doses, 10 mg/kg weekly for 1 month, 
1 mg/kg weekly for 10 weeks, or 10 mg/kg monthly for 6 months was generally well tolerated, 

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without significant clinical, immunotoxicological, or histopathological findings (Investigator 
Brochure, 2011).  However, when ipilimumab was administered in combination with another 
immunomodulatory antibody (BMS-663513, a fully human anti-CD137 mAb) and simian 
immunodeficiency virus (SIV) DNA, two immune-related adverse events (irAEs) were observed:  
severe colitis requiring euthanasia in one monkey and reversible dermatitis/rash in the inguinal 
area and peripheral lymphadenopathy in another monkey. 
 
Complete information on the pre-clinical toxicology studies can be found in the Ipilimumab 
Investigator Brochure (IB).  Non-clinical toxicity assessments included in vitro cynomolgus 
monkeys alone and in the presence of vaccines.  Low to moderate ADCC activity was noted at 
concentrations up to 50 mcg/mL.  These data are consistent with the requirement of high levels 
of antigen expression on the surface of target cells for efficient ADCC or CDCC.  No mortality 
or signs of toxicity were observed in three independent 14-day intravenous (IV) toxicology 
studies in cynomolgus monkeys at multiple doses up to 30 mg/kg/dose.  Furthermore, 
ipilimumab was evaluated in sub-chronic and chronic toxicology studies in cynomolgus 
monkeys with and without Hepatitis B (HepB) Vaccine and Melanoma Vaccine.  Ipilimumab 
was well tolerated alone or in combination in all studies.  There were no significant changes in 
clinical signs, body weight values, clinical pathology values or T-cell activation markers. In 
addition, there were no significant histopathology changes in the stomach or colon. 
 
2.1.2.6 Clinical Development of Ipilimumab (Part B) 
 
Company-Sponsored Studies 
BMS and Medarex (acquired by BMS in September 2009) have co-sponsored an extensive 
clinical development program for ipilimumab, encompassing more than 4000 subjects in several 
cancer types in 33 completed and ongoing studies (Investigator Brochure, 2011).  The focus of 
the clinical program is in melanoma, prostate cancer, and lung cancer, with advanced melanoma 
being the most comprehensively studied indication.  Ipilimumab is being investigated both as 
single agent and in combination with other modalities such as chemotherapy, radiation therapy, 
and other immunotherapy. 
 
Phase 3 programs are ongoing in melanoma and prostate cancer (Investigator Brochure, 2011).  
In the phase 3 combination study of ipilimumab with glycoprotein 100 (gp100) peptide vaccine 
(melanoma study MDX010-20), ipilimumab was administered at a dose of 3 mg/kg of body 
weight, with or without gp100 q3w for up to four treatments50.  The median overall survival (OS) 
in the ipilimumab plus gp100 group was 10.0 months (95% confidence interval [CI], 8.5 to 11.5 
months) compared with 6.4 months (95% CI, 5.5 to 8.7 months) in the gp100-alone group 
(hazard ratio [HR] for death, 0.68; p<0.001).  Grade 3 or 4 immune related events (irAEs) 
occurred in 10 to 15% of patients treated with ipilimumab and in 3% of patients treated with 
gp100 alone.   
 
A second phase 3 study in melanoma was reported for ipilimumab in combination with 
dacarbazine versus dacarbazine alone in previously untreated advanced melanoma.  CA184024 
evaluated the addition of 10 mg/kg ipilimumab to dacarbazine in patients with previously 
untreated, metastatic melanoma.  A total of 502 patients were randomized to receive up to 8 
cycles of dacarbazine 850 mg/m2 q3w, with either ipilimumab 10 mg or placebo for cycles 1-4 

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and as maintenance after completion of chemotherapy. Ipilimumab AEs were consistent with 
previous studies and predominantly affected skin, GI tract, liver, and the endocrine system.  
Events were managed with established guidelines and were generally responsive to dose 
interruption/discontinuation, corticosteroids and/or other immunosuppresants.   
 
Checkmate 227 (NCT02477826) is an ongoing phase III trial of nivolumab, nivolumab plus 
ipilimumab, nivolumab plus platinum doublet therapy or platinum doublet chemotherapy in 
treatment naïve stage IV non-small cell lung cancer.   
 
There are two ongoing studies of ipilimumab as adjuvant monotherapy for high-risk Stage III 
melanoma (CA184029) and ECOG 1609 (www.clinicaltrials.gov).  
 
Two phase 3 studies are ongoing in subjects with castration-resistant prostate cancer who have 
received prior chemotherapy (CA184043, ipilimumab in combination with radiation therapy) and 
those who are chemotherapy-naïve (CA184095, ipilimumab monotherapy). 
 
An ongoing large phase 2 study (CA184041) is investigating the addition of ipilimumab to 
carboplatin and paclitaxel using two different schedules (concurrent and phased) in subjects with 
non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) (Investigator Brochure, 
2011).  The results demonstrate an improved immune-related progression-free survival (irPFS) 
for the subjects who received combination therapy with paclitaxel/carboplatin/ipilimumab 
compared with paclitaxel/carboplatin alone.  The improvement in irPFS with the combination 
therapy met the prespecified protocol criteria for significance for both the concurrent and phased 
schedules (p=0.0935 and 0.0258, respectively).  Using modified World Health Organization 
(mWHO) criteria (a key secondary endpoint), PFS was significantly improved with the phased 
schedule but not the concurrent schedule (p=0.0240 and 0.2502, respectively). 
 
2.1.2.7 Other Clinical Studies with Ipilimumab (Part B) 
 
Renal cell carcinoma (RCC) 
Yang and colleagues presented data on a phase 2 study of ipilimumab conducted in patients with 
metastatic RCC51.   Sequential cohorts received either 3 mg/kg followed by 1 mg/kg or all doses 
at 3 mg/kg q3w.  One of 21 patients receiving the lower dose had a PR.  Five of 40 patients at the 
higher dose had PRs (95% CI, cohort response rate 4 to 27%) and responses were seen in 
patients who had previously not responded to IL-2.  Thirty-three percent of patients experienced 
grade 3 or 4 irAEs.  There was a highly significant association between autoimmune events and 
tumor regression (response rate = 30% with AE, 0% without AE).  The authors concluded that 
CTLA-4 blockade with ipilimumab induced cancer regression in some patients with metastatic 
clear cell renal cancer, even if they had not responded to other immunotherapies.   
 
Melanoma (ipilimumab plus bevacizumab) 
At the 2011 ASCO meeting Hodi and colleagues presented results on 21 evaluable patients (22 
patients enrolled) with unresectable stage III or stage IV melanoma treated with the combination 
of 10 mg/kg ipilimumab and 7.5 mg/kg bevacizumab on a phase 1 study52.  AEs included giant 
cell arteritis (1), hypophysitis (3), thyroiditis (4), grade 3-4 hepatitis (2), bilateral uveitis (2), and 
grade 2 colitis (2); 5 patients required systemic steroids and stopped treatment.  All toxicities 

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were resolved.  Eight PRs and 6 SDs were observed.  All responses were durable (>6 months).  
Post-treatment biopsies in 12 patients revealed activated vessel endothelium with extensive T-
cell trafficking non-productive central angiogenesis, and peripheral blood monitoring revealed a 
marked increase in CD4/CCR7/CD45RO central memory cells in the majority of patients,  not 
seen with ipilimumab alone.  The authors concluded that the combination of ipilimumab with 
bevacizumab can be safely administered with clinical activity and correlatives suggesting 
synergistic effects.  
 
Bladder cancer 
Carthon and colleagues reported immunodulatory effects following a brief exposure of anti-
CTLA-4 in patients with urothelial carcinoma of the bladder requiring surgery (BMS study 
CA184027)53.  12 patients were enrolled (6 patients received 3 mg/kg/dose of ipilimumab and 
another 6 patients received 10 mg/kg/dose for two doses prior to surgery).  The treatment was 
found to be tolerable in the cohort of patients with 11 of 12 patients receiving both doses of 
antibody.  Grade 1-2 diarrhea and rash were the most common drug-related AEs.  The only noted 
grade 3 irAEs were ischemic papillopathy and diarrhea, which were both responsive to treatment 
with steroids.   
 
Liakou and colleagues found that CD4 T cells from peripheral blood and tumor tissues of all 
bladder cancer patients treated with anti-CTLA-4 antibody had markedly increased expression of 
inducible costimulator (ICOS)54.  These CD4+ICOShi T cells produced interferon-gamma (IFN-γ) 
and could recognize the tumor antigen NY-ESO-1.  Increase in CD4+ICOShi cells led to an 
increase in the ratio of effector to regulatory T cells.  The authors indicated that these 
immunologic changes were reported in both tumor tissues and peripheral blood as a result of 
treatment with anti-CTLA-4 antibody, and they may be used to guide dosing and scheduling of 
this agent to improve clinical responses.  A sustained increased frequency of CD4+ICOShi T cells 
may serve as a biomarker of anti-CTLA-4 activity and/or of clinical benefit for patients who are 
being treated with this novel agent53.   
 
Pancreatic cancer 
Royal and colleagues presented the results on 27 patients (metastatic disease: 20 and locally 
advanced: 7)55.  Three subjects experienced ≥ grade 3 irAEs (colitis:1, encephalitis:1, 
hypophysitis:1).  One subject experienced a delayed response after initial progressive disease.  In 
this subject, new metastases after 2 doses of ipilimumab established progressive disease.  
However, continued administration of the agent per protocol resulted in significant delayed 
regression of the primary lesion and 20 hepatic metastases with normalization of tumor markers 
and clinically significant improvement of performance status.  The investigators concluded that 
single agent ipilimumab at 3.0 mg/kg/dose was ineffective for the treatment of advanced 
pancreatic cancer.  However, a significant delayed response in one subject of this trial suggests 
that immunotherapeutic approaches to pancreatic cancer deserve further exploration. 
 
CTEP-Sponsored Studies 
The DCTD, NCI, has sponsored nine studies with ipilimumab including one pilot study (NCI 
#5744, lymphoma), three phase 1 studies (5708 [ovarian], 6082 [solid tumors], and 7458 [solid 
tumors]), one phase 1/2 study (6359 [non-Hodgkin’s lymphoma]) with single agent ipilimumab, 
two phase 1 combination studies in prostate cancer with GM-CSF (6032) and with prostate-

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specific antigen (PSA)-TRICOM vaccine (7207), one phase 2 combination study of ipilimumab 
with GM-CSF (E1608, melanoma) and one phase 3 study (E1609) of adjuvant ipilimumab 
therapy versus high-dose interferon alpha-2b in patients with resected high-risk melanoma.   
 
Results from 11 patients (colon, n=3; non-Hodgkin’s lymphoma, n=4; prostate, n=4) who 
received ipilimumab on study 5744 included tumor regression in 2 patients with lymphoma; 1 of 
whom (follicular lymphoma patient) had a partial response (PR) of 14-month duration56.  
Ipilimumab was well tolerated with predominantly grade 1/2 toxicities.  One drug-related grade 3 
AE was observed.  Tregs, as detected by expression of CD4+CD25+CD62L+, declined at early 
time points but rebounded to levels at or above baseline values at the time of the next infusion.  
The investigators concluded that ipilimumab treatment depressed Treg numbers at early time 
points in the treatment cycle but was not accompanied by an increase in vaccine-specific CD8+ 
T-cell responses in these patients previously treated with a variety of investigational anticancer 
vaccines.   
 
Hodi and colleagues reported preliminary results on 20 patients (11 metastatic melanoma 
patients and 9 metastatic ovarian carcinoma patients) on study 570857.  None of the 11 patients 
from the metastatic melanoma cohort manifested grade 3 or 4 inflammatory toxicities; however, 
all subjects revealed mild inflammatory pathologies associated with low-level constitutional 
symptoms.  The most common toxicity (10/11 subjects) was a grade 1-2 reticular and 
erythematous rash on the trunk and/or extremities that arose between 3 days and 3 weeks after 
antibody administration and then gradually resolved without specific intervention.  Biopsies of 
involved skin revealed low-grade interface dermatitis, minor to moderate mononuclear infiltrates 
surrounding the superficial dermal vasculature, and increased mucin deposition in the papillary 
and reticular dermis.  These pathologic features resembled those observed in mild cutaneous 
forms of systemic lupus erythematosis.  Three PRs (range, 21-34+ months) and five events of 
stable disease (SD) (range, 4-25 months) were observed.  One PR and three SDs (2, 4, and 6+ 
months) were observed in the ovarian carcinoma group.  The investigators concluded that 
selective targeting of antitumor regulatory T cells (Treg) may constitute a complementary 
strategy for combination of ipilimumab and GM-CSF-based antigen tumor cell vaccine therapy.  
 
Results from 29 patients with malignancies that were recurrent or progressive after allogeneic 
hematopoietic cell transplantation (allo-HCT) demonstrated that drug was well tolerated at single 
doses up to 3 mg/kg58.  Four patients experienced organ-specific irAEs of reversible grade 3 
arthritis, grade 2 hyperthyroidism, dyspnea, and grade 4 pneumonitis.  Three patients had 
objective responses: one PR lasting for 2 months, and two durable complete responses (CRs).  
Two additional patients with Hodgkin’s disease who had evidence of rapid disease progression 
prior to ipilimumab treatment achieved SD for 3 and 6 months, following infusion at the 3 mg/kg 
dose level.  Median OS was 24.7 months.  At a 3.0 mg/kg dose, active serum concentrations of 
ipilimumab were maintained for 30 days following a single infusion.  Zhou and colleagues 
reported immunophenotypes of peripheral blood T cells, including T-cell reconstitution, 
activation, and Treg expression, in 29 patients before and after a single-dose infusion of 
ipilimumab59.  CTLA-4 blockade by a single infusion of ipilimumab increased CD4+ and 
CD4+HLA-DR+ T lymphocyte counts and intracellular CTLA-4 expression at the highest dose 
level (3.0 mg/kg).  There was no significant change in Treg cell numbers after ipilimumab 
infusion.  These data demonstrate that significant changes in T-cell populations occur on 

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exposure to a single dose of ipilimumab.   
 
Harzstark and colleagues reported results on 36 patients with hormone refractory metastatic 
prostate cancer60.  Of six patients treated with ipilimumab at a dose of 3 mg/kg, three patients 
had confirmed PSA declines of ≥50%, with a time to progression (TTP) of 22, 26, and 103 
weeks.  One of these patients had a PR in hepatic metastases.  Grade 3 IrAEs consisted of rash in 
five patients, panhypopituitarism in one patient, temporal arteritis in one patient, and diarrhea in 
three patients.  Non-irAEs included grade 3 and 4 cerebrovascular events (one patient each), 
grade 3 angina (one patient), grade 3 atrial fibrillation (one patient), grade 3 fatigue (four 
patients), and grade 5 pulmonary embolism (one patient).  One patient treated at 10 mg/kg had a 
PSA decline of ≥50% with a TTP of 39 weeks.  Higher doses of treatment with MDX-010 + 
GM-CSF induced the expansion of activated circulating CD25+, CD69+, and CD8+ T cells more 
frequently than was seen in patients who received the same doses of either MDX-010 or GM-
CSF alone61.  The sera screening with protein arrays showed that the treatment can induce 
antibody responses to the testicular antigen NY-ESO-1.   
 
Patients with metastatic prostate cancer were treated with ProstVac vaccine and ipilimumab 
before chemotherapy.  The median OS for all patients on study was 31.8 months with a 74% 
survival probability at 24 months62.  The median Halabi predicted OS for all patients was 18.5 
months.  There was no significant difference in OS at different dose levels of antibody (range 1-
10 mg).  A unique effect of the vaccines on the rate of tumor growth may be a novel method to 
evaluate the anti-tumor effects of the vaccine63.  The authors suggested that the addition of 
immune checkpoint inhibition may augment the clinical benefit of vaccines.  
 
Ansell and colleagues reported data on 18 treated patients with NHL64.  Two clinical responses 
were observed:  one patient with diffuse large B-cell lymphoma (BCL) had an ongoing CR (>31 
months), and one with follicular lymphoma had a PR lasting 19 months.  In 5 of 16 cases tested, 
T-cell proliferation to recall antigens was >2 fold increased after ipilimumab therapy.  The 
investigators have found that blockade of CTLA-4 signaling with the use of ipilimumab is well 
tolerated at the doses used.  Ipilimumab has antitumor activity in patients with BCL, resulting in 
durable responses in a minority of patients.  Ipilimumab at 3 mg/kg monthly for 4 months can be 
given safely and is the dose that recommended for future combination studies.   
 
2.1.2.8 Clinical Safety   
 
Safety Experience  
The most common treatment-related AEs (those considered possibly, probably, or definitely 
related to study drug by the investigator) associated with the use of ipilimumab were immune 
related irAEs (Investigator Brochure, 2011).  The irAEs primarily involved the gastrointestinal 
(GI) tract (e.g., diarrhea and colitis) and skin (e.g., pruritus and rash), and less frequently, the 
liver, endocrine glands (including the thyroid, pituitary, and adrenal glands) and nervous system.  
IrAEs were generally managed with either symptomatic therapy (grade 1-2 events), systemic 
corticosteroids (grade 3-4 events), or other immunosuppressants (e.g., infliximab, mycophenolate 
mofetil) for steroid-unresponsive GI or hepatic irAEs, as appropriate.  Management of irAEs was 
usually paired with omission of dosing for mild or moderate events and permanent 
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reactions due to T-cell activation and proliferation.  Fatalities due to GI perforation, hepatic 
failure, toxic epidermal necrolysis, and Guillain-Barré syndrome have been reported in clinical 
trials of ipilimumab.   
 
Clinical trials are conducted under widely varying conditions so that extrapolation to novel 
settings and combinations regarding rates and severity of events may be unreliable.  Given the 
expected rate of toxicity which may require stopping study drug but may also be related to a 
therapeutic immunologic response alternative DLT criteria are discussed in Section 6.  
 
Min and colleagues reported three patients who received ipilimumab alone or combined with 
bevacizumab therapy and developed thyroiditis, and the first report of euthyroid Graves’ 
ophthalmopathy65.  They recommend that all patients on ipilimumab alone or combined with 
bevacizumab therapy have baseline thyroid function tests and careful monitoring for new onset 
of thyroid disease, particularly during the first 3 months of treatment. See specific events in 
Section 5.  
 
Safety Profile of Ipilimumab at a Dose of 10 mg/kg (Phase 2 data) 
The safety profile of ipilimumab as monotherapy over multiple doses at a dose of 10 mg/kg in 
325 subjects was determined from 4 completed melanoma studies.  Overall, the incidence of 
grade 3/4 AEs attributable to study drug was 31%.  The target organ system, the incidence, and 
the severity of the most commonly observed irAEs vary among studies and with drug 
combinations.  Typically, the severity but not necessarily the overall incidence increases with 
dose.  Additional information on specific events is provided in Section 7.1 and the IB:   
 
Summary of irAE Safety Data for 10 mg/kg in Melanoma 
 
Total 
Low-grade 
(Grade 1 - 2) 
(%) 
High-grade 
(Grade 3 - 4) 
(%) 
Median Time to 
Resolution of 
Grade 2 - 4 
irAEs (weeks) 
All irAEs 
72.3 
46.2 
25.2 
- 
Skin (e.g., rash, pruritus) 
52.0 
49.2 
2.8 
6.14 
GI (e.g., colitis, diarrhea) 
37.2 
24.9 
12.3 
2.29 
Liver (e.g., LFT 
elevations) 
8.0 
0.9 
6.8 
4.0 
Endocrine (e.g., 
hypophysitis, 
hypothyroid) 
6.2 
3.7 
2.5 
20.1 
 
Pregnancy 
Preliminary results are available in cynomolgus monkeys.  Pregnant monkeys received 
ipilimumab every 21 days from the onset of organogenesis in the first trimester through delivery, 
at dose levels either 2.6 or 7.2 times higher than the clinical dose of 3 mg/kg of ipilimumab (by 
AUC).  No treatment-related adverse effects on reproduction were detected during the first two 
trimesters of pregnancy.  Beginning in the third trimester, the ipilimumab groups experienced 
higher incidences of abortion, stillbirth, premature delivery (with corresponding lower birth 
weight), and higher incidences of infant mortality in a dose-related manner compared to controls.  

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Based on animal data, ipilimumab may cause fetal harm.  The use of ipilimumab during human 
pregnancy has not been formally studied in clinical trials.  There have been 7 known pregnancies 
during ipilimumab treatment: in 3 female subjects and in the partners of 4 male study subjects.  
Two (2) of the 3 female pregnancies ended with elected terminations.  The third female subject 
had a history of seizures and delivered the baby at 36 weeks gestation.  The baby had respiratory 
complications that resolved by birth week 16.  Three (3) of the 4 partners of male study subjects 
had full term, normal babies.  The fourth baby had small ureters, which are expected to grow as 
the baby matures.  Although these outcomes do not indicate that stillbirths or other severe 
abnormalities will occur, pregnancy should be avoided during treatment with ipilimumab.  
 
Immunogenicity 
In clinical studies, 1.1% of 1024 evaluable patients tested positive for binding antibodies against 
ipilimumab in an electrochemiluminescent (ECL) based assay.  This assay has substantial 
limitations in detecting anti-ipilimumab antibodies in the presence of ipilimumab. Infusion-
related or peri-infusional reactions consistent with hypersensitivity or anaphylaxis were not 
reported in these 11 patients nor were neutralizing antibodies against ipilimumab detected.  
Because trough levels of ipilimumab interfere with the ECL assay results, a subset analysis was 
performed in the dose cohort with the lowest trough levels. In this analysis, 6.9% of 58 evaluable 
patients, who were treated with 0.3 mg/kg dose, tested positive for binding antibodies against 
ipilimumab.  These results are highly dependent on methodology, and comparison of incidence 
of antibodies to ipilimumab with the incidences of antibodies to other products may be 
misleading. 
 
Study Results and Clinical Efficacy  
The clinical efficacy of ipilimumab as a single agent at a dose of 3 mg/kg administered q3w for 4 
doses has been established in MDX010-20 (a randomized, controlled study in second line, 
locally advanced/metastatic melanoma), which led to approval of ipilimumab by the FDA.  In 
study CA184024, the addition of 10 mg/kg ipilimumab to dacarbazine led to a prolongation of 
overall survival in patients with previously untreated melanoma.  
 
In melanoma studies, disease stabilization in subjects receiving ipilimumab is characteristic of 
anti-tumor activity.  Stable disease, sometimes of long duration, or slow steady decline of tumor 
lesion size over long periods of time, has been observed.  Some subjects demonstrate initial 
tumor volume increase before response, possibly due to T-cell infiltration as shown by biopsies 
or to the time required for immunologic activation.  Consequently, an initial determination of 
progressive disease and consequently PFS may not capture all patterns response and may 
underestimate the clinical activity of ipilimumab.  Please see section “Considerations for Using 
Immune-Related Tumor Assessment Criteria (irRC).”  
 
MDX010-20 (Phase 3, 3 mg/kg, previously treated melanoma) 
MDX010-20, a randomized (3:1:1), double-blind, double-dummy study included 
676 randomized subjects with unresectable or metastatic melanoma previously treated with one 
or more of the following: aldesleukin, dacarbazine, temozolomide, fotemustine, or carboplatin.  
Of these 676 subjects, 403 were randomized to receive ipilimumab at 3 mg/kg in combination 
with an investigational peptide vaccine with incomplete Freund’s adjuvant (gp100), 137 were 
randomized to receive ipilimumab at 3 mg/kg, and 136 were randomized to receive gp100 alone.  

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The study enrolled only subjects with HLA A2*0201 genotype; this HLA genotype facilitates 
the immune presentation of the investigational peptide vaccine.  The study excluded subjects 
with active autoimmune disease or those receiving systemic immunosuppression for organ 
transplantation. The OS results are shown in the table below. 
 
MDX010-20 Overall Survival Results 
 
Ipilimumab 
n = 137 
Ipilimumab + 
gp100 
n = 403 
gp100 
n = 136 
Hazard Ratio (vs gp100) 
 
(95% CI) 
0.66 
(0.51, 0.87) 
0.68 
(0.55, 0.85) 
 
 
p-value 
p = 0.0026a 
p = 0.0004 
 
Hazard Ratio (vs 
ipilimumab) 
 
(95% CI) 
 
1.04 
(0.83, 1.30) 
 
Median (months) 
 
(95% CI) 
10 
(8.0, 13.8) 
10 
(8.5, 11.5) 
6 
(5.5, 8.7) 
aNot adjusted for multiple comparisons. 
 
CA184024 (Phase 3, previously untreated melanoma, 10 mg/kg) 
CA184024 evaluated the addition of 10 mg/kg ipilimumab to dacarbazine in patients with 
previously untreated, metastatic melanoma. A total of 502 patients were randomized to receive 
up to 8 cycles of dacarbazine 850 mg/m2 q3w, with either ipilimumab 10 mg/kg or placebo 
cycles 1-4, and as maintenance after completion of chemotherapy. 
 
Patients on the ipilimumab arm received a median of 3 ipilimumab induction doses, versus 4 
placebo induction doses on the placebo arm. A total of 17.4% and 21.1% of patients continued to 
receive maintenance ipilimumab or placebo, for a median of 4 and 2 doses, respectively. The 
number of patients who received all 8 dacarbazine doses was 12.2% in the ipilimumab arm, and 
21.5% in the placebo arm. 
 
The study met its primary end-point of prolonging overall survival in patients treated with 
ipilimumab (HR 0.72 [95% CI, 0.59 – 0.87], median OS 11.2 vs 9.1 months, p = 0.0009).  
 
One, two, and three year survival rates were 47.3%, 28.5%, and 20.8% in the ipilimumab arm, 
and 36.3%, 17.9%, and 12.2% in the placebo arm. 
 
PFS, a secondary end-point, was also prolonged by the addition of ipilimumab, HR 0.76 (95% 
CI, 0.63 - 0.93). The median PFS was 2.8 months in the ipilimumab vs 2.6 months in the placebo 
arm, p = 0.006. 
 
Best overall response rate (BORR) was increased from 10.3% in the placebo arm to 15.2% in the 
ipilimumab arm. More importantly, duration of response was more than twice as long in the 
ipilimumab arm (19.3 months) than in the placebo arm (8.1 months). 
 
10 mg/kg Dosing with Ipilimumab 

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Several additional trials studied the efficacy and safety of 10 mg/kg dosing, and additional 
information gained from these trials is listed below: 
A dose of 10 mg/kg may be necessary to ensure blockade of the CTLA-4 pathway; in vitro, a 
concentration of 20 mcg/mL of ipilimumab was the minimal concentration able to fully abrogate 
the binding of CTLA-4 to B7.1 and B7.2.  With a dose of 3 mg/kg q3w, 30% achieved a trough 
concentration of ipilimumab greater than 20 g/mL, compared to 95% of subjects treated at 10 
mg/kg q3w. 
 
In addition, in all ipilimumab trials examined to date, mean Absolute Lymphocyte Count (ALC) 
increased after ipilimumab treatment throughout the 12-week induction-dosing period, in a dose-
dependent manner.  In an analysis of ipilimumab at 0.3, 3, or 10 mg/kg in melanoma studies 
CA184007, CA184008, and CA184022 combined, the rate of change in ALC after ipilimumab 
treatment was significantly associated with dose (p = 0.0003), with the largest rate at 10 mg/kg 
ipilimumab.  Moreover, the rate of change in ALC over the first half of the induction-dosing 
period was significantly associated with clinical activity in these studies (p = 0.009), where 
clinical activity was defined as CR, PR, or prolonged SD (i.e., SD lasting at least 6 months from 
first dose). Although these analyses alone could not determine whether the rate of change in 
ALC was specifically associated with clinical activity in response to ipilimumab treatment, as 
opposed to being generally prognostic, these results do suggest a potential benefit to higher rates 
of ALC increase after ipilimumab treatment.  Among the 3 doses evaluated, 10 mg/kg 
ipilimumab led to the greatest such rates. 
 
In the 3 primary studies conducted in advanced melanoma (CA184007, CA184008, and 
CA184022), subjects treated with 10 mg/kg single agent ipilimumab had the highest response, 
disease control rates, median OS as well as 1-year and 2-year survival rates compared to lower  
doses. The CA184022 data are summarized in the table below. 
 
Summary of Phase 2 Response Data in Melanoma (CA184022 see IB ) 
 
10 mg/kg 
(n = 72) 
3 mg/kg 
(n = 72) 
0.3 mg/kg 
(n = 73) 
BORR (mWHO) – % 
(95% CI) 
11.1 
(4.9 - 20.7) 
4.2 
(0.9 - 11.7) 
0 
(0.0 - 4.9) 
DCR (mWHO) – % 
(95% CI) 
29.2 
(19.0 - 41.1) 
26.4 
(16.7 - 38.1) 
13.7 
(6.8 - 23.8) 
Survival rate at 1 year - 
% 
%, 95% CI 
48.64 
(36.84, 60.36) 
39.32 
(27.97, 50.87) 
39.58 
(28.20, 51.19) 
Survival rate at 2 year - 
% 
%, 95% CI 
29.81 
(19.13, 41.14) 
24.20 
(14.42, 34.75) 
18.43 
(9.62, 28.22) 
Overall median survival 
95%CI (months) 
11.43 
(6.90, 16.10) 
8.74 
(6.87, 12.12) 
8.57 
(7.69, 12.71) 
 
Dose, Schedule, and Regimen 
While optimal doses and schedules for ipilimumab have not yet been determined, in proposed 
proof of principle studies demonstration of efficacy at 10 mg/kg would allow future studies to 

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explore biologic and clinical efficacy at lower doses with reduced toxicity. For most studies in 
new combinations or settings, a short phase 1 component at 3 mg would be appropriate with a 5 
or 6 mg/kg dose added as an additional cohort if needed.    
 
A recommended dose of 10 mg/kg is proposed by the manufacturer for most studies of 
ipilimumab.  In melanoma, a similar survival benefit was demonstrated in phase 3 trials at the 3 
mg/kg and at 10 mg/kg with DTIC.  However, the incidence of grade >3 toxicity was 15 and 
25% respectively.   
 
Based on Phase 2 studies, response rates of ipilimumab appear to be dose dependent up to 10 
mg/kg. Exposure-response analyses [Cminss Analysis of PK data from patients treated with 
ipilimumab at 0.3 mg/kg (N=47), 3 mg/kg (N=60) and 10 mg/kg (N=311)], showed that the 
target Cminss target threshold of 20 mcg/ml was exceeded in 0%, 30% and 95% subjects 
respectively.  The slope of change in absolute lymphocyte count (ALC) correlated with clinical 
benefit and T-cell activation markers such as HLA-class II expression may also be dose 
dependent.  Responses have not been compared systematically in randomized phase 2 or phase 3 
studies in patients with tumor types other than melanoma.  
 
Regarding schedule, the typical schedule for advanced melanoma at present is once q3w for four 
doses followed by a maintenance phase of four doses every 12 weeks.  Of interest, ipilimumab 
was evaluated in NSCLC and SCLC using a dose of 10 mg/kg given concomitantly or following 
initial paclitaxel/cisplatin.  When used in the phased schedule, 10 mg significantly improved 
irPFS and mWHO defined responses but not PFS determined by Response Evaluation Criteria in 
Solid Tumors (RECIST).  There also was a trend for an improvement in OS in both indications.  
Doses less than 10 mg/kg have not been evaluated in either NSCLC or SCLC.  
 
