MATCH Treatment Subprotocol Z1D: Nivolumab in Patients With Tumors With Mismatch Repair Deficiency
Phase: PHASE2
Conditions: Advanced Lymphoma, Advanced Malignant Solid Neoplasm, Hematopoietic and Lymphoid Cell Neoplasm, Refractory Lymphoma, Refractory Malignant Solid Neoplasm, Refractory Plasma Cell Myeloma
Sponsor: National Cancer Institute (NCI)

i 
28 State Street, Suite 1100, Boston, MA 02109 
p: 857.504.2900 
f: 617.589.0914 
ecog-acrin.org 
 
 
 
 
October 12, 2018 
 
Martha Kruhm, MS RAC 
Head, Protocol and Information Office 
Quality Assurance Section 
CTEP, DCT, NCI 
6130 Executive Blvd, EPN Room 7000 
Bethesda, MD 20892 
Dear Ms. Kruhm: 
Enclosed is Addendum #16 to EAY131-Z1D, Molecular Analysis for Therapy Choice (MATCH): 
MATCH Treatment Subprotocol Z1D: Nivolumab in Patients with Tumors with Mismatch Repair 
Deficiency. 
The following are ECOG-ACRIN’s responses to CTEP’s “Review of Amendment #23 of Protocol 
#EAY131-Z1D” dated October 5, 2018. Please note the Principal Investigator’s comment 
appears in bold below: 
I.  Comments Requiring a Response– Administrative & Editorial Issues: 
Section 
Comments 
1. Appendix V 
As a reminder, Food and Drug Administration (FDA) regulations and 
National Cancer Institute (NCI) policy requires all persons participating in 
any NCI-sponsored clinical trial to register. 
Our records indicate: 
• 
Evisa Gjini, PhD (A-603314)- Registration will expire on October 10, 
2018 and also needs to be advanced to Associate Plus (AP) person 
type. 
• 
Scott Rodig, MD, PhD (IVR-  needs to be claimed on the ECOG-
ACRIN roster. 
• 
Mariano Severgnini, PhD (A-558565) has a suspended registration 
status in RCR. This needs to be renewed and advanced to Associate 
Plus (AP) person type. 
To register, please log into the NCI Registration and Credential 
Repository (RCR) at https://ctepcore.nci.nih.gov/rcr/ using your CTEP-
IAM username and password and complete the required information.  
If you need to update your CTEP-IAM account information, please visit: 
https://ctepcore.nci.nih.gov/iam . 
For guidance on registering via RCR, please visit: 
https://ctep.cancer.gov/investigatorResources/default.htm . 
If you have questions or encounter difficulties, please contact the CTEP 
Registration and Credential Repository (RCR) Team at 
RCRHelpDesk@nih.gov at any time. 
Thank you for your cooperation. If you have any questions, please feel free 
to contact the Pharmaceutical Management Branch by phone at (240) 276-

ii 
Section 
Comments 
6575 Monday through Friday from 8:30 am to 4:30 pm Eastern Time or by 
email at RCRHelpDesk@nih.gov . 
PI Response: Updates to registrations listed above are in progress. 
 
II. Recommendations: 
Section 
Comments 
2. 
 
Please include the recommended changes below at the earliest 
opportunity.  The changes are based on information from the latest 
nivolumab IB (version 17). 
3. 
5.1.4 
Add the following after the first sentence:   The unopened vials can be 
stored at room temperature (up to 25°C, 77°F) and room light for up to 48 
hours. 
PI Response: The sentence has been added as requested. 
4. 
5.1.5 
Replace the current section with the following: 
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 dihydrate, sodium chloride, mannitol, 
diethylenetriaminepentacetic acid (pentetic acid), 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:  change “butyl” with “fluoropolymer film-laminated”. 
PI Response: The requested changes have been made. 
5. 
5.1.6 
Replace section with the following: 
Preparation:  Nivolumab injection can be infused undiluted (10 mg/mL) or 
diluted with 0.9% Sodium Chloride Injection, USP or 5% Dextrose.  When 
the dose is based on patient weight (i.e., mg/kg), nivolumab injection can 
be infused undiluted or diluted 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. During drug product preparation and handling, 
vigorous mixing or shaking is to be avoided. 
PI Response: The section has been replaced as requested. 
6. 
5.1.7 
Add “Intravenous infusion over 30 minutes.” 
PI Response: The first sentence has been modified appropriately. 
Please see responses to the CIRB Early Phase Emphasis Review below. 
 
December 11, 2018 
 
Re: CIRB Approval Pending Modification of Amendment Review 
Study ID, Study Title: EAY131, Molecular Analysis for Therapy Choice (MATCH) 
Protocol Version Date: October 12, 2018 
 

iii 
Study Chair: Keith Flaherty, M.D., B.S. 
 
Dear NCI Adult CIRB - Early Phase Emphasis, 
 
Thank you for reviewing EAY131-Z1D (Protocol Version Date 10/12/18) protocol at the meeting 
of the NCI Adult CIRB - Early Phase Emphasis held on December 4, 2018. The CIRB 
determined that the regulatory and CIRB SOP requirements for approval are met, but the CIRB 
requests minor, directed modifications as described below. 
 
Stipulations: 
 
 
Section 
Comments 
1.  Appendix V 
Appendix VI under the Methodology section revise the sentence, “All specimens will 
be de-identified/coded prior to distribution to the investigator” to remove reference 
to de-identification as it is inaccurate. Add information regarding the code and code-
key process. 
 
PI Response: The term “de-identified/coded” refers to the process of 
stripping PHI from the samples and inventories (de-identified) and replacing 
with codes (coded).  The key to link the codes back to the patient identifiers 
are held by the BioBank, the ECOG-ACRIN Operations Office and the MATCH 
statisticians.  So there is no confusion regarding the term “de-identified”, the 
text has been replaced with the following: 
 
“No patient identifiers will be provided to investigators with the samples. The 
trial-specific patient ID will be replaced with a unique randomized number and 
specimens will be labeled with a barcode not related to any PHI. The following 
entities have access to the key:  the ECOG-ACRIN biobank, the ECOG-ACRIN 
Operations Office, and the MATCH statisticians.” 
Additional Changes by Principal Investigator: 
 
Section 
Comments 
1.  ICD: Page 1 
Updated version date. 
2.  Protocol 
Cover Page 
Updated version date. 
3.  Appendix V 
Removed Dr. Gjini as a Principal Investigator of Correlative Proposal CS-
MATCH-0005. 
The following revisions to EAY131-Z1D protocol have been made in this addendum: 
 
Section 
Change 
1. 
Cover Page 
Updated version date and title. 
2. 
5.1.4 
Updated storage information. 
3. 
5.1.5 
Updated dosing and supply information. 
4. 
5.1.6 
Updated preparation information. 
5. 
5.1.7 
Updated drug administration information. 

iv 
6. 
Appendix V 
Added Correlative Proposal CS-MATCH-0005. 
The following revisions to EAY131-Z1D Informed Consent Document have been made in this 
addendum: 
 
Section 
Change 
1. 
Page 1 
Updated version date and title. 
If you have any questions regarding this addendum, please contact kpollard@ecog-acrin.org or 
857-504-2900. 
We request review and approval of this addendum to EAY131-Z1D so ECOG-ACRIN may 
activate it promptly.   
Thank you.  
Sincerely, 
Pamela Cogliano 
Senior Director of Protocol Development 
Enclosure 
CC: Nilofer Azad, MD 
Michael James Overman, MD  
Jonathan Schoenfeld, MD 
Alice Chen, MD 
Keith Thomas Flaherty, MD 
Peter O’Dwyer, MD 
Mickey Williams, PhD 
Stanley Hamilton, MD 
Lisa McShane, PhD  
Larry Rubinstein, PhD 
Robert Gray, PhD 
Shuli Li, PhD 
Lalitha Shankar, MD, PhD 
Susanna Lee, MD, PhD 
Constantine Gastonis, PhD 
Paolo Caimi, MD 
Shaji Kumar, MD 
Carlos Arteaga, MD 
Edith Mitchell, MD 
John J. Wright, MD, PhD 
Lyndsay Harris, MD 
James Tricoli, PhD 
 
 
Bruce Giantonio, MD 
Donna Marinucci 
Gayle Ipock 
Kerry Higgins 
Jean MacDonald 
Carol Chami, R.N. 
Juanita Andrews 
Julianne Human 
Elocine Elie 
Kelly Redmond 
Jennifer VanCamp 
Dan Reeve 
Becky Fillingham 
Jeffrey Zhang 
Kevin Pollard 
Amy Li 
Michael T. Balco 
Lauren Lambert 
Margaret Cavenagh 
Cayden Maican 
Russell McDaniel 
Alexandra Sachs 
Ben Kim 
 