Studies comparing doses in non-melanoma and combinations have not been widely done.  There 
are also no clear data that peak levels, Cmin, AUC, exposure and number of doses given, or the 
occurrence of autoimmune events, predict responses in individual patients.  We note that the 
incidence of specific events such as hypophysitis may vary from study to study and with 
different combinations of agents.  The severity and possibly time to onset but not necessarily the 
frequency of events increases with dose.  In addition, there are rare but serious events such as 
toxic epidermal necrolysis (TEN) for which a dose relationship has not been established. Case 
report forms should include data on the prior treatment, timing, number of doses, duration of 
event, response to treatment, and complications to allow comparisons among studies.   
 
Considerations in Using Immune-Related Tumor Assessment Criteria (irRC) 
New end point definitions for trials of immunologic agents have been proposed based on novel 
patterns of clinical activity in malignant melanoma66,67. These alternative definitions allow time 
for immunologically mediated effectors to develop that may result in late tumor responses even 
after initial progression by RECIST.  Also, in some patients, tumor necrosis and inflammation 
may increase tumor size radiographically prior to response.  Changing the definitions of OR and 
PD may alter (increase) the number patients achieving responses and the duration of PFS.  
 
On a protocol by protocol basis, we would consider allowing study treatment to continue during 
initial progression up to the 12-16 week assessment to allow time for responses to be observed, if 

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the patient is clinically stable, there is no deterioration in PS, and there is no need for immediate 
additional treatment.  While maintaining standard definitions of progression and response, we 
would allow new lesions and some progression beyond 20% increases in tumor measurements 
during the initial treatment period to allow time for responses to develop (these delayed tumor 
responses may be seen in 10-20% of melanoma patients who initially progress during the initial 
treatment cycles and evaluation).  We do not have experience with response patterns with 
combination therapy nor in diseases other than melanoma.  Please use standard response 
definitions as the primary end point in these studies.   
 
Note that the proposed irRC may be incorporated as secondary end points to compare to standard 
criteria and evaluate alternative patterns of response in various disease setting and treatment 
regimens.  A copy of the proposed criteria is presented in Appendix D.  
 
Patients who demonstrate mixed responses, stable disease, or objective responses by standard 
RECIST following initial progression may be identified separately as “delayed SD, PR, or CR”.   
 
Overall Risk/Benefit Assessment 
The unique immune-based mechanism of action is reflected in the clinical patterns of anti-cancer 
activity in some patients.  Ipilimumab affects tumor cells indirectly, and measurable clinical 
effects emerge after the immunological effects.  Tumor infiltration with lymphocytes and the 
associated inflammation (documented by biopsy in some subjects) is likely the cornerstone of the 
effect of ipilimumab and can manifest in various patterns of clinical activity leading to tumor 
control.  In some cases, response may be preceded by an apparent increase in initial tumor 
volume and/or the appearance of new lesions, which may be mistaken for tumor progression on 
radiological evaluations.  Therefore, in subjects who are not experiencing rapid clinical 
deterioration, confirmation of progression is recommended, at the investigator’s discretion, to 
better understand the prognosis as well as to avoid unnecessarily initiating potentially toxic 
alternative therapies in subjects who might be benefitting from treatment.  Immune-related (ir) 
response criteria were developed based on these observations to systematically categorize novel 
patterns of clinical activity and are currently being prospectively evaluated in clinical studies. 
 
In metastatic diseases, stabilization is more common than response, and in some instances is 
associated with slow, steady decline in tumor burden over many months, sometimes improving 
to partial and/or complete responses.  Thus, the immune-based mechanism of action of 
ipilimumab results in durable disease control, sometimes with novel patterns of response, which 
contribute to its improvement in OS. 
 
The immune-based mechanism of action is also reflected in the safety profile.  The most 
common drug-related AEs are immune-mediated, consistent with the mechanism of action of the 
drug and generally medically manageable with topical and/or systemic immunosuppressants.  As 
previously discussed, the immune-mediated adverse reactions primarily involve the GI tract, 
skin, liver, endocrine glands, and nervous system. 
 
The early diagnosis of immune-mediated adverse reactions is important to initiate therapy and 
minimize complications.  Immune-mediated adverse reactions are generally manageable using 
symptomatic or immunosuppressive therapy as recommended through detailed diagnosis and 

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management guidelines, as described fully in the current IB.  The management guidelines for 
general immune-mediated adverse reactions and ipilimumab-related GI toxicities, hepatotoxicity, 
endocrinopathy, and neuropathy are provided in the appendices of the current IB. 
 
2.2 Rationale (Parts A+B) 
 
The development of SCCA of the anal canal is often triggered by enduring infection with high-
risk strands of human papillomavirus (HPV), most commonly HPV-16 and HPV-1868-71.  
Approximately 75% of all sexually active adults will become infected with HPV during their 
lifetimes72.  While most will clear infection of HPV without any required intervention73, the virus 
may persist in a fraction of patients and cause cancer when viral DNA becomes incorporated into 
the host genome.  The oncoproteins E6 and E7, translated products of HPV DNA integrated into 
the host cell, promote oncogenesis in mucosal squamous cells of anal canal through multiple 
mechanisms.  For example, E6 binds p53 to generate a complex which results in ubiquitin-
mediated p53 degradation74-76.  Likewise, the interaction between E7 and the retinoblastoma 
protein Rb triggers a phosphorylation of Rb and allows continuation of cell cycle progression 
through the loss of tumor suppressor function by Rb77-79.   
 
The presence of high-risk HPV has been detected in approximately 75-95% of all reported cases 
of SCCA of the anal canal68-71.  In addition, this virus has also been correlated to the 
development of other malignancies like SCCAs of the head and neck (over 70% of which are 
HPV-positive tumors), cervix (> 90%), penis (>60%), vulva (~70%), and vagina (75%) 16,30,80-85.  
A clear association between altered immunity and the development of SCCA is apparent, and a 
functional immune system may be responsible for clearing HPV infection.  Immunosuppression 
from HIV/AIDS, use of immunosuppressive medications following organ transplantation, and 
coexisting autoimmune disease are all recognized risk factors for developing SCCA19,23,28,68,86.  
Furthermore, the HPV-generated oncoproteins E6 and E7 are immunogenic and trigger an anti-
tumor host immune response via activation of tumor infiltrating lymphocytes (TILs) 87-91.  These 
neoantigens are examples of “non-self” molecules recognized by T cells which are produced 
following viral incorporation into the host genome.  However, suppression of such anti-tumor 
activity may allow a tumor to evade an immune response and survive in HPV-related 
malignancies.  This notion has been verified in invasive cervical cancer, in which HPV-specific 
CD8+ cytotoxic T cells and CD4+ helper T-cells are lost and inhibitory HPV-specific regulatory 
T cells92-94 become activated.   
   
In a preventative trial, vaccination with a quadrivalent HPV vaccine (covering HPV-16) showed 
fewer cases of precancerous high-grade anal intraepithelial neoplasia when compared to people 
who received placebo, and no cases of invasive carcinoma95.  Likewise, similar results in 
preventing development of pre-cancerous cervical dysplasia have been described by vaccinating 
young women with the quadrivalent HPV vaccine87.  Despite such clinical promise, recent 
studies have suggested that fewer than 30% of eligible females in the United States have 
received the complete series of HPV vaccinations, and this proportion is even lower in males96.  
In underdeveloped countries, where the prevalence of HPV infection and HPV-associated 
malignancies are higher than in developed nations, providing access to these preventative 
vaccinations are currently in development, so that at present the majority of citizens do not 
receive these vaccines.  In addition, patients with HPV-associated anogenital malignancies are 

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exposed to an increased risk of developing a second HPV-related cancer from the effects of field 
cancerization caused by HPV infection97.  Therefore, for these reasons it is expected that the 
incidence of SCCA of the anal canal will continue to rise and will continue to worsen in global 
prevalence for several decades.  
 
Tumor cells express the programmed death receptor ligand-1 (PD-L1) as a means to down 
regulate T cell activation and thwart the local anti-tumor immune response by binding the 
inhibitory receptor programmed death-1(PD-1) on the surface of T cells21,98.  Expression of PD-
L1 on resected oral squamous cell tumors demonstrated a negative correlation with intratumoral 
CD8+ TIL density99.  Additionally, in one study of HPV+ tumors of the oropharynx, 14 of 20 
specimens (70%) demonstrated increased PD-L1 expression by immunohistochemistry (IHC), 
whereas only 2 of 7 (29%) HPV- tumors stained positive for PD-L1100.  These results support the 
notion that HPV+ tumors may utilize the PD-1: PD-L1 interaction between the tumor cell and 
nearby T cell in order to disrupt an antitumor host immune response. 
 
Nivolumab is a monoclonal antibody against PD-1.  In vitro, disruption of PD-1: PD-L1 
communication by nivolumab perpetuates T-cell activity against tumor cells101,102.  When 
translated to a phase I study of nivolumab as a single agent, objective responses have been 
observed in over 20% of patients with refractory metastatic melanoma, non-small cell lung 
cancer, and renal cell carcinoma32.  Additionally, among tumor specimens for which PD-L1 
expression could be evaluated by IHC, a positive correlation between PD-L1 expression and 
response to nivolumab was detected, and no tumors that lacked PD-L1 expression demonstrated 
a response to nivolumab.   
 
The prognostic impact of HPV in patients with metastatic SCCA of the anal canal remains 
unreported.  In a series of 72 patients with metastatic SCCA of the anal canal who had undergone 
biopsy and/or surgical resection of their tumors at MD 
Anderson for survival outcomes103,  tumors were tested 
for the presence of HPV using in-situ hybridization and 
by immunohistochemistry with monoclonal antibodies 
against P16, a protein regarded as a surrogate biomarker 
for HPV infection104 with upregulated expression 
following loss of E7-mediated Rb function105,106.   HPV 
was detected in 68 of the 72 patients (95%) analyzed.  
Per Figure 1, no differences in survival were measured 
between the HPV-positive and HPV-negative cohorts, 
although a trend towards improved survival was seen in 
the HPV-positive patients.  This result may be attributed 
to an under powering to detect true differences because 
the overwhelming majority of patients with metastatic 
SCCA of the anal canal treated at M.D. Anderson Cancer 
Center have detectable HPV. 
 
Rationale for the combination (Part B):  
The immunogenicity of tumor cells renders them susceptible to immune recognition and attack 
by cytotoxic T cells.  In response, multiple adaptive mechanisms within the tumor 

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microenvironment, through inhibitory interactions of the intratumoral T cells with both the tumor 
cell antigen-presenting cells, can thwart the anti-tumor immune response.   For example, binding 
of B7 on APCs with CTLA-4 on activated T cells can downregulate the effector function of 
cytotoxic T cells specific to a given tumor 40-42.  Ipilimumab is a monoclonal antibody against 
CTLA-4 which impairs this B7:CTLA-4 interaction at the T cell surface and retains T cell 
activation 43,44.  This drug has received FDA approval for the treatment of metastatic melanoma 
following multiple positive phase III trials 45,46, and recent reports have demonstrated that 20% 
of patients with this disease remain alive after three years following treatment with ipilimumab47.  
Therefore, manipulation of the immune response for advanced tumors like melanoma improve 
patient outcomes when standard cytotoxic therapies are less effective and generate significant 
optimism for these findings to be similar in other immunogenic tumors like metastatic SCCA. 
 
Other surface proteins on the tumor cells themselves like  programmed death receptor ligand-1 
(PD-L1) can be expressed to downregulate T cell activation by binding the inhibitory receptor  
PD-1 on the surface of T cells 48,49.  Expression of PD-L1 on resected oral squamous cell tumors 
demonstrated a negative correlation with intratumoral CD8+ TIL density50.  Additionally, in one 
study of HPV+ tumors of the oropharynx, 14 of 20 specimens (70%) demonstrated increased 
PD-L1 expression by immunohistochemistry (IHC), whereas only 2 of 7 (29%) HPV- tumors 
stained positive for PD-L151.  These results support the notion that HPV+ tumors may likewise 
utilize the PD-1:PD-L1 interaction between the tumor cell and nearby T cell in order to disrupt 
an antitumor hose immune response.  Nivolumab is a monoclonal antibody against PD-1, and in 
vitro, disruption of PD-1:PD-L1 communication by nivolumab perpetuates T-cell activity against 
tumor cells52,53.  As a single agent, nivolumab has demonstrated significant clinical activity for 
patients with refractory metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, 
bladder cancer, and Hodgkin’s lymphoma54.bladder cancer, and Hodgkin’s lymphoma54. 
 
Anti-PD1 therapy has activity in patients with previously treated metastatic SCCA: We 
completed a single-arm, multi-institutional ETCTN phase II trial of nivolumab for patients with 
refractory SCCA (NCI9673)107.   This trial accrued all 37 patients within 6 months.  Objective 
responses were noted in 9 of 37 treated patients (response rate 24%), with a median progression-
free survival of 4.1 months.  The responses rate met the predefined statistical significance for 
efficacy in this study. The median number of administered doses of nivolumab was 6 
(interquartile range, 3-10).  Treatment was overall safe and well tolerated.  These findings 
suggest that anti-PD1 therapy may be a novel effective treatment approach for patients with 
metastatic SCCA. 
 
Based on our preliminary findings suggesting that durable responses to anti-PD-1 therapy can be 
observed for patients with metastatic SCCA in the presence of an inflammatory peritumoral 
milieu, it is feasible that additional agents targeting the immune checkpoint axes can further 
deepen the anti-tumor response and improve clinical outcomes in this setting.  While nivolumab 
has earned FDA approval as a single-agent therapy for advanced malignancies like melanoma 
and non-small cell lung cancer (NSCLC), the combination of nivolumab with ipilimumab 
appears to improve response rates further for affected patients.  Indeed, in a phase II trial 
comparing nivolumab and ipilimumab revealed improved clinical outcomes for patients treated 
with the combination relative to ipilimumab alone.  Analogously, recent results from the 
CheckMate-012 study reported response rates of 39% in patients with NSCLC who received 

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nivolumab 3 mg/kg every 2 weeks and ipilimumab 1 mg/kg every 6 weeks.  The combination of 
immune checkpoint agents targeting PD-1 and CTLA-4 together appeared to have improved 
clinical outcomes for NSCLC when compared to prior studies with the response rates of single 
agent nivolumab of approximately 20%.  Recent data supports the PD-1 and CTLA-4 
combination in two phase III trials in treatment naïve advanced melanoma patients108. 
NCT02231749 (Checkmate 214) is an ongoing phase III trial of nivolumab + ipilimumab versus 
sunitinib in treatment-naïve advanced or metastatic renal cell carcinoma patients109. 
 
Collectively, these findings suggest that dual blockade of non-overlapping immune checkpoint 
targets may augment the anti-tumor immune response as manifested by improved clinical 
outcomes for patients treated with anti-PD-1 and anti-CTLA-4 agents together. 
 
To expand upon the success of nivolumab as a single-agent observed in the NCI 9673 study, we 
have amended NCI9673 for a randomized phase II trial of nivolumab plus/minus ipilimumab for 
patients with previously treated metastatic anal cancer.  We have demonstrated through the 
aforementioned NCI 9673 trial that patients with this disease can successfully be accrued 
through collaboration between multiple institutions within the ETCTN network.  Based on the  
findings of this study, we envision future trials in which nivolumab, with/or without ipilimumab 
(pending the efficacy of the immunotherapy combination), could be compared to traditional 
cytotoxic chemotherapeutic regimens commonly used in the management of this disease in order 
to identify the optimal treatment approach for patients  with this disease. 
 
2.3 Rationale for modified dosing for nivolumab and ipilimumab (Part B) 
 
Nivolumab 
Nivolumab monotherapy has been extensively studied in multiple tumor types, including 
melanoma, NSCLC, renal cell carcinoma (RCC), classical Hodgkin’s lymphoma (cHL), head 
and neck (H&N) and urothelial carcinoma (UC), using body weight normalized dosing (mg/kg), 
and has been safely administered at doses up to 10 mg/kg Q2W. Nivolumab is currently 
approved in the US for use in melanoma, NSCLC, RCC, and UC as 240 mg Q2W, and in cHL 
and H&N as 3 mg/kg Q2W. 
 
The nivolumab dose of 480 mg Q4W was selected for this study based on clinical data and 
modeling and simulation approaches using population PK (PPK) and exposure-response (ER) 
analyses examining relationships between nivolumab exposures and efficacy (e.g. OS, OR) and 
safety responses, using data from studies in multiple tumor types (melanoma, NSCLC, and RCC) 
with body weight-normalized dosing (mg/kg). The PPK analyses have shown that exposure to 
nivolumab increased dose proportionally over the dose range of 0.1 to 10 mg/kg administered 
Q2W, and no clinically meaningful differences in PK across ethnicities and tumor types were 
observed. Nivolumab clearance and volume of distribution were found to increase as body weight 
increases but less than proportionally with increasing weight, indicating that milligram-per-
kilogram dosing represents an over-adjustment for the effect of body weight on nivolumab PK. 
 
Using the PPK and ER models, nivolumab exposures and probabilities of efficacy responses and 
risks of AEs were predicted following nivolumab 480 mg Q4W and compared to those following 
nivolumab 3 mg/kg Q2W. The overall distributions of average nivolumab steady state exposures 

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(Cavgss) are comparable following administration with either nivolumab 3 mg/kg Q2W or 
nivolumab 480 mg Q4W. Nivolumab 480 mg Q4W is predicted to result in approximately 43% 
greater steady state peak concentrations (Cmaxss) compared to nivolumab 3 mg/kg Q2W; 
however, these exposures are predicted to be lower than the exposure ranges observed at doses up 
to nivolumab 10 mg/kg Q2W used in the nivolumab clinical program. Although the Cmaxss of 
nivolumab is expected to be greater following nivolumab 480 mg Q4W compared to nivolumab 3 
mg/kg Q2W, the predicted Cmaxss following nivolumab 480 mg Q4W is well below the median 
Cmaxss achieved following administration of nivolumab 10 mg/kg Q2W, a safe and tolerable dose 
level. 
 
Exposure-safety analysis demonstrated that the exposure margins for safety are maintained 
following nivolumab 480 mg Q4W, and the predicted  risks of AE-DC/D, AE Grade 3+, and  AE-
IM Grade 2+, are predicted to be similar following nivolumab 480 mg Q4W relative to nivolumab 
3 mg/kg Q2W across tumor types. Safety analyses using available data following nivolumab 3 
mg/kg Q2W and 10 mg/kg Q2W administration indicated there were no differences in AE profiles 
across body weight groups. Finally, initial evidence demonstrates that following administration of 
nivolumab 480 mg Q4W, nivolumab has been shown to be well tolerated. 
 
Nivolumab 480 mg Q4W is predicted to be approximately 16% lower steady-state trough 
concentrations (Cminss) compared to nivolumab 3 mg/kg Q2W. While these exposures are 
predicted to be lower, they are on the flat part of the exposure response curves and are not predicted 
to affect efficacy, Exposure-efficacy analyses of multiple PK measures and efficacy endpoints 
(e.g. OS, OR) indicated that following administration of nivolumab 480 mg Q4W efficacy is 
predicted to be similar to that following administration of nivolumab 3 mg/kg Q2W across multiple 
tumor types. Based on these data, nivolumab 480 mg Q4W is expected to have similar efficacy 
and safety profiles to nivolumab 240 mg Q2W or nivolumab 3 mg/kg Q2W. 
 
 
Ipilimumab 
In CA209012, ipilimumab 1 mg/kg using Q6W and Q12W schedules were assessed and were 
found to be safe in combination with nivolumab 3 mg/kg Q2W. Both the Q6W and Q12W arms 
were associated with improved and manageable tolerability compared to the arms with more 
frequent ipilimumab dosing (Q3W).  
 
Rationale for shorter infusion time:  
 
Administration of nivolumab using a 30-minute infusion time has been evaluated in subjects 
with cancer. Previous clinical studies of nivolumab monotherapy for the treatment of cancer have 
used a 60-minute infusion duration wherein nivolumab has been safely administered up to 10 
mg/kg over long treatment periods. Infusion reactions including high-grade hypersensitivity 
reactions have been uncommon across the nivolumab clinical program. In CA209010, a dose 
association was observed for infusion site reactions and hypersensitivity reactions (1.7% at 0.3 
mg/kg, 3.7% at 2 mg/kg and 18.5% at 10 mg/kg). All the events were Grade 1-2 and were 
manageable. An infusion duration of 30 minutes for 3 mg/kg nivolumab (30% of the dose 
provided at 10 mg/kg) is not expected to present any safety concerns compared to the prior 
experience at 10 mg/kg nivolumab dose infused over a 60-minute duration. The safety of 
nivolumab 3 mg/kg administered as a 30-min infusion was assessed in CA209153 in patients 

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(n=322) with previously treated advanced NSCLC. Overall, there were no clinically meaningful 
differences in the frequency of hypersensitivity/infusion-related reactions (of any cause or 
treatment-related) in cancer patients administered nivolumab over a 30 min infusion compared 
with that reported for patients with the 60 min infusion. Thus, it was shown that nivolumab can 
be safely infused over 30 min in subjects with cancer. 
 
 
•       Similarly, ipilimumab at 10 mg/kg has been safely administered over 90 minutes. In the 
CA184022 study, where ipilimumab was administered up to a dose of 10 mg/kg, on-study drug-
related hypersensitivity events (Grade 1-2) were reported in 1 (1.4%) participant in the 0.3 mg/kg 
and in 2 (2.8%) participants in the 10 mg/kg group. There were no drug-related hypersensitivity 
events reported in the 3 mg/kg group. Across the 3 treatment groups, no Grade 3-4 drug-related 
hypersensitivity events were reported, and there were no reports of infusion reactions. 
Ipilimumab 10 mg/kg monotherapy has also been safely administered as 90-minute infusion in 
large Phase 3 studies in prostate cancer (CA184043) and as adjuvant therapy for stage 3 
melanoma (CA184029), with no infusion reactions occurring in participants. Administering 1 
mg/kg of ipilimumab represents one-tenth of the 10 mg/kg dose.  
 
 
Overall, infusion reactions including high-grade hypersensitivity reactions have been uncommon 
across nivolumab or ipilimumab clinical studies or the combination of nivolumab and 
ipilimumab. Furthermore, a 30-minute break after the first infusion for the combination cohort 
will ensure the appropriate safety monitoring before the start of the second infusion. Overall, a 
change in safety profile is not anticipated with 30-minute infusion of nivolumab, ipilimumab, or 
combination. 
 
 
 
2.4 Correlative Studies Background (Part A) 
 
2.4.1 
Role of PD-L1 and PD-1 in the response of SCCA of the anal canal to nivolumab 
 
2.4.1.1 
Rationale 
 
While PD-L1 expression has been reported in a variety of solid tumors21,29,85,110,111, no data exist 
describing the expression of this protein in SCCA of the anal canal.   
 

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2.4.1.2 
Background  
Unpublished data from a small cohort of surgically resected tumors from patients with metastatic 
SCCA of the anal canal was collected for PD-L1 expression using an assay at MD Anderson 
which had previously been optimized to detect 
successfully cell surface PD-L1 expression in HPV-
positive cervical and penile cancers.  Tumors were 
stained using a monoclonal antibody against PD-L1 
(Epitomics; Burlingame, CA) and defined as “PD-L1 
positive” if at least 5% of tumor cells demonstrated 
PD-L1 surface expression (based on criteria used in 
prior published studies with nivolumab)32.  As 
exemplified in Figure 2, staining in PD-L1 was seen in 
two of the four (50%) tumors analyzed.  However, all 
four tumors exhibited peritumoral lymphocytes, a 
feature which has been associated with a more avid 
anti-tumor immune response in patients with other 
solid malignancies112.  These data demonstrate that PD-
L1 may be expressed in patients with HPV-positive 
metastatic SCCA of the anal canal. 
 
2.4.1.3 
Hypothesis 
 
Patients with metastatic SCCA of the anal canal will respond to therapy with nivolumab if their 
tumors stain positive for PD-L1 prior to treatment.  Durable responses to therapy may be 
observed in patients whose tumors maintain PD-L1staining on immunohistochemistry after two 
cycles of therapy.  Analogously, we hypothesize that the detection of PD-1, present on T-cells to 
interact with PD-L1, will also be associated with an anti-tumor response for patients treated with 
nivolumab. 
 
2.4.2 
Role of CD8+ Infiltrating T-cells in the response of SCCA of 
the anal canal to nivolumab 
 
2.4.2.1 
Rationale 
 
The associations between the presence of CD8+ infiltrating T cells and (1) responses to 
nivolumab and (2) survival outcomes have never been described in a prospective study for 
patients with metastatic SCCA of the anal canal.  This analysis would not only inform on the 
relevance of these immune cells in this particular disease for the first time, but could also be used 
in understanding further the role of CD8+  infiltrating T cells as a potential biomarker for immune 
checkpoint inhibitors like nivolumab across various HPV-related malignancies. The role of 
HPV-specific CD8+ T cells after nivolumab treatment may be of especial importance for clinical 
response. It will also be easy to assay since we already know their specificity (see 2.3.4. below). 
 
2.4.2.2 
Background 
 
Tumor specimens from a series of 38 patients with locoregional, non-metastatic SCCA of the 

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anal canal were reviewed retrospectively for a correlation between the presence of CD8+  
infiltrating T cells113.   No correlation between the higher numbers of these T cells and clinical 
outcomes were observed.  However, patients with metastatic disease were not included in this 
analysis, and descriptions of an association between CD8+ infiltrating T cells,  and not only 
overall survival in patients with metastatic SCCA of the anal canal but  also responses to anti-
PD-L1 therapy have not been described.   
 
2.4.2.3 
Hypothesis 
 
Given the cytotoxic nature of CD8+ infiltrating T cells against tumors, we hypothesize that a 
positive correlation between CD8+ infiltrating T cells and response to nivolumab will be observed 
in this study.  However, we recognize that the sample size for this phase II study is 
underpowered to detect a statistically relevant association, and therefore we plan to incorporate 
these findings as the foundation for larger studies in the future. 
 
2.4.3 
Role of CD4+ Infiltrating T-cells in the response of SCCA of 
the anal canal to nivolumab 
 
2.4.3.1 
Rationale 
 
The associations between the presence of CD4+  infiltrating T cells and (1) responses to 
nivolumab and (2) survival outcomes have never been described in a prospective study for 
patients with metastatic SCCA of the anal canal.  This analysis would not only inform on the 
relevance of these immune cells in this particular disease for the first time, but could also be used 
in understanding further the role of CD4+  infiltrating T cells as a potential biomarker for immune 
checkpoint inhibitors like nivolumab across various HPV-related malignancies. 
 
2.4.3.2 
Background 
 
A correlation between the presence of CD4+ infiltrating T-cells and improved clinical  outcomes 
has been previously described in a cohort of patients with squamous cell carcinoma of the head 
and neck 114, an important finding given that a large proportion of these patients have tumors 
driven by HPV infection.  However, whether or not an association may exist between the 
presence of CD4+ infiltrating T-cells and (1) response to nivolumab therapy and (2) survival 
outcomes remains unknown for patients with metastatic SCCA of the anal canal. 
 
2.4.3.3 
Hypothesis 
 
Given that CD4+ infiltrating T-cells assist CD8+ infiltrating T-cells in maintaining an anti-tumor 
response in the tumor microenvironment, we hypothesize that patients with metastatic SCCA of 
the anal canal whose tumors contain CD4+  infiltrating T cells may respond to nivolumab in this 
study.  However, we recognize that the sample size for this phase II study is underpowered to 
detect a statistically relevant association, and therefore we plan to incorporate these findings as 
the foundation for larger studies in the future. We will also study the potential of antigen-specific 
CD4+ T regulatory cells to decrease the response to nivolumab in this study (see 2.3.4., below). 
 

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2.4.4 Role of anti-HPV CD8+ TILs, CD4+ TILs and anti-HPV regulatory T cells in the response 
of SCCA of the anal canal to nivolumab 
 
2.4.4.1 
Rationale 
 
While the presence of CD8+ and CD4+ infiltrating T-cells appear important in the anti-tumor 
response for HPV-associated malignancies, it is important to ensure that these immune cells are 
specific to tumor cells expressing immunogenic, viral neoepitopes introduced on the cell surface 
following incorporation of viral HPV DNA into the host cell genome.    
 
2.4.4.2 
Background 
 
The presence of intratumoral anti-HPV T-cells has been described in other HPV-associated 
malignancies like cervical cancer and squamous cell carcinoma of the head and neck 94,114 and 
are associated with locoregional control of disease.  However, any associations between the 
presence of immune cells specific to HPV-infected cells and clinical outcomes have not been 
described for patients with metastatic SCCA of the anal canal. 
 
2.4.4.3 
Hypothesis 
 
We hypothesize that patients with metastatic SCCA of the anal canal whose blood samples show 
an increase in frequency of anti-HPV CD8+ and/or CD4+ infiltrating T-cells may respond to 
nivolumab in this study.  Likewise, no change or a decrease in the frequency of anti-HPV CD8+ 
and/or CD4+ infiltrating T-cells may be associated with a lack of response to nivolumab for 
patients with previously treated metastatic SCCA of the anal canal.  We also hypothesize that a 
negative correlation may exist between the presence of anti-HPV regulatory T cells and response 
to nivolumab. However, we recognize that the sample size for this phase II study is 
underpowered to detect a statistically relevant association, and therefore we plan to incorporate 
these findings as the foundation for larger studies in the future. 
 
2.5 Correlative Studies Background (Part B) 
 
In NCI9673, higher tumor PD-1:PD-L1 expression on activated CD8+ T cells at baseline were 
associated with response to nivolumab: Twelve patients at MD Anderson who enrolled on NCI 
9673 participated in an optional translational component for collection of two paired tumor 
biopsies, one pretreatment and the latter after 3 doses of nivolumab.  Nine of these patients had 
adequate tissue available for both biopsies.  Tissue was analyzed by immunohistochemistry and 
by flow cytometry to compare expression levels of various immune makers for patients with and 
without responses to nivolumab.  Patients with responses to nivolumab demonstrated higher 
expression in baseline biopsies of CD8 (P=.01) and granzyme B (P=.005) by 
immunohistochemistry56.  In addition, responders had higher levels of baseline PD-1 and PD-L1 
expression by immunohistochemistry (P=.02 and P=.005, respectively) and by flow cytometry 
(P=.004 and P=.04, respectively) when compared to non-responders. Higher pretreatment levels 
of CD8+ T cells expressing both PD-1 and PD-L1 (Figure 2) at baseline were seen among the 
responders to single agent nivolumab.  In addition, when flow cytometry was used to quantify 
CD8+ T cells co-expressing multiple inhibitory immune checkpoint proteins, higher levels of 

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dual PD1+/TIM3+ and dual PD1+/LAG3+ populations were seen among the patients with 
responses to anti-PD1 therapy (Figures 3).  These cells trended down after three doses of 
nivolumab as seen at the time of second biopsy.  Collectively, these results support the 
hypothesis that (1) the presence of a PD-1:PD-L1 axis is amenable to be blocked and (2) the 
presence of activated T cells amid a more inflamed tumor at baseline may together be associated 
with a response to anti-PD-1 therapies targeting immune checkpoint blockade. 
 
 
 
Figure 2: Increased CD8 + T cells expressing both PD-1 and PD-L1 in responders vs. nonresponders 
 
 
Figure 3: Increased dual PD1+/TIM3+ and PD1+/LAG3+ populations in responders vs. nonresponders 
 
3. PATIENT SELECTION 
 
3.1 Eligibility Criteria (Parts A+B) 
 
3.1.1 Patients must have histologically or cytologically confirmed previously treated metastatic 
squamous cell carcinoma of the anal canal. 
 

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3.1.2 Patients must have measurable disease according to the standard RECIST version 1.1. 
See Section 12 for the evaluation of measurable disease. CT scans or MRIs used to assess 
the measurable disease must have been completed within 28 days prior to study drug 
initiation. 
 
3.1.3 Patients must have been treated with at least one prior systemic treatment for incurable 
advanced or metastatic SCCA of the anal canal.  Prior treatment for metastatic disease is 
not required for patients who develop new metastatic lesions during or within 6 months 
of completion of chemoradiation for limited-stage disease.  Patients who receive 
chemotherapy for incurable advanced or metastatic SCCA of the anal canal must wait a 
minimum > 28 days (6 weeks for nitrosoureas or mitomycin C) after the date of 
completion of chemotherapy prior to initiating treatment with nivolumab on this study.  
Patients who undergo palliative radiotherapy to a site of tumor must wait a minimum > 
28 days from the date of completion of radiotherapy prior to initiating treatment with 
nivolumab (Part A and B) or nivolumab +/- Ipilimumab (Part B) on this study.   
 
3.1.4 Patients must be of age ≥18 years at the time of study registration.  Because no dosing or 
adverse event data are currently available on the use of nivolumab in patients <18 years 
of age, children are excluded from this study, but will be eligible for future pediatric 
trials. 
 
3.1.5 ECOG performance status 0 or 1 (Karnofsky ≥80%, see Appendix A). 
 
3.1.6 
Patients must have normal organ and marrow function as 
defined below: 
 
 
− 
leukocytes 
≥2,000/mcL 
− 
absolute neutrophil count 
≥1,500/mcL 
− 
hemoglobin                            ≥ 9.0 gm/dL 
− 
platelets 
≥100,000/mcL 
− 
total bilirubin 
≤1.5× institutional upper limit of normal (ULN) 
(except patients with Gilbert Syndrome, who can have total bilirubin <3.0 mg/dL) 
− 
AST(SGOT)/ALT(SGPT) 
≤ 2.5× ULN 
− 
Serum creatinine 
≤1.5× ULN  
OR 
− 
creatinine clearance (CrCl) 
≥50 mL/min (if using the Cockcroft-Gault formula 
below): 
Female CrCl = (140 - age in years) x weight in kg x 0.85 
72 x serum creatinine in mg/dL 
Male CrCl = (140 - age in years) x weight in kg x 1.00  
72 x serum creatinine in mg/dL  
 

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3.1.7 
Before study enrollment, women of child bearing potential 
must be advised of the importance of avoiding pregnancy during study 
participation and the potential risk factors for an unintentional pregnancy. 
The subject must sign an informed consent form documenting this 
discussion 
 
 
Part A+B: The effects of nivolumab and ipilimumab on the developing human fetus are 
unknown.  For this reason, women of child-bearing potential (WOCBP) and men must 
agree to use adequate contraception (hormonal or barrier method of birth control; 
abstinence) prior to study entry and for the duration of study participation.  Women of 
childbearing potential MUST have a negative serum or urine pregnancy test (minimum 
sensitivity 25 IU/L or equivalent units of HCG) within 24 hours prior to the start of 
nivolumab with or without ipilimumab.  The minimum sensitivity of the pregnancy test 
must be 25 IU/L or equivalent units of HCG. If the pregnancy test is positive, the subject 
must not receive nivolumab with or without ipilimumab and must not be enrolled in the 
study. 
 
Women of childbearing potential (WOCBP) is defined as any female who has 
experienced menarche and who has not undergone surgical sterilization (hysterectomy or 
bilateral oophorectomy) or who is not postmenopausal.  Menopause is defined clinically 
as 12 months of amenorrhea in a woman over 45 in the absence of other biological or 
physiological causes.   
 
WOCBP receiving nivolumab (Parts A+B) or nivolumab and ipilimumab (Part B) will be 
instructed to adhere to contraception for a period of 5 months after the last dose of 
investigational product.  Men receiving nivolumab  (Parts A+B) or nivolumab and 
ipilimumab (Part B only) and who are sexually active with WOCBP will be instructed to 
adhere to contraception for a period of 7 months after the last dose of investigational 
product.  These durations have been calculated using the upper limit of the half-life for 
nivolumab (25 days) and are based on the protocol requirement that WOCBP use 
contraception for 5 half-lives plus 30 days and men who are sexually active with 
WOCBP use contraception for 5 half-lives plus 90 days. 
 
Should a woman become pregnant or suspect she is pregnant while she or her partner is 
participating in this study, she (or the participating partner) should inform the treating 
physician immediately. 
 
If, following initiation of the investigational product, it is subsequently discovered that a 
study subject is pregnant or may have been pregnant at the time of investigational product 
exposure, including during at least 6 half-lives after product administration, the 
investigational product will be permanently discontinued in an appropriate manner (e.g., 
dose tapering if necessary for subject safety). The investigator must immediately notify 
BMS of this event and record the pregnancy on the Pregnancy Surveillance Form (not an 
SAE form). Initial information on a pregnancy must be reported immediately to BMS, 

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and the outcome information provided once the outcome is known. Completed Pregnancy 
Surveillance Forms must be forwarded to BMS according to SAE reporting procedures. 
 
Any pregnancy that occurs in a female partner of a male study participant should be 
reported to the sponsor. Information on this pregnancy will be collected on the Pregnancy 
Surveillance Form. 
 
Protocol-required procedures for study discontinuation and follow-up must be performed 
on the subject unless contraindicated by pregnancy (e.g., X-ray studies). Other 
appropriate pregnancy follow-up procedures should be considered if indicated. In 
addition, the investigator must report and follow-up on information regarding the course 
of the pregnancy, including perinatal and neonatal outcome. Infants should be followed 
for a minimum of 8 weeks. 
 
3.1.8 
Ability to understand and the willingness to sign a written 
informed consent document. 
 
3.1.9 
Brain metastases are allowed if they have been adequately 
treated with radiotherapy or surgery and have been stable for at least three 
months prior to registration.   Eligible subjects should be neurologically 
asymptomatic.   There is no magnetic resonance imaging (MRI) evidence 
of progression for a minimum of 4 weeks after treatment is complete and 
within 28 days prior to the first dose of nivolumab administration.  There 
must also be no requirement for immunosuppressive doses of systemic 
corticosteroids (>10 mg/day prednisone equivalents) for at least 2 weeks 
prior to study drug administration. 
 
3.1.10 
Willingness for evaluation of cardiac function including EKG 
and ECHO cardiogram for any patients with a history of CHF or at risk 
because of underlying cardiovascular disease or  
exposure to 
cardiotoxic drugs as clinically indicated.  
 
3.1.11 
All patients must be willing to undergo testing for HIV testing 
if not tested within the past 6 months.  
 
3.1.12 
If HIV+ positive, all patients infected with Human 
Immunodeficiency Virus (HIV) may be eligible for study provided that 
their CD4+ count ≥ 300/μL; their viral load is undetectable; they are 
currently receiving Highly Active Antiretroviral Therapy (HAART).  
  
3.1.13 
All HIV+ patients will be under the care of an Infectious 
Diseases specialist.  If a relationship with an Infectious Diseases specialist 
is not established, Infectious Disease specialist will be consulted. Records 
of all viral counts and peripheral T-cell counts must be sent to the Study 
Coordinator in order to follow these values over the course of treatment. 
 

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3.1.14 
All patients must be willing to be tested for Hepatitis 
screening.  Patients co-infected with hepatitis B virus and/or hepatitis C 
virus may be included in this study provided that their liver function tests 
remain within the limits listed above.  Patients must be followed by a 
hepatologist during the course of this study. 
 
3.2 Exclusion Criteria (Parts A+B) 
 
3.2.1 
Patients who have had chemotherapy or radiotherapy within 4 
weeks (6 weeks for nitrosoureas or mitomycin C) prior to entering the 
study or those who have not recovered from adverse events (AEs) due to 
agents administered more than 4 weeks earlier (i.e., grade ≥ 2 AE present).  
Palliative (limited-field) radiation therapy is permitted, as long as the 
lesion being considered for palliative radiation is not a target lesion. 
 
3.2.2 
Patients who are receiving any other investigational agents. 
 
3.2.3 
Patients should be excluded if they have had prior treatment 
with an anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4 antibody, or any 
other antibody or drug specifically targeting T-cell co-stimulation or 
immune checkpoint pathways.   
 
3.2.4 
History of allergic reactions attributed to compounds of similar 
chemical or biologic composition to nivolumab (Parts A+ B) and/or 
ipilimumab (Part B). 
 
3.2.5 
History of severe hypersensitivity reaction to any monoclonal 
antibody. 
 
3.2.6 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements. 
 

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3.2.7 
Patients with active autoimmune disease or history of 
autoimmune disease that might recur, which may affect vital organ 
function or require immune suppressive treatment including chronic 
prolonged systemic corticosteroids (defined as corticosteroid use of 
duration one month or greater), should be excluded.  These include but are 
not limited to patients with a history of immune related neurologic disease,  
multiple sclerosis, autoimmune (demyelinating)  neuropathy, Guillain-
Barre syndrome, myasthenia gravis; systemic autoimmune disease such as 
SLE, connective tissue diseases, scleroderma, inflammatory bowel disease 
(IBD), Crohn’s, ulcerative colitis, and patients with a history of toxic 
epidermal necrolysis (TEN), Stevens-Johnson syndrome, or anti-
phospholipid syndrome should be excluded because of the risk of 
recurrence or exacerbation of disease.   
 
3.2.8 Patients should be excluded if they have a condition requiring systemic treatment with 
either corticosteroids (>10 mg daily prednisone equivalents) or other immunosuppressive 
medications within 14 days of study drug administration.  Inhaled or topical steroids and 
adrenal replacement doses <10 mg daily prednisone equivalents are permitted in the 
absence of active autoimmune disease.  Patients are permitted to use topical, ocular, intra-
articular, intranasal, and inhalational corticosteroids (with minimal systemic absorption).  
Physiologic replacement doses of systemic corticosteroids are permitted, even if <10 
mg/day prednisone equivalents.  A brief course of corticosteroids for prophylaxis (e.g., 
contrast dye allergy) or for treatment of non-autoimmune conditions (e.g., delayed-type 
hypersensitivity reaction caused by contact allergen) is permitted. 
 
3.2.9 No other prior malignancy is allowed except for the following: adequately 
treated basal cell or squamous cell skin cancer, in situ cervical cancer, 
adequately treated Stage I or II cancer from which the patient is currently 
in complete remission, or any other cancer from which the patient has 
been disease free for at least three years. 
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. 
 
4. REGISTRATION PROCEDURES  
 
4.1 
Investigator and Research Associate Registration with CTEP 
 
4.1.1 CTEP Registration Procedures 
 
Food and Drug Administration (FDA) regulations and National Cancer Institute (NCI) policy 
require all individuals contributing to NCI-sponsored trials to register and to renew their 
registration annually.  To register, all individuals must obtain a Cancer Therapy Evaluation 
Program (CTEP) Identity and Access Management (IAM) account 
(https://ctepcore.nci.nih.gov/iam).  In addition, persons with a registration type of Investigator 

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Each investigator or group of investigators at a clinical site must obtain Institutional Review 
Board (IRB) approval for this protocol and submit all required regulatory documents to the 
CTSU Regulatory Office before they can be approved to enroll patients. Assignment of site 
registration status in the CTSU Regulatory Support System (RSS) uses extensive data to make a 
determination of whether a site has fulfilled all regulatory criteria including but not limited to the 
following:  
• An active Federal Wide Assurance (FWA) number 
• An active roster affiliation with the Lead Network or a participating organization 
• A valid IRB approval 
• Compliance with all protocol specific requirements 
 
In addition, the site-protocol Principal Investigator (PI) must meet the following criteria: 
• 
Active registration status 
• 
The IRB number of the site IRB of record listed on their Form FDA 1572 
• 
An active status on a participating roster at the registering site 
Sites participating on the NCI CIRB initiative that are approved by the CIRB for this study are 
not required to submit IRB approval documentation to the CTSU Regulatory Office. For sites 
using the CIRB, IRB approval information is received from the CIRB and applied to the RSS in 
an automated process.  Signatory Institutions must submit a Study Specific Worksheet for Local 
Context (SSW) to the CIRB via IRBManager to indicate their intent to open the study locally.  
The CIRB’s approval of the SSW is then communicated to the CTSU Regulatory Office.  In 
order for the SSW approval to be processed, the Signatory Institution must inform the CTSU 
which CIRB-approved institutions aligned with the Signatory Institution are participating in the 
study. 
 
4.2.1 Downloading Regulatory Documents 
 
• Go to https://www.ctsu.org and log in using your CTEP IAM username and 
password 
• Click on the Protocols tab in the upper left of your screen  
• Either enter the protocol # in the search field at the top of the protocol tree, or 
• Click on the By Lead Organization folder to expand, then select LAO-TX035, and 
protocol #9673. 
• Click on LPO Documents, select the Site Registration documents link, and 
download and complete the forms provided.  (Note: For sites under the CIRB 
initiative, IRB data load to RSS as described above.) 
 
4.2.2 Submitting Regulatory Documents 
 
Submit required forms and documents to the CTSU Regulatory Office, where they will 
be entered and tracked in the CTSU RSS.   

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Regulatory Submission Portal: www.ctsu.org  (members’ area) → Regulatory Tab 
→Regulatory Submission  
 
When applicable, original documents should be mailed to: 
CTSU Regulatory Office 
1818 Market Street, Suite 3000 
Philadelphia, PA 19103 
 
Institutions with patients waiting that are unable to use the Portal should alert the CTSU 
Regulatory Office immediately at 1-866-651-2878 in order to receive further instruction 
and support 
 
• IRB approval (For sites not participating via the NCI CIRB; local IRB 
documentation, an IRB-signed CTSU IRB Certification Form, Protocol of Human 
Subjects Assurance Identification/IRB Certification/Declaration of Exemption Form, 
or combination is accepted ) 
 
4.2.3 Checking Site Registration Status 
 
You can verify your site registration status on the members’ section of the CTSU 
website.   
 
• Go to https://www.ctsu.org and log in to the members’ area using your CTEP- 
IAM username and password 
• Click on the Regulatory tab at the top of your screen 
• Click on the Site Registration tab 
• Enter your 5-character CTEP Institution Code and click on Go 
 
Note: The status given only reflects compliance with IRB documentation and institutional 
compliance with protocol-specific requirements as outlined by the Lead Network. It does 
not reflect compliance with protocol requirements for individuals participating on the 
protocol or the enrolling investigator’s status with the NCI or their affiliated networks. 
 
 
4.3 
Patient Registration 
 

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4.3.1 OPEN / IWRS 
 
Patient enrollment will be facilitated using the Oncology Patient Enrollment Network 
(OPEN).  OPEN is a web-based registration system available to users on a 24/7 basis.  It 
is integrated with the CTSU Enterprise System for regulatory and roster data interchange 
and with the Theradex Interactive Web Response System (IWRS) for retrieval of patient 
registration/randomization assignment.  Patient enrollment data entered by Registrars in 
OPEN / IWRS will automatically transfer to the NCI’s clinical data management system, 
Medidata Rave. 
 
The OPEN system will provide the site with a printable confirmation of registration and 
treatment information.  Please print this confirmation for your records.  
 
4.3.2 OPEN/IWRS User Requirements 
 
OPEN/IWRS users must meet the following requirements: 
• Have a valid CTEP-IAM account (i.e., CTEP username and password). 
• To enroll patients:  Be on an ETCTN Corresponding or Participating Organization 
roster with the role of Registrar. Registrars must hold a minimum of an AP 
registration type.   
• To access cohort management:  Be identified to Theradex as the “Client Admin” 
for the study.  
• Have regulatory approval for the conduct of the study at their site. 
 
Prior to accessing OPEN/IWRS, site staff should verify the following: 
• All eligibility criteria have been met within the protocol stated timeframes. Site 
staff should use the registration forms provided on the CTSU web site as a tool to 
verify eligibility. 
• If applicable, all patients have signed an appropriate consent form and HIPAA 
authorization form. 
 
4.3.3 OPEN/IWRS Questions? 
 
Further instructional information on OPEN is provided on the OPEN tab of the CTSU 
website at https://www.ctsu.org or at https://open.ctsu.org.  For any additional questions 
contact the CTSU Help Desk at 1-888-823-5923 or ctsucontact@westat.com.  
 
 
 
Theradex has developed a Slot Reservations and Cohort Management User Guide, which is 
available on the Theradex website:  http://www.theradex.com/clinicalTechnologies/?National-
Cancer-Institute-NCI-11 
  
This link to the Theradex website is also on the CTSU website OPEN tab. For questions about 
the use of IWRS for slot reservations, contact the Theradex Helpdesk: 609-619-7862 or 

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Theradex main number 609-799-7580; CTMSSupport@theradex.com. 
 
4.4 
General Guidelines 
 
Following registration, patients should begin protocol treatment within 7 days.  Issues that would 
cause treatment delays should be discussed with the Principal Investigator.  If a patient does not 
receive protocol therapy following registration, the patient’s registration on the study may be 
canceled.  The Study Coordinator should be notified of cancellations as soon as possible. 
 
5. TREATMENT PLAN 
 
5.1 Treatment with nivolumab (Parts A+B) 
 
Treatment will be administered on an outpatient basis.  For patients randomized to Part B, 
nivolumab will be administered prior to ipilimumab.  Reported adverse events and potential risks 
are described in Section 7.  Appropriate dose modifications are described in Section 6.  No 
investigational or commercial agents or therapies other than those described below may be 
administered with the intent to treat the patient's malignancy. 
 
5.1.1 Nivolumab 
 
There are no premedications recommended for nivolumab on the first dose.  Subjects should be 
carefully monitored for infusion reactions during nivolumab administration.  If an acute infusion 
reaction is noted, subjects should be managed according to Section 5.8. 
  
5.1.2 Other Modalities or Procedures 
 
N/A 
 
5.2 
Nivolumab Administration 
 
Part A: Nivolumab will be given intravenously every two weeks (±3 days) at a dose of 3 mg/kg 
IV.  Patients may be dosed no fewer than 12 days from the previous dose of drug.  
 
Part B: Nivolumab will be given intravenously every four weeks (±3 days) at a dose of at a flat 
dose of 480 mg IV over 30 minutes.  Patients may be dosed no fewer than 12 days from the 
previous dose of drug.  
 
Nivolumab is to be administered as an IV infusion through a 0.2-micron to 1.2- micron 
pore size, low-protein binding (polyethersulfone membrane) in-line filter at the protocol-
specified doses and infusion times. It is not to be administered as an IV push or bolus 
injection. When the dose is based on patient weight (ie, mg/kg), nivolumab injection can be 
infused undiluted (10 mg/mL) or diluted with 0.9% Sodium Chloride Injection, USP or 
5% Dextrose Injection, USP to protein concentrations as low as 0.35 mg/mL. When the 
dose is fixed (eg, 240 mg, 360 mg, or 480 mg flat dose), nivolumab injection can be 
infused undiluted or diluted so as not to exceed a total infusion volume of 160 mL. 

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For patients weighing less than 40 kilograms (kg), the total volume of infusion must not 
exceed 4 mL per kg of patient weight. 
 
 
5.3 
General Concomitant Medication and Supportive Care Guidelines (Parts A+B) 
 
Although there is not a potential for interaction of nivolumab with other concomitantly 
administered drugs through the cytochrome P450 system, the case report form must still capture 
the concurrent use of all other drugs, over-the-counter medications, or alternative therapies.   
 
5.4 
Duration of Therapy (Parts A+B) 
 
In the absence of treatment delays due to adverse event(s), treatment may continue until one of 
the following criteria applies: 
 
o 
Disease progression, 
 
o 
Intercurrent illness that prevents further administration of treatment, 
 
o 
Unacceptable adverse event(s) which include the following (see also Section 7 and 
specific algorithms in Appendix D): 
 
o Any grade 4 events except as noted below. 
 
o Grade 3 drug-related autoimmune or inflammatory events including uveitis, 
pneumonitis, diarrhea, colitis, neurologic adverse events, hypersensitivity 
reaction, or infusion reaction of any duration requires discontinuation except as 
noted below: 
 
o Any other grade 3 non-skin, drug-related AE lasting < 7 days including 
fatigue. 
 
o Any grade 3 or 4 drug-related laboratory abnormality or electrolyte 
abnormality, not associated with underlying organ pathology that does not 
require treatment except for electrolyte replacements does not require 
treatment discontinuation.  
 
o Grade 3 or 4 amylase or lipase abnormalities that are not associated with 
diabetes mellitus (DM), associated liver or gall bladder inflammation or 
clinical manifestations of pancreatitis and which decrease to < Grade 2 within 
1 week of onset may resume study treatment when resolved.   
 
o Any grade 2 drug-related uveitis or eye pain or blurred vision that does not 
respond to topical therapy and does not improve to Grade 1 severity within the re-
treatment period OR requires systemic treatment.  
 

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o Patients requiring > two dose delays for the same type of event should go off 
protocol therapy. 
 
o Any adverse event, laboratory abnormality, or intercurrent illness which, in the 
judgment of the investigator, presents a substantial clinical risk to the subject with 
continued study drug dosing  
 
o Any dosing interruption lasting > 6 weeks, with the following exceptions:   
 
• Patients being tapered after high dose corticosteroids over one month 
followed by a two-week observation period will be allowed an additional two 
weeks to restart treatment (a maximum  eight week interruption). Dosing 
interruptions >6 weeks that occur for non-drug-related reasons may be 
allowed if approved by the Investigator.  Prior to re-initiating treatment in a 
subject with a dosing interruption lasting >6 weeks, the Principal Investigator 
must be consulted. 
 
o 
Tumor assessments should continue as per protocol even if dosing is interrupted.  
 
o 
Any patients who require additional immune suppressive treatment beyond 
steroids should go off study treatment. 
 
o 
Patient decides to withdraw from the study. 
 
o 
General or specific changes in the patient's condition render the patient 
unacceptable for further treatment in the judgment of the investigator. 
 
5.5 
Duration of Follow Up 
 
All patients will be followed for adverse events for 100 days after last dose of nivolumab +/- 
ipilimumab.  Patients who discontinue treatment for unacceptable adverse event(s) will be 
followed until resolution or stabilization of the adverse event.  If a patient stops treatment due to 
unacceptable adverse event(s) but has not demonstrated disease progression, then the patient will 
be followed with imaging studies every 6 weeks until the time of progression radiographically 
according to RECIST 1.1 criteria.  In the event that a radiographic response is detected, then this 
event will be included as a response in the final analysis, and the time of progression used in 
calculation of the survival analysis. 
 
Patients will be followed for survival status every 3 months for 2 years after treatment  
discontinuation or until death, whichever occurs first.   
 
5.6 
Criteria for Removal from Study (Parts A+B) 
 
Patients will be removed from study when any of the applicable criteria, including progressive 
disease, adverse events, patient withdrawal or inability to follow study protocol as listed in 

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Section 5.4.  The reason for study removal and the date the patient was removed must be 
documented in the Case Report Form. 
 
5.7 
Criteria to Resume Treatment (Parts A+B) 
 
Some patients may continue to benefit from treatment, maintaining or improving responses after 
progression including those treated with steroids. 
 
Restarting applies only to grade 2 events and some grade 3 events (skin rash and thyroiditis). 
 
For non-autoimmune or non-inflammatory events patients may resume treatment with study drug 
when the drug-related AE(s) resolve to Grade ≤1 or baseline value, with the following 
exceptions: 
• Patients may resume treatment in the presence of Grade 2 fatigue 
• Evaluation to exclude any additional immune mediated events endocrine, GI, and liver / 
pancreas function as clinically indicated must be made prior to restarting.  
• Non-drug-related toxicity including hepatic, pulmonary toxicity, diarrhea, or colitis, must 
have resolved to baseline before treatment is resumed. 
 
If the criteria to resume treatment are met, the patient should restart treatment at the earliest 
convenient point that is within the six week delay period.  
 
If treatment is delayed for >6 weeks, (>8 weeks for patients on a steroid taper), the patient must 
be permanently discontinued from study therapy, except as specified in Section 5.4 (Duration of 
Therapy). 
 
For patients treated with corticosteroids: 
 
Grade 2 events must resolve to < Grade 1 before considering retreatment.  
 
All patients treated with steroids for grade ≥2 events should have study treatment held until 
resolution to < Grade 1 for at least 2 weeks following complete removal from steroid treatment 
except for maintenance replacement doses for adrenal insufficiency (preferably no greater than 
10mg prednisone equivalent daily).   
 
All patients treated with steroids for grade ≥3 events should have study treatment discontinued.  
Patients with grade 3 thyroiditis and skin rash may continue therapy as for grade 2 events with 
resolution and stable replacement treatment. 
 
Patients with hepatitis, pancreatitis, pneumonitis, and colitis are at risk for exacerbation with 
retreatment if there is residual inflammation and should resolve to Grade 0 or baseline before 
retreatment.  Baseline can mean the initial grade i.e. grade <1 where permitted on study. 
 
Patients with thyroiditis or hypopituitarism who are stable as above may be restarted with 
replacement hormones including thyroid hormone and physiologic doses only of corticosteroids.  
 

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Please note that grading and for hypophysitis with symptoms of headache, visual or neurologic 
changes or radiologic evidence of pituitary enlargement and other CNS events such as aseptic 
meningitis or encephalitis should be considered grade 3 events.  
 
New immune related events or exacerbation of existing events during steroid treatment or taper 
suggest the presence of ongoing immune activation and should require permanent 
discontinuation of nivolumab.  
 
A patient who is treated with steroids, evaluated, and found to not have an autoimmune or 
inflammatory event requiring steroid treatment, may be restarted if asymptomatic off steroids for 
2 weeks and other restarting criteria are met.  
 
Prior to starting corticosteroids or hormone replacement for any reason, appropriate endocrine 
testing including cortisol, ACTH, TSH and T4 must be drawn if clinically feasible  to document 
baseline function and distinguish the pituitary  from  peripheral organ dysfunction and later from 
steroid (or thyroid)  treatment associated  ACTH ( or TSH)  suppression.  Steroids should be 
started prior to obtaining results based on clinical indications.  
 
5.8 
Treatment of Nivolumab-Related Infusion Reactions (Parts A+B) 
 
Since nivolumab contains only human immunoglobulin protein sequences, it is unlikely to be 
immunogenic and induce infusion or hypersensitivity reactions.  However, if such a reaction 
were to occur, it might manifest with fever, chills, rigors, headache, rash, urticaria, angioedema, 
pruritis, arthralgias, hypo- or hypertension, bronchospasm, or other symptoms.  
 
All Grade 3 or 4 infusion reactions should be reported as an SAE if criteria are met.  Infusion 
reactions should be graded according to NCI CTCAE version 5.0 guidelines. 
 
Treatment recommendations are provided below and may be modified based on local treatment 
standards and guidelines as medically appropriate: 
 
For Grade 1 symptoms:  (Mild reaction; infusion interruption not indicated; intervention not 
indicated) 
 
Remain at bedside and monitor subject until recovery from symptoms.  Infusion rate may be 
slowed.  If the infusion is interrupted, then restart the infusion at 50% of the original infusion 
rate when symptoms resolve; if no further complications ensue after 30 minutes, the rate may be 
increased to 100% of the original infusion rate.  Monitor patient closely.   
 
The following prophylactic premedications are recommended for future infusions:  
diphenhydramine 50 mg (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen) at 
least 30 minutes before additional nivolumab administrations, slowing infusion rate as above. 
 
For Grade 2 symptoms:  (Moderate reaction requires therapy or infusion interruption but 
responds promptly to symptomatic treatment [e.g., antihistamines, non-steroidal anti-
inflammatory drugs, narcotics, corticosteroids, bronchodilators, IV fluids]; close observation for 

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recurrence and treatment  medications may need to be continued for 24-48 hours).   
 
Stop the nivolumab infusion, begin an IV infusion of normal saline, and treat the subject with 
diphenhydramine 50 mg IV (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen); 
remain at bedside and monitor patient until resolution of symptoms.  Corticosteroid or 
bronchodilator therapy may also be administered as appropriate.  If the infusion is interrupted, 
then restart the infusion at 50% of the original infusion rate when symptoms resolve; if no further 
complications ensue after 30 minutes, the rate may be increased to 100% of the original infusion 
rate.  Monitor patient closely.  If symptoms recur, then no further nivolumab will be 
administered at that visit.  Administer diphenhydramine 50 mg IV, and remain at bedside and 
monitor the patient until resolution of symptoms.  The amount of study drug infused must be 
recorded on the electronic case report form (eCRF).  The following prophylactic premedications 
are recommended for future infusions: diphenhydramine 50 mg (or equivalent) and 
(acetaminophen) (or paracetamol) 325 to 1000 mg should be administered at least 30 minutes 
before additional nivolumab administrations.  If necessary, corticosteroids (recommended dose: 
up to 25 mg of IV hydrocortisone or equivalent) may be used. 
 
For Grade 3 symptoms: (Severe reaction), Grade 3 symptoms: prolonged [i.e., not rapidly 
responsive to symptomatic medication and/or brief interruption of infusion]; recurrence of 
symptoms following initial improvement; hospitalization indicated for other clinical sequelae 
[e.g., renal impairment, pulmonary infiltrates]).   
 

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Grade 4 symptoms: (life threatening; pressor or ventilatory support indicated).  
 
Immediately discontinue infusion of nivolumab.  Begin an IV infusion of normal saline, and treat 
the subject as follows.  Recommend bronchodilators, epinephrine 0.2 to 1 mg of a 1:1,000 
solution for subcutaneous administration or 0.1 to 0.25 mg of a 1:10,000 solution injected slowly 
for IV administration, and/or diphenhydramine 50 mg IV with methylprednisolone 100 mg IV 
(or equivalent), as needed.   
 
Patient should be monitored until the investigator is comfortable that the symptoms will not 
recur.  Nivolumab will be permanently discontinued.  Investigators should follow their 
institutional guidelines for the treatment of anaphylaxis.  Remain at bedside and monitor patient 
until recovery from symptoms.  In the case of late-occurring hypersensitivity symptoms (e.g., 
appearance of a localized or generalized pruritis within 1 week after treatment), symptomatic 
treatment may be given (e.g., oral antihistamine, or corticosteroids). 
 
Please note that late occurring events including isolated fever and fatigue may represent the 
presentation of systemic inflammation.  Please evaluate accordingly.  
 
5.9 
Ipilimumab Administration (Part B) 
 
Ipilimumab will be administered IV over 30 minutes at 1 mg/kg every 8 weeks until progression, 
unacceptable toxicity, or other reasons specified in the protocol. The dose of ipilimumab does 
not have to be recalculated at each cycle unless patient has a weight change greater than 10% at 
the time, as per FDA. 
 