 

EAY131-Z1D 
1 
Molecular Analysis for Therapy Choice (MATCH) 
MATCH Treatment Subprotocol Z1D: Nivolumab in 
Patients with Tumors with Mismatch Repair Deficiency 
NIVOLUMAB TREATMENT SUBPROTOCOL CHAIR: 
Nilofer Azad, MD 
NIVOLUMAB TREATMENT SUBPROTOCOL CO-CHAIR: 
Michael James Overman, MD 
NIVOLUMAB TRANSLATIONAL CHAIR: 
Jonathan Schoenfeld, MD 
Version Date:  December 11, 2018 
NOTE: This subprotocol (EAY131-Z1D) should 
be used in conjunction with the 
MATCH Master Protocol (EAY131). 
NOTE: As of 11/17, all protocol changes will 
be noted by addendum number. Please 
reference the activation memo for the 
addendum activation date. 
SUBPROTOCOL ACTIVATION DATE 
May 31, 2016 (Incorporated in Addendum #3) 
Addendum #4 – 7/16 
Addendum #5 – 12/16 
Addendum #6 – 1/17 
Addendum #7 – 3/17 
Addendum #13 
Addendum #15 
Addendum #16 
Agent 
IND# 
NSC# 
Supply 
Nivolumab 
NCI Supplied 
Rev. Add13 
Rev. Add16 

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Version Date: December 11, 2018 
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Table of Contents 
Molecular Analysis for Therapy Choice (MATCH) ...................................................... i 
MATCH Treatment Subprotocol Z1D: Nivolumab in Patients with Tumors with 
Mismatch Repair Deficiency ............................................................................... 1 
Table of Contents ...................................................................................................... 2 
Schema ...................................................................................................................... 4 
1. Introduction ........................................................................................................ 5 
1.1 Nivolumab (BMS-936558) ......................................................................... 5 
1.2 Supporting Preliminary Data ..................................................................... 7 
1.3 Justification for the collection of research blood ..................................... 8 
2. Selection of Patients ........................................................................................... 9 
2.1 Registration to Treatment ......................................................................... 9 
3. Nivolumab Treatment Plan ............................................................................... 12 
3.1 Administration Schedule ......................................................................... 12 
3.2 Adverse Event Reporting Requirements ................................................. 12 
3.3 Comprehensive Adverse Events and Potential Risks List (CAEPR) for 
BMS-936558 (Nivolumab, MDX-1106, NSC 748726) .............................. 15 
3.4 Dose Modifications ................................................................................. 20 
3.5 Supportive Care ....................................................................................... 25 
3.6 Duration of Agent-specific treatment ..................................................... 25 
3.7 Treatment Past Progression .................................................................... 26 
3.8 Duration of Follow-Up ............................................................................. 26 
4. Study Parameters .............................................................................................. 27 
4.1 Therapeutic Parameters for Nivolumab Treatment ............................... 27 
5. Drug Formulation and Procurement ................................................................. 29 
5.1 Nivolumab (NSC #748726) ...................................................................... 30 
6. Specimen Submissions ...................................................................................... 33 
6.1 Specimen Submissions ............................................................................ 33 
6.2 ECOG-ACRIN Sample Tracking System .................................................... 34 
6.3 Use of Specimens in Research ................................................................ 35 
6.4 Sample Inventory Submission Guidelines ............................................... 35 
7. References ........................................................................................................ 36 
Appendix I  Actionable Mutations for Sub-Protocol EAY131-Z1D ........................... 37 
Appendix II  Management Algorithms For Endocrinopathy, Gastrointestinal, 
Hepatic, Neurological, Pulmonary, Renal, And Skin Adverse Events ................ 38 
Appendix III  EAY131-Z1D Collection and Shipping Kit Order Form ........................ 46 
Appendix IV  Patient Drug Information Handout and Wallet Card ......................... 47 
Appendix V  CS-MATCH-0005 .................................................................................. 49 
 

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Version Date: December 11, 2018 
3 
 
TREATMENT SUBPROTOCOL CHAIR 
Nilofer Azad, MD 
Sidney Kimmel Comprehensive Cancer Center 
at John Hopkins 
1650 Orleans Street 
Suite 4M10  
Baltimore, Maryland, 21287 
Tel: 410-502-2995 
Fax: 410-614-9006 
Email: Nazad2@jhmi.edu 
 
 
 
TREATMENT SUBPROTOCOL CO-CHAIR 
Michael Overman MD 
MD Anderson Cancer Center 
Houston, Texas, 77030 
Tel: 713-745-4317 
Fax: 713-745-1163 
Email: moverman@mdanderson.org 
 
 
 
TREATMENT TRANSLATIONAL CHAIR 
Jonathan Schoenfeld, MD MPH 
Brigham and Women’s / Dana-Farber Cancer Center  
Boston, MA 02115 
Tel: 617-632-3591 
Fax: 617-632-4247 
Email: jdschoenfeld@partners.org 
 
 
 
 

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EAY131-Z1D 
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Version Date: December 11, 2018 
4 
Schema 
Long-Term Follow-Up
Nivolumab 240 mg IV days 1 
and 15 of each cycle
After 4 Cycles of therapy, 
480 mg IV day 1 of each 
cycle.
Until Progression
Cycle = 28 days
Accrual Goal: 70
 
Rev. 3/17 

ECOG-ACRIN 
EAY131-Z1D 
Cancer Research Group 
Version Date: December 11, 2018 
5 
1. Introduction 
1.1 
Nivolumab (BMS-936558) 
Nivolumab (also known as BMS-936558 or anti-PD-1) is a fully human, IgG4 
(kappa) isotype monoclonal antibody (mAb) PD-1 receptor blocker. 
1.1.1 
Nonclinical Development of Nivolumab 
In intravenous (IV) repeat-dose toxicology studies in cynomolgus 
monkeys, nivolumab alone was well tolerated (Investigator Brochure, 
2014).  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. 
1.1.2 
Clinical Development of Nivolumab 
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 indications (Investigator Brochure, 2014).  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.   
1.1.3 
Pharmacokinetics 
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 level (Investigator Brochure, 2014).  
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.   

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1.1.4 
Efficacy 
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 doses (Sznol et al., 2013).  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, respectively (Brahmer et 
al., 2013).  In the RCC cohort, median duration of response was 12.9 
months for both doses with 5 of the 10 responses lasting ≥1 year 
(Drake et al., 2013).   
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) 
(Wolchok et al., 2013).  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-doublet (Rizvi et al., 
2013).  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. 
In addition, an overall survival benefit has been demonstrated with 
nivolumab treatment in squamous cell NSCLC, non-squamous cell 
NSCLC, renal cell cancer, melanoma, and head and neck cancer. 
1.1.5 
Toxicology 
A maximum tolerated dose (MTD) of nivolumab was not defined 
(Topalian et al., 2012).  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 group (Wolchok et al., 2013), 
grade 3 or 4 treatment-related events were noted in 53% of patients.  
Skin rash represents the majority of these events.   
1.1.6 
Pharmacodynamics/Biomarkers 
Tumor-cell expression (melanoma) of PD-L1 was characterized in 
combination with ipilimumab with the use of IHC staining and 

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pharmacodynamics changes in the peripheral-blood absolute 
lymphocyte count (Wolchok et al., 2013).  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. 
1.2 
Supporting Preliminary Data  
Rationale for anti-PD1 therapy in mismatch repair deficient tumors 
Germline DNA repair defects, e.g. mismatch repair deficiencies, are associated 
with higher risk of cancers of certain organs.  They have also been associated 
with higher number of mutations within tumors compared to those without 
mismatch repair deficiency (Timmerman et al. 2010, Eshleman et al. 1995 TCGA 
2012).  Although unselected colorectal cancer patients did not respond to PD-1 
inhibitors, an exceptional responder was found to have a mismatch repair 
deficiency. A recent phase 2 study (Le et al., 2015) noted that pembrolizumab, 
and anti-PD1 agent, had an immune related and RECIST response rate of 62% 
(N=13) in colorectal tumors that had mismatch repair deficiency. In patients with 
tumors other than colorectal cancer, who had mismatch repair, the response rate 
was similar (6 out of 10 patients, including one CR).  In this study, high somatic 
mutation loads assessed with whole exome sequencing correlated with 
prolonged progression free survival.  The investigators determined mismatch 
repair status with the Promega MSI Analysis system, evaluating selected 
microsatellite sequences. In this study, the numbers of tumor infiltrating CD8+ T-
cells and PD-L1 expression were not significantly associated with progression 
free survival or overall survival, though the study was not adequately powered to 
assess this. 
Lynch syndrome is an autosomal dominant inherited condition caused by 
germline mutation in one of the mismatch repair genes (MLH1, MSH2, MSH6, 
PMS2, EPCAM). The germline mutations increase the risk of colorectal, 
endometrial, breast, prostate and other cancers, which usually occur at an earlier 
age than sporadic cancers. The mismatch repair phenotype can also occur in 
sporadic cancers, usually as a result of epigenetic silencing of MMR genes.  The 
diagnosis of mismatch repair deficiency can be challenging, due in part to the 
fact that MMR proteins bind to one another, and there can be false positives with 
loss of MSH6.  It is common for both PCR and IHC to be used to diagnose Lynch 
syndrome in clinical practice. This subprotocol will not restrict eligibility to those 
with Lynch syndrome, but will restrict eligibility to those with MMR deficiency. 