Ipilimumab is given undiluted or further diluted in 0.9% NaCl Injection, USP or 5% Dextrose 
Injection, USP in concentrations between 1 mg/mL and 4 mg/mL.  Ipilimumab is stable in a 
polyvinyl chloride (PVC), non-PVC/non DEHP (di-(2-ethylhexyl) phthalate) IV bag or glass 
container up to 24 hours refrigerated at (20 to 80 C) or at room temperature/ room light.  
 
Can use a volumetric pump to infuse ipilimumab at the protocol-specific dose(s) and rate(s) via a 
PVC IV infusion set with an in-line, sterile, non-pyrogenic, low-protein-binding filter (0.2 
micron to 1.2 micron). 
 
Recommended safety measures for preparation and handling include protective clothing, gloves, 
and safety cabinets. 
 
5.9.1 Dose Delays 
 
Decisions to skip an ipilimumab dose must be made on specified safety criteria.  Treatment with 
ipilimumab will be skipped or discontinued if the subject experiences at least one adverse event, 
specified below, considered by the investigator to be “possibly,” probably,” or “definitely” 
related to ipilimumab treatment.  
 
Any adverse event that will prompt a skipped dose or discontinuation of ipilimumab must be 
reported.  

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Co-investigators and/or research team members at every participating site will participate 
in a teleconference once monthly with the PI and with the NCI to review AE’s over the 
course of the study.  
 
The investigator should contact the study PI or drug monitor to discuss any questions.  
 
The following criteria will be used to determine dose skipping, restarting doses, or discontinuing 
ipilimumab. 
 
It is necessary to avoid study drug dosing and initiate appropriate evaluation and/or 
treatment for the following adverse events: 
• Any ≥ grade 3 skin related adverse event regardless of causality. 
• Any ≥ grade 2 non-skin related adverse event (including immune-mediated adverse 
reactions), except for easily correctly laboratory abnormalities that do not reflect 
underlying organ pathology. 
• Any ≥ grade 3 laboratory abnormality. 
• Any adverse event, laboratory abnormality or intercurrent illness that, in the judgment of 
the investigator, presents a substantial clinical risk to the subject with continued dosing. 
It may be necessary to hold study drug to evaluate Grade 1 events that suggest ongoing or 
incipient autoimmune disease including GI toxicity, diarrhea, pancreatitis, hepatitis, pituitary 
insufficiency, early evidence of neurologic events, skin toxicity until diagnosis and progression 
are determined. 
3    Criteria to Resume Treatment 
 
Ipilimumab may be restarted if/when the adverse event(s) resolve(s) to severity or returns 
to baseline within 2 weeks of initial dose administration: 
• If the adverse event has been determined not be related to ipilimumab or is not an 
autoimmune/inflammatory event.  If >1 dose is to be skipped or > 2 week delay is 
expected due to current events not related to study the dosing schedule modifications 
must be discussed with the principal investigator prior to implementation.  
• If the adverse event has resolved to ≤ grade 1, ipilimumab dosing may be restarted at the 
next scheduled time point per protocol.  Please follow guidelines for specific events. 
Please note that re-initiating treatment may be associated with recurrence or exacerbation 
of autoimmune or inflammatory events.  In some instances clinical resolution of events 
such as colitis may be associated with residual pathologic changes and should require 
evaluation of complete resolution prior to restarting therapy.  
• If the adverse event has not resolved in the protocol-specified dosing window (3 weeks 
[±3 days]), the next scheduled dose will be skipped and dosing will be resumed at the 
subsequently scheduled dose. 
• Events which require intervention with immunosuppressant therapy, steroids, surgery, or 
hormone replacement generally require permanently stopping study treatment.  Consult 
guidelines for exceptions and specific events.   
• Autoimmune/inflammatory events are presumably related to the mechanisms of action of 
ipilimumab and potentially to a therapeutic effect.  The incidence and severity of these 
events may be dose related, but once initiated, there is no evidence that lowered doses 

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can be administered without continued autoimmune activity and there has so far been no 
demonstrable benefit to continuing ipilimumab after an autoimmune event during the 
initial treatment.  The significance and benefit of toxicity or continued treatment in the 
maintenance phase has not been determined.  Typically no dose modification is used for 
ipilimumab.  
 
5.9.2 Immune-Related Adverse Events (irAEs) Reactions and Immune-mediated Adverse 
Reactions: Definition, Monitoring, and Treatment 
 
For the purposes of this study, an immune-related adverse reaction is defined as an adverse 
reaction of unknown etiology associated with drug exposure and consistent with an immune 
phenomenon. Efforts should be made to rule out neoplastic, infectious, metabolic, toxin or other 
etiologic causes prior to labeling an event an irAEs.  Serologic, immunologic, and histologic 
(biopsy) data should be used to support the diagnosis of an immune-related toxicity. Suspected 
immune-related adverse reactions must be documented on an AE or SAE form. Another term for 
an irAE is an immune-mediate adverse reaction, as it is termed in the Ipilimumab US Prescribing 
Information. Both terms may be used in this protocol document.  
 
Patients should be informed of and carefully monitored for evidence of clinically significant 
systemic immune-mediated adverse reactions (e.g., systemic lupus erythematosus-like diseases) 
or organ-specific immune-mediated adverse reaction (e.g., rash, colitis, uveitis, hepatitis or 
thyroid disease).  If an immune-mediated adverse reaction is noted, appropriate work-up 
(including biopsy if possible) should be performed, and steroid therapy may be considered if 
clinically necessary.   
 
It is unknown if systemic corticosteroid therapy has an attenuating effect on ipilimumab activity. 
However, clinical anti-tumor responses have been maintained in patients treated with 
corticosteroids and discontinued from ipilimumab. If utilized, corticosteroid therapy should be 
individualized for each patient. Prior experience suggests that colitis manifested as ≥ grade 3 
diarrhea requires corticosteroid treatment. 
 
Recommended guidelines for specific immune-mediated adverse reactions are included in 
Section 6.2.4 below, in Appendix C, and the package insert.  These recommendations should be 
utilized as clinically appropriate for the treatment of individual patients.  
 
Please contact the PI or drug monitor for any questions.   
 
5.9.3 
Permanent Discontinuation of ipilimumab for Related Adverse 
Events (Part B) 
 
Permanently discontinue ipilimumab for any of the following: 
• Persistent adverse reactions that requires holding more than 2 treatment doses.   Any 
grade 3 or 4 event (see exceptions below)  
• Any event that requires immunosuppressive treatment or systemic steroids  
 
Severe or life-threatening adverse reactions, including any of the following: 

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o Colitis with abdominal pain, fever, ileus, or peritoneal signs; increase in stool 
frequency (7 or more over baseline), stool incontinence, need for IV hydration for 
more than 24 hours, gastrointestinal hemorrhage, and gastrointestinal perforation 
o Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >5 times the 
upper limit of normal or total bilirubin >3 times the upper limit of normal 
o Stevens-Johnson syndrome, toxic epidermal necrolysis, or rash complicated by full 
thickness dermal ulceration, or necrotic, bullous, or hemorrhagic manifestations 
o Severe motor or sensory neuropathy, Guillain-Barré syndrome, or myasthenia gravis 
o Severe immune-mediated reactions involving any organ system (e.g., nephritis, 
pneumonitis, pancreatitis, non-infectious myocarditis) 
o Immune-mediated ocular disease that is unresponsive to topical immunosuppressive 
therapy 
o Any adverse event, laboratory abnormality or intercurrent illness which, in the 
judgment of the investigator, presents organ specific injury and/or a substantial 
clinical risk to the patient with continued dosing.  
 
The following neurological adverse event requires permanent discontinuation of ipilimumab and 
defines unacceptable neurotoxicity: 
• Any motor neurologic toxicity >/= grade 3 regardless of causality 
• Any >/= grade 3 treatment related sensory neurologic toxicity 
 
Please refer to Appendix C and the IB for specific treatment algorithms. 
 
5.9.4 
Exceptions to Permanent Discontinuation of Ipilimumab  
 
• Potentially reversible inflammation (< grade 4), attributable to a local anti-tumor reaction and 
a potential therapeutic response. This includes inflammatory reactions at sites of tumor 
resections or in draining lymph nodes, or at sites suspicious for, but not diagnostic of 
metastasis. 
• Hospitalization for ≤ grade 2 adverse events where the primary reason for hospitalization is 
to expedite the clinical work-up. 
• Patients with the following conditions where in the investigator’s opinion continuing study 
drug administration is justified based on the potential for continued clinical benefit:  
o Patients treated with systemic steroids for less than 2 weeks without evidence of 
autoimmune disease requiring steroids treatment  
o Grade 2 skin rash treated with topical steroids for less than 4 weeks  
o Grade 2 Ocular toxicity that has completely responded to topical therapy within 4 
weeks 
o Endocrinopathies where clinical symptoms are controlled with appropriate hormone 
replacement therapy. Note: Ipilimumab may not be restarted while the patient is 
being treated with systemic corticosteroids except for patients on stable doses of 
hormone replacement therapy such as hydrocortisone.  
 
 
 

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The following guidance is provided for the management of ipilimumab treatment related events.  
These recommendations, treatment algorithms in Appendix D, and further information in the IB, 
should be considered in the context of appropriate medical treatment for each patient.   
 
5.9.5 
Treatment of Infusion Reactions Associated with Ipilimumab 
 
Since ipilimumab contains only human protein sequences, it is less likely that any allergic 
reaction will be seen in patients.  However, it is possible that infusion of ipilimumab will induce 
a cytokine release syndrome that could be evidenced by fever, chills, rigors, rash, pruritus, 
hypotension, hypertension, bronchospasm, or other symptoms.  No prophylactic pre-medication 
should be given unless indicated by previous experience in an individual patient. Reactions 
should be treated based upon the following recommendations.  
 
For mild symptoms (e.g., localized cutaneous reactions such as mild pruritus, flushing, rash): 
o Decrease the rate of infusion until recovery from symptoms, remain at bedside and 
monitor patient. 
o Complete the ipilimumab infusion at the initial planned rate. 
o Diphenhydramine 50 mg IV may be administered at the discretion of the treating 
physician and patients may receive additional doses with close monitoring. 
o Premedication with diphenhydramine may be given at the discretion of the 
investigator for subsequent doses of ipilimumab. 
• For moderate symptoms (any symptom not listed above [mild symptoms] or 
below [severe symptoms] such as generalized pruritus, flushing, rash, dyspnea, 
hypotension with systolic BP >80 mmHg):  
o Interrupt ipilimumab. 
o Administer diphenhydramine 50 mg IV. 
o Monitor patient closely until resolution of symptoms. 
o Corticosteroids may abrogate any beneficial immunologic effect, but may be 
administered at the discretion of the treating physician. 
o Resume ipilimumab infusion after recovery of symptoms. 
o At the discretion of the treating physician, ipilimumab infusion may be resumed at 
one half the initial infusion rate, then increased incrementally to the initial infusion 
rate. 
o If symptoms develop after resumption of the infusion, the infusion should be 
discontinued and no additional ipilimumab should be administered that day. 
o The next dose of ipilimumab will be administered at its next scheduled time and may 
be given with pre-medication (diphenhydramine and acetaminophen) and careful 
monitoring, following the same treatment guidelines outlined above. 
o At the discretion of the treating physician additional oral or IV antihistamine may be 
administered prior to dosing with ipilimumab. 
• For severe symptoms (e.g., any reaction such as bronchospasm, generalized 
urticaria, systolic blood pressure 80 mm Hg, or angioedema): 
o Immediately discontinue infusion of ipilimumab, and disconnect infusion tubing from 
the subject. 
o Consider bronchodilators, epinephrine 1 mg IV or subcutaneously, and/or 
diphenhydramine 50 mg IV, with solumedrol 100 mg IV, as needed. 

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o Patients should be monitored until the investigator is comfortable that the symptoms 
will not recur. 
o No further ipilimumab will be administered. 
• In case of late-occurring hypersensitivity symptoms (e.g., appearance within 
one week after treatment of a localized or generalized pruritus), symptomatic 
treatment may be given (e.g., oral antihistamine, or corticosteroids). 
5.9.6 Treatment of Ipilimumab-Related Isolated Drug Fever 
 
In the event of isolated drug fever, the investigator must use clinical judgment to determine if the 
fever is related to the ipilimumab or to an infectious etiology.  If a patient experiences isolated 
drug fever, for the next dose, pre-treatment with acetaminophen or non-steroidal anti-
inflammatory agent (investigator discretion) should be instituted and a repeated antipyretic dose 
at 6 and 12 hours after ipilimumab infusion, should be administered.  The infusion rate will 
remain unchanged for future doses.  If a patient experiences recurrent isolated drug fever 
following premedication and post dosing with an appropriate antipyretic, the infusion rate for 
subsequent dosing should be decreased to 50% of the previous rate.  If fever recurs following 
infusion rate change, the investigator should assess the patient’s level of discomfort with the 
event and use clinical judgment to determine if the patient should receive further ipilimumab. 
 
5.9.7 
Monitoring and Management of Immune-mediated Adverse 
Reactions 
 
Immune-mediated enterocolitis 
• The clinical presentation of GI immune-related AEs included diarrhea, increase 
in the frequency of bowel movements, abdominal pain, or hematochezia, with 
or without fever. However inflammation may occur in any part of the GI tract 
including esophagitis and gastritis.  Fatalities due to GI perforation have been 
reported in clinical trials of ipilimumab. Patients should be carefully monitored 
for GI symptoms that may be indicative of immune-related colitis, diarrhea, or 
GI perforation.  Diarrhea or colitis occurring after initiation of ipilimumab 
therapy should be evaluated to exclude infectious or alternate etiologies. In 
clinical trials, immune-related colitis was associated with evidence of mucosal 
inflammation, with or without ulcerations, and lymphocytic infiltration. 
 
• Monitor patients for signs and symptoms of enterocolitis (such as diarrhea, 
abdominal pain, mucus or blood in stool, with or without fever) and bowel 
perforation (such as peritoneal signs and ileus).  In symptomatic patients, rule 
out infectious etiologies and consider endoscopic evaluation to establish 
etiology and for persistent or severe symptoms.  C.difficile toxin has been 
detected in several patients with colitis and may be an independent entity or 
may co-exist with ipilimumab induced inflammatory colitis.   
 
• Withhold ipilimumab dosing for any patients with enterocolitis pending 
evaluation; administer anti-diarrheal treatment and, if persistent evaluate with 
colonoscopy and initiate systemic corticosteroids at a dose of 0.5 mg/kg/day 
prednisone or equivalent. 

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• Permanently discontinue ipilimumab in patients with severe enterocolitis and 
initiate systemic corticosteroids at a dose of 1 to 2 mg/kg/day of prednisone or 
equivalent. Upon improvement to grade 1 or less, initiate corticosteroid taper 
and continue to taper over at least one month. In clinical trials, rapid 
corticosteroid tapering has resulted in recurrence or worsening symptoms of 
enterocolitis in some patients.  
 
• Patients have been treated with anti-TNF agents for persistent colitis not 
responding to steroids. 
 
• Please note autoimmune pancreatitis may cause abdominal pain and should be 
included in all evaluations. Enteritis may occur occasionally with other 
autoimmune events including hepatitis, pancreatitis, and endocrine 
insufficiency, which should be evaluated as clinically indicated.  
 
Immune-mediated Hepatitis and Pancreatitis  
• Hepatic immune-related AEs were mostly clinically silent and manifested as 
transaminase or bilirubin laboratory abnormalities.  Fatal hepatic failure has been 
reported in clinical trials of ipilimumab.  Serum transaminase and bilirubin and lipase 
levels must be evaluated before each dose of ipilimumab as early laboratory changes 
may be indicative of emerging immune-related hepatitis/ pancreatitis and elevations 
in liver function tests (LFTs) may develop in the absence of clinical symptoms.  
Increase in LFT or total bilirubin should be evaluated to exclude other causes of hepatic 
injury, including infections, disease progression, or other medications, and monitored 
until resolution.  Liver biopsies from patients who had immune-related hepatotoxicity 
showed evidence of acute inflammation (neutrophils, lymphocytes, and macrophages). 
 
• Monitor liver function tests (hepatic transaminase and bilirubin levels, lipase) and assess 
patients for signs and symptoms of hepatotoxicity/ pancreatitis before each dose of 
ipilimumab.  In patients with hepatotoxicity, rule out infectious or malignant causes and 
increase frequency of liver function test monitoring until resolution.  Withhold 
ipilimumab in patients with grade 2 hepatotoxicity.  
 
• Permanently discontinue ipilimumab in patients with grade 3–5 
hepatotoxicity/pancreatitis and administer systemic corticosteroids at a dose of 1 to 2 
mg/kg/day of prednisone or equivalent.  When liver function tests show sustained 
improvement or return to baseline, initiate corticosteroid tapering and continue to taper 
over 1 month.  Across the clinical development program for ipilimumab, mycophenolate 
treatment has been administered in patients who have persistent severe hepatitis despite 
high-dose corticosteroids.  
 
Immune-mediated Dermatitis 
• Skin immune-related AEs presented mostly frequently as a rash and/or pruritus.  Some 
subjects reported vitiligo associated with ipilimumab administration.  Fatal toxic 
epidermal necrolysis has been reported in clinical trials of ipilimumab.  

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• Monitor patients for signs and symptoms of dermatitis such as rash and pruritus.  Unless 
an alternate etiology has been identified, signs or symptoms of dermatitis should be 
considered immune-mediated.   
 
• Permanently discontinue ipilimumab in patients with Stevens-Johnson syndrome, toxic 
epidermal necrolysis, or rash complicated by full thickness dermal ulceration, or necrotic, 
bullous, or hemorrhagic manifestations.  Administer systemic corticosteroids at a dose of 
1 to 2 mg/kg/day of prednisone or equivalent.  When dermatitis is controlled, 
corticosteroid tapering should occur over a period of at least 1 month.  Withhold 
ipilimumab dosing in patients with moderate to severe signs and symptoms.  
 
• For mild to moderate dermatitis, such as grade 2 localized rash and pruritus, treat 
symptomatically. For persistent grade 2, grade 3, or greater, topical steroids may be 
administered.  Administer topical or systemic corticosteroids as indicated if there is no 
improvement of symptoms within 1 week. 
 
Immune-related Neurological Events  
• Fatal Guillain-Barré syndrome has been reported in clinical trials of ipilimumab. Patients 
may present with muscle weakness and myasthenia gravis, cranial nerve palsy (n VII 
Bell’s palsy), and aseptic meningitis, encephalopathy.  Unexplained motor neuropathy, 
muscle weakness, or sensory neuropathy lasting more than 4 days should be evaluated 
and non-inflammatory causes such as disease progression, infections, metabolic 
syndromes, nerve entrapment, and medications should be excluded as causes.  
 
• Withhold ipilimumab dosing in patients with any evidence of neuropathy pending 
evaluation.   
 
• Monitor for symptoms of motor or sensory neuropathy such as unilateral or bilateral 
weakness, sensory alterations, or paresthesia.  Permanently discontinue ipilimumab in 
patients with severe neuropathy (interfering with daily activities) such as Guillain-Barré-
like syndromes.  Institute medical intervention as appropriate for management of 
neuropathy and other neurologic events.  Consider initiation of systemic corticosteroids at 
a dose of 1 to 2 mg/kg/day prednisone or equivalent for severe neuropathies.  
 
Immune-mediated Endocrinopathies 
• Ipilimumab can cause inflammation of endocrine organs including thyroid (Hashimoto’s 
thryroiditis with positive antibodies) and adrenal glands, hypophysitis, hypopituitarism, 
and resulting thyroid and adrenal insufficiency, low ADH, prolactin, FSH, LH.  
Hyperthyroid with Graves’ disease and positive antibody has been reported.  Patients 
may present with subtle and nonspecific symptoms.  The most common clinical 
presentation includes headache and fatigue.  Symptoms may also include visual field 
defects, behavioral changes, and electrolyte disturbances including hyponatremia and 
hypotension.  Adrenal crisis as a cause of the patient’s symptoms should be excluded.  
Based on the available data with known outcome, most of the subjects symptomatically 
improved with hormone replacement therapy.  Long-term hormone replacement therapy 

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with HC and synthroid will typically be required for subjects developing 
hypophysitis/hypopituitarism after treatment with ipilimumab.  Some patients have 
regained partial function following steroid treatment.  
 
• Monitor patients for clinical signs and symptoms of hypophysitis, adrenal insufficiency 
(including adrenal crisis), and hyper- or hypothyroidism.  Headache is often the first 
symptoms of hypophysitis.  Patients may present with fatigue, headache, mental status 
changes, loss of libido, abdominal pain, unusual bowel habits, and hypotension, or 
nonspecific symptoms which may resemble other causes such as brain metastasis or 
underlying disease.  Unless an alternate etiology has been identified, signs or symptoms 
of endocrinopathies should be considered immune-mediated and drug withheld pending 
evaluation.  Patients may demonstrate both central (hypophysitis) and peripheral adrenal 
and thyroid insufficiency.  Evaluation of hypophysitis should include pituitary MRI.  
 
• *Endocrine evaluation, including TSH, should be performed at baseline prior to 
initial treatment.  Monitor thyroid function tests and clinical chemistries at the start of 
treatment and hold blood for possible evaluation should clinical events require 
determining baseline function and anti-thyroid antibodies.  A plan for evaluating 
endocrine function at each visit either by history or monitoring TSH should be included 
in the protocol with further evaluation as clinically indicated.  Endocrine evaluation, 
including TSH, should be performed at baseline prior to treatment, and the protocol must 
include a plan for continued monitoring of endocrine and pituitary function.  The package 
insert for ipilimumab includes a recommendation for monitoring TSH prior to each 
infusion; as an early indication for pituitary dysfunction and hypophysitis, clinical 
monitoring of symptoms may be equally or more sensitive as an initial presentation.  
Clinical monitoring is required for all protocols as above, and should include any 
requirements per protocol for laboratory evaluation, both periodically or as clinically 
indicated, consistent with good medical practice.  In a limited number of patients, 
hypophysitis was diagnosed by imaging studies through enlargement of the pituitary 
gland.   
 
• Withhold ipilimumab dosing in patients symptomatic for hypophysitis.  Initiate systemic 
corticosteroids at a dose of 1 to 2 mg/kg/day of prednisone or equivalent, and initiate 
appropriate hormone replacement therapy.  
 
Other Immune-mediated Adverse Reactions, Including Ocular Manifestations  
• Ocular inflammation, manifested as grade 2 or grade 3 episcleritis or uveitis, was 
associated with concomitant diarrhea in a few subjects (<1%) and occasionally occurred 
in the absence of clinically apparent GI symptoms.  Other presumed immune-related AEs 
reported include, but were not limited to, arthritis/arthralgias, pneumonitis, pancreatitis, 
autoimmune (aseptic) meningitis, autoimmune nephritis, pure red cell aplasia, 
noninfective myocarditis, polymyositis, and myasthenia gravis, of which were 
individually reported for <1% of subjects.  
 
• The following clinically significant immune-mediated adverse reactions were seen in less 
than 1% of ipilimumab-treated patients in Study 1: nephritis, pneumonitis, pulmonary 

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granuloma resembling sarcoidosis, meningitis, pericarditis, uveitis, iritis, ITP, 
neutropenia and hemolytic anemia. 
 
• Across the clinical development program for ipilimumab, the following likely immune-
mediated adverse reactions were also reported with less than 1% incidence: myocarditis, 
angiopathy, temporal arteritis, vasculitis, polymyalgia rheumatica, conjunctivitis, 
blepharitis, episcleritis, scleritis, leukocytoclastic vasculitis, erythema multiforme, 
psoriasis, pancreatitis, arthritis, and autoimmune thyroiditis.   
 
• Permanently discontinue ipilimumab for clinically significant or severe immune-
mediated adverse reactions.  Initiate systemic corticosteroids at a dose of 1 to 2 
mg/kg/day prednisone or equivalent for severe immune-mediated adverse reactions. 
 
• Administer corticosteroid eye drops to patients who develop uveitis, iritis, or episcleritis.  
Permanently discontinue ipilimumab for immune-mediated ocular disease that is 
unresponsive to local immunosuppressive therapy. 
 
• Overall, immune-related AEs commonly started within 3 to 10 weeks from first dose, 
were successfully managed in most instances by omitting doses, discontinuing dosing, 
and/or through administering symptomatic or immunosuppressive therapy, including 
corticosteroids, as mentioned above and detailed in Section 7.  Immune-related AEs 
generally resolved within days to weeks in the majority of subjects.  
 
5.9.8 
Precautions 
 
Combination therapy may result in unexpected toxicity especially in novel combinations with 
other immune modifying agents.  A striking example in macaques is presented in Vaccari, et al. 
2012. 
 
Please note that while unproven, there is a suggestion that autoimmune events, including 
hepatitis, may occur more frequently at sites of metastases or prior injury.  
 
Caution is advised when considering treatment with high-dose IL-2 in patients who have 
previously been administered ipilimumab, particularly in patients who experienced ipilimumab-
related diarrhea/colitis.   
 
Colonoscopy or sigmoidoscopy with biopsy may be advisable prior to IL-2 administration once 
the patient is no longer receiving ipilimumab.  The management guidelines for general 
inflammatory AEs and ipilimumab-related GI toxicities, hepatotoxicity, endocrinopathy, and 
neuropathy (Investigator Brochure, 2011) are provided in Appendix C. 
 
Patients who have received ipilimumab may potentially develop autoimmune disease with 
subsequent therapy including the appearance of colitis, hypophysitis or adrenal insufficiency.  
 
5.10  Prohibited and Restricted Therapies 
 

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• Patients in this study may use standard vaccines.  Where possible, routine vaccination for 
influenza, pneumococcal pneumonia should be given prior to the start of therapy but may 
be administered during treatment when clinically indicated. Vaccination should be given 
when there is enough separation to distinguish any vaccine reactions from drug toxicity.  
There is no experience using live attenuated vaccination during ipilimumab therapy, so 
that live vaccine should be used cautiously during treatment.   
 
5.11 
Treatment Beyond Progression 
 
A minority of subjects treated with immunotherapy may derive clinical benefit either delayed 
responses, stable disease, or increased overall survival despite initial evidence of progressive 
disease (PD) with nivolumab +/- ipilimumab.   
 
Patients may be permitted to continue treatment beyond initial RECIST 1.1-defined PD 
occurring during the initial treatment period (up to 12 weeks) as long as they meet the following 
criteria: 
 
• No more than 4 new lesions, total sum of the longest diameter (SHORT diameter 
for LN) cannot exceed  40% of the initial sum including new lesions   
• Patients must be clinically stable with no change in performance status due to 
disease progression 
• No indication for immediate alternative treatment 
• Patient [assessed by the investigator] is showing clinical benefit and tolerates 
study drug.  The assessment of clinical benefit should take into account whether 
the subject is clinically stable or deteriorating and  likely or unlikely to receive 
further benefit from continued treatment.  
• The time of progression is noted from the first assessment that exceeds standard 
criteria 
 
New lesions are considered measurable at the time of initial progression if the longest diameter is 
at least 10 mm (except for pathological lymph nodes, which must have a short axis of at least 15 
mm).  Any new lesion considered non-measurable at the time of initial progression may become 
measurable and therefore included in the tumor burden measurement if the longest diameter 
increases to at least 10 mm (except for pathological lymph nodes, which must have an increase 
in short axis to at least 15 mm). 
 
Patients are allowed to continue treatment for 1 additional dose (4‐8 weeks) and reassessed.  
Treatment may continue up to an additional 10% or 30% total single diameter increased over 
baseline.  New measureable lesions are not permitted with this schema.  
 
5.12 
Discontinuation of Study Therapy 
 
Subjects MUST be discontinued from study therapy AND withdrawn from the study for the 
following reasons: 
• The development of progression PD by RECIST with modifications to allow continued 
treatment defined in Section 5.11.  Treatment may continue under the guidelines 

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discussed in Section 5.7.  While patients maintain minimal progression and performance 
status unless further benefit from ipilimumab, dosing is unlikely or requires a change of 
therapy. 
• Withdrawal of informed consent (subject’s decision to withdraw for any reason). 
• Any clinical adverse event, laboratory abnormality or intercurrent illness which, in the 
opinion of the investigator, indicates that continued treatment with study therapy is not in 
the best interest of the subject. 
• Termination of the study by the sponsor. 
• Inability to continue treatment.  
• Pregnancy 
o All WOCBP should be instructed to contact the investigator immediately if they 
suspect they might be pregnant (e.g., missed or late menstrual period) at any time 
during study participation. Institutional policy and local regulations should determine 
the frequency of on study pregnancy tests for WOCBP enrolled in the study. 
o The investigator must immediately notify CTEP in the event of a confirmed 
pregnancy in a patient participating in the study. 
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
6.1 
Dosing Modifications 
 
There will be no dose modifications allowed for management of toxicities.    
 
6.2 
Dosing Delays 
 
ALL OTHER 
EVENTS*^ 
Management/Next Dose for Nivolumab +/- Ipilimumab 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1 OR baseline Resume at same dose level. 
Grade 3 
Hold* until ≤ Grade 1 continue at investigator discretion  
Grade 4 
Off protocol therapy 
* Not agent related,  or agent related non-immunologically mediated 
^Per investigator discretion, unrelated Grade 2 anemia does not require dose delay.  
Recommended management:  As clinically indicated  
ALL OTHER 
EVENTS** 
Management/Next Dose for Nivolumab +/- Ipilimumab 
≤ Grade 1 
No change in dose  
Grade 2 
Hold until ≤ Grade 1 OR baseline* When resolved  <  or  following 
steroids resume at same dose level . 
Grade 3 
 Off protocol therapy (exceptions noted in 5.4)  
Grade 4 
 Off protocol therapy 
* *immunologically mediated, with the exception of the immune-related events mentioned 
on pages 64-65 
Recommended management:  As clinically indicated  
 

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Skin Rash and Oral 
Lesions 
 
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1-2 
Refer to Skin Adverse Event management algorithm (Appendix D). 
Grade 3 
Delay therapy.  Refer to Skin Adverse Event management algorithm 
(Appendix D). 
Grade 4 
Off protocol therapy 
*Patients with purpuric or bullous lesions must be evaluated for vasculitis, Steven-Johnson syndrome, 
TEN, and autoimmune bullous disease including oral lesions of bullous pemphigus/ pemphigoid.  
Pruritus may occur with or without skin rash and should be treated symptomatically if there is no 
associated liver or GI toxicity.  Note skin rash typically occurs early and may be followed by additional 
events particularly during steroids tapering.   
Rule out non-inflammatory causes.  If non-inflammatory cause, then treat accordingly and continue 
nivolumab therapy. 
 
Renal Function 
 
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1-2 
Refer to Renal Adverse Event management algorithm (Appendix D). 
Grade 3 
Delay therapy.  Refer to Renal Adverse Event management algorithm 
(Appendix D). 
Grade 4 
Off protocol therapy 
 
Liver Function 
(AST, ALT, total 
bilirubin)  
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1 
Refer to Hepatic Adverse Event management algorithm (Appendix D).   
Grade 2 
Delay therapy.  Refer to Hepatic Adverse Event management algorithm 
(Appendix D).   
Grade 3 
Delay therapy.  Refer to Hepatic Adverse Event management algorithm 
(Appendix D).   
Grade 4 
Off protocol therapy. 
Continued treatment of active immune mediated hepatitis may exacerbate ongoing inflammation.  
Holding drug to evaluate LFT changes and to rule out non-inflammatory causes.  Early treatment is 
recommended.   
LFT changes may occur during steroid tapers from other events and may occur together with other GI 
events including cholecystitis/pancreatitis. 
 