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The study mentioned above enrolled a small number of patients with presumed 
Lynch syndrome.  Yet in this small cohort, 60% responded. This proposal will 
explore the activity of a different PD-1 inhibitor, nivolumab, in patients with 
tumors that have mismatch repair deficiency in order to ascertain the response 
rate and duration of response in these patients.  IHC for MLH-1 and MSH-2 
captures 90% of Lynch syndrome patients, as well as those with sporadic MSI. 
Because several studies are underway testing anti-PD1 agents in colorectal 
cancer (CRC) with MSI, we will not enroll such patients in this study.  About 15% 
of CRC, gastric and endometrial cancers will have MSI (Yamamoto et al. 2015).  
Hereditary MMR defects account for about 10% of epithelial ovarian cancer 
(Kobayashi et al. 2014).  Other cancers have not been as well characterized as 
to their MMR status, but reports have documented adrenocortical tumors, 
peritoneal mesothelioma, pancreatic tumors, MFH, prostate cancer, anaplastic 
thyroid cancer, melanoma, various sarcomas, breast cancer and others as 
having lost expression of MLH1 or MSH2 with or without microsatellite instability 
(Karamurzin et al. 2012). 
To date, there are no known effective therapies targeting patients with mismatch 
repair deficiencies.  As mismatch repair deficiencies can be found across many 
solid tumors, if treatment with nivolumab is successful, this treatment would be a 
major advance. In addition, the responses observed in the early study of PD1 
inhibition in MMR-deficient tumors demonstrated compelling durability of 
responses, with 5/6 responders maintaining continued response at the time of 
reporting [median not reached (Le et al. 2015)]. This speaks to the realistic 
possibility of checkpoint inhibitor therapy converting MMR-deficient cancers into 
truly chronic diseases. 
Based on the preliminary clinical data described above, patients with mismatch 
repair deficiency as determined by the microsatellite instability assay 
incorporated in the MATCH diagnostic panel will be assigned to this treatment 
arm in preference to any other potential treatment assignment regardless of co-
occurring actionable molecular alterations of interest. 
1.3 
Justification for the collection of research blood 
There is great utility to identifying mechanisms of response and resistance to PD-
1 blockade as well as evaluating potential biomarkers that may be associated 
with clinical benefit. Based on prior preliminary studies conducted in patients 
across several disease types, systemic immune responses engendered by 
immunologic checkpoint blockade therapy may be predictive of response (Herbst 
et al. Nature 2014; Posto et al. N Engl J Med 2012; Schoenfeld et al. Cancer Res 
2010).  However, it is unknown to what degree circulating factors such as specific 
T-cell populations, myeloid derived suppressor cells, and immunologic cytokines 
may change in response to PD-1 blockade in patients with mismatch repair 
deficient tumors, and to what degree these factors are associated with response 
and clinical benefit. 
Performing serial blood collections will allow for the analyses of circulating 
immunologic factors and changes in these factors engendered over the course of 
treatment.  Specific analyses will be reviewed and approved by the MATCH 
Correlative Science committee prior to inclusion for this study.  Results of such 
studies may also serve as the basis for combinatorial immune therapies to further 
improve response rates. 

ECOG-ACRIN 
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Cancer Research Group 
Version Date: December 11, 2018 
9 
2. 
Selection of Patients 
Each of the criteria in the checklist that follows must be met, along with the eligibility in 
the MATCH Master Protocol, in order for a patient to be considered eligible for this 
study. Use the checklist to confirm a patient’s eligibility. For each patient, this checklist 
must be photocopied, completed and maintained in the patient’s chart.  
In calculating days of tests and measurements, the day a test or measurement is 
done is considered Day 0. Therefore, if a test is done on a Monday, the Monday 
four weeks later would be considered Day 28.  
ECOG-ACRIN Patient No. ____________________________ 
Patient’s Initials (L, F, M) _______________________ 
Physician Signature and Date ___________________ 
NOTE: 
Policy does not allow for the issuance of waivers to any protocol specified 
criteria (http://ctep.cancer.gov/protocolDevelopment/policies_deviations.htm). 
Therefore, all eligibility criteria listed in Section 2 must be met, without 
exception. The registration of individuals who do not meet all criteria listed in 
Section 2 can result in the participant being censored from the analysis of the 
study, and the citation of a major protocol violation during an audit.  All 
questions regarding clarification of eligibility criteria must be directed to the 
Group's Executive Officer (EA.Execofficer@jimmy.harvard.edu) or the 
Group's Regulatory Officer (EA.RegOfficer@jimmy.harvard.edu). 
NOTE: 
Institutions may use the eligibility checklist as source documentation if it has 
been reviewed, signed, and dated prior to registration/randomization by the 
treating physician. 
NOTE: 
All patients must have signed the relevant treatment consent form 
2.1 
Registration to Treatment  
2.1.1 
Patients must fulfill all eligibility criteria outlined in Section 3.1 of 
MATCH Master Protocol (excluding Section 3.1. 4 and 3.1.6) at the 
time of registration to treatment step (Step 1, 3, 5, 7) 
2.1.2 
Patients must have mismatch repair deficiency as determined via the 
MATCH Master Protocol. See Appendix I for a list of the mismatch 
repair genes whose absence results in mismatch repair deficiency 
and corresponding Levels of Evidence. 
2.1.3 
Patients must not have known hypersensitivity to nivolumab or 
compounds of similar chemical or biologic composition. 
2.1.4 
No prior therapy with anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, 
anti-OX-40, anti-CD40 or anti-CTLA-4 antibodies (or any other 
antibody targeting T cell co-regulatory pathways). 
2.1.5 
Patients with cancers for which nivolumab is approved or becomes 
approved are excluded (e.g: colorectal cancer, locally advanced or 
metastatic urothelial carcinoma, unresectable or metastatic 
melanoma, metastatic non-small cell lung cancer, advanced renal cell 
carcinoma, classical Hodgkin lymphoma, and recurrent or metastatic 
squamous cancer of the head and neck). 
_____
_____
_____
_____
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Rev. 3/17 
Rev. Add13 

ECOG-ACRIN 
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2.1.6 
Must not have received any of the following therapies within four 
weeks prior to the first dose of the study drug: IL-2, interferon, or other 
non-study immunotherapy regimens or immunosuppressive 
agents.The master protocol eligibility criterion regarding wash-out 
period from prior therapy is also applicable (See Section 3.1.13 of the 
Master Protocol). 
2.1.7 
Must not have a history of toxic epidermal necrolysis (Stevens-
Johnson syndrome). 
2.1.8 
Must not have received growth factors, including but not limited to 
granulocyte-colony stimulating factor (G-CSF), granulocyte 
macrophage-colony stimulating factor (GM-CSF), erythropoietin, etc. 
within 2 weeks of study drug administration. Use of such agents while 
on study is also prohibited. Prior use of growth factors should be 
documented in the patient’s medical history. 
2.1.9 
Must not have a history of any autoimmune disease: inflammatory 
bowel disease, (including ulcerative colitis and Crohn’s Disease), 
rheumatoid arthritis, systemic progressive sclerosis (scleroderma), 
systemic lupus erythematosus (SLE) autoimmune vasculitis (e.g., 
Wegener’s Granulomatosis), CNS or motor neuropathy considered to 
be of autoimmune origin (e.g., Guillian-Barre Syndrome, Myasthenia 
Gravis, Multiple Sclerosis). Patients are permitted to enroll if they 
have vitiligo, type I diabetes mellitus, residual hypothyroidism due to 
autoimmune condition only requiring hormone replacement, psoriasis 
not requiring systemic treatment, or conditions not expected to recur 
in the absence of an external trigger (precipitating event). Entry of 
patients with autoimmune diagnoses not listed here must be approved 
by the protocol chair. 
2.1.10 
Must not be on supplemental home oxygen.  
2.1.11 
Must not have evidence of interstitial lung disease 
2.1.12 
Patients with a requirement for steroid treatment or other 
immunosuppressive treatment:  Patients will be excluded if they have 
a condition requiring systemic treatment with either corticosteroids 
(>10 mg daily prednisone equivalents) 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. 
2.1.13 
No history of severe hypersensitivity reaction to any monoclonal 
antibody. 
2.1.14 
Women of childbearing potential (WOCBP) receiving nivolumab must 
agree to use adequate contraception (hormonal or double barrier 
method of birth control; abstinence) from one week prior to study 
treatment starting, during treatment, and for a period of 5 months after 
the last dose of nivolumab. Men receiving nivolumab and who are 
sexually active with WOCBP must agree to use adequate 
contraception (hormonal or double barrier method of birth control; 
abstinence) from one week prior to study treatment starting, during 
treatment, and for a period of 7 months after the last dose of 
nivolumab. 
_____
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Rev. 3/17 

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2.1.15 
Patients with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) 
infection may be eligible provided they have the following: 
• 
There must be no evidence of clinically significant hepatic injury 
from hepatitis virus infection. 
• 
For HBV, patients must be on suppressive therapy and have 
undetectable HBV viral load. 
• 
For HCV, patients must either be on suppressive therapy for HCV 
or have already completed therapy thought to have eradicated 
HCV. 
 
 
 
 
 
 
Physician Signature 
 
Date 
OPTIONAL: 
This signature line is provided for use by institutions wishing to 
use the eligibility checklist as source documentation. 

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3. 
Nivolumab Treatment Plan 
3.1 
Administration Schedule 
Nivolumab will be given Days 1 and 15 (every two weeks ± 2 days) at a dose of 
240 mg. Patients may be dosed no less than 12 days from the previous dose of 
drug. After 4 cycles of therapy, patients will be transitioned to 480 mg flat dose 
every 4 weeks (± 2 days) for the remainder of their time on study. 
Nivolumab is to be administered as an approximately 60-minute IV infusion (± 10 
minutes) or can be safely infused over 30 minutes, using a volumetric pump with 
a 0.2-1.2 micron in-line filter at the protocol-specified dose. The drug can be 
diluted with 0.9% normal saline for delivery but the total drug concentration of the 
solution cannot be below 0.35 mg/mL or not to exceed 120 mL. It is not to be 
administered as an IV push or bolus injection. At the end of the infusion, flush the 
line with a sufficient quantity of normal saline. 
Repeat cycles every 28 days until progression. 
3.2 
Adverse Event Reporting Requirements 
The Adverse Event Reporting Requirements for all EAY131 subprotocols are 
outlined in the MATCH MASTER protocol.  Please refer to those guidelines when 
determining if an event qualifies as a Serious Adverse Event (SAE) and requires 
expedited reporting via CTEP’s Adverse Event Reporting System (CTEP-AERS). 
In addition, the following section outlines agent specific requirements and must 
be followed to ensure all reporting requirements are met. 
3.2.1 
Additional instructions, requirements and exceptions for protocol 
EAY131 – Subprotocol Z1D 
Additional Instructions 
For instructions on how to specifically report events that result in 
persistent or significant disability/incapacity, congenital anomaly, or 
birth defect events via CTEP-AERS, please contact the AEMD Help 
Desk at aemd@tech-res.com  or 301-897-7497.  This will need to be 
discussed on a case-by-case basis. 
 
EAY131 – Subprotocol Z1D specific expedited reporting 
requirements: 
• 
Pregnancies:  Pregnancies and suspected pregnancies 
(including a positive or inconclusive pregnancy test, regardless of 
age or disease state) occurring while the subject is on nivolumab, 
or within 28 days of the subject’s last dose of nivolumab, are 
considered immediately reportable events. The pregnancy, 
suspected pregnancy, or positive/ inconclusive pregnancy test 
must be reported via CTEP-AERS within 24 hours of the 
Investigator’s knowledge.  Please refer to Appendix VIII in 
MATCH Master Protocol for detailed instructions on how to report 
the occurrence of a pregnancy as well as the outcome of all 
pregnancies. 
 
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Version Date: December 11, 2018 
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EAY131 – Subprotocol Z1D specific expedited reporting 
exceptions: 
For Subprotocol Z1D, the adverse events listed below do not require 
expedited reporting via CTEP-AERS: 
• 
If an AE meets the reporting requirements of the protocol, and it 
is listed on the SPEER, it should ONLY be reported via CTEP-
AERS if the grade being reported exceeds the grade listed in the 
parentheses next to the event.  
3.2.2 
Second Primary Cancer Reporting Requirements 
All cases of second primary cancers, including acute myeloid 
leukemia (AML) and myelodysplastic syndrome (MDS), that occur 
following treatment on NCI-sponsored trials must be reported to 
ECOG-ACRIN using Medidata Rave 
• 
A second malignancy is a cancer that is UNRELATED to any 
prior anti-cancer treatment (including the treatment on this 
protocol).  Second malignancies require ONLY routine reporting 
as follows: 
1. Complete a Second Primary Form in Medidata Rave within 14 
days. 
2. Upload a copy of the pathology report to ECOG-ACRIN via 
Medidata Rave confirming the diagnosis. 
3. If the patient has been diagnosed with AML/MDS, upload a 
copy of the cytogenetics report (if available) to ECOG-ACRIN 
via Medidata Rave. 
• 
A secondary malignancy is a cancer CAUSED BY any prior anti-
cancer treatment (including the treatment on this protocol). 
Secondary malignancies require both routine and expedited 
reporting as follows: 
1. Complete a Second Primary Form in Medidata Rave within 14 
days 
2. Report the diagnosis via CTEP-AERS at http://ctep.cancer.gov  
Report under a.) leukemia secondary to oncology 
chemotherapy, b.) myelodysplastic syndrome, or c.) 
treatment related secondary malignancy 
3. Upload a copy of the pathology report to ECOG-ACRIN via 
Medidata Rave and submit a copy to NCI/CTEP confirming the 
diagnosis. 
4. If the patient has been diagnosed with AML/MDS, upload a 
copy of the cytogenetics report (if available) to ECOG-ACRIN 
via Medidata Rave and submit a copy to NCI/CTEP. 
NOTE: 
The Second Primary Form and the CTEP-AERS report 
should not be used to report recurrence or development of 
metastatic disease. 
NOTE: 
If a patient has been enrolled in more than one NCI-
sponsored study, the Second Primary Form must be 

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submitted for the most recent trial. ECOG-ACRIN must be 
provided with a copy of the form and the associated 
pathology report and cytogenetics report (if available) even 
if ECOG-ACRIN was not the patient's most recent trial. 
NOTE: 
Once data regarding survival and remission status are no 
longer required by the protocol, no follow-up data should 
be submitted via CTEP-AERS or by the Second Primary 
Form. 

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3.3 
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/aeguide
lines.pdf for further clarification. Frequency is provided based on 2069 patients. 
Below is the CAEPR for BMS-936558 (Nivolumab, MDX-1106). 
NOTE: 
If an AE meets the reporting requirements of the protocol, and it is 
listed on the SPEER, it should ONLY be reported via CTEP-AERS if 
the grade being reported exceeds the grade listed in the parentheses 
next to the event in the SPEER. 
Version 2.3, June 18, 20181 
 
Adverse Events with Possible 
Relationship to BMS-936558 (Nivolumab, MDX-1106) 
(CTCAE 5.0 Term) 
[n= 2069] 
 
 
Specific Protocol 
Exceptions to 
Expedited Reporting 
(SPEER) 
 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
 
Anemia 
 
 Anemia (Gr 2) 
CARDIAC DISORDERS 
  
 
 
Cardiac disorders - Other 
(cardiomyopathy) 
  
 
 
Myocarditis 
  
 
 
Pericardial tamponade2 
  
 
 
Pericarditis 
  
ENDOCRINE DISORDERS 
  
 
Adrenal insufficiency3 
 
  
 
Hypophysitis3 
 
  
 
Hyperthyroidism3 
 
  
 
Hypothyroidism3 
 
  
EYE DISORDERS 
  
 
 
Blurred vision  
  
 
 
Dry eye 
  
 
 
Eye disorders - Other 
(diplopia)3 
  
 
 
Eye disorders - Other 
(Graves ophthalmopathy)3 
  
 
 
Eye disorders - Other (optic 
neuritis retrobulbar)3 
  
 
Uveitis 
 
  
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
 Abdominal pain (Gr 2) 
 