Diarrhea/ Colitis  
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1 
Refer to GI Adverse Event management algorithm (Appendix D).   
Grade 2 
Delay therapy.  Refer to GI Adverse Event management algorithm 
(Appendix D).   
Grade 3 
Delay therapy.  Refer to GI Adverse Event management algorithm 
(Appendix D).   
Grade 4 
Off protocol therapy. 
See GI AE Algorithm for management of symptomatic colitis.  
Patients with grade 2 symptoms but normal colonoscopy and biopsies may be retreated after resolution.  
Evaluation for all patients for additional causes includes C. diff, acute and self-limited infectious and 

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Diarrhea/ Colitis  
Management/Next Dose for Nivolumab +/- Ipilimumab 
foodborne illness, ischemic bowel, diverticulitis, and IBD.  
Recommended management:  see GI AE management Algorithm 
 
Pancreatitis 
Amylase/Lipase  
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1 
Hold dose until grade 0 
Grade 2 
Hold dose until Grade 0.  Resume at same dose level if asymptomatic. 
 
Grade 3-4 
Hold* dose until Grade 0.  Resume at same dose level if asymptomatic. 
Patients who develop symptomatic pancreatitis or DM should be taken 
off treatment. * 
*Patients may develop symptomatic and radiologic evidence of pancreatitis as well as DM and 
DKA.  Lipase elevation may occur during the period of steroid withdrawal and with other 
immune mediated events or associated with colitis, hepatitis, and patients who have 
asymptomatic lipase elevation typically have self-limited course and may be retreated.  
For treatment management of symptomatic pancreatitis please follow the Hepatic Adverse 
Event Management Algorithm  
 
Pneumonitis 
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1 
Hold dose pending evaluation and resolution to Grade 0 or baseline 
including baseline pO2.*   
Grade 2 
Hold dose pending evaluation. Refer to Pulmonary Adverse Event 
management algorithm (appendix D). 
Grade 3 
Hold dose pending evaluation. Refer to Pulmonary Adverse Event 
management algorithm (appendix D). 
Grade 4 
Off protocol therapy. 
Distinguishing inflammatory pneumonitis is often a diagnosis of exclusion for patients who do not 
respond to antibiotics and have no causal organism identified including influenza.  Most patients with 
respiratory failure or hypoxia will be treated with steroids.  Bronchoscopy may be required and analysis 
of lavage fluid for lymphocytic predominance may be helpful.  Patients with new lung nodules should 
be evaluated for sarcoid like granuloma.  Please consider recommending seasonal influenza killed 
vaccine for all patients. 
*Note: this is more conservative management than the Pulmonary Adverse Event Management 
algorithm (appendix D) suggests for Grade 1 pneumonitis 
 
Other GI 
Nausea/Vomiting  
Management/Next Dose for Nivolumab +/- Ipilimumab 
≤ Grade 1 
No change in dose.  
Grade 2 
Hold pending evaluation for gastritis duodenitis and other immune 
adverse events or other causes.  Resume at same dose level after 
resolution to ≤ Grade 1.   
Grade 3 
Hold pending evaluation   until ≤ Grade 1.  Resume at same dose level. 
If symptoms do not resolve within 7 days with symptomatic treatment  patients 
should go off protocol therapy 
Grade 4 
Off protocol therapy 
Patients with grade 2 or 3 N-V should be evaluated for upper GI inflammation and other immune related 

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Other GI 
Nausea/Vomiting  
Management/Next Dose for Nivolumab +/- Ipilimumab 
events.   
 
Fatigue  
Management/Next Dose for Nivolumab +/- Ipilimumab 
≤ Grade 1 
No change in dose. 
Grade 2 
No change in dose 
Grade 3 
Hold until ≤ Grade 2.  Resume at same dose level 
Grade 4 
Off protocol therapy 
Fatigue is the most common adverse event associated with immune checkpoint therapy.  Grade 
2 or greater fatigue should be evaluated for associated or underlying organ involvement 
including pituitary, thyroid, and hepatic, or muscle (CPK) inflammation  
 
Neurologic events  
 
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1 
Hold dose pending evaluation and observation. Resume with no change 
in dose. *  Refer to Neurological Adverse Event management algorithm 
(Appendix D). 
Grade 2 
Hold dose pending evaluation and observation.*  Refer to Neurological 
Adverse Event management algorithm (Appendix D). 
Grade 3-4 
Off protocol therapy  
*Patients with any CNS events including aseptic meningitis, encephalitis, symptomatic 
hypophysitis, or myopathy, peripheral demyelinating neuropathy, cranial neuropathy (other 
than peripheral n. VII), GB syndrome, or myasthenia gravis should be off study.   
Recommended management:  See Neurologic Adverse Event Management Algorithm  
 
Endocrine 
Hypophysitis or 
Adrenal 
Insufficiency  
 
Management/Next Dose for Nivolumab +/- Ipilimumab 
Grade 1-2 
Asymptomatic TSH elevation *  Hold pending evaluation, endocrine 
consult .  Refer to Endocrinopathy Adverse Event management 
algorithm (Appendix D). 
Grade 3-4 
Off protocol therapy 
Note all patients with symptomatic pituitary enlargement, exclusive of hormone deficiency, but 
including severe headache or enlarged pituitary on MRI should be considered grade 3 events.  Isolated 
thyroid or testosterone deficiency may be treated as grade 2 if there are no other associated deficiencies 
and adrenal function is monitored.  
Please evaluate pituitary function before beginning steroid therapy or replacement therapy of any kind.  
*Note patients with thyroiditis may be retreated on replacement therapy.  Patients must be evaluated to 
rule out pituitary disease prior to initiating thyroid replacement. 
Recommended management: See Endocrine Management Algorithm  
 
 

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Fever  
Management/Next Dose for Nivolumab +/- Ipilimumab  
≤ Grade 1 
Continue therapy at same dose. If no improvement after 5-7 days or 
worsening, then treat as Grade 2. 
Grade 2 
Hold until ≤ Grade 1.  Resume at same dose level. 
Grade 3 
Hold until ≤ Grade 1.  Resume at same dose level. 
Grade 4 
Off treatment 
Patients with fever should be evaluated as clinically appropriate.  Patients may experience 
isolated fever during infusion reactions or up to several days after infusion.  Evaluation over the 
course of 1-2 weeks should be done for other autoimmune events that may present as fever  
See section 5.    infusion reactions   
 
Cardiac*  
Management/Next Dose for (Nivolumab) + Ipilimumab Cardiac 
Toxicities 
≤ Grade 1 
Hold dose pending evaluation and observation.**  Evaluate for signs 
and symptoms of CHF, ischemia, arrhythmia or myositis.  Obtain 
history EKG, CK (for concomitant myositis), CK-MB.  Repeat troponin, 
CK and EKG 2-3 days.  If troponin and labs normalize may resume 
therapy.   If labs worsen or symptoms develop then treat as below.  Hold 
pending evaluation 
 
Grade >2 with 
suspected 
myocarditis 
Hold dose.**  Admit to hospital.  Cardiology consult.  Rule out MI and 
other causes of cardiac disease.  Cardiac Monitoring.  Cardiac Echo.  
Consider cardiac MRI and cardiac biopsy.  Initiate high dose 
methylprednisolone.  If no improvement within 24 hours, add either 
infliximab, ATG or tacrolimus.  Resume therapy if there is a return to 
baseline and myocarditis is excluded or considered unlikely. 
 
Grade >2 with 
confirmed 
myocarditis   
Off protocol therapy.  Admit to CCU (consider transfer to nearest 
Cardiac Transplant Unit).  Treat as above.  Consider high dose 
methylprednisolone.   
Add ATG or tacrolimus if no improvement. 
 
 
 *Including CHF, LV systolic dysfunction, Myocarditis, CPK, and troponin  
**Patients with evidence of myositis without myocarditis may be treated according as “other 
event”   
Note:  The optimal treatment regimen for immune mediated myocarditis has not been 
established.  Since this toxicity has caused patient deaths, an aggressive approach is 
recommended.   
 
If treatment is delayed >6 weeks ( >8 weeks for patients on high dose steroids) with 
recommended 4 weeks taper and 2 week observation, the patient must be permanently 
discontinued from study therapy, except as specified in Section 5.7 (Criteria to Resume 
Treatment.) 
  
Patients requiring a delay of >6 weeks (>8 weeks for patients on high dose steroids with required 

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4 weeks minimal taper and 2 week observation), should go off protocol therapy.  
Patients requiring > two dose delays for the same event should go off protocol therapy. 
 
Prior to starting corticosteroids or hormone replacement for any reason, appropriate endocrine 
testing including cortisol, ACTH, TSH and T4 must be obtained to document baseline.  
 
Patients may be dose-delayed for evaluation and restarted depending on results.  
 
Any patient started on corticosteroids initially who is determined to not require steroids 
treatment for an autoimmune adverse event may resume therapy after a 2 week observation 
period without further symptoms at the discretion of the PI or investigator. 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of AEs (Section 7.1) and the characteristics of an observed AE (Section 7.2) will 
determine whether the event requires expedited reporting via the CTEP Adverse Event Reporting 
System (CTEP-AERS) in addition to routine reporting. 
 
7.1 
Comprehensive Adverse Events and Potential Risks List (CAEPR) 
 
The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 
presentation of events by body system.  In addition to the comprehensive list, a subset of AEs, 
the Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column 
and is identified with bold and italicized text.  The SPEER is a list of events that are protocol-
specific exceptions to expedited reporting to NCI (except as noted below).  Refer to the 'CTEP, 
NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/adverse effects.htm for further clarification.  
 
NOTE:  The highest grade currently reported is noted in parentheses next to the AE in the 
SPEER.  Report ONLY AEs higher than this grade expeditiously.   
 
7.1.1 CAEPR for CTEP IND Agent Nivolumab (Parts A+B) 
 
Comprehensive Adverse Events and Potential Risks list (CAEPR) 
for 
BMS-936558 (Nivolumab, MDX-1106, NSC 748726) 
 
The Comprehensive Adverse Events and Potential Risks list (CAEPR) provides a single list of reported and/or 
potential adverse events (AE) associated with an agent using a uniform presentation of events by body system. In 
addition to the comprehensive list, a subset, the Specific Protocol Exceptions to Expedited Reporting (SPEER), 
appears in a separate column and is identified with bold and italicized text. This subset of AEs (SPEER) is a list of 
events that are protocol specific exceptions to expedited reporting to NCI (except as noted below).  
Refer to the 'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/electronic applications/docs/aeguidelines.pdf for further clarification. 
Frequency is provided based on 2678 patients. Below is the CAEPR for Ipilimumab (MDX-010).  

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4Pancreatitis may result in increased serum amylase and/or more frequently lipase. 
 
5Cytokine release syndrome may manifest as hemophagocytic lymphohistiocytosis with accompanying fever and 
pancytopenia. 
 
6Complications including hyperacute graft-versus-host disease (GVHD), some fatal, have occurred in patients 
receiving allo stem cell transplant (SCT) after receiving Nivolumab.  These complications may occur despite 
intervening therapy between receiving Nivolumab and allo-SCT. 
 
7Infusion reactions, including high-grade hypersensitivity reactions which have been observed following 
administration of nivolumab, may manifest as fever, chills, shakes, itching, rash, hypertension or hypotension, or 
difficulty breathing during and immediately after administration of nivolumab. 
 
 
Adverse events reported on Nivolumab trials, but for which there is insufficient evidence to suggest that there 
was a reasonable possibility that Nivolumab caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Leukocytosis 
CARDIAC DISORDERS - Atrial fibrillation; Atrioventricular block complete; Heart failure; Ventricular 
arrhythmia 
EAR AND LABYRINTH DISORDERS - Vestibular disorder 
EYE DISORDERS - Eye disorders - Other (iridocyclitis); Optic nerve disorder; Periorbital edema 
GASTROINTESTINAL DISORDERS - Constipation; Duodenal ulcer; Flatulence; Gastrointestinal disorders - 
Other (mouth sores); Vomiting 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Chills; Edema limbs; Malaise; 
Pain 
HEPATOBILIARY DISORDERS - Bile duct stenosis 
IMMUNE SYSTEM DISORDERS - Anaphylaxis; Immune system disorders - Other (autoimmune thrombotic 
microangiopathy); Immune system disorders - Other (limbic encephalitis) 
INFECTIONS AND INFESTATIONS - Bronchial infection; Lung infection; Sepsis; Upper respiratory infection 
INVESTIGATIONS - Blood lactate dehydrogenase increased; GGT increased; Investigations - Other (protein total 
decreased); Lymphocyte count increased; Weight loss 
METABOLISM AND NUTRITION DISORDERS - Dehydration; Hyperuricemia; Hypoalbuminemia; 
Hypocalcemia; Hyponatremia; Hypophosphatemia 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Back pain; Musculoskeletal and 
connective tissue disorder - Other (musculoskeletal pain); Musculoskeletal and connective tissue disorder - Other 
(polymyalgia rheumatica); Myalgia; Pain in extremity 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - Neoplasms 
benign, malignant and unspecified (incl cysts and polyps) - Other (Histiocytic necrotizing lymphadenitis) 
NERVOUS SYSTEM DISORDERS - Dizziness; Headache; Intracranial hemorrhage 
PSYCHIATRIC DISORDERS - Insomnia 
RENAL AND URINARY DISORDERS - Hematuria; Renal and urinary disorders - Other (tubulointerstitial 
nephritis) 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Bronchospasm; Cough; Dyspnea; 
Hypoxia 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Dry skin; Hyperhidrosis; Pain of skin; 
Photosensitivity; Rash acneiform; Skin and subcutaneous tissue disorders - Other (rosacea) 
VASCULAR DISORDERS - Flushing; Hypertension; Hypotension; Vasculitis 
 
Note: Nivolumab in combination with other agents could cause an exacerbation of any adverse event currently 
known to be caused by the other agent, or the combination may result in events never previously associated with 
either agent. 
 
7.1.2  
Comprehensive Adverse Events and Potential Risks list (CAEPR) 

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3Late bowel perforations have been noted in patients receiving MDX-010 (ipilimumab) in association with 
subsequent IL-2 therapy. 
 
4Complications including hyperacute graft-versus-host disease (GVHD), may occur in patients receving allo stem 
cell transplant (SCT) after receiving Ipilimumab (MDX-010).  These complications may occur despite intervening 
therapy between receiving Ipilimumab (MDX-010) and allo-SCT. 
 
5In rare cases diplopia (double vision) has occurred as a result of muscle weakness (Myasthenia gravis). 
 
6Gastrointestinal hemorrhage includes Anal hemorrhage, Cecal hemorrhage, Colonic hemorrhage, Duodenal 
hemorrhage, Esophageal hemorrhage, Esophageal varices hemorrhage, Gastric hemorrhage, Hemorrhoidal 
hemorrhage, Ileal hemorrhage, Intra-abdominal hemorrhage, Jejunal hemorrhage, Lower gastrointestinal 
hemorrhage, Oral hemorrhage, Pancreatic hemorrhage, Rectal hemorrhage, Retroperitoneal hemorrhage, and Upper 
gastrointestinal hemorrhage under the GASTROINTESTINAL DISORDERS SOC. 
 
7Infection includes all 75 sites of infection under the INFECTIONS AND INFESTATIONS SOC. 
 
 
Adverse events reported on Ipilimumab (MDX-010) trials, but for which there is insufficient evidence to 
suggest that there was a reasonable possibility that Ipilimumab (MDX-010) caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Anemia; Blood and lymphatic system disorders - Other 
(pure red cell aplasia)2; Febrile neutropenia 
CARDIAC DISORDERS - Conduction disorder; Restrictive cardiomyopathy 
EYE DISORDERS - Extraocular muscle paresis5; Eye disorders - Other (retinal pigment changes) 
GASTROINTESTINAL DISORDERS - Colonic ulcer; Dyspepsia; Dysphagia; Gastrointestinal disorders - Other 
(gastroenteritis); Gastrointestinal hemorrhage6; Proctitis 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Flu like symptoms; Non-cardiac 
chest pain 
HEPATOBILIARY DISORDERS - Hepatic failure2 
IMMUNE SYSTEM DISORDERS - Allergic reaction 
INFECTIONS AND INFESTATIONS - Infection7 
INVESTIGATIONS - Creatinine increased; Investigations - Other (rheumatoid factor); Lipase increased; Platelet 
count decreased; Serum amylase increased; White blood cell decreased 
METABOLISM AND NUTRITION DISORDERS - Tumor lysis syndrome 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Back pain; Joint range of motion 
decreased; Myalgia; Pain in extremity 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - Tumor pain 
NERVOUS SYSTEM DISORDERS - Dizziness; Dysphasia; Ischemia cerebrovascular; Seizure 
PSYCHIATRIC DISORDERS - Anxiety; Confusion; Depression; Insomnia 
RENAL AND URINARY DISORDERS - Proteinuria 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Allergic rhinitis; Cough; Dyspnea; 
Laryngospasm 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Dry skin; Hyperhidrosis; Skin 
hypopigmentation 
VASCULAR DISORDERS - Flushing; Hypertension; Vascular disorders - Other (temporal arteritis) 
 
 
Note: Ipilimumab (BMS-734016; MDX-010 Transfectoma-derived) in combination with other agents could cause 
an exacerbation of any adverse event currently known to be caused by the other agent, or the combination may result 
in events never previously associated with either agent. 
 
 

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7.2 
Adverse Event Characteristics  
 
• CTCAE term (AE description) and grade:  The descriptions and grading scales found in 
the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 
will be utilized for AE reporting.  All appropriate treatment areas should have access to a 
copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic applications/ctc.htm. 
 
• For expedited reporting purposes only:   
- 
AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the 
SPEER column, Section 7.1.1) should be reported through CTEP-AERS, however 
CTCAE version 5.0 must be used for serious AE reporting through CTEP-AERS,  
only if the grade is above the grade provided in the SPEER. 
- 
Other AEs for the protocol that do not require expedited reporting are outlined in 
section 7.3.4. 
 
• Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 
Expedited Adverse Event Reporting 
 
7.3.1 Expedited AE reporting for this study must use CTEP-AERS (CTEP Adverse Event 
Reporting System), accessed via the CTEP Web site (https://eapps-
ctep.nci.nih.gov/ctepaers).  The reporting procedures to be followed are presented in the 
“NCI Guidelines for Investigators: Adverse Event Reporting Requirements for DCTD 
(CTEP and CIP) and DCP INDs and IDEs” which can be downloaded from the CTEP 
Web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm
).  These requirements are briefly outlined in the tables below (Section 7.3.3). 
 
In the rare occurrence when Internet connectivity is lost, a 24-hour notification is to be 
made to CTEP by telephone at 301-897-7497.  Once Internet connectivity is restored, the 
24-hour notification phoned in must be entered electronically into CTEP-AERS by the 
original submitter at the site. 
 
7.3.2 CTEP-AERS is programmed for automatic electronic distribution of reports to the 
following individuals:  Principal Investigator and Adverse Event Coordinator(s) (if 
applicable) of the Corresponding Organization, the local treating physician, and the 
Reporter and Submitter.  CTEP-AERS provides a copy feature for other e-mail recipients.  
 
The Coordinating Center of the Corresponding Organization is responsible for submitting 

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o 
“10 Calendar Days” - A complete expedited report on the AE must be submitted electronically within 10 
calendar days of learning of the AE. 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 5 calendar days for: 
• 
All Grade 4, and Grade 5 AEs 
Expedited 10 calendar day reports for: 
• 
Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
• 
Grade 3 adverse events 
2For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, 
rounded UP to the nearest whole day, after the agent/intervention was last administered.  Footnote “1” above 
applies after this reporting period. 
Effective Date:  May 5, 2011 
 
7.3.4 Additional Protocol-Specific Expedited Adverse Event Reporting Exclusions 
 
N/A 
 
7.4 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions.  AEs reported 
expeditiously through CTEP-AERS must also be reported in routine study data 
submissions. 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are 
done to ensure the safety of patients enrolled in the studies as well as those who will enroll in 
future studies using similar agents.  AEs are reported in a routine manner at scheduled times 
during the trial using Medidata Rave.  For this trial the Adverse Event CRF is used for routine 
AE reporting in Rave. 
 
7.5 
Secondary Malignancy 
 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation or chemotherapy). A secondary 
malignancy is not considered a metastasis of the initial neoplasm. 
 
CTEP requires all secondary malignancies that occur following treatment with an agent under an 
NCI IND/IDE be reported expeditiously via CTEP-AERS.  Three options are available to 
describe the event:  
 
• Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia 
[AML])  
• Myelodysplastic syndrome (MDS)  
• Treatment-related secondary malignancy  

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Any malignancy possibly related to cancer treatment (including AML/MDS) should also be 
reported via the routine reporting mechanisms outlined in each protocol. For this trial the 
Adverse Event CRF is used for routine AE reporting in Rave.  Submission of the pathology 
report is not required.  
 
7.6 
Second Malignancy 
 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT a 
metastasis from the initial malignancy).  Second malignancies require ONLY routine AE 
reporting unless otherwise specified. 
 
7.7 
 Safety Monitoring Plan 
 
All participants will be carefully followed for safety.  Participants are seen by their study doctor 
and research nurse before each dose of nivolumab (every 2 weeks). Safety evaluations at this 
time include a physical exam, vital signs, performance status assessment, and safety laboratory 
tests.  The study team will continuously monitor participants for treatment side effects.  
Participants are instructed to inform their study doctor right away if they notice or feel anything 
different so the study doctor can check for side effects.  The study doctor may be able to provide 
treatment for side effects. The study doctor may temporarily hold the study drug to reduce side 
effects. The study doctor will permanently stop the study drug if side effects are too severe 
and/or long lasting. All participants will be followed for side effects for 100 days from their last 
dose of nivolumab. Participants with ongoing side effects will continue to be followed until 
resolution or stabilization of the side effects.  Because it is not known if nivolumab will be 
effective against anal cancer, to enrollment will stop after 12 participants are treated with 
nivolumab if none of them have their tumors shrink.  Study team conferences will be held 
monthly or more frequently if needed. 
 
8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational agent 
administered in this study can be found in Section 7.1. 
 
8.1 
CTEP IND Agent 
 
8.1.1 Nivolumab (NSC 748726) (Part A and B) 
 
Amino Acid Sequence:  4 polypeptide chains, which include 2 identical heavy chains 
with 440 amino acids and 2 identical light chains. 
 
Other Names:  BMS-936558, MDX1106 
 
Classification:  Anti-PD-1MAb 
 
M.W.:  146,221 daltons 

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Mode of Action:  Nivolumab targets the programmed death–1 (PD-1, cluster of 
differentiation 279 [CD279]) cell surface membrane receptor.  PD-1 is a negative 
regulatory receptor expressed by activated T and B lymphocytes.  Binding of PD-1 to its 
ligands, programmed death–ligand 1 (PD-L1) and 2 (PD-L2), results in the down-
regulation of lymphocyte activation.  Nivolumab inhibits the binding of PD-1 to PD-L1 
and PD-L2.  Inhibition of the interaction between PD-1 and its ligands promotes immune 
responses and antigen-specific T-cell responses to both foreign antigens as well as self-
antigens. 
 
Description:  Nivolumab Injection is a clear to opalescent, colorless to pale yellow 
liquid; light (few) particulates may be present.  The drug product is a sterile, 
nonpyrogenic, single-use, isotonic aqueous solution formulated in sodium citrate, sodium 
chloride, mannitol, diethylenetriamine pentacetic acid (pentetic acid) and polysorbate 80 
(Tween® 80), and water for injection.  Dilute solutions of hydrochloric acid and/or 
sodium hydroxide may be used for pH adjustment (pH 5.5-6.5). 
 
How Supplied:  Nivolumab is supplied by Bristol-Myers Squibb and distributed by the 
Pharmaceutical Management Branch, CTEP/DCTD/NCI as 100 mg vials (10 mg/mL) 
with a 0.7 mL overfill.  It is supplied in 10 mL type I flint glass vials, with fluoropolymer 
film-laminated rubber stoppers and aluminum seals. 
 
Preparation:   
Nivolumab injection is to be administered as an IV infusion through a 0.2-micron 
to 1.2- micron pore size, low-protein binding (polyethersulfone membrane) in-line 
filter at the protocol-specified doses and infusion times. It is not to be administered 
as an IV push or bolus injection. When the dose is based on patient weight (ie, 
mg/kg), nivolumab injection can be infused undiluted (10 mg/mL) or diluted with 
0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP to protein 
concentrations as low as 0.35 mg/mL. When the dose is fixed (eg, 240 mg, 360 
mg, or 480 mg flat dose), nivolumab injection can be infused undiluted or 
diluted so as not to exceed a total infusion volume of 160 mL. For patients 
weighing less than 40 kilograms (kg), the total volume of infusion must not 
exceed 4 mL per kg of patient weight. 
 
 
Storage and Stability:  The administration of nivolumab infusion must be completed 
within 24 hours of preparation. If not used immediately, the infusion solution may be 
stored under refrigeration conditions (2°C to 8°C, 36°F to 46°F) for up to 24 hours, and 
a maximum of 8 hours of the total 24 hours can be at room temperature (20°C to 
25°C, 68°F to 77°F) and room light. The maximum of 8 hours under room 
temperature and room light conditions includes the product administration period. The 
unopened vials can be stored at room temperature (up to 25°C, 77°F) and room light for 
up to 48 hours. 
 

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Stability:  Shelf-life surveillance of the intact vials is ongoing. 
 
CAUTION:  The single-use dosage form contains no antibacterial preservative or 
bacteriostatic agent.  Therefore, it is advised that the product be discarded 8 hours after 
initial entry.  
 
Route of Administration:  Intravenous infusion over 30 minutes. Nivolumab can be 
safely infused over 30 minutes in subjects with cancer (Part B only).  Do not administer as 
an IV push or bolus injection. 
 
Method of Administration:  Administer through a 0.2 micron to 1.2 micron pore size, 
low-protein binding polyethersulfone membrane in-line filter. 
 
Potential Drug Interactions:  No incompatibilities between nivolumab injection and 
polyvinyl chloride (PVC), non-PVC/non-DEHP (di[2-ethylhexyl]phthalate) IV 
components, or glass bottles have been observed. 
 
Availability 
 
Nivolumab is an investigational agent supplied to investigators by the Division of Cancer 
Treatment and Diagnosis (DCTD), NCI. 
 
Nivolumab is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator and the DCTD, NCI (see Section 12.3). 
 
8.1.2 Ipilimumab (NSC 732442) 
 
Chemical Name or Amino Acid Sequence: 4 polypeptide chains, 2 identical heavy 
chains with 447 amino acids and 2 identical light chains consisting of 215 amino acids. 
 
Other Names: MDX-CTLA-4, MDX-010, Yervoy™ 
 
Classification: Human monoclonal antibody 
 
M.W.: 147,991 Daltons 
 
Mode of Action: Ipilimumab is specific for the CTLA4 antigen expressed on a subset of 
activated T-cells. CTLA4 interaction with the B7 molecule, one of its ligands expressed 
on professional antigen presenting cells, can down-regulate T-cell response. Ipilimumab 
is, thought to act by blocking the interaction of CTLA4 with the B7 ligand, resulting in a 
blockade of the inhibitory effect of T-cell activation.  The CTLA4/B7 creates the 
interaction.  
 
Description: Ipilimumab is a fully human immunoglobulin (IgG1) with two 
manufacturing processes – ongoing trials have been using substances manufactured using 
Process B.  New clinical trials will be using ipilimumab that is manufactured by Process 

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C. The Process C has been developed using a higher producing sub-clone of the current 
Master Cell Bank, and modified cell culture and purification steps.  
  
How Supplied: Bristol-Myers-Squibb (BMS) supplies Ipilimumab to the DCTD/NCI.  
Ipilimumab injection, 200 mg/40 mL (5 mg/mL), is formulated as a clear to slightly 
opalescent, colorless to pale yellow, sterile, nonpyrogenic, single-use, isotonic aqueous 
solution that may contain particles. 
 
Each vial is a Type I flint glass vial with gray butyl stoppers and sealed with aluminum 
seals. 
 
Preparation:  Do not shake.  Allow the vials to stand at room temperature for 
approximately 5 minutes prior to preparation of infusion.  Ipilimumab is given undiluted 
or further diluted in 0.9% NaCl Injection, USP or 5% Dextrose Injection, USP in 
concentrations between 1 mg/mL and 4 mg/mL.  Ipilimumab is stable in a polyvinyl 
chloride (PVC), non-PVC/non DEHP (di-(2-ethylhexyl) phthalate) IV bag or glass 
container up to 24 hours refrigerated at (2° to 8° C) or at room temperature/ room light.  
 
Recommended safety measures for preparation and handling include protective clothing, 
gloves, and safety cabinets. 
  
Storage: Store intact vials refrigerated at (2° to 8° C), protected from light.  Do not 
freeze. 
 
Stability: Shelf-life surveillance of the intact vials is ongoing.  Solution as described 
above is stable up to 24 hours refrigerated at (2° to 8° C) or at room temperature/ room 
light.  
CAUTION:  Ipilimumab does not contain antibacterial preservatives.  Use prepared IV 
solution immediately. Discard partially used vials.  
 
Route(s) of Administration: Intravenous infusion over 30 minutes.  Do not administer 
ipilimumab as an IV push or bolus injection. 
 
Method of Administration:  Can use a volumetric pump to infuse ipilimumab at the 
protocol-specific dose(s) and rate(s) via a PVC IV infusion set with an in-line, sterile, 
non-pyrogenic, low-protein-binding filter (0.2 micron to 1.2 micron). 
 
Availability:  Ipilimumab is an investigational agent supplied to investigators by the 
Division of Cancer Treatment and Diagnosis (DCTD), NCI. 
 

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Ipilimumab is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator and the DCTD, NCI (see Section 12.3). 
 
8.1.3 Agent Ordering and Agent Accountability 
 
8.1.3.1 
 NCI -supplied agents may be requested by eligible participating investigators (or 
their authorized designee) at each participating institution.  The CTEP-assigned 
protocol number must be used for ordering all CTEP-supplied investigational agents.  
The eligible participating investigators at each participating institution must be 
registered with CTEP, DCTD through an annual submission of FDA Form 1572 
(Statement of Investigator), NCI Biosketch, Agent Shipment Form, and Financial 
Disclosure Form (FDF).  If there are several participating investigators at one 
institution, CTEP-supplied investigational agents for the study should be ordered 
under the name of one lead participating investigator at that institution. 
 
No starter supplies will be provided. Study agents must be ordered after the patient is 
enrolled on the assigned treatment arm. If expedited shipment is required, sites should 
provide an express courier account through the Online Agent Order Processing 
(OAOP) application. 
 
Active CTEP-registered investigators and investigator-designated shipping designees 
and ordering designees can submit agent requests through the PMB Online Agent 
Order Processing (OAOP) application.  Access to OAOP requires the establishment 
of a CTEP Identity and Access Management (IAM) account and the maintenance of 
an “active” account status, and a “current” password, and active person registration 
status.  For questions about drug orders, transfers, returns, or accountability, call or 
email PMB any time.  Refer to the PMB’s website for specific policies and guidelines 
related to agent management.   
 
8.1.3.2 
Agent Inventory Records – The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the inventory and disposition of all 
agents received from DCTD using the NCI Drug Accountability Record Form 
(DARF).  (See the NCI Investigator’s Handbook for Procedures for Drug 
Accountability and Storage.) 
 
8.1.3.3 
Investigator Brochure Availability – The current versions of the IBs for the agents 
will be accessible to site investigators and research staff through the PMB OAOP 
application.  Access to OAOP requires the establishment of a CTEP IAM account and 
the maintenance of an “active” account status, a “current” password and active person 
registration status.  Questions about IB access may be directed to the PMB IB 
Coordinator via email. 
 
8.1.3.4 
Useful Links and Contacts 
• CTEP Forms, Templates, Documents:  http://ctep.cancer.gov/forms/ 
• NCI CTEP Investigator Registration:  RCRHelpDesk@nih.gov 

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• PMB policies and guidelines: 
http://ctep.cancer.gov/branches/pmb/agent_management.htm  
• PMB Online Agent Order Processing (OAOP) application: 
https://ctepcore.nci.nih.gov/OAOP/  
• CTEP Identity and Access Management (IAM) account: 
https://ctepcore.nci.nih.gov/iam/  
• CTEP IAM account help:  ctepreghelp@ctep.nci.nih.gov  
• IB Coordinator:  IBCoordinator@mail.nih.gov  
• PMB email:  PMBAfterHours@mail.nih.gov 
PMB phone and hours of service: (240) 276-6575 Monday through Friday between 8:30 am and 
4:30 pm (ET) 
 
 

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9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES (PART A ONLY, SEE 
SECTION 10 FOR PART B) 
 
9.1 
Integral Laboratory or Imaging Studies 
 
Not applicable 
 
9.2 
Integrated Correlative Studies 
 
Not applicable 
 
9.3 
Exploratory Correlative Studies 
 
9.3.1 Exploratory Studies – Methodology 
 
Providers must offer and encourage optional tissue biopsies for tumor tissue for conduction of 
exploratory studies to all patients on this trial.  Blood collections for correlative studies will be 
mandatory. Tumor tissue and blood samples will be collected for immune monitoring as 
previously published54,116-118, under the supervision of the Immunotherapy Platform at MD 
Anderson. In tumor tissues, immunohistochemical studies will be performed to evaluate CD4+ 
and CD8+ T cells and regulatory T cells. In peripheral blood, we will also evaluate T cell 
populations including, but not limited to, CD4 cells, CD8+ cells, and regulatory T cells in pre- 
and post-therapy samples. 
 
Peripheral blood  
Up to 100 mL (within 24 hours) of peripheral blood will be collected for testing of biomarkers 
described in this clinical protocol at the following time points:  
• At screening (any time prior to first dose of nivolumab) 
• Before doses 2, 4, and 6  
• At treatment discontinuation  
 
For those patients who come off study due to reasons other than progression while on treatment 
(e.g., excessive toxicity, prolonged treatment break, withdrawal of consent), blood at the time of 
treatment discontinuation will not be required.  The patient’s hemoglobin concentration must be 
≥ 10.0 g/dL in order for the blood sample to be collected.  The treating physician or designee will 
have the option to cancel the laboratory protocol collection for patient safety without protocol 
deviation. 
 
Please refer to Appendix E For additional details regarding sample analyses. 
 
9.3.2 Blood Processing and Specimen Storage Management 
 
The MD Anderson Immunotherapy Platform (IMT) has worked on a number of methods testing 
different factors affecting the yield and quality of peripheral blood mononuclear cells (PBMCs) 
from blood samples of cancer patients. We have arrived at an optimal method that involves 
collecting blood in heparinized “Green Top” tubes, processing the blood within 24 hours of the 

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blood draw and diluting the blood 5 times with D-PBS. All samples are maintained at ambient 
temperature (room temperature) during transport and processing up until freezing or cell 
cryopreservation. This method ensures maximum PBMC recovery, especially with patients who 
are lymphopenic. 
 
An important aspect of our PBMC isolation and manipulation of cells, after thawing for 
experiments, is the use of an automated viable cell counting instrument (Cellometer; Nexcelom 
Bioscience, USA) instead of a hemocytometer. This instrument has been shown to be essential 
for consistent cell counting. We have also tracked the viability of the PBMC after thawing 
samples processed and frozen from melanoma patients to verify the quality of our 
cryopreservation techniques. The viabilities have been consistently good (80%-98%) after 
thawing. At the same time, we have also monitored the time periods between blood draws and 
processing in the lab and found that shipping cells at room temperature (25oC) greatly improves 
cell recovery when processing is carried out within 24 hours. 
 
We have on hand an interactive web-based and powerful software system developed with Aptia 
Systems (Houston, TX) to track all the cryopreserved cell and serum samples in this trial. The 
software, called the “Visual Specimen Manager” or “VSM” for short, has its own independent 
server, backed up hourly, and allows each authorized user to place in or take out vials of samples 
in a Windows-based interface. It is CFR 21 Part 11 compliant, with only specific users allowed 
access with passwords. The VSM system is capable of storing all relevant information on each 
sample stored by simply moving over it with a mouse. In addition, it allows rapid vial labeling 
and identification of samples by automatically generating a unique bar code. It can also print the 
specimen information on a cryolabel as the user enters it in a set template. The bar code system 
then allows for quick identification and localization of the specimens. The system works for 
liquid nitrogen (LN) tanks as well as -80 freezers. 
 
Immune monitoring screening for blood specimens is mandatory for this study and must occur: 
(1) Pre-Treatment: Within 14 days prior to treatment start.  
(2) During Treatment: Before nivolumab treatment doses 2, 4, and 6. 
(3) At study discontinuation (for patients who come off study due to disease progression while 
on treatment). 
 
Kits for the immune monitoring part of the protocol will be shipped by the MD Anderson 
Immunotherapy Platform (IMT) to the enrolling institution prior to enrollment. A total of 2 kits 
will be sent. All kits are for blood draws as per the protocol and are interchangeable from patient 
to patient.  Samples must be processed as per the immune-specific processing instructions below. 
The collection, processing, and shipping conditions must be followed.  
 
NOTE: Samples must be shipped to the IMT during the weekdays (Monday-Thursday morning) 
by overnight priority shipping/courier at room/ambient temperature (20-30 oC).  Blood and 
serum should not be collected from Thursday afternoon through Friday to avoid an over-the-
weekend delay in receipt by the IMT that will compromise sample quality.  Under no 
circumstances can immune monitoring blood products collected at any site be frozen or 
placed in any fixative reagent. 
 

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9.3.3 
Due to the limitations of fresh tissue sampling in this high impact study, residual 
blood samples from MDACC and from our ETCTN collaborators will be considered for a 
noninvasive approach in mutation analysis to optimize all available information we have from all 
collaborators.  For the purpose of this analysis, all correlative blood work is exploratory and will 
not be used for the purpose of treatment decision-making. 
 
9.3.4 
Description of the Guardant Health Assay (Part A only) 
Guardant360 is a next-generation sequencing (NGS) panel of 70 clinically actionable onco- and 
tumor suppressor genes utilizing digital sequencing of cell-free circulating tumor DNA (cfDNA) 
isolated from a simple, non-invasive blood draw. It is medically indicated for the prevention of a 
repeat invasive biopsy in advanced cancer patients when the initial biopsy is insufficient (QNS) 
or unavailable/unobtainable as well as when cancer has progressed or recurred despite treatment. 
The test detects single nucleotide variants via complete exon sequencing in 70 genes, copy 
number amplifications in 16 genes, small indels in EGFR, ERBB2 and MET exon 14 skipping, 
and fusions in ALK, FGFR2, FGFR3, RET, ROS1 and NTRK1. The genes are selected because 
mutations in these genes have FDA-approved matched therapies or are eligible for late phase 
clinical trials, as well as non-druggable genes with high prevalence alterations that may be 
helpful in monitoring for molecular response/non-response such as TP53. The panel also 
includes genomic markers of acquired resistance that may require a change in pharmacotherapy, 
e.g. EGFR T790M, ALK or ESR1 mutations. 
 
Guardant360 is an advanced diagnostic laboratory test (ADLT) offered by a sole source 
laboratory certified by the Clinical Laboratory Improvement Amendments (CLIA) for high 
complexity (molecular pathology) testing and accredited by the College of American Pathology 
(CAP). Due to high rates of false positives with traditional NGS assays when tumor DNA is in 
low concentrations, the majority of “liquid biopsy” methods interrogating cell-free DNA have 
been limited to hotspot analyses. In contrast, the ultra-high specificity (> 99.9999%) of the 
digital sequencing method enables the sequencing of long, targeted regions (146,000 base pairs) 
without false positives. Complete exons are sequenced for all exons in 30 genes and the critical 
exons (those reported as having a somatic mutation in COSMIC) in 40 additional genes. Thus, its 
key differentiating characteristic from other “liquid biopsy” methods is the ability to sequence 
complete exons in many genes, in contrast to gene hotspot testing. 
 
Advantages of the Guardant360 cell-free DNA (cfDNA) NGS methodology versus solid tumor 
tissue-based NGS are: 
1. 
An invasive needle or surgical biopsy is avoided with cfDNA, reducing 
costs and complications. 
2. 
CfDNA provides a quantitative measure (concentration or mutant allele 
frequency) of mutations present whereas solid tumor biopsy typically 
provides a qualitative result (mutation either present or not detected). The 
quantitative cfDNA result may be followed over time to monitor response 
to treatment and evolution of acquired resistance. 
3. 
CfDNA sequencing identifies both germline and somatic mutations in the 
same sample. 
4. 
The assay failure rate is for cfDNA is less than 0.5% (in the first 9,000+ 
samples) compared to 15%-25% failure rates of tissue-based NGS related 

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to insufficient quantity of tissue (QNS). 
 
Guardant360 utilizes algorithmic methods to encode and ultimately decode inputs and outputs 
from massively parallel deep sequencing analysis. By leveraging signal transduction processing 
technology where voice or image data is digitally encoded before transmission and then decoded 
post-transmission, this NGS method, known as Digital SequencingTM, enables signal 
interference to be reduced by two orders of magnitude or more (88).  Four validation studies have 
been published with concordance to tissue biopsy-based genomic testing (88-91). With high 
enough sensitivity and specificity to robustly quantitate ctDNA from blood, this approach has the 
potential to evaluate the multiple genomic targets required in NCCN guidelines, to act as a 
“summary” of the different tumor clones in patients with intra-tumor and inter-tumor 
heterogeneity, and to prevent the time delays, costs and complications inherent in invasive 
biopsies. The analytical and clinical validation of Guardant360 is conducted in conformance with 
evidentiary standards established by the Standards for Reporting of Diagnostic Accuracy 
(STARD), REporting of tumor MARKer Studies (REMARK), Evaluation of Genomic 
Applications in Practice and Prevention (EGAPP), and the recent Next-generation Sequencing: 
Standardization of Clinical Testing (Nex-StoCT) biomarker guidelines (92-95). 
 
Methodology 
 
The gene panel was selected to focus on those genomic alterations that are currently actionable 
defined as being targets of sensitivity or resistance to an FDA-approved matched therapy and/or 
a targeted therapy in clinical trials. The test simultaneously sequences the 70 cancer-related 
genes to an average depth of coverage of greater than 8,000X. To summarize, cell-free DNA is 
extracted from plasma and genomic alterations are analyzed by massively parallel paired end 
synthesis-by-sequencing of amplified target genes utilizing an Illumina Next-Seq platform 
complemented by systematic end-to-end process optimization including conversion of cell-free 
DNA fragments into digital sequences, improvements in the Illumina next generation sequencing 
process itself, followed by bioinformatics algorithms which enable ctDNA to be measured as a 
quantitative percentage of total cell-free DNA.  
 
Two 10mls of whole blood are collected in Streck Cell-Free DNA Blood Collection (Streck) 
tubes, which contain a proprietary formaldehyde-free preservative in that stabilizes white blood 
cells, preventing the release of genomic DNA and allowing shipping and stability for seven days 
without need for refrigeration, cold bricks or preliminary centrifugation prior to shipping.  
 
After digital libraries are produced, the sample is sequenced and post-sequencing data is 
processed using bioinformatics algorithms to quantify the absolute number of unique DNA 
fragments at a given nucleotide position. This proprietary process is referred to as Digital 
SequencingTM and enables reporting of the fractional concentration (mutant allele frequency) of 
a given SNV. Circulating cell-free DNA is mostly derived from leukocyte lysis (germline) and 
generally a much smaller amount of tumor DNA is derived from cancer cell apoptosis/necrosis. 
All of the cell-free DNA fragments, including leukocyte-derived and tumor-derived, are 
simultaneously sequenced with up to single molecule sensitivity. In other words, both tumor 
DNA and “normal”/germline DNA are sequenced and measured in the same sequencing assay. 
The fractional concentration or mutant allele frequency for a given mutation is calculated as the 

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fraction of circulating tumor DNA harboring that mutation in a background of wild-type cell-free 
DNA fragments. The analytic sensitivity reaches detection of 1-2 single mutant cell-free DNA 
molecules from a 10 ml blood sample. 
 
Gene list and genomic alterations in the Guardant360 Panel 
 
 
Guardant360 Panel 2015 
All NCCN Somatic Genomic Targets in a Single Test 
POINT MUTATIONS - Complete* or Critical Exon Coverage in 70 Genes 
 
 
AKT1 
ALK 
APC 
AR 
ARAF 
ARID1A 
ATM 
BRAF 
BRCA1 
BRCA2 
CCDN1 
CCND2 
CCNE1 
CDH1 
CDK4 
CDK6 
CDKN2A 
CDKN2B 
CTNNB1 
EGFR 
ERBB2 
ESR1 
EZH2 
FBXW7 
FGFR1 
FGFR2 
FGFR3 
GATA3 
GNA11 
GNAQ 
GNAS 
HNF1A 
HRAS 
IDH1 
IDH2 
JAK2 
JAK3 
KIT 
KRAS 
MAP2K1 
MAP2K2 
MET 
MLH1 
MPL 
MYC 
NF1 
NFE2L2 
NOTCH1 
NPM1 
NRAS 
NTRK1 
PDGFRA 
PIK3CA 
PTEN 
PTPN11 
RAF1 
RB1 
RET 
RHEB 
RHOA 
RIT1 
ROS1 
SMAD4 
SMO 
SRC 
STK11 
TERT 
TP53 
TSC1 
VHL 
AMPLIFICATIONS  
AR 
BRAF 
CCNE1 
CDK4 
CDK6 
EGFR 
ERBB2 
FGFR1 
FGFR2 
KIT 
KRAS 
MET 
MYC 
PDGFRA 
PIK3CA 
RAF1 
FUSIONS 
ALK 
FGFR2 
FGFR3 
RET 
ROS1 
NTRK1 
INDELS 
EGFR exons 19/20 
ERBB2 exons 19/20 
MET exon 14 skipping 

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Serum: 1 Red Top Tube (10 ml) 
 
1. Label tube with Protocol number, collection date/time, protocol time-point collected (e.g. 
pretreatment, post-treatment), and clearly mark specimen as “serum”. 
2. Allow one red top tube to clot for 30 minutes at room temperature. 
3. Spin in a standard clinical centrifuge at ~2500 RPM for 10 minutes at 4 oC (preferred). If sites 
are unable to process samples at 4 oC then spinning at room temperature is acceptable if done 
within 2 hours of draw but must be noted. 
4. Place tube into Styrofoam container (together with the Green top tubes mentioned below), 
then into a biohazard bag and then place the bag into the Cardboard shipping (outer) box.  
5. Ship to the address below the same day using overnight courier (FedEx), early morning 
delivery option. 
 
Under no circumstances are collected blood specimens to be frozen or put in any sort of 
fixative. 
 
PLEASE MAKE SURE THAT EVERY SPECIMEN IS LABELED and include collection time 
point on the Specimen Transmittal Form (STF). 
 
Whole Blood:  8-9 Green Top Tubes (90 ml) 
 
1. After collection, invert tube(s) multiple times to ensure adequate mixing of Sodium Heparin. 
2. Label tube with protocol number, collection date/time, protocol time-point collected (e.g. 
pretreatment, post-treatment), and clearly mark specimens as “PBMC.” 
3. Place tubes into Styrofoam container (together with the Red top tube mentioned above), then 
into a biohazard bag and then place the bag into the Cardboard shipping (outer) box.  
4. Ship to the address below the same day using overnight courier (FedEx), early morning 
delivery option. 
Under no circumstances are collected blood specimens to be frozen or put in any sort of 
fixative. 
 
PLEASE MAKE SURE THAT EVERY SPECIMEN IS LABELED and include collection time 
point on the STF. 
 
9.3.3 Multicolor Flow Cytometry-Based Lymphocyte Phenotyping 
 
To support the flow cytometry assays in this clinical trial, the IMT has developed robust 
multicolor flow cytometry (FACS) staining protocols for analyzing lymphocyte subsets in 
peripheral blood and in tumor isolates. The IMT has developed a standard research protocol 
(SRP) that details a comprehensive flow cytometry staining panel using validated antibodies (a 
copy of this SRP can be supplied on request).   
 
Multicolor flow cytometry-based lymphocyte phenotyping will be carried out at: 1) Before 
nivolumab treatment and 2) Prior to the 2nd,  4th, 6th administration of nivolumab and at 
progression/discontinuation (if the patient receives as many doses prior to discontinuation).  
 

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9.4 
Immunohistochemical (IHC) Staining and Analysis for PD-L1, PD-1, CD8+,  and 
CD4+ Tumor-Infiltrating Lymphocytes (Parts A and B) 
 
Tissue biopsies for exploratory correlative studies are to be offered to each patient on study, yet 
are optional for enrollment and treatment.  Patients who develop thrombocytopenia (platelet 
count < 50,000/ µL) or are otherwise deemed to be at high risk for bleeding from biopsy by the 
investigator may have the second biopsy delayed by two additional weeks/ rescheduled to prior 
to receiving the next dose of nivolumab.  If the patient is still deemed to be at too high risk for 
bleeding, then they may proceed with additional treatment without undergoing the second 
biopsy.  Patients who choose not to undergo tissue biopsy will still be permitted to remain on 
study, as will patients who are unable to complete a second tissue biopsy due to high risk for 
bleeding.  
 
Core biopsies under radiographic (ultrasound, CT, or MRI) – guided biopsy will be performed 
using a lumen no smaller than an 18-gauge needle pretreatment and 2 weeks following initiation 
of treatment with nivolumab (up to 3 days prior to 3rd dose of nivolumab).  5-8 core samples 
should ideally be taken, and stored as fresh frozen tissue and as paraffin-embedded tissue, 
alternating between the two with each successive tissue core biopsy. 
 
Detection of the selected markers for this study will be performed using immunohistochemistry 
with Dako kits (Agilent Tehcnologies) provided by BMS.  These kits will included all necessary 
reagents including, but not limited to, primary and secondary antibodies, needed in order to 
perform exploratory biomarker studies.  After the staining, the slides will be digitally scanned in 
an Aperio AT system (Aperio™, Leica Biosystems™) to convert the IHC slides into digital 
pathology files for posterior analysis.  IHC expression analyses of markers consider a thorough 
staining pattern evaluation including distribution (percentage of positive cells) and intensity in 
the form of H-score, and evaluating the IHC expression in the proper subcellular location (i.e. 
membrane, cytoplasm or nucleus).  IHC analysis and scoring will be performed by a certified 
pathologist at MD Anderson using an image analysis software (Image Toolbox, Aperio™). 
 
Outcome from the analysis will be calculated according to the H-score.  The H-score ranges from 
0 to 300, and it considers both intensity of the IHC (from 0 to 3) and distribution (percentage of 
the target cells positive, from 0 to 100).  The scoring is the addition of the percentages of cells 
with intensity 0 + intensity 2 + intensity 3, thus the addition of the final percentage is 100% and 
the scoring will range from 0 to 300. Hence, the H score will incorporate both   percentage of 
positive cells and intensity of marker expression. 
 
9.5 
Specimen Submission Summary  
 
9.5.1 Submission of Blood Specimens 
 

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9.5.1.1 
 Handling Instructions 
 
Specimens for Immune Monitoring (required) 
for shipment to MD Anderson—Immunotherapy Platform: See Section 9.4 
Specimens taken 
from patient: 
Collected when: 
 
Submitted as: 
Shipped: Same day 
as Blood draw. 
SERUM: 10 mL of whole 
blood in 1 red-top tube and 
centrifuge 
Prior to nivolumab treatment 
start -at screening or any time 
prior to first dose of 
nivolumab), before doses 2,4, 
6, and at study end/disease 
progression 
Whole blood samples in 
red top tubes 
Sent at room temperature  
via overnight carrier to MD 
Anderson Immunotherapy 
Platform 
WHOLE BLOOD:  90 mL of 
whole blood in sodium 
heparin tubes 
Prior to nivolumab treatment 
start -at screening (or any 
time prior to first dose of 
nivolumab), before doses 2, 4, 
6,, and at study end/disease 
progression 
Whole blood samples in 9 
green top tubes (Na 
Heparin) 
Sent at room temperature  
via overnight carrier to MD 
Anderson Immune 
Monitoring Lab 
*If the patient discontinues therapy prior to dose 6, then only the blood draws prior to 
treatment administration and at progression/discontinuation will be required. 
 
9.5.1.2 
Shipping Instructions for Blood Specimens 
 

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1. Place the blood tubes (red top, green top and PAXgene) in the Styrofoam container as 
shown in the figure. This figure shows only the positioning of the tubes: the color top 
does not depict what you will have. 
 
2. Place the rubber band around the Styrofoam container as shown in this figure (this is 
only for added stability) 
 
3. Place the Styrofoam container into the cardboard shipping (outer) box, and then place 
the cardboard box into a biohazard bag together with the Absorbent shipping materials. 

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4. Place everything into the FedEx shipping back and send to the address below (blood 
spesimens only): 
Immunotherapy Platform 
MD Anderson Cancer Center 
Attention: Karen Millerchip/Jaimol Peedikayil 
SCR3.3208  
7455 Fannin St.  
                  Houston, TX 77054-1901 
 
9.5.2 Submission of Slides for IHC Analysis 
 
9.5.2.1 
Handling Instructions 
 
Specimens for Central Pathology Review  
for shipment to MD Anderson—Jorge Blando: See Section 9.5.2.2 
Specimens taken 
from patient: 
Collected when: 
 
Submitted as: 
Shipped: after 
paraffin 
embedding/slice 
cutting. 
6-8 unstained tumor 
slides per biopsy or 
time point  
Two separate 
biopsies: one prior to 
treatment initiation 
and another after 2 
doses of treatment 
with nivolumab (i.e., 
up to 3 days prior to 
3rd dose of 
nivolumab).  
Paraffin-embedded 
unstained tumor 
slides 
Overnight mail to Dr. 
Jorge Blando 
 
9.5.2.2 
Shipping address for IHC samples only:  
 
Immunopathology Laboratory 
Immunotherapy Platform (IMT) 
UT MD Anderson Cancer Center,  
Life Science Plaza Building 
2130 W. Holcombe Blvd, 
Unit 2951, Houston, TX 77030 
 
9.6 
Special Studies - Analysis for the Presence of HPV 
 
When available, HPV testing, if not previously performed, will be conducted on archival tissue 
for each patient at the participating site treating each particular patient.  The availability of 
remaining tissue for HPV testing is not required for participation on this study.  Testing will 
include detection of HPV DNA by in-situ hybridization and/or detection of p16 by 
immunohistochemistry.  Formalin-fixed, paraffin-embedded samples will be deparaffinized in 
preparation for DNA extraction.  To determine whether or not HPV had been incorporated into 

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the host genome, in-situ hybridization will be utilized to assess for positive hybridization in the 
tumor cell nuclei using probes for various oncogenic subtypes of HPV.  To determine whether or 
not p16 is expressed in tumor cells, IHC analysis using monoclonal antibodies against the p16 
protein will performed.  Tumors will considered to express p16 if 5% or greater of tumor cells 
demonstrate p16 staining on IHC.  Patients will be considered to have HPV-positive tumors if 
HPV DNA is detected by in-situ hybridization and/or p16 is detected in the tumor cells by 
IHC.  Patients will be deemed to have HPV-negative tumors if HPV DNA is absent and p16 is 
not detected.   
 
9.7 
Optional Banking of Residual Tissue and Blood Samples for Future Research  
 
All patients who sign consent for this study also will be asked to consider participation in this 
optional part of the study. In consenting patients, any remaining blood and tissue samples not 
used for biomarker assessment will be banked for potential future research.  The samples will be 
banked in the Immunotherapy Platform Laboratory, The University of Texas MD Anderson 
Cancer Center, South Campus Research Bldg SCR3.3208, 7455 Fannin Street, Houston, TX 
77054.    
 
IRB approval will be obtained prior to the use of the banked samples for any research not 
described in this protocol. The samples will be given a code number.  No identifying information 
will be directly linked to the samples. Only the research team in charge of the bank will have 
access to the code numbers and be able to link the samples to the subject. If a patient withdraws 
his/her consent for banking of samples, the banked samples will be destroyed. However, if any 
samples were previously used for research prior to the withdrawal of consent, the samples will 
not be able to be destroyed. 
 
10. PART B – CORRELATIVE AND BLOOD WORK:   
 
We will quantitatively assess multiple immune markers in FFPE tumor histology sections, 
including immunohistochemistry (IHC) PD-L1 expression in malignant and inflammatory cells, 
as well as by immunofluorescence (mIF) TIL’s, macrophages and myeloid cell sub-populations. 
All antibodies used will be optimized for IHC/MIF by examination of positive and negative 
controls and testing of the biomarker, correlative, and special studies. 
 
10.1 
Integral Laboratory or Imaging Studies 
 
N/A 
 
10.2 
Integrated Correlative Studies 
 
N/A 
 
10.3 
Exploratory Correlative Studies 
 
10.3.1 Exploratory Studies – Methodology 
 

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Providers must offer and encourage optional tissue biopsies for tumor tissue for conduction of 
exploratory studies to all patients on this trial.  Blood collections for correlative studies will be 
mandatory. Tumor tissue and blood samples will be collected for immune monitoring as 
previously published54,116-118, under the supervision of the Translational Immunotherapy 
Platform/Molecular Pathology Platform at MD Anderson. In tumor tissues, 
immunohistochemical studies will be performed to evaluate CD4+ and CD8+ T cells and 
regulatory T cells. In peripheral blood, we will also evaluate T cell populations including, but not 
limited to, CD4 cells, CD8+ cells, and regulatory T cells in pre- and post-therapy samples. 
 
Peripheral blood  
Up to 70 mL (within 24 hours) of peripheral blood will be collected for testing of biomarkers 
described in this clinical protocol at the following time points:  
• At screening. 
• After cycle #1 (between weeks 9-10, one cycle = 8 weeks). 
• At treatment discontinuation.  
 
For those patients who come off study due to reasons other than progression while on treatment 
(e.g., excessive toxicity, prolonged treatment break, withdrawal of consent), blood at the time of 
treatment discontinuation will not be required.  The patient’s hemoglobin concentration must be 
≥ 10.0 g/dL in order for the blood sample to be collected.  The treating physician or designee will 
have the option to cancel the laboratory protocol collection for patient safety without protocol 
deviation. 
 
Please refer to Appendix E For additional details regarding sample analyses. 
 
10.3.2 Tissue Collection and processing 
 
Fresh and formalin-fixed and paraffin-embedded (FFPE) core needle biopsies (CNBs) tumor 
tissues will be used for immune and molecular profiling. Core biopsy is typically performed 
using 21-18 gauge needles and, with conditions permitting, up to 5 cores should be collected, 
including two cores (#1-2) for overnight FFPE processing, and 3 additional cores (#3-5) as fresh 
tumor tissue that will be immediately frozen. In at least 20 cases, one fresh tumor tissue core 
(core #3) will be processed for TILs isolation and flow cytometry analysis. Up to 60 ml of whole 
blood will be collected at each time point and processed for isolation of plasma (for future 
analysis of, for example, circulating tumor DNA), and PMBCs for 14-color flow cytometry using 
methods previously published. All tissue specimens collected will be reviewed by reference 
pathologists. The following quality control activities for specimens collected will be performed: 
a) histology/cytology examination of the tissues and cells to confirm the presence of tumor cells, 
as well as their abundance; b) assessment of fresh tissue specimens for extraction of DNA and 
RNA, and to prepare histology specimens for immunohistochemistry (IHC), multiplex 
Immunofluorescence (mIF) and gene expression analysis of immune-oncology panels; and, c) 
assessment of DNA/RNA extracted. All histology stained slides will be scanned and digital 
images will be available for review.  
 
Core needle biopsy (CNB):  If FFPE and a fresh frozen diagnostic core needle biopsy (CNB) 
specimen is not sufficient for biomarker analysis, a fresh CNB will obtained solely with the 

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purpose of research studies (pre-treatment samples) and sent to the MD Anderson Institutional 
Tissue Bank at the Department of Translational Molecular Pathology (TMP, Ignacio I Wistuba) 
immediately after collection. The number of cores obtained will be affected by the patient 
clinical condition at the time of biopsy and determined by the radiologist who is performing the 
procedure. Up to 5 tissue cores should be collected, including 2 for clinical processing and 3 
additional passes will be attempted to obtain the research core samples. These cores will be 
processed for: 
Cores 1 and 2: Immediate and overnight fixation in 10% buffered formalin for paraffin 
embedding, usually within 20-24 hour after fixation. For biopsies performed on Friday, fixation 
time may extend to 48 hours (FFPE samples) 
 
Cores 3 - 5: Flash freezing in liquid nitrogen for RNA-sequencing. If an additional core is 
available will be flash freezing in liquid nitrogen as back up. In at least 20 cases, core #3 will be 
processed fresh for flow cytometry analysis. 
When the amount of tissue is more limited, priority will be assigned to FFPE analyses and RNA 
sequencing (at least 2 FFPE cores and at least 1 fresh frozen core). The FFPE sample is 
important as it also provides a histological confirmation for the presence and cellularity of tumor 
cells. FFPE samples will also be prioritized for immune gene expression profiling by exploratory 
immunohistochemistry assay, multiplex immunofluorescence (IF) and biobanking for other to be 
determined assays. 
For patients NOT participating in optional tissue collection, submission of ARCHIVAL 
tissue specimens are recommended:  
For patients who do NOT consent to undergo a biopsy (or are unable to do so) as part of this trial 
or for whom adequate tumor specimen is unavailable from the trial biopsies, archival FFPE 
tissue (tumor with or without normal tissue) may be submitted for translational studies.   
• Either (1) one H&E slide with 10-15 unstained slides – all 4 micron thickness 
 OR 
• (2) an FFPE block of MALIGNANT tumor may be mailed (to the address listed below) 
for further genomic and immune biomarker analysis.   
Here, a tumor specimen with the largest amount of malignant tumor present should be 
prioritized and selected for optional specimen submission.   
In addition, (1) one H&E slide with 10-15 unstained slides – all 4 micron thickness – or (2) an 
FFPE block of normal, NON-MALIGNANT tissue should be submitted if available.   
Archival samples from patients who are deceased at the time of retroactive sample request may 
also be submitted to the address below for further analysis (due to waiver of consent). 