Colitis3 
 
  
Rev. 3/17 
Rev. Add15 

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EAY131-Z1D 
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Version Date: December 11, 2018 
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Adverse Events with Possible 
Relationship to BMS-936558 (Nivolumab, MDX-1106) 
(CTCAE 5.0 Term) 
[n= 2069] 
 
 
Specific Protocol 
Exceptions to 
Expedited Reporting 
(SPEER) 
 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
 
 
Colonic perforation3 
  
 
Diarrhea 
 
 Diarrhea (Gr 3) 
 
Dry mouth 
 
 Dry mouth (Gr 2) 
 
 
Gastritis 
  
 
 
Mucositis oral 
  
 
Nausea 
 
 Nausea (Gr 2) 
 
Pancreatitis4 
 
  
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
Fatigue 
 
 
 Fatigue (Gr 3) 
 
Fever 
 
 Fever (Gr 2) 
 
Injection site reaction 
 
 Injection site reaction (Gr 
2) 
IMMUNE SYSTEM DISORDERS 
  
 
 
Allergic reaction3 
  
 
 
Autoimmune disorder3 
  
 
 
Cytokine release syndrome5   
 
 
Immune system disorders - 
Other (GVHD in the setting 
of allotransplant)3,6  
  
 
 
Immune system disorders - 
Other (sarcoid granuloma)3    
INJURY, POISONING AND PROCEDURAL COMPLICATIONS 
  
 
Infusion related reaction7 
 
  
INVESTIGATIONS 
  
 
Alanine aminotransferase 
increased3 
 
 Alanine aminotransferase 
increased3 (Gr 3)  
 
Aspartate aminotransferase 
increased3 
 
 
Aspartate 
aminotransferase 
increased3 (Gr 3)  
 
Blood bilirubin increased3 
 
 Blood bilirubin increased3 
(Gr 2)  
 
Creatinine increased 
 
  
 
Lipase increased 
 
  
 
Lymphocyte count 
decreased 
 
 Lymphocyte count 
decreased (Gr 2) 
  
Neutrophil count decreased  
  
 
Platelet count decreased 
 
  
 
Serum amylase increased 
 
  
METABOLISM AND NUTRITION DISORDERS 
  
 
Anorexia 
 
  
 
 
Hyperglycemia 
 Hyperglycemia (Gr 2) 
 
 
Metabolism and nutrition 
disorders - Other (diabetes 
mellitus with ketoacidosis)3 
  
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia 
 
  
 
 
Musculoskeletal and 
connective tissue disorder - 
Other (polymyositis) 
  

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Version Date: December 11, 2018 
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Adverse Events with Possible 
Relationship to BMS-936558 (Nivolumab, MDX-1106) 
(CTCAE 5.0 Term) 
[n= 2069] 
 
 
Specific Protocol 
Exceptions to 
Expedited Reporting 
(SPEER) 
 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
 
 
Myositis 
  
 
 
Rhabdomyolysis 
  
NERVOUS SYSTEM DISORDERS 
  
 
 
Encephalopathy3 
  
 
 
Facial nerve disorder3 
  
 
 
Guillain-Barre syndrome3 
  
 
 
Myasthenia gravis3 
  
 
 
Nervous system disorders - 
Other (demyelination 
myasthenic syndrome) 
  
 
 
Nervous system disorders - 
Other (encephalitis)3 
  
 
 
Nervous system disorders - 
Other (meningoencephalitis)   
 
 
Nervous system disorders - 
Other (meningoradiculitis)3   
 
 
Nervous system disorders - 
Other (myasthenic 
syndrome) 
  
 
 
Peripheral motor 
neuropathy 
  
 
 
Peripheral sensory 
neuropathy 
  
 
 
Reversible posterior 
leukoencephalopathy 
syndrome3 
  
RENAL AND URINARY DISORDERS 
  
 
 
Acute kidney injury3 
  
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
Pleural effusion3 
 
  
 
Pneumonitis3 
 
  
 
 
Respiratory, thoracic and 
mediastinal disorders - 
Other (bronchiolitis 
obliterans with organizing 
pneumonia)3 
  
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
  
 
 
Erythema multiforme3 
  
 
Pruritus3 
 
 Pruritus3 (Gr 2)  
 
Rash maculo-papular3 
 
 Rash maculo-papular3 (Gr 
2)  
 
 
Skin and subcutaneous 
disorders -Other (bullous 
pemphigoid)  
  
 
Skin and subcutaneous 
disorders - Other (Sweet’s 
Syndrome)3 
 
  
 
Skin hypopigmentation3 
 
  
 
 
Stevens-Johnson syndrome   
 
 
Toxic epidermal necrolysis   

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EAY131-Z1D 
Cancer Research Group 
Version Date: December 11, 2018 
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1This table will be updated as the toxicity profile of the agent is revised.  Updates will be distributed to all 
Principal Investigators at the time of revision.  The current version can be obtained by contacting 
PIO@CTEP.NCI.NIH.GOV.  Your name, the name of the investigator, the protocol and the agent should 
be included in the e-mail. 
2 Pericardial tamponade may be related to possible inflammatory reaction at tumor site. 
3 BMS-936558 (Nivolumab, MDX-1106) being a member of class of agents involved in the inhibition of 
“immune checkpoints”, may result in severe and possibly fatal immune-mediated adverse events 
probably due to T-cell activation and proliferation.  This may result in autoimmune disorders that can 
include (but are not limited to) autoimmune hemolytic anemia, acquired anti-factor VIII immune 
response, autoimmune aseptic meningitis, autoimmune hepatitis, autoimmune nephritis, autoimmune 
neuropathy, autoimmune thyroiditis, bullous pemphigoid, exacerbation of Churg-Strauss Syndrome, drug 
rash with eosinophilia systemic symptoms [DRESS] syndrome, facial nerve disorder (facial nerve 
paralysis), limbic encephalitis, hepatic failure, pure red cell aplasia, pancreatitis, ulcerative and 
hemorrhagic colitis, endocrine disorders (e.g., autoimmune thyroiditis, hyperthyroidism, hypothyroidism, 
autoimmune hypophysitis/hypopituitarism, thyrotoxicosis, and adrenal insufficiency), sarcoid granuloma, 
myasthenia gravis, polymyositis, and Guillain-Barre syndrome. 
4 Pancreatitis may result in increased serum amylase and/or more frequently lipase. 
5 Cytokine release syndrome may manifest as hemophagocytic lymphohistiocytosis with accompanying 
fever and pancytopenia. 
6 Complications including hyperacute graft-versus-host disease (GVHD), some fatal, have occurred in 
patients receiving allo stem cell transplant (SCT) after receiving BMS-936558 (Nivolumab, MDX-1106).  
These complications may occur despite intervening therapy between receiving BMS-936558 
(Nivolumab, MDX-1106) and allo-SCT. 
7 Infusion 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 BMS-936558 (Nivolumab, MDX-1106) trials, but for which 
there is insufficient evidence to suggest that there was a reasonable possibility that 
BMS-936558 (Nivolumab, MDX-1106) 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; 

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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: 
BMS-936558 (Nivolumab, MDX-1106) 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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Version Date: December 11, 2018 
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3.4 
Dose Modifications  
All toxicity grades below are described using the NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 4.0. 
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 website (http://ctep.cancer.gov). 
Please refer to the Nivolumab Investigator Brochure or Appendix II to the 
protocol for toxicity management algorithms which include specific 
treatment guidelines.  These algorithms should be followed unless there 
are specific clinical circumstances for which the treating physician decides 
an alternative treatment approach is clinically appropriate.  Consultation 
with the study chair is recommended. 
In all of the tables for dose modification and holds in this section, the 
guidelines are for adverse events thought at least possibly attributed to 
study drug. Generally we strongly encourage early evaluation while 
withholding drug, and appropriate treatment as indicated in the 
management tables and event specific guidelines. 
ALL OTHER EVENTS 
Management/Next Dose for Nivolumab  
≤ Grade 1 
No change in dose  
Grade 2 
Hold until ≤ Grade 1 OR baseline (exceptions as noted below) 
Grade 3 
Off protocol therapy (exceptions as noted below) 
Grade 4 
Off protocol therapy 
Recommended management:  As clinically indicated  
• 
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 should go off protocol 
treatment 
• 
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 should go off protocol treatment. 
• 
Any grade 2-4 drug-related laboratory abnormality or electrolyte abnormality, 
that can be managed independently from underlying organ pathology with 
electrolyte replacement, hormone replacement, transfusion or insulin or that 
does not require treatment does not require hold/discontinuation. 
• 
Tolerable and clinically stable grade 2 toxicities attributed to study drug may 
remain on study with PI approval (e.g. thrombosis that is being 
anticoagulated) 
Rev. 12/16 
Rev. 3/17 
Rev. 3/17 
Rev. 3/17 