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All FFPE tissue should be mailed to: 
University of Texas-MD Anderson Cancer Center 
Ship AMBIENT To: MDACC 
Attn: Institutional Tissue Bank (ITB) 
1515 Holcombe Blvd, Rm G1.3586 
Houston, TX 77030 
T: 713.745.7047 
 
10.3.3 Blood Processing and Specimen Storage Management 
 
The MD Anderson Immunotherapy Platform (IMT) has worked on a number of methods testing 
different factors affecting the yield and quality of peripheral blood mononuclear cells (PBMCs) 
from blood samples of cancer patients. We have arrived at an optimal method that involves 
collecting blood in heparinized “Green Top” tubes, processing the blood within 24 hours of the 
blood draw and diluting the blood 5 times with D-PBS. All samples are maintained at ambient 
temperature (room temperature) during transport and processing up until freezing or cell 
cryopreservation. This method ensures maximum PBMC recovery, especially with patients who 
are lymphopenic. 
 
An important aspect of our PBMC isolation and manipulation of cells, after thawing for 
experiments, is the use of an automated viable cell counting instrument (Cellometer; Nexcelom 
Bioscience, USA) instead of a hemocytometer. This instrument has been shown to be essential 
for consistent cell counting. We have also tracked the viability of the PBMC after thawing 
samples processed and frozen from melanoma patients to verify the quality of our 
cryopreservation techniques. The viabilities have been consistently good (80%-98%) after 
thawing. At the same time, we have also monitored the time periods between blood draws and 
processing in the lab and found that shipping cells at room temperature (25oC) greatly improves 
cell recovery when processing is carried out within 24 hours. 
 
We have on hand an interactive web-based and powerful software system developed with Aptia 
Systems (Houston, TX) to track all the cryopreserved cell and serum samples in this trial. The 
software, called the “Visual Specimen Manager” or “VSM” for short, has its own independent 
server, backed up hourly, and allows each authorized user to place in or take out vials of samples 
in a Windows-based interface. It is CFR 21 Part 11 compliant, with only specific users allowed 
access with passwords. The VSM system is capable of storing all relevant information on each 
sample stored by simply moving over it with a mouse. In addition, it allows rapid vial labeling 
and identification of samples by automatically generating a unique bar code. It can also print the 
specimen information on a cryolabel as the user enters it in a set template. The bar code system 
then allows for quick identification and localization of the specimens. The system works for 
liquid nitrogen (LN) tanks as well as -80 freezers. 
 
Immune monitoring screening for blood specimens is mandatory for this study and must occur: 
(1) Pre-Treatment: Within 14 days prior to treatment start.  
(2) During Treatment: Before cycle #2. 
(3) At Progression of Disease. 

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Kits for the immune monitoring part of the protocol will be shipped by the Department of 
Translational Molecular Pathology at MD Anderson Cancer Center (Houston, Texas) to the 
enrolling institution prior to enrollment. To request for kits, email Beatriz Sanchez Espiridion 
(bsanchez2@mdanderson.org). Include in the email your CTEP site code, PI, receiving 
individual, and address.  A total of 2 kits will be sent. All kits are for blood draws as per the 
protocol and are interchangeable from patient to patient.  Samples must be processed as per the 
immune-specific processing instructions below. The collection, processing, and shipping 
conditions must be followed.  
 
NOTE: Samples must be shipped to the Institutional Tissue Bank, Department of Translational 
Molecular Pathology, at MD Anderson Cancer Center during the weekdays (Monday-Thursday 
morning) by overnight priority shipping/courier at room/ambient temperature (20-30 oC).  Blood 
and serum should not be collected from Thursday afternoon through Friday to avoid an over-the-
weekend delay in receipt by the Institutional Tissue Bank that will compromise sample quality.  
Under no circumstances can immune monitoring blood products collected at any site be 
frozen or placed in any fixative reagent. 
 

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For the purpose of this analysis, all correlative blood work is exploratory and will not be used for 
the purpose of treatment decision-making. Serum: 1 Red Top Tube (10 ml) 
 
1. Label tube with Protocol number, collection date/time, protocol time-point collected (e.g. 
pretreatment, post-treatment), and clearly mark specimen as “serum”. 
2. Allow one red top tube to clot for 30 minutes at room temperature. 
3. Spin in a standard clinical centrifuge at ~2500 RPM for 10 minutes at 4 oC (preferred). If sites 
are unable to process samples at 4 oC then spinning at room temperature is acceptable if done 
within 2 hours of draw but must be noted. 
4. Place tube into Styrofoam container (together with the Green top tubes mentioned below), 
then into a biohazard bag and then place the bag into the Cardboard shipping (outer) box.  
5. Ship to the address below the same day using overnight courier (FedEx), early morning 
delivery option. 
 
Under no circumstances are collected blood specimens to be frozen or put in any sort of 
fixative. 
 
PLEASE MAKE SURE THAT EVERY SPECIMEN IS LABELED and include collection time 
point on the Specimen Transmittal Form (STF). 
 
Whole Blood:  6 Green Top Tubes (60 ml) 
 
1. After collection, invert tube(s) multiple times to ensure adequate mixing of Sodium Heparin. 
2. Label tube with protocol number, collection date/time, protocol time-point collected (e.g. 
pretreatment, post-treatment), and clearly mark specimens as “PBMC.” 
3. Place tubes into Styrofoam container (together with the Red top tube mentioned above), then 
into a biohazard bag and then place the bag into the Cardboard shipping (outer) box.  
4. Ship to the address below the same day using overnight courier (FedEx), early morning 
delivery option. 
Under no circumstances are collected blood specimens to be frozen or put in any sort of 
fixative. 
 
PLEASE MAKE SURE THAT EVERY SPECIMEN IS LABELED and include collection time 
point on the STF. 
 
10.3.4 Correlative Biomarker Sample Analyses 
 
Histology: H&E-stained sections from CNBs will be used to confirm the presence of tumor cells, 
as well as their abundance (tumor cellularity), stromal components and lymphocytic infiltrates. 
Hematoxylin and eosin (H&E)-stained sections from all FFPE diagnostic slides (tumor, normal 
and lymph nodes) will be scanned in Aperio™ digital pathology scanner analysis for pathological 
evaluation and selection of 1 or 2 blocks (depending on tumor availability) for biomarker analysis.  
From the CNB specimens, the following pathological analysis will be performed: 1) tumor 
diagnosis; 2) lowest degree of tumor differentiation; 3) percentage of areas of necrosis; 4) 
percentage of areas of fibrosis; 5) percentage of viable tumor tissue; and, 6) percentage of viable 
malignant cells. 

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Central Review: The scanned H&E-stained slides will be available for pathology analysis at the 
TMP Pathology and Biomarker Lab (TMP-IL) chaired by Dr. Ignacio I. Wistuba, MD Anderson 
Cancer Center.  
 
Quality Control (QC): All tissue specimens collected will be reviewed by reference pathologists. 
At least, three types of QC activities for specimens collected will be performed: a) 
histology/cytology examination of the tissues and cells; b) tissue quality assessment of fresh 
specimens for extraction of DNA, RNA and proteins, and to prepare histology specimens such as 
whole sections for immunohistochemistry and immunofluorescence; and, c) quality assessment of 
DNA, RNA and protein extracted. All histology stained samples will be scanned and digital images 
will be available for review. 
 
HPV Testing: PCR-based HPV detection and genotyping (Cervista assay, Holoantibodies 
standard methods, including Western blotting.  
 
Immune Biomarker Testing: All pathology slides will be scanned into a digital image scanner 
and analyzed using image analysis software: IHC analysis will be performed using the Aperio 
Image Toolbox™ (Leica Biosystems) and IF analysis using the Vectra Inform™ (Perkin-Elmer) 
software. Five random 1-mm square areas within the tumor region will be selected for analysis. 
PD-L1 expression of markers in malignant cells will be evaluated using the Aperio™ digital H-
score system which includes the percentage of positive cells (0 to 100) and intensity (0 to 3+), 
with a total score ranging from 0 to 300. For mIF analysis, we will use the Opal chemistry and 
multispectral microscopy Vectra system (Perkin-Elmer) and run two Vectra mIF panels. On the 
multiplex IHC system, each multiplex panel includes 6 to 7 antibodies that are stained on the 
same slide and labeled using a tyramide-signal amplification (TSA)-based kit (PerkinElmer). The 
scoring of PD-L1 is expressed as percentage of positive cells in the tumor cell and in the tumor 
associated macrophages compartments defined by immune-labeling, e.g., tumor epithelial cells 
labeled with cytokeratins (AE1/AE3+ PDL1+) and macrophages (CD68+ PDL1+).  T-cell 
subpopulations are evaluated as cell density (number of positive cells by mm square of tumor 
area) and also defined by co-expression of specific markers, e.g., T-helper (CD3+CD4+), T-
cytotoxic (CD3+CD8+).  Additional immune-markers are also expressed as cell density. Finally, 
the data is consolidated using Spotfire software (PerkinElmer) for the final report including 
percentage of tumor cells expressing PDL-1, percentage of tumor associated macrophages 
expressing PDL-1, and cell density for intratumoral T-cell infiltrates (including PD1, T-reg, T-
memory, NK and cytotoxic) for each tumor case.       
 
Nanostring Immune Oncology Pancancer Panel: Immune-related genes will be examined using 
RNA extracted from FFPE tumor tissue histology sections using 200ng of RNA, as previously 
published119. This panel includes  770  gene  and  combines  markers  for  24  different  immune  
cell  types  and populations, 30 common cancer antigens and genes that represent all categories 
of immune response including key checkpoint blockade genes. 
 
Whole exome sequencing (WES) and RNA-sequencing: Nucleic acids extraction from fresh 
frozen tumor tissue and PMBCs will be performed using standard methods. DNA and RNA 
quantity and integrity will be assessed using NanoDrop 1000 spectrophotometer (Nanodrop 

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103 
 
Technologies) and Pico-green analyses.  WES will be conducted to a minimum of 200-fold 
sequencing depth. Somatic mutations will be called using Mutect for point mutations \ and Pindel 
for insertions and deletions. A subset of variants will be re-sequenced using NGS of targeted PCR 
products to assess quality and false positive rates as previously described. Mutations will be 
further analyzed in silico to predict whether they would produce potentially immunogenic 
neoantigens using NetMHCpan. mRNA from each tumor will be extracted and sequenced by 
Illumina HiSeq, and gene expression will be quantified for the transcript models corresponding 
to the TCGA GAF2.1, using RSEM.  
 
Flow cytometry: a 14-color panels will focus on a) delineation of major immune cell types (T cells, 
B cells, NK cells, DC); b) determination of T cell differentiation status and limited functionality 
(IFN, TNF, GB); and, c) defining the expression level of co-stimulatory and co-inhibitory 
molecules on T cells and their respective receptors such as PD-L1 on infiltrating myeloid cells or 
tumor. Briefly, 50cc of heparinized peripheral blood will be processed fresh (within 24h of being 
drawn) for PBMCs isolation by MDACC ITB. PBMCs will be cryopreserved and stored in liquid 
nitrogen until use for cytometric analysis. All time points belonging to a patient will be stained 
and acquired at the same time to avoid any technical variation. In a subset of fresh tumor tissues, 
flow cytometry will be performed. Tumor tissue will be stored in HBSS for up to 24h before 
processing for flow cytometry. Fresh tissue will be mechanically disaggregated or digested 
according to needs and panel design. The cells will be processed as a single cell suspension tissue 
samples will be stained for surface and/or intracellular markers using standard procedures. Stained 
samples will be fixed and acquired on a flow cytometer (BD Fortessa X-20 or Canto II, BD 
Biosicence, San Jose, CA) within one week and analyzed using FlowJo software.   
 
 
 
 
 
 
 

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104 
 
10.4 
Specimen Submission Summary 
 
Specimens for Immune Monitoring (required) 
for shipment to Institutional Tissue Banks (ITB) MD Anderson—Department of 
Translational Molecular Pathology 
Specimens taken 
from patient: 
Collected when: 
 
Submitted as: 
Shipped: Same day 
as Blood draw. 
SERUM: 10 mL of whole 
blood in 1 red-top tube 
and centrifuge 
Prior to 
nivolumab/ipilimumab 
treatment start -at 
screening or any time 
prior to first dose  and 
before doses 2,and at 
study end/disease 
progression 
Whole blood samples in 
red top tubes 
Sent at room temperature 
via overnight carrier to 
MD Anderson 
(Institutional Tissue 
Bank).  
 
WHOLE BLOOD:  60  
mL of whole blood in 
sodium heparin tubes 
Prior to 
nivolumab/ipilimumab 
treatment start -at 
screening (or any time 
prior to first dose of 
nivolumab), before dose 2, 
and at study end/disease 
progression 
Whole blood samples in 6 
green top tubes (Na 
Heparin) 
Sent at room temperature  
via overnight carrier to 
MD Anderson ( 
Institutional Tissue Bank) 
Specimens taken 
from patient: 
Collected when: 
 
Submitted as: 
Shipped: after 
paraffin 
embedding/slice 
cutting. 
10 unstained tumor slides 
or FFPE block per biopsy 
or time point  
Two separate biopsies: 
one prior to treatment 
initiation and another after 
2 doses of treatment with 
nivolumab (i.e., up to 3 
days prior to 3rd dose of 
nivolumab).  
Paraffin-embedded 
unstained tumor slides 
Sent at room temperature  
via overnight carrier to 
MD Anderson 
(Institutional Tissue 
Bank)  
(1)10-15 unstained slides 
of malignant and 10-15 
unstained  normal slides 
or (2) FFPE blocks from 
archival malignant and 
normal tissue 
Archival specimens (for 
patients not participating 
in the optional biopsies or 
for patients in whom 
adequate malignant tissue 
is not available for 
biomarker analysis) 
Paraffin-embedded 
unstained slides or block 
Sent at room temperature  
via overnight carrier to 
MD Anderson 
(Institutional Tissue 
Bank) 
 
 
 
 
 
 
 
 
 
 

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105 
 
 
 
 
 
 
 
10.4.1 Shipping Instructions for Blood Specimens 
 
 
5. Place the blood tubes (red top, green top and PAXgene) in the Styrofoam container as 
shown in the figure. This figure shows only the positioning of the tubes: the color top 
does not depict what you will have. 

NCI Protocol #9673 
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106 
 
 
6. Place the rubber band around the Styrofoam container as shown in this figure (this is 
only for added stability) 
 
7. Place the Styrofoam container into the cardboard shipping (outer) box, and then place 
the cardboard box into a biohazard bag together with the Absorbent shipping materials. 
8. Place everything into the FedEx shipping back and send to the address below (blood 
specimens only): 
 
10.4.2 Submission of Slides for IHC Analysis 
 
University of Texas-MD Anderson Cancer Center 
Ship AMBIENT To: MDACC 
Attn: Institutional Tissue Bank (ITB) 
1515 Holcombe Blvd, Rm G1.3586 
Houston, TX 77030 
T: 713.745.7047 
10.5 
Optional Banking of Residual Tissue and Blood Samples for Future Research 
 
All patients who sign consent for this study also will be asked to consider participation in this 
optional part of the study. In consenting patients, any remaining blood and tissue samples not 
used for biomarker assessment will be banked for potential future research.  The samples will be 
banked in the Institutional Tissue Bank (ITB), at The University of Texas MD Anderson Cancer 
Center, South Campus Research Bldg SCR3.3208, 7455 Fannin Street, Houston, TX 77054.    
 
IRB approval will be obtained prior to the use of the banked samples for any research not 
described in this protocol. The samples will be given a code number.  No identifying information 
will be directly linked to the samples. Only the research team in charge of the bank will have 
access to the code numbers and be able to link the samples to the subject. If a patient withdraws 
his/her consent for banking of samples, the banked samples will be destroyed. However, if any 
samples were previously used for research prior to the withdrawal of consent, the samples will 
not be able to be destroyed. 

11. STUDY CALENDAR  
 
11.1 
Part A:  
 
Baseline evaluations are to be conducted within 1 week prior to start of protocol therapy.  Scans and x-rays must be done <4 weeks 
prior to the start of therapy.  Unless otherwise noted, a standard window of -1 day to + 2 days will be considered acceptable for all 
testing and evaluations (will not be considered study deviation). In the event that the patient’s condition is deteriorating, laboratory 
evaluations should be repeated within 48 hours prior to initiation of the next dose of therapy. 
 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Treatment 
Discontinuat
ion Visit 
28 Days 
from Last 
Dose of 
Nivolumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
Follow-up 
Nivolumab 
 
A 
 
A 
 
A 
 
A 
 
A 
 
A 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concurrent medications 
X 
Xg 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
Physical exam 
X 
Xg 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
Vital signs 
X 
Xg 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
Performance status 
X 
Xg 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
CBC w/diff, plts 
X 
Xg 
  
X 
 
X 
 
X 
  
X 
 
X 
 
X 
X 
 
 
Serum chemistrya 
X 
Xg 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
Carcinoembryonic antigen 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
Thyroid stimulating hormoneb 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
HIV Antibodyc 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV Viral Loadd 
X 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
 
 

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Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Treatment 
Discontinuat
ion Visit 
28 Days 
from Last 
Dose of 
Nivolumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
Follow-up 
Hepatitis B Panele 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hepatitis C Viral Antibody 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG (as indicated) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG and ECHO p 
Xp 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG, CPK, troponin, ECHO q 
Xq 
Xq 
 
Adverse event evaluation 
 
X---------------------------------------------------------------------------------------------X 
X 
X 
Xj 
Xj 
Tumor measurements 
X 
Tumor measurements are repeated every 6 weeks.  Documentation (radiologic) must 
be provided for patients removed from study for progressive disease.  
X 
 
 
 
Radiologic evaluation:  CT or 
MRI of Chest, Abdomen,  
Pelvis & other known or 
suspected sites of disease 
X 
Radiologic measurements should be performed every 6 weeks 
X 
 
 
 
B-HCG 
Xf 
Serum or urine pregnancy test  must be performed every 6 weeks 
Xf 
Xf 
 
HPV testing 
Xo 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for Correlative 
Studiesm 
X 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
 
 
 
Tumor Biopsies for 
Correlative Studiesn 
X 
 
 
 
 
Xl 
 
 
 
 
 
 
 
 
 
 
 
Survival status 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xk 
 
A: 
Nivolumab IV 3 mg/kg given every 2 weeks +/- 3 days. 
a: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], 
SGPT [ALT], sodium. 
b:      Patients with TSH > 10.0 U/mL or TSH < 0.5 U/mL must be referred to an endrocrinologist with correction in a range between 0.5-10.0 U/mL prior to treatment 
initiation. 
c:      If not tested within past 6 months. 
d: 
Only for patients with  HIV infection as documented by the presence of a positive HIV antibody test. 
e:      Hepatitis B surface antibody, Hepatitis B surface antigen, hepatitis B core antibody. 
f:      Serum or urine pregnancy test (women of childbearing potential) must be performed with 24 hours prior to start of nivolumab, then every 8weeks. After discontinuation 
from nivolumab these should be repeated at approximately 30 days and approximately 70 days.  
g.      Tests/evaluations do not need to be repeated prior to first nivolumab dose if performed within past 7 days.  
h.      Nivolumab may continue to be given every 4weeks until any of the criteria outlined in section 5 require it be stopped. 
i:       Off-treatment  evaluation, only for patients who discontinue treatment due to disease progression.   
j.       All patients will be followed for adverse events for 100 days from last dose of nivolumab via phone call. Patients with ongoing adverse event(s) will continue to be 
 

NCI Protocol #9673 
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109 
 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Treatment 
Discontinuat
ion Visit 
28 Days 
from Last 
Dose of 
Nivolumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
Follow-up 
followed via phone call(s) until resolution or stabilization of the adverse event. Patients who discontinue therapy due to unacceptable toxicity but have not demonstrated 
radiographic disease progression will continue to be followed on schedule every 8 weeks with imaging studies until disease progression is documented. 
k.      Patients will be followed for survival status every 3 months for 2 years after treatment discontinuation or until death, whichever occurs first.  Follow up may be 
accomplished by clinic visit, medical record review, phone contact or email. 
l.       2 weeks after initiation  of treatment with nivolumab (up to 3 days prior to 3rd dose of nivolumab). 
m.     Peripheral blood will be collected as described in section 9.3 at screening (any time prior to first dose of nivolumab) and before doses 2, 4, 6, and at treatment 
discontinuation (for patients who progress while on nivolumab).   Blood should not be collected if the hemoglobin concentration is < 10.0 g/dL.   
n.      Optional, but not required, for participation on study.   
o.      If not previously performed, DNA in-situ  hybridization and/or  p16 by IHC  testing if sufficient archival tissue is available will be performed at enrolling site.  
p.      For any patients with a history of CHF or at risk because of underlying cardiovascular disease or exposure to cardiotoxic drugs as clinically indicated. 
q.      For patients with evidence of CHF, MI, cardiomyopathy, or myositis cardiac evaluation including lab tests and cardiology consultations as clinically indicated including 
EKG, CPK, troponin, ECHO cardiogram. 
 
 
11.2  Part B:  
 
Baseline evaluations are to be conducted within 1 week prior to start of protocol therapy.  Scans and x-rays must be done <4 weeks 
prior to the start of therapy.  Unless otherwise noted, a standard window of -1 day to + 2 days will be considered acceptable for all 
testing and evaluations (will not be considered study deviation). In the event that the patient’s condition is deteriorating, laboratory 
evaluations should be repeated within 48 hours prior to initiation of the next dose of therapy. Note: The below study calendar presents 
an example of the first 13 weeks on treatment, which will be repeated every 13 weeks. For example, Week 14 should follow Week 1 
schedule, Week 15 should follow Week 2 schedule, and so forth until criteria for treatment discontinuation is met. 
 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Wk  
13 
Treatment 
Discontinuation 
Visiti 
28 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
Follow-up 
Nivolumab 
 
A 
 
 
 
A 
 
 
 
A 
 
 
 
A 
 
 
 
 
Ipilimumab* 
 
B 
 
 
 
 
 
 
 
B 
 
 
 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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110 
 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Wk  
13 
Treatment 
Discontinuation 
Visiti 
28 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
Follow-up 
Concurrent medications 
X 
Xg 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Physical exam 
X 
Xg 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
 
Vital signs 
X 
Xg 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
 
Performance status 
X 
Xg 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
 
CBC w/diff, plts 
X 
Xg 
  
 
 
X 
 
 
  
X 
 
 
 
X 
X 
X 
 
 
Carcinoembryonic antigen 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
 
Serum chemistrya 
X 
Xg 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
 
 
Thyroid panelb 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
HIV Antibodyc 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV Viral Loadd 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
 
 
Hepatitis B Panele 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hepatitis C Viral Antibody 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG (as indicated) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG and ECHO p 
Xp 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG, CPK, troponin, ECHO q 
Xq 
 
Xq 
 
Adverse event evaluation 
 
X---------------------------------------------------------------------------------------------X 
 
X 
X 
Xj 
Xj 
Tumor measurements 
X 
Tumor measurements are repeated every 8 weeks.  Documentation (radiologic) must 
be provided for patients removed from study for progressive disease.  
 
X 
 
 
 
Radiologic evaluation:  CT or 
MRI of Chest, Abdomen,  
Pelvis & other known or 
X 
Radiologic measurements should be performed every 8 weeks 
 
X 
 
 
 

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111 
 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Wk  
13 
Treatment 
Discontinuation 
Visiti 
28 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
Follow-up 
suspected sites of disease 
B-HCG 
Xf 
 
Serum or urine pregnancy test  must be performed every 8 weeks 
Xf 
Xf 
 
HPV testing 
Xo 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for Correlative 
Studiesm 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
Tumor Biopsies for 
Correlative Studiesn 
X 
 
 
 
 
 
 
 
 
Xl 
 
 
 
 
X 
 
 
 
Survival status 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xk 
 
 
A: 
Nivolumab IV 480 mg IV given every 4 weeks +/- 3 days. 
B:     Ipilimumab IV 1 mg/kg IV given every 8 weeks +/- 3 days.  
*:      For patients randoimized to receive ipilimumab in combination with nivolumab.  
a: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], 
SGPT [ALT], sodium. 
b:      Thyroid panel including TSH, Free T3, and Free T4. Patients with TSH > 10.0 U/mL or TSH < 0.5 U/mL must be referred to an endrocrinologist with correction in a 
range between 0.5-10.0 U/mL prior to treatment initiation. 
c:      If not tested within past 6 months. 
d: 
Only for patients with  HIV infection as documented by the presence of a positive HIV antibody test. 
e:      Hepatitis B surface antibody, Hepatitis B surface antigen, hepatitis B core antibody. 
f:      Serum or urine pregnancy test (women of childbearing potential) must be performed with 24 hours prior to start of nivolumab, then every 8weeks. After discontinuation 
from nivolumab these should be repeated at approximately 30 days and approximately 70 days.  
g.      Tests/evaluations do not need to be repeated prior to first nivolumab dose if performed within past 7 days.  
h.      Nivolumab may continue to be given every 4 weeks until any of the criteria outlined in section 5 require it be stopped. 
i:      Off-treatment  evaluation, only for patients who discontinue treatment due to disease progression.   
j.      All patients will be followed for adverse events for 100 days from last dose of treatment on study via phone call. Patients with ongoing adverse event(s) will continue to 
be followed via phone call(s) until resolution or stabilization of the adverse event. Patients who discontinue therapy due to unacceptable toxicity but have not 
demonstrated radiographic disease progression will continue to be followed on schedule every 8 weeks with imaging studies until disease progression is documented. 
k.      Patients will be followed for survival status every 3 months for 2 years after treatment discontinuation or until death, whichever occurs first.  Follow up may be 
accomplished by clinic visit, medical record review, phone contact or email. 
l.      8weeks after initiation of treatment with nivolumab (up to 3 days prior to 3rd dose of nivolumab). 
m.    Peripheral blood will be collected as described in section 10.3 at screening (any time prior to first dose of treatment), after cycle 1, 8 weeks, and at treatment 
discontinuation (for patients who progress while on treatment). Blood should not be collected if the hemoglobin concentration is < 10.0 g/dL.   
n.     Optional, but not required, for participation on study.   
o.     If not previously performed, DNA in-situ hybridization and/or p16 by IHC testing if sufficient archival tissue is available will be performed at enrolling site.  
p.     For any patients with a history of CHF or at risk because of underlying cardiovascular disease or exposure to cardiotoxic drugs as clinically indicated. 
q.     For patients with evidence of CHF, MI, cardiomyopathy, or myositis cardiac evaluation including lab tests and cardiology consultations as clinically indicated including 
EKG, CPK, troponin, echocardiogram (transthoracic). 
 
 

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Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11h 
Wk 
12 
Wk  
13 
Treatment 
Discontinuation 
Visiti 
28 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
30 Days 
from End of 
Treatment  
Follow-up 
100 Days 
from Last 
Dose of 
Nivolumab 
and 
Ipilimumab 
Follow-up 
 
 
 

111 
12.  MEASUREMENT OF EFFECT 
 
12.1 
Antitumor Effect – Solid Tumors 
 
For the purposes of this study, patients should be re-evaluated for response every 8 weeks. In 
addition to a baseline scan, confirmatory scans should also be obtained 8 weeks following initial 
documentation of objective response. 
 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1)120.  Changes in the largest diameter (unidimensional measurement) of the tumor 
lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST 
criteria. 
 
12.1.1 Definitions 
 
Evaluable for toxicity.  All patients will be evaluable for toxicity from the time of their 
first treatment with nivolumab. 
 
Evaluable for objective response.  Only those patients who have measurable disease 
present at baseline, have received at least one dose of therapy, and have had their disease 
re-evaluated will be considered evaluable for response.  These patients will have their 
response classified according to the definitions stated below.  (Note:  Patients who exhibit 
objective disease progression after the first dose will also be considered evaluable.) 
 
Evaluable Non-Target Disease Response.  Patients who have lesions present at baseline 
that are evaluable but do not meet the definitions of measurable disease, have received at 
least one dose of therapy, and have had their disease re-evaluated will be considered 
evaluable for non-target disease.  The response assessment is based on the presence, 
absence, or unequivocal progression of the lesions.  
 
12.1.2 Disease Parameters 
 
Measurable disease.  Measurable lesions are defined as those that can be accurately 
measured in at least one dimension (longest diameter to be recorded) as ≥20 mm (≥2 cm) 
by chest x-ray or as ≥10 mm (≥1 cm) with CT scan, MRI, or calipers by clinical exam.  
All tumor measurements must be recorded in millimeters (or decimal fractions of 
centimeters). 
 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a 
lymph node must be ≥15 mm (≥1.5 cm) in short axis when assessed by CT scan (CT scan 
slice thickness recommended to be no greater than 5 mm [0.5 cm]).  At baseline and in 
follow-up, only the short axis will be measured and followed. 
 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions 
(longest diameter <10 mm [<1 cm] or pathological lymph nodes with ≥10 to <15 mm [≥1 

NCI Protocol #9673 
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114 
 
to <1.5 cm] short axis), are considered non-measurable disease.  Bone lesions, 
leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis 
cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by 
CT or MRI), are considered as non-measurable. 
 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts 
should not be considered as malignant lesions (neither measurable nor non-measurable) 
since they are, by definition, simple cysts. 
 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable 
lesions, if they meet the definition of measurability described above. However, if non-
cystic lesions are present in the same patient, these are preferred for selection as target 
lesions. 
 
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 
lesions in total, representative of all involved organs, should be identified as target 
lesions and recorded and measured at baseline.  Target lesions should be selected on the 
basis of their size (lesions with the longest diameter), be representative of all involved 
organs, but in addition should be those that lend themselves to reproducible repeated 
measurements.  It may be the case that, on occasion, the largest lesion does not lend itself 
to reproducible measurement in which circumstance the next largest lesion which can be 
measured reproducibly should be selected.  A sum of the diameters (longest for non-
nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and 
reported as the baseline sum diameters.  If lymph nodes are to be included in the sum, 
then only the short axis is added into the sum.  The baseline sum diameters will be used 
as reference to further characterize any objective tumor regression in the measurable 
dimension of the disease. 
 
Non-target lesions.  All other lesions (or sites of disease) including any measurable 
lesions over and above the 5 target lesions should be identified as non-target lesions and 
should also be recorded at baseline.  Measurements of these lesions are not required, but 
the presence, absence, or in rare cases unequivocal progression of each should be noted 
throughout follow-up.  
 
12.1.3 Methods for Evaluation of Measurable Disease 
 
All measurements should be taken and recorded in metric notation using a ruler or 
calipers.  All baseline evaluations should be performed as closely as possible to the 
beginning of treatment and never more than 4 weeks before the beginning of the 
treatment. 
 
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging-based 
evaluation is preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam. 
 

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Clinical lesions  Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes) and ≥10 mm (≥1 cm) diameter 
as assessed using calipers (e.g., skin nodules).  In the case of skin lesions, documentation 
by color photography, including a ruler to estimate the size of the lesion, is 
recommended.  
 
Chest x-ray  Lesions on chest x-ray are acceptable as measurable lesions when they are 
clearly defined and surrounded by aerated lung.  However, CT is preferable.  
 
Conventional CT and MRI  This guideline has defined measurability of lesions on CT 
scan based on the assumption that CT slice thickness is 5 mm (0.5 cm) or less.  If CT 
scans have slice thickness greater than 5 mm (0.5 cm), the minimum size for a 
measurable lesion should be twice the slice thickness.  MRI is also acceptable in certain 
situations (e.g. for body scans).   
 