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Skin Rash and 
Oral Lesions 
Management/Next Dose for Nivolumab 
≤ Grade 1 
No change in dose * 
Grade 2 
Treatment may continue at investigator discretion. A short course of steroids is 
permitted. If treatment is held, resume at same dose level. 
Grade 3 
Hold* until ≤ Grade 1.  Resume at same level at investigator discretion  
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/pemphagoid.  
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.   
Recommended management:  AE management guidelines 
 
Liver Function AST, ALT, Bilirubin   
Management/Next Dose for Nivolumab 
≤ Grade 1 
Treatment may be continued at investigator discretion. Dose 
level is maintained. 
Grade 2 
Hold until UNL or baseline.  Resume at same dose level.  
Grade 3 
Off protocol therapy  
Grade 4 
Off protocol therapy 
* I-O therapy may be delayed rather than discontinued if AST/ALT ≤ 8 x ULN and T.bili ≤ 5 x ULN 
Continued treatment of active immune mediated hepatitis may exacerbate ongoing inflammation. Holding 
drug to evaluate LFT changes and early treatment are recommended.   
** LFT changes may occur during steroid tapers from other events and may occur together with other GI 
events including cholecystitis/pancreatitis.  
Recommended management:  see Hepatic AE management algorithm 
 
Diarrhea/ Colitis  
Management/Next Dose for Nivolumab 
≤ Grade 1 
Symptomatic treatment but continue therapy. No change in dose 
Grade 2 
Hold until baseline. No change in dose 
Grade 3 
Hold until baseline. No change in dose. 
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.  
Please evaluate pituitary function prior to starting steroids if possible without compromising acute care.   
Evaluation for all patients for additional causes includes C. diff, acute and self-limited infectious and 
foodborne illness, ischemic bowel, diverticulitis, and IBD.  
Recommended management:  see GI AE management Algorithm 
 
Rev. 3/17 
Rev. 3/17 

ECOG-ACRIN 
EAY131-Z1D 
Cancer Research Group 
Version Date: December 11, 2018 
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Symptomatic 
Pancreatitis  
Management/Next Dose for Nivolumab 
≤ Grade 1 
Continue therapy. 
Grade 2 
Continue therapy. 
Grade 3 
Patients who develop symptomatic pancreatitis or DM should be taken off 
treatment  
Grade 4 
Patients who develop symptomatic pancreatitis or DM should be taken off 
treatment  
Patients with any grade lipase/amylase elevation may continue treatment at investigator discretion if 
asymptomatic. 
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 
≤ Grade 1 
Hold dose pending evaluation and resolution to baseline including baseline pO2.  
Resume no change in dose after pulmonary and/or ID consultation excludes lymphocytic 
pneumonitis.  
Grade 2 
Hold dose pending evaluation. Resume no change in dose after pulmonary and/or ID 
consultation excludes nivolumab and associated lymphocytic pneumonitis as the cause 
of the pneumonitis. Off study if steroids are required. ^  
Grade 3 
Hold dose pending evaluation. Refer to Appendix II for diagnostic and treatment 
guidelines. 
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. 
Recommended management:  See Pulmonary Adverse Event Management Algorithm  
 
Fatigue  
Management/Next Dose for Nivolumab 
≤ Grade 1 
No change in dose. 
Grade 2 
No change in dose 
Grade 3 
Hold until ≤ Grade 2.  Resume at same dose level 
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  
 

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Neurologic events  
Management/Next Dose for Nivolumab 
≤ Grade 1 
Hold dose pending evaluation and observation.  Resume with no change in dose 
when resolved to baseline.  
Grade 2 
Hold dose pending evaluation and observation. # Hold until ≤ Grade 1. Off 
protocol therapy if treatment with steroids is required.  Resume at same dose level 
for peripheral isolated n. VII (Bell’s palsy) 
Grade 3 
Off protocol therapy  
Grade 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, myasthenia gravis should be off study.   
Recommended management:  See Neurologic Adverse Event Management Algorithm  
 
Endocrine 
Hypophysitis 
Adrenal Insufficiency  
Management/Next Dose for Nivolumab 
≤ Grade 1 
Asymptomatic TSH elevation * May continue therapy pending evaluation, 
consider endocrine consult 
Grade 2 
Hold until patients are on a stable replacement hormone regimen.  If treated 
with steroids patients must be stable off steroids for two weeks. Resume at 
same dose level.  
Grade 3 
See Appendix II for diagnostic and management algorithm. 
Grade 4 
See Appendix II for diagnostic and management algorithm. 
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  
 
Renal   
Management/Next Dose for Nivolumab combination 
≤ Grade 1 
May continue therapy per investigator discretion. 
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 
 
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Infusion reaction  
Management/Next Dose for Nivolumab combination 
≤ Grade 1 
May continue therapy per investigator discretion. 
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  
 
Fever  
Management/Next Dose for Nivolumab combination 
≤ Grade 1 
Evaluate and continue at same dose level 
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 BMS-936558 (Nivolumab) 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.   Off treatment. 
* 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. 
• 
Drug will be held for grade 2 cardiac dysfunction pending evaluation 
• 
Drug will be permanently discontinued for grade  3 or 4 cardiac dysfunction 
and grade 2 events that do not recover to baseline or that reoccur 
• 
Treatment with steroids as clinically indicated 
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If treatment is delayed >6 weeks for an adverse event, the study PI must be 
consulted for any consideration of further therapy. 
Patients with grade 3 thyroiditis and skin rash may continue therapy as for grade 
2 events with resolution and stable replacement treatment. 
Patients with thyroiditis or hypopituitarism who are stable as above may be 
restarted with replacement hormones including thyroid hormone and physiologic 
doses of corticosteroids.  
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.  
Any patients who require additional immune suppressive treatment beyond 
steroids should go off study treatment 
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 should be 
obtained to document baseline.  However, if urgent steroid use is clinically 
required, steroid treatment should not be delayed for bloodwork. 
Please note that in some cases the treatment algorithms recommend steroids if 
symptoms do not resolve in 7 days. However, this recommendation is not meant 
to delay steroid treatment at any time it is clinically indicated.   
Patients may be dose-delayed for evaluation and restarted depending on results.  
Any patient started on corticosteroids initially who is determined to not require 
steroid 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. 
3.5 
Supportive Care  
3.5.1 
Appendix II details required supportive care and management 
guidelines for immune-related toxicity 
3.6 
Duration of Agent-specific treatment  
In the absence of treatment delays due to adverse event(s), treatment may 
continue until one of the following criteria applies: 
• 
Extraordinary Medical Circumstances: If at any time the constraints of this 
protocol are detrimental to the patient's health, protocol treatment should be 
discontinued. In this event submit forms according to the instructions in the 
MATCH Forms Packet. 
• 
Patient withdraws consent. 
• 
Patient experiences unacceptable toxicity. 
• 
Non-protocol therapies are administered. Patients who require palliative 
radiation therapy may be considered to remain on study after discussion and 
approval by the subprotocol chair(s). Treatment with nivolumab should be 
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• 
Any dosing interruption lasting >6 weeks, with the following exceptions: 
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. Tumor assessments should continue as per protocol even 
if dosing is interrupted.  
• 
Disease progression 
• 
Please see Section 3.7 for details regarding treatment past progression 
3.7 
Treatment Past 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 or combination 
treatment.   
Patients may be permitted to continue treatment beyond initial RECIST 1.1-
defined PD occurring during 24 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). 
3.8 
Duration of Follow-Up 
Refer to the MATCH Master Protocol for specifics on the duration of follow-up. 