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which 
greatly impact image quality, lesion conspicuity, and measurement.  Furthermore, the 
availability of MRI is variable globally.  As with CT, if an MRI is performed, the 
technical specifications of the scanning sequences used should be optimized for the 
evaluation of the type and site of disease.  Furthermore, as with CT, the modality used at 
follow-up should be the same as was used at baseline and the lesions should be 
measured/assessed on the same pulse sequence.  It is beyond the scope of the RECIST 
guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body 
parts, and diseases.  Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior scans.  Body scans 
should be performed with breath-hold scanning techniques, if possible. 
 
PET-CT  At present, the low dose or attenuation correction CT portion of a combined 
PET-CT is not always of optimal diagnostic CT quality for use with RECIST 
measurements.  However, if the site can document that the CT performed as part of a 
PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), 
then the CT portion of the PET-CT can be used for RECIST measurements and can be 
used interchangeably with conventional CT in accurately measuring cancer lesions over 
time.  Note, however, that the PET portion of the CT introduces additional data which 
may bias an investigator if it is not routinely or serially performed.   
 
Ultrasound  Ultrasound is not useful in assessment of lesion size and should not be used 
as a method of measurement.  Ultrasound examinations cannot be reproduced in their 
entirety for independent review at a later date and, because they are operator dependent, it 
cannot be guaranteed that the same technique and measurements will be taken from one 
assessment to the next.  If new lesions are identified by ultrasound in the course of the 
study, confirmation by CT or MRI is advised.  If there is concern about radiation 
exposure at CT, MRI may be used instead of CT in selected instances. 
 
Endoscopy, Laparoscopy  The utilization of these techniques for objective tumor 

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evaluation is not advised.  However, such techniques may be useful to confirm complete 
pathological response when biopsies are obtained or to determine relapse in trials where 
recurrence following complete response (CR) or surgical resection is an endpoint. 
 
Tumor markers  Tumor markers alone cannot be used to assess response.  
 
Cytology, Histology  These techniques can be used to differentiate between partial 
responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor 
types, such as germ cell tumors, where known residual benign tumors can remain). 
 
The cytological confirmation of the neoplastic origin of any effusion that appears or 
worsens during treatment when the measurable tumor has met criteria for response or 
stable disease is mandatory to differentiate between response or stable disease (an 
effusion may be a side effect of the treatment) and progressive disease. 
 
FDG-PET  While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis 
of FDG-PET imaging can be identified according to the following algorithm:  
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of 
PD based on a new lesion. 
b. No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive FDG-
PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD.  If 
the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, 
additional follow-up CT  scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal 
FDG-PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing 
site of disease on CT that is not progressing on the basis of the anatomic images, this 
is not PD. 
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy 
in cases where a residual radiographic abnormality is thought to represent fibrosis or 
scarring.  The use of FDG-PET in this circumstance should be prospectively 
described in the protocol and supported by disease-specific medical literature for the 
indication.  However, it must be acknowledged that both approaches may lead to false 
positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity. 
 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake 
greater than twice that of the surrounding tissue on the attenuation corrected image. 
 
12.1.4 Response Criteria:  
 
12.1.4.1 
Evaluation of Target Lesions 
 
Complete Response (CR):  Disappearance of all target lesions.  Any pathological 
lymph nodes (whether target or non-target) must have reduction in short axis to <10 
mm (<1 cm). 

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Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters. 
 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of 
target lesions, taking as reference the smallest sum on study (this includes the 
baseline sum if that is the smallest on study).  In addition to the relative increase of 
20%, the sum must also demonstrate an absolute increase of at least 5 mm (0.5 cm).  
(Note:  the appearance of one or more new lesions is also considered progressions). 
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 
study. 
 
12.1.4.2 
Evaluation of Non-Target Lesions 
 
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
[<1 cm] short axis). 
 
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits. 
 
Progressive Disease (PD):  Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions.  Unequivocal progression 
should not normally trump target lesion status.  It must be representative of overall 
disease status change, not a single lesion increase.     
 
Although a clear progression of “non-target” lesions only is exceptional, the opinion 
of the treating physician should prevail in such circumstances, and the progression 
status should be confirmed at a later time by the review panel (or Principal 
Investigator). 
 
12.1.4.3 
Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).  The patient's best 
response assignment will depend on the achievement of both measurement and 
confirmation criteria. 
 
Responses will be assessed using CT scans or magnetic resonance imaging according 
to standard RECIST 1.1 criteria in order to assess disease progression.  These criteria 
will also allow for patients who experience an initial disease flare, and as some 
patients who will have a delayed response may experience an initial disease flare, we 
will allow patients with radiographic disease progression to continue on the trial 

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provided that they have a stable ECOG performance status, no need for immediate 
alternative treatment, and progression of no more than 40% with three or fewer new 
lesions.  If, at the next evaluation, these patients demonstrate further disease 
progression, then they will be labeled as non-responders to therapy.  However, if they 
do meet the criteria for response according to RECIST 1.1, then they will be deemed 
as a responder and counted accordingly in the tabulations for determining whether or 
not to proceed to a second stage or achieving the study end point. 
 
For Patients with Measurable Disease (i.e., Target Disease) 
 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-CR/Non-
PD 
No 
PR 
>4 wks. Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-CR/Non-
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
**    Only for non-randomized trials with response as primary endpoint. 
***  In exceptional circumstances, unequivocal progression in non-target lesions may be 
accepted as disease progression. 
 
Note:  Patients with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.”  Every effort should be made to document the 
objective progression even after discontinuation of treatment. 
 
For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
 
‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign this 
category when no lesions can be measured is not advised 

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12.1.5 Duration of Response 
 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first 
date that recurrent or progressive disease is objectively documented (taking as reference 
for progressive disease the smallest measurements recorded since the treatment started). 
 
The duration of overall CR is measured from the time measurement criteria are first met 
for CR until the first date that progressive disease is objectively documented. 
120 
 
Duration of stable disease:  Stable disease is measured from the start of the treatment 
until the criteria for progression are met, taking as reference the smallest measurements 
recorded since the treatment started, including the baseline measurements.  
 
12.1.6 Progression-Free Survival 
 
Part A: PFS is defined as the duration of time from start of treatment to time of 
progression or death, whichever occurs first. 
 
Part B: PFS is defined as the time period from the date of randomization to the date of PD 
or death whichever occurred first. 
 
12.1.7 Response Review 
 
All responses will be reviewed by an expert independent of the study at the study’s 
completion.  Simultaneous review of the patients’ files and radiological images is the best 
approach.  
 
12.2 
Other Response Parameters 
 
12.2.1 Overall Survival (OS) 
 
Part A: OS is defined as the duration of time from start of treatment to time of death. 
Part B:  OS will be measured from the time of randomization to the time of death. 
 
13. STUDY OVERSIGHT AND DATA REPORTING / REGULATORY 
REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
13.1 
Study Oversight 
 
This protocol is monitored at several levels, as described in this section.  The Protocol Principal 
Investigator is responsible for monitoring the conduct and progress of the clinical trial, including 
the ongoing review of accrual, patient-specific clinical and laboratory data, and routine and 

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serious adverse events; reporting of expedited adverse events; and accumulation of reported 
adverse events from other trials testing the same drug(s).  The Protocol Principal Investigator and 
statistician have access to the data at all times through the CTMS web-based reporting portal.   
 
During the Phase 2 portion of the study, the Protocol Principal Investigator will have, at a 
minimum, quarterly conference calls with the Study Investigators and the CTEP Medical 
Officer(s) to review accrual, progress, and pharmacovigilance.  Decisions to proceed to the 
second stage of a Phase 2 trial will require sign-off by the Protocol Principal Investigator and the 
Protocol Statistician. 
 
All Study Investigators at participating sites who register/enroll patients on a given protocol are 
responsible for timely submission of data via Medidata Rave and timely reporting of adverse 
events for that particular study.  This includes timely review of data collected on the electronic 
CRFs submitted via Medidata Rave. 
 
All studies are also reviewed in accordance with the enrolling institution’s data safety monitoring 
plan. 
 
13.2 
Data Reporting 
 
Data collection for this study will be done exclusively through Medidata Rave.  Access to the 
trial in Rave is granted through the iMedidata application to all persons with the appropriate 
roles assigned in the Regulatory Support System (RSS).  To access Rave via iMedidata, the site 
user must have an active CTEP IAM account (check at < https://ctepcore.nci.nih.gov/iam  >) and 
the appropriate Rave role (Rave CRA, Read-Only, RAVE CRA, Lab Admin, SLA or Site 
Investigator) on either the LPO or participating organization roster at the enrolling site. To the 
hold Rave CRA role or RAVE CRA (Lab Admin) role, the user must hold a minimum of an AP 
registration type.  To hold the Rave Site Investigator role, the individual must be registered as an 
NPIVR or IVR.  Associates can hold read-only roles in Rave.   
 
Upon initial site registration approval for the study in RSS, all persons with Rave roles assigned 
on the appropriate roster will be sent a study invitation e-mail from iMedidata.  To accept the 
invitation, site users must log into the Select Login (https://login.imedidata.com/selectlogin) 
using their CTEP-IAM user name and password, and click on the “accept” link in the upper 
right-corner of the iMedidata page.  Please note, site users will not be able to access the study in 
Rave until all required Medidata and study specific trainings are completed.  Trainings will be in 
the form of electronic learnings (eLearnings), and can be accessed by clicking on the link in the 
upper right pane of the iMedidata screen.   
 
Users that have not previously activated their iMedidata/Rave account at the time of initial site 
registration approval for the study in RSS will also receive a separate invitation from iMedidata 
to activate their account. Account activation instructions are located on the CTSU website, Rave 
tab under the Rave resource materials (Medidata Account Activation and Study Invitation 
Acceptance).  Additional information on iMedidata/Rave is available on the CTSU members’ 
website under the Rave tab or by contacting the CTSU Help Desk at 1-888-823-5923 or by e-
mail at ctsucontact@westat.com. 

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13.2.1 Method 
 
CTMS Routine Monitoring:  This study will be monitored by the Clinical Trials 
Monitoring Service (CTMS). Data is to be submitted to CTMS at least once every two 
weeks via Medidata Rave (or other modality if approved by CTEP).  Information on 
CTMS reporting is available at:  
http://www.theradex.com/clinicalTechnologies/?National-Cancer-Institute-NCI-11.  On-
site audits will be conducted on a 18-36 month basis as part of routine cancer center site 
visits. More frequent audits may be conducted if warranted by accrual or due to concerns 
regarding data quality or timely submission. For CTMS monitored studies, after users 
have activated their accounts, please contact the Theradex Help Desk at (609) 799-7580 
or by email at CTMSSupport@theradex.com for additional support with Rave and 
completion of CRFs. 
 
13.2.2 Responsibility for Data Submission 
 
For ETCTN trials, it is the responsibility of the PI(s) at the site to ensure that all 
investigators at the ETCTN Sites understand the procedures for data submission for each 
ETCTN protocol and that protocol specified data are submitted accurately and in a timely 
manner to the CTMS via the electronic data capture system, Medidata Rave.   
 
Data are to be submitted via Medidata Rave to CTMS on a real-time basis, but no less 
than once every 2 weeks.  The timeliness of data submissions and timeliness in resolving 
data queries will be tracked by CTMS.  Metrics for timeliness will be followed and 
assessed on a quarterly basis.  For the purpose of Institutional Performance Monitoring, 
data will be considered delinquent if it is greater than 4 weeks past due. 
 
Data from Medidata Rave and CTEP-AERS is reviewed by the CTMS on an ongoing 
basis as data is received.  Queries will be issued by CTMS directly within Rave.  The 
queries will appear on the Task Summary Tab within Rave for the CRA at the ETCTN to 
resolve.  Monthly web-based reports are posted for review by the Drug Monitors in the 
IDB, CTEP.  Onsite audits will be conducted by the CTMS to ensure compliance with 
regulatory requirements, GCP, and NCI policies and procedures with the overarching 
goal of ensuring the integrity of data generated from NCI-sponsored clinical trials, as 
described in the ETCTN Program Guidelines, which may be found on the CTEP 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm
)  and CTSU websites. 
 
An End of Study CRF is to be completed by the PI, and is to include a summary of study 
endpoints not otherwise captured in the database, such as (for phase 1 trials) the 
recommended phase 2 dose (RP2D), and a description of any dose-limiting toxicities 
(DLTs).  CTMS will utilize a core set of eCRFs that are Cancer Data Standards Registry 
and Repository (caDSR) compliant (http://cbiit.nci.nih.gov/ncip/biomedical-informatics-
resources/interoperability-and-semantics/metadata-and-models).  Customized eCRFs will 
be included when appropriate to meet unique study requirements.  The PI is encouraged 

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to review the eCRFs, working closely with CTMS to ensure prospectively that all 
required items are appropriately captured in the eCRFs prior to study activation.  CTMS 
will prepare the eCRFs with built-in edit checks to the extent possible to promote data 
integrity. 
 
CDUS data submissions for ETCTN trials activated after March 1, 2014, will be carried 
out by the CTMS contractor, Theradex.  CDUS submissions are performed by Theradex 
on a monthly basis.  The trial’s lead institution is responsible for timely submission to 
CTMS via Rave, as above. 
 
Further information on data submission procedures can be found in the ETCTN Program 
Guidelines 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm
). 
 
See Section 12.1.1 for details on CDUS reporting.  As the data management center for 
this trial, Theradex is responsible for compiling and submitting CDUS data to CTEP for 
all participants and for providing the data to the Principal Investigator for review.   
13.3 
CTEP Multicenter Guidelines 
 
N/A 
 
13.4 
Collaborative Agreements Language 
 
The agent supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI under 
a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical Company 
(hereinafter referred to as “Collaborator(s)”) and the NCI Division of Cancer Treatment and 
Diagnosis.  Therefore, the following obligations/guidelines, in addition to the provisions in the 
“Intellectual Property Option to Collaborator”  
(http://ctep.cancer.gov/industryCollaborations2/intellectual property.htm) contained within the 
terms of award, apply to the use of the Agent(s) in this study: 
 
1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can 
Agent(s) be transferred or licensed to any party not participating in the clinical study.  
Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall 
be maintained as such by the investigators.  The protocol documents for studies utilizing 
Agents contain confidential information and should not be shared or distributed without the 
permission of the NCI.  If a copy of this protocol is requested by a patient or patient’s family 
member participating on the study, the individual should sign a confidentiality agreement.  A 
suitable model agreement can be downloaded from: http://ctep.cancer.gov. 
 
2. For a clinical protocol where there is an investigational Agent used in combination with 
(an)other Agent(s), each the subject of different Collaborative Agreements, the access to and 
use of data by each Collaborator shall be as follows (data pertaining to such combination use 
shall hereinafter be referred to as "Multi-Party Data”): 

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a. NCI will provide all Collaborators with prior written notice regarding the existence and 
nature of any agreements governing their collaboration with NCI, the design of the 
proposed combination protocol, and the existence of any obligations that would tend to 
restrict NCI's participation in the proposed combination protocol. 
 
b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial 
by any other Collaborator solely to the extent necessary to allow said other Collaborator 
to develop, obtain regulatory approval or commercialize its own Agent. 
 
c. Any Collaborator having the right to use the Multi-Party Data from these trials must 
agree in writing prior to the commencement of the trials that it will use the Multi-Party 
Data solely for development, regulatory approval, and commercialization of its own 
Agent. 
 
3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement 
will be made available to Collaborator(s), the NCI, and the FDA, as appropriate and unless 
additional disclosure is required by law or court order as described in the IP Option to 
Collaborator (http://ctep.cancer.gov/industryCollaborations2/intellectual property.htm).  
Additionally, all Clinical Data and Results and Raw Data will be collected, used and 
disclosed consistent with all applicable federal statutes and regulations for the protection of 
human subjects, including, if applicable, the Standards for Privacy of Individually 
Identifiable Health Information set forth in 45 C.F.R. Part 164. 
 
4. When a Collaborator wishes to initiate a data request, the request should first be sent to the 
NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group 
studies, or PI for other studies) of Collaborator's wish to contact them. 
 
5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the 
guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a 
DMC for this clinical trial. 
 
6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the 
Group office for Cooperative Group studies or by the principal investigator for non-
Cooperative Group studies for immediate delivery to Collaborator(s) for advisory review and 
comment prior to submission for publication.  Collaborator(s) will have 30 days from the 
date of receipt for review.  Collaborator shall have the right to request that publication be 
delayed for up to an additional 30 days in order to ensure that Collaborator’s confidential and 
proprietary data, in addition to Collaborator(s)’s intellectual property rights, are protected.  
Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy 
review as soon as possible and preferably at least three (3) days prior to submission, but in 
any case, prior to presentation at the meeting or publication in the proceedings.  Press 
releases and other media presentations must also be forwarded to CTEP prior to release.  
Copies of any manuscript, abstract and/or press release/ media presentation should be sent to: 
 
Email:  ncicteppubs@mail.nih.gov 

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The Regulatory Affairs Branch will then distribute them to Collaborator(s).  No publication, 
manuscript or other form of public disclosure shall contain any of Collaborator’s confidential/ 
proprietary information. 
 
14. STATISTICAL CONSIDERATIONS 
 
14.1 
Study Design/Endpoints (Part A) 
 
14.1.1 Primary Endpoint 
 
Overall response rate is the primary endpoint for this study.   
 
14.1.2 Study Design 
This study is a two-staged, Simon Optimal phase II clinical trial of nivolumab as a single 
agent for the treatment of patients with locally advanced/unresectable or metastatic 
squamous cell carcinoma of the anal canal who have progressed through at least one prior 
line of therapy.  Responses will be assessed using CT scans or magnetic resonance 
imaging according to standard RECIST 1.1 criteria in order to assess disease progression.  
These criteria will also allow for patients who experience an initial disease flare, and as 
some patients who will have a delayed response may experience an initial disease flare, 
we will allow patients with radiographic disease progression to continue on the trial 
provided that they have a stable ECOG performance status, no need for immediate 
alternative treatment, and progression of no more than 40% with three or fewer new 
lesions.  If, at the next evaluation, these patients demonstrate further disease progression, 
then they will be labeled as non-responders to therapy.  However, if they do meet the 
criteria for response according to RECIST 1.1, then they will be deemed as a responder 
and counted accordingly in the tabulations for determining whether or not to proceed to a 
second stage or achieving the study end point.  If no response is observed among the 12 
patients treated in the first stage at six months, then the trial will be stopped for futility.  
However, if one or more responses are observed, then the trial will be expanded to 
include an additional 25 patients, with 4 or more responses needed among the 37 total 
participants to declare an efficacy of this single-agent therapy. 
 
14.1.3 Null/Alternative Hypotheses 
 
For this Simon Optimal, two-stage phase II study, we propose a null hypothesis Ho: p ≤ 
0.05 and an alternative hypothesis Ha: p ≥ 0.20, where p represents the percentage of 
patients with metastatic SCCA of the anal canal demonstrating either a partial response or 
a complete response according to RECIST 1.1 criteria when treated with nivolumab.  If 
the null hypothesis is rejected and the alternative hypothesis is accepted based on the 
results of this study, then a phase III trial comparing nivolumab to best supportive care in 
patients with refractory metastatic squamous cell carcinoma of the anal canal will be 
proposed. 
 

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Overall survival 
 
Kaplan-Meier analysis will be performed to estimate the median overall survival with a 
90% confidence interval.  Overall survival will be calculated as the time from initiation 
of treatment with nivolumab until death. 
 
 
Toxicity 
 
Adverse events will be monitored and recorded according to grade for each patient from 
the time of treatment initiation until 30 days (±7 days) after radiographic progression and 
end of treatment via phone call or visit. The frequency of each serious adverse event will 
be measured relative to the total number of patients treated. Toxicities will be tabulated 
by type and grade. All patients will be followed for adverse events for 100 days from last 
dose of treatment on study via phone call. Patients with ongoing adverse event(s) will 
continue to be followed via phone call(s) until resolution or stabilization of the adverse 
event. 
 
14.5 
Analyses of Exploratory Endpoints:  
 
 
14.5.1 
Descriptive Statistics of IHC and Blood Sample Studies. 
 
Descriptive statistics including plots, mean, median and standard deviations will be used to 
summarize data. For continuous outcomes, t-test and ANOVA will be used to compare 
outcome measures across patient characteristics. Dunnett’s and Tukey’s test that properly 
adjust for multiplicity in multiple tests will be implemented. Pair-wise comparisons will be 
performed using pre- and post-therapy samples from each patient . The chi-square (c2) test or 
Fisher’s exact test will be used to test the association between two categorical variables such 
as disease state and performance status. Both univariate and multivariate logistic regressions 
will be performed to model prognostic factors.  
 
14.6 
Study Design/Endpoints (Part B) 
 
The primary endpoint of part B is progression-free survival (PFS). The event is progression of 
disease or death, whichever occurs first. The PFS time is defined as the time period from the date 
of randomization to the date of PD or death whichever occurred first. Patients who are alive 
without PD will be censored, and their PFS time is defined as the time period from the date of 
randomization to the date of last follow-up. 
 
14.6.1 
Study Design 
 
This is a randomized phase II trial.  A group sequential design with one interim analysis for 
futility will be implemented for this randomized study.  Sample size calculation is based on 
comparison of PFS between the two treatment arms with the following assumptions: 
 
• 1:1 randomization Median PFS of 4 months for nivolumab only arm 
• Median PFS of 7 months for nivolumab + ipilimumab arm 

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An interim analysis will be performed when 50% of events (i.e.  44 events) occurs. If the 
p value ≥ 0.586 the study will stopped early for futility corresponding to a boundary for 
the HR being 1.068.   
 
A. Definition of analysis populations: 
 
 
The safety analysis set consists of all subjects who take at least one dose of study 
 
drug. The safety analysis set will be used for safety analyses. The full analysis set 
 
consists of all randomized subjects. The full analysis set will be used for efficacy 
 
analyses. 
 
14.6.4 
Analysis of secondary endpoints 
 
Secondary endpoints for part B include overall response rates, overall survival, and grade 3/4/5 
adverse events.  These endpoints will be analyzed as described in part A. 
 
14.7 
Reporting and Exclusions 
 
14.7.1 
Evaluation of Toxicity 
 
All patients will be evaluable for toxicity from the time of their first treatment with 
nivolumab. 
 
14.7.2 
Evaluation of Response 
 
All eligible patients included in the study must be assessed for response to treatment, 
even if there are major protocol treatment deviations.  Each patient will be assigned one 
of the following categories:  1) complete response, 2) partial response, 3) stable disease, 
4) progressive disease, 5) early death from malignant disease, 6) early death from 
toxicity, 7) early death because of other cause, or 9) unknown (not assessable, insufficient 
data).  [Note:  By arbitrary convention, category 9 usually designates the “unknown” 
status of any type of data in a clinical database.] 
 
All of the patients who met the eligibility criteria (with the possible exception of those 
who received no study medication) should be included in the main analysis of the 
response rate.  Patients in response categories 4-9 should be considered to have a 
treatment failure (disease progression).  Thus, an incorrect treatment schedule or drug 
administration does not result in exclusion from the analysis of the response rate.  Precise 
definitions for categories 4-9 will be protocol specific.  
 
All conclusions should be based on all eligible patients.  Subanalyses may then be 
performed on the basis of a subset of patients, excluding those for whom major protocol 
deviations have been identified (e.g., early death due to other reasons, early 
discontinuation of treatment, major protocol violations, etc.).  However, these 
subanalyses may not serve as the basis for drawing conclusions concerning treatment 

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efficacy, and the reasons for excluding patients from the analysis should be clearly 
reported.  The overall response (CR and PR) rate along with the 95% confidence interval 
will be estimated.  
 
15. STUDY STATUS UPDATES AND STUDY CLOSURE 
 
15.1 
Definitions of Study Status Changes 
 
15.1.1 
Temporarily Closed to Accrual 
 
The study status is Temporarily Closed to Accrual when no patient slots are currently 
available, but there is the possibility that the trial will re-open for accrual (patient slots 
become available).  Sites are not permitted to accrue additional patients until CTEP is 
notified of Re-Activation. 
 
Study status will need to be changed to Temporarily Closed to Accrual when any of the 
following criteria are met: 
 
• Sites are notified by CTEP (via Request for Rapid Amendment [RRA]) of 
changes in the risk/benefit ratio that necessitate changes to the patient Informed 
Consent document.  Requested changes will be specified in the RRA and must be 
reviewed by the study’s IRB. 
• CTEP and the lead investigator agree that unacceptable toxicities necessitate a 
discussion to change the dosing/regimen. 
• A protocol-defined benchmark has been achieved (such as an interim analysis 
before proceeding to the next stage).  
 
15.1.2 
Closed to Accrual 
 
The study status is (permanently) Closed to Accrual when no more patient enrollment 
slots are available, and at least one patient is still actively receiving the study treatment.  
Sites are no longer permitted to enroll additional patients. 
 
Patient slots are no longer available when the following criteria are met: 
 
• The pre-specified number of evaluable patients has been successfully enrolled, 
treated, and evaluated. 
• The study treatment has failed to meet the pre-specified efficacy goal at the stage 
1 interim analysis. 
• CTEP and the investigators agree that unacceptable toxicities preclude further 
enrollment. 
 
15.1.3 
Closed to Accrual and Treatment 
 
The study status is Closed to Accrual and Treatment when no more patient enrollment 
slots are available and no patients are currently receiving the study treatment.  Patients 

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may still be enrolled on the protocol only for the purposes of follow-up. 
 
Patient accrual and treatment will be permanently halted when any of the following 
criteria are met: 
 
• Enrollment was previously closed (study status of “Closed to Accrual”), and no 
patients are receiving the study treatment. 
• CTEP and the investigators agree that unacceptable toxicities preclude further 
enrollment.  In this case, CTEP and the investigators must collaborate to alter the 
regimen or to halt the study treatment altogether as soon as it can be safely done 
for patients currently receiving treatment.  
 
CTEP and Theradex must be notified when patients are no longer receiving treatment 
[i.e., when the last patient(s) to be receiving treatment is/are no longer receiving the study 
regimen for any reason]. 
 
15.1.4 
Closed to Follow-Up 
 
The study is considered Closed to Follow-Up when all protocol-defined follow-up 
procedures have been completed for all patients who have not been removed from the 
study for other reasons.  That is, there are no outstanding follow-up procedures to be 
performed as mandated by the protocol. 
 
CTEP does not need to be notified of a status change to “Closed to Follow Up.” 
 
15.1.5 
Complete 
 
Study is considered Complete if it has been at least thirty (30) days since the last patient 
follow-up evaluation.   
 
A citation to a final study report (manuscript, meeting abstract, etc.) is required with the 
submission of the Protocol Status Update Form to CTEP PIO.   
 
15.2 
Responsibility for Filing Protocol Status Update Forms 
 
CTEP must be notified of all study status changes in Section 14.1 (except for Closed to Follow-
Up) by the Corresponding Organization via Protocol Status Update Form, available from the 
CTEP website at http://ctep.cancer.gov/protocolDevelopment/default.htm#amendments.    
 
Theradex must be notified as soon as all patients are off treatment (i.e., when study status 
changes to Closed to Accrual and Treatment).  Theradex will produce a report within 90 days of 
this notification. 
 
 

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APPENDIX A 
PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no evidence 
of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of disease. 
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). 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active 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. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
50 
Requires considerable assistance and 
frequent medical care. 
3 
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
4 
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 
 
 
 

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APPENDIX B 
CTEP MULTICENTER GUIDELINES FOR NON-ETCTN TRIALS 
 
N/A 
 
 
 
 

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APPENDIX C 
BIOASSAY TEMPLATES 
 
No integrated or integral biomarkers will be used for this study. 

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APPENDIX D 
MANAGEMENT ALGORITHMS FOR ENDOCRINOPATHY, 
GASTROINTESTINAL, HEPATIC, NEUROLOGICAL, PULMONARY, RENAL, AND 
SKIN ADVERSE EVENTS 
 
 
Please note that the abbreviation “I-O” in the following pages stands for “Immunotherapy-
Oncology”

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APPENDIX E  ANALYSES OF SAMPLES FOR EXPLORATORY STUDIES 
 
Fresh frozen tumor samples, if available, will be analyzed by RT-PCR to assess for mRNA 
expression of genes that may contribute to anti-tumor immune responses, e.g. IFN-γ and 
other cytokines. Tumor tissues will also be analyzed by immunohistochemistry (IHC) for 
markers that may include, but are not limited to, CD4, CD8, FoxP3, and Granzyme.  
 
Fresh tumor samples, if available and assessed by physician adequate for selection, from 
routine surgical procedures, will be used for flow cytometry analysis of tumor infiltrating 
lymphocytes (TILs) when possible.  
  
Lymphocytes from collected blood or from tumor infiltrating cells will be analyzed using 17 
core panels by flow cytometry to identify sub-populations of T-cells (for example, CD4+ 
helper T-cells, CD8+ cytotoxic T-cells, CD4+, FoxP3+ regulatory T-cells). These studies 
require 20 cc of blood per blood draw. 
   
Lymphocytes from collected blood will be analyzed by ELISPOT analyses, if possible, to 
identify T-cells that are functionally reactive against a HPV-specific antigens E6 and E7. For 
example, in an ELISPOT assay that uses HPV as the target antigen, a patient’s T-cell will be 
analyzed to determine if a cytokine, such as interferon- gamma, is produced by the T-cell, in 
response to recognition of the HPV antigen in the context of an antigen-presenting cell. These 
studies require a minimum of 80 cc of blood per blood draw. 
  
Funding for these tests will be provided by MD Anderson Cancer Center institutional sources 
and philanthropic donations.  
 
 

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APPENDIX F PARTICIPANT WALLET CARD 
 
 
 
 
 
 
 
 
 
 
INFORMATION FOR PATIENTS AND PRESCRIBERS  
You are enrolled on a clinical trial using experimental agents 
nivolumab with or without ipilimumab.  This clinical trial is sponsored 
by the NCI.  It is very important to: 
➢ Take this card with you to the emergency room or any healthcare 
provider other than your study doctor.  Tell all healthcare providers 
that you are being treated with nivolumab with or without 
ipilimumab and SHOW THEM THIS CARD.  Tell your doctors if you 
stop taking regular medicine or if you start taking a new medicine.  
Do not take over-the-counter medications, dietary supplements, or 
prescription medications without the approval of your study doctor. 
 
➢ Tell all of your prescribers (doctor, physicians’ assistant, nurse 
practitioner, pharmacist) that you are taking part in a clinical trial. 
 
➢ See your study doctor for help managing symptoms.  Doctors who 
are not familiar with nivolumab or ipilimumab may not be aware of 
the appropriate management or side effects. 
➢ If you have any of these signs or symptoms, call your study doctor 
or nurse right away: new or worsening shortness of breath, cough, 
or wheezing; diarrhea or any increase in the amount or number of 
bowel movements above normal, blood in stool or dark, tarry, 
sticky stools; stomach pain/cramps; yellowing of the white part of 
the eye(s); blurry vision, double vision, or other vision problems; 
eye pain or redness; severe rash, itching, or peeling; yellowing of 
the skin; fever; decreased strength or energy; dizziness; 
headaches; muscle weakness; numbness or tingling in the hands 
or feet; changes in behavior; dark urine; palpitations, or fast or 
irregular heartbeats. 
 
➢ Before prescribing new medicines, your regular prescribers should 
contact your study doctor.   
 
➢ Your study doctor’s name is _____________________________ 
and can be contacted at _________________________________.