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4. 
Study Parameters 
4.1 
Therapeutic Parameters for Nivolumab Treatment  
NOTE: 
In addition to the study parameters listed in the MATCH Master Protocol, the below parameters must also be 
performed for patients receiving nivolumab treatment. 
NOTE: 
All assessments required prior to registration to treatment should be done ≤ 4 weeks prior to registration to 
Steps 1, 3, 5, 7, excluding the radiologic evaluation and electrocardiogram (ECG). 
Test/Assessment 
Prior to Registration to 
Treatment 
Treatment 
End of 
Treatment 
Follow UpF 
Every Cycle, prior 
to treatment 
Every 2 
Cycles 
H&P, Weight, Vital signsA 
X 
 XI 
 
 
X 
Performance status 
X 
 XI 
 
 
X 
CBC w/diff, pltsB 
X 
 XI 
 
 
X 
Serum chemistryB 
X 
 XI 
 
 
X 
Thyroid function testing  
[TSH (with reflex to free T4 ± free T3 if abnormal)] 
X 
X 
 
 
X 
Radiologic evaluationD 
X 
 
 XD 
 
 XF 
-HCGC 
X 
 
 
 
 
Toxicity AssessmentG 
 
X 
 
X 
 XF 
ECG 
X 
XH 
 
 
 
ECHO 
XH 
 
 
 
 
Tumor biopsy and blood sample for MATCH Master ProtocolE 
 
 
X 
 X 
 
Research Blood (See Section 6)J 
 
XJ 
 
XJ 
 
Archival tumor block or 20 (at least 5 micron thick) unstained 
slides, if available 
 XK 
 
 
 
 
A. History and physical, including vital signs and weight at the start of each cycle (up to 3 days before start of new cycle). A contraceptive, 
menstrual, and sexual history be obtained with each H&P for all women of childbearing potential and sexually active men. 
B. Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, creatinine, glucose, phosphorus, potassium, SGOT[AST], SGPT[ALT], 
sodium, magnesium, TSH (with reflexive Free T4 ± Free T3 if abnormal), serum tumor markers (prior to each cycle, not each dose). For eligibility 
purposes, participants with creatinine levels above eligibility, Cockcroft-Gault will be used to calculate creatinine clearance. CBC w/diff, platelets 
and serum chemistries should be performed on cycle 1, day 1 (or up to 7 days prior), and at the start of each subsequent dose (up to 3 days 
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before start of next dose) while tumor markers are performed prior to day 1 of a cycle (up to 3 days before). CBC with differential will be 
performed more frequently in patients with grade 4 neutropenia or thrombocytopenia until resolution to ≤ grade 3. CBC and serum chemistries 
are only required in follow-up until values return to pre-treatment levels or until progressive disease. 
C. Blood pregnancy test (women of childbearing potential) required prior to beginning treatment. 
D. Disease measurements are repeated every 2 cycles for the first 26 cycles, and every 3 cycles thereafter until PD or start of another MATCH 
treatment step. The baseline evaluation should be performed as closely as possible to the beginning of treatment and never more than 6 weeks 
before registration to treatment step. For multiple myeloma patients, please refer to Section 6.4 of the MATCH Master Protocol for additional 
information on myeloma response criteria and the required disease assessments. Documentation (radiologic) must be provided for patients 
removed from study for progressive disease. 
E. Additional blood specimens and/or biopsies are to be submitted from consenting patients per Section 9.3.2 of the MATCH Master Protocol. 
Submit at the following time points, as applicable: 
• 
Blood specimens are to be submitted at the end of Cycle 2 (prior to start of Cycle 3 treatment). If patient progresses or treatment is 
discontinued prior to Cycle 3, collect the blood at that time instead. On-treatment kits for blood sample collections will be automatically 
shipped to sites upon registration to the treatment step. 
• 
Screening biopsies for additional aMOI assessments after registration to appropriate screening step, if applicable (Step 2 or Step 4). 
• 
At end of all MATCH study treatments, blood specimens and/or research biopsy after consent and registration to Step 8 
Please refer to Section 4 of the MATCH Master Protocol to determine whether the patient proceeds to the next screening step or to follow-up 
(with a potential end of treatment biopsy for research purposes on Step 8). Samples are to be submitted as outlined in Section 9 of the MATCH 
Master Protocol. To order Step 2/4 Screening or Step 8 kits, complete the EAY131 Collection and Shipping Kit Order Form (See Appendix XII of 
the MATCH Master Protocol) and fax to 713-563-6506. 
F. Every 3 months if patient is < 2 years from study entry, and every 6 months for year 3. Toxicity assessments and radiologic evaluations are not 
required to be done during Follow Up if progression has been previously reported; however if an adverse event occurs post treatment that meets 
the SAE reporting requirements, it still must be reported via CTEP-AERS, even if progression has occurred. 
G. Site personnel should evaluate for toxicity and discuss treatment compliance with the patient in order to ensure the medication is taken correctly; 
this evaluation may be conducted by telephone or in person. The Toxicity Assessment is not required prior to Cycle 1, but is required every 
subsequent cycle. 
H. Evaluate cardiac function including EKG and ECHO for any patients with a history of CHF or at risk because of underlying cardiovascular 
disease or exposure to cardiotoxic drugs, as clinically indicated. 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. 
I. 
For Cycle 1, if the following tests/assessments occurred within 7 days of Day 1, they do not need to be repeated at this timepoint: H&P, Weight, 
Vital Signs; Performance Status; CBC w/diff, plts; Serum chemistry; Concomitant Medications. 
J. From consenting patients, collection prior to start of treatment and end of cycle 2 (prior to start of cycle 3) and End of treatment. 
K. MANDATORY: Submit per EAY131 Master Protocol (Section 9.3 and Section 9.4) prior to or within 8 weeks following registration to EAY131-
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5. 
Drug Formulation and Procurement 
This information has been prepared by the ECOG-ACRIN Pharmacy and Nursing 
Committees. 
Availability 
NO STARTER SUPPLIES MAY BE ORDERED. Subjects must be enrolled and assigned 
to the treatment subprotocol prior to submitting the clinical drug request to PMB. 
Drug Ordering: NCI supplied agents may be requested by eligible participating 
Investigators (or their authorized designee) at each participating institution.  
Pharmaceutical Management Branch (PMB) policy requires that drug be shipped directly 
to the institution where the patient is to be treated. PMB does not permit the transfer of 
agents between institutions (unless prior approval from PMB is obtained – see general 
information) 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 investigator at that institution. 
Submit agent requests through the PMB Online Agent Order Processing (OAOP) 
application (https://ctepcore.nci.nih.gov/OAOP).  Access to OAOP requires the 
establishment of a CTEP Identity and Access Management (IAM) account 
(https://ctepcore.nci.nih.gov/iam/) and the maintenance of an “active” account status, a 
“current” password, and an active person registration status. 
NCI Supplied Agent(s) – General Information 
Questions about drug orders, transfers, returns, or accountability should be 
addressed to the PMB by calling 240-276-6575 Monday through Friday between 
8:30 AM and 4:30 PM Eastern Time or email PMBAfterHours@mail.nih.gov 
anytime. 
Drug Returns: All undispensed drug supplies should be returned to the PMB. When it is 
necessary to return study drug (e.g., sealed bottles remaining when PMB sends a stock 
recovery letter), investigators should return the study drug to the PMB using the NCI 
Return Agent Form available on the NCI home page (http://ctep.cancer.gov). 
Drug Accountability: The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the receipt, disposition, and return of 
agent received from the PMB using the NCI Investigational Agent Accountability Record 
Form available on the NCI home page (http://ctep.cancer.gov). Maintain separate NCI 
Investigational Agent Accountability Records for each agent, strength, formulation and 
ordering investigator. 
Investigator Brochure Availability: The current versions of the IBs for PMB-supplied 
agents will be accessible to site investigators and research staff 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, a “current” password, and active person 
registration status. Questions about IB access may be directed to the PMB IB 
coordinator at IBCoordinator@mail.nih.gov. 
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5.1 
Nivolumab (NSC #748726) 
5.1.1 
Other Names  
BMS-936558, MDX1106 
5.1.2 
Classification  
Anti-PD-1MAb 
5.1.3 
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. 
5.1.4 
Storage and Stability  
Storage: 
Vials of Nivolumab injection must be stored at 2°-8°C (36°-46°F) and 
protected from light and freezing. The unopened vials can be stored at 
room temperature (up to 25°C, 77°F) and room light for up to 48 
hours. 
If a storage temperature excursion is identified, promptly return 
Nivolumab to 2°C-8°C and quarantine the supplies.  Provide a detailed 
report of the excursion (including documentation of temperature 
monitoring and duration of the excursion) to 
PMBAfterHours@mail.nih.gov for determination of suitability. 
Stability:   
Shelf-life surveillance of the intact vials is ongoing. The administration 
of undiluted and diluted solutions of Nivolumab must be completed 
within 24 hours of preparation. If not used immediately, the infusion 
solution may be stored up to 24 hours in a refrigerator at 2°-8°C (36°-
46°F) and a maximum of 8 hours of the total 24 hours can be at room 
temperature (20°-25°C, 68°-77°F) and room light.  The maximum 8-
hour period under room temperature and room light conditions 
includes the product administration period. 
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. 
5.1.5 
Dose Specifics  
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 dihydrate, sodium chloride, 
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mannitol, diethylenetriaminepentacetic 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 0.7mL overfill.  It is 
supplied in 10 mL type I flint glass vials, with fluoropolymer film-
laminated rubber stoppers and aluminum seals. 
5.1.6 
Preparation  
Preparation:  Nivolumab injection can be infused undiluted (10 
mg/mL) or diluted with 0.9% Sodium Chloride Injection, USP or 5% 
Dextrose.  When the dose is based on patient weight (i.e., mg/kg), 
nivolumab injection can be infused undiluted or diluted 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. During drug product preparation and handling, vigorous 
mixing or shaking is to be avoided. 
5.1.7 
Route of Administration  
Intravenous infusion over 30 minutes. 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. 
5.1.8 
Potential Drug Interactions 
The indirect drug-drug interaction potential of nivolumab was 
assessed using systemic cytokine modulation data for cytokines 
known to modulate CYP enzymes. There were no meaningful 
changes in cytokines known to have indirect effects on CYP enzymes 
across all dose levels of nivolumab. This lack of cytokine modulation 
suggests that nivolumab has no or low potential for modulating CYP 
enzymes, thereby indicating a low risk of therapeutic protein-drug 
interaction. 
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5.1.9 
Patient Care Implications: Women of childbearing potential (WOCBP) 
receiving nivolumab must agree to use adequate contraception 
(hormonal or double barrier method of birth control; abstinence) from 
one week prior to study treatment starting, during treatment, and for a 
period of 5 months after the last dose of nivolumab. Men receiving 
nivolumab and who are sexually active with WOCBP must agree to 
use adequate contraception (hormonal or double barrier method of 
birth control; abstinence) from one week prior to study treatment 
starting, during treatment, and for a period of 7 months after the last 
dose of nivolumab. 
5.1.10 
Side Effects  
See Section 3.3 for side effects. 
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6. 
Specimen Submissions 
Peripheral blood for research studies will be obtained from patients on EAY131-Z1D who 
consent “Yes” to “I agree to provide additional blood for research." These blood 
collections will allow for potential analyses of circulating immunologic factors and 
changes in these factors engendered over the course of treatment and other possible 
research studies. 
Kits are available to order for the collection and submission of the peripheral blood 
samples and will contain the supplies and instructions for collecting, processing, and 
shipping the samples. To order kits, complete the EAY131-Z1D Collection and Shipping 
Kit Order Form (Appendix III) and Fax as instructed. Kits will arrive within 48-72 hours 
from when the order was placed. 
Requirements and guidelines for the submission of archived FFPE tumor tissue for 
confirmation of MLH1 or MSH2 testing are in Section 9 of the MATCH Master Protocol 
as they are considered part of the screening assessments. Tissue is to be submitted 
prior to or within 8 weeks following registration to EAY131-Z1D. If the results of these 
additional assessments do not confirm the initial screening assessment and are received 
after registration to treatment on EAY131-Z1D, treatment on EAY131-Z1D can continue 
per physician discretion. 
6.1 
Specimen Submissions 
If you have any questions concerning sample collection and shipment, please 
contact the ECOG-ACRIN CBPF at (844) 744-2420. 
Logging and Tracking of Samples: It is required that all samples submitted on 
this EAY131-Z1D be entered and tracked using the ECOG-ACRIN Sample 
Tracking System (see Section 6.1.4). An STS shipping manifest form is to be 
included with every submission. 
Labeling of Samples: All samples must be clearly labeled with the ECOG-
ACRIN protocol number (EAY131-Z1D), ECOG-ACRIN five-digit patient case 
number, patient initials, date of collection and sample type. 
6.1.1 
Sample Collection and Submission Schedule 
Peripheral blood samples are to be submitted at ambient on day of 
collection. Samples are to be collected at the following time points for 
each tube type: 
• 
Prior to start of treatment 
• 
Post-Cycle 2 (prior to start of Cycle 3) 
• 
Discontinuation of treatment 
6.1.2 
Specimen Collection Guidelines 
Peripheral blood samples are to be collected  
• 
Draw 32mL of whole blood into four (4) CPT tubes (blue/black top, 
provided in the kit) – Note: Tube should be gently inverted 5-10 
times following collection 
• 
Draw 7mL of whole blood into one (1) red top tube (provided in the 
kit) 

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6.1.3 
Shipping Procedures 
The integrity of the blood samples requires they be processed within 
24 hours of collection. Peripheral blood samples are to be shipped 
Monday-Thursday at ambient via overnight courier the day of 
collection. Do not ship samples the day before a weekend or 
holiday. 
Ship using the ECOG-ACRIN CBPF’s FedEx account using the 
FedEx on-line Ship Manager. 
Access to the shipping account for shipments to the ECOG-ACRIN 
CBPF can only be obtained by logging into fedex.com with an account 
issued by the ECOG-ACRIN CBPF. For security reasons, the account 
number will no longer be given out in protocols, over the phone, or via 
email. If your site needs to have an account created, please contact 
the ECOG-ACRIN CBPF by email at eacbpf@mdanderson.org. 
Ship to: 
ECOG-ACRIN Central Biorepository and Pathology Facility 
MD Anderson Cancer Center 
Department of Pathology, Unit 085 
Tissue Qualification Laboratory for ECOG-ACRIN 
Room G1.3598 
1515 Holcombe Boulevard 
Houston, TX 77030  
Toll Free Phone: (844) 744-2420 (713-745-4440 Local or 
International Sites) 
Fax: (713) 563-6506 
Email: eacbpf@mdanderson.org 
6.2 
ECOG-ACRIN Sample Tracking System  
It is required that all specimens submitted on this trial be entered and tracked 
using the ECOG-ACRIN Sample Tracking System (STS). The software will allow 
the use of either 1) an ECOG-ACRIN user-name and password previously 
assigned (for those already using STS), or 2) a CTSU username and password.   
When you are ready to log the collection and/or shipment of the samples 
required for this study, please access the Sample Tracking System software by 
clicking https://webapps.ecog.org/Tst.  
Important: Please note that the STS software creates pop-up windows, so you 
will need to enable pop-ups within your web browser while using the 
software. A user manual and interactive demo are available by 
clicking this link: http://www.ecog.org/general/stsinfo.html 
Please take a moment to familiarize yourself with the software prior to using the 
system. 
An STS generated shipping manifest form should be shipped with all sample 
submissions. 
Please direct your questions or comments pertaining to the STS to 
ecog.tst@jimmy.harvard.edu.  

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Study Specific Notes 
Generic Specimen Submission Form (#2981v3) will be required only if STS is 
unavailable at time of sample submission. Notify the laboratory of the shipment 
by faxing a copy of the completed form to the laboratory. 
• 
ECOG-ACRIN Central Biorepository and Pathology Facility 
Retroactively enter all sample collection and shipping information when STS is 
available. 
6.3 
Use of Specimens in Research 
From the heparinized whole blood, peripheral blood mononuclear cells (PBMC) 
will be prepared by Ficoll-Hypaque density gradient centrifugation by standard 
laboratory procedures, transferred to cryogenic vials, and stored in a liquid 
nitrogen freezer for use in approved research projects. Aliquots of plasma will 
also be stored in 1mL cryogenic vials. 
Whole blood Serum Tube will be processed using standard laboratory 
procedures.  Aliquots of 1 mL of serum will be transferred to cryogenic vials and 
stored at ≤ -70°C or below. 
Specimens will be retained in an ECOG-ACRIN designated central repository. 
For this trial, specimens will be retained at the ECOG-ACRIN Central 
Biorepository and Pathology Facility.  
Specific analyses will be reviewed and approved by the MATCH Correlative 
Science committee and NCI prior to inclusion for this study. 
If consent for use in research is denied or withdrawn by the patient, the 
specimens will be removed from consideration for use in any future research 
study and the blood specimens destroyed per guidelines of the respective 
repository. 
6.4 
Sample Inventory Submission Guidelines  
Inventories of all samples submitted from institutions will be tracked via the 
ECOG-ACRIN STS and receipt and usability verified by the receiving laboratory.  
Inventories of samples forwarded and utilized for approved laboratory research 
studies will be submitted by the investigating laboratories to the ECOG-ACRIN 
Operations Office - Boston on a monthly basis in an electronic format defined by 
the ECOG-ACRIN Operations Office - Boston. 

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7. 
References 
1. 
Brahmer, J.R., L. Horn, S.J. Antonia, et al.  (2013). Survival and long-term follow-
up of the phase I trial of nivolumab (Anti-PD-1; BMS-936558; ONO-4538) in 
patients (pts) with previously treated advanced non-small cell lung cancer 
(NSCLC).  J Clin Oncol. 31:A8030.  
2. 
Cancer Genome Atlas Network. Comprehensive molecular characterization of 
human colon and rectal cancer. Nature 2012; 487: 330-7. 
3. 
Drake, C.G., D.F. McDermott, M. Sznol, et al. (2013). Survival, safety, and 
response duration results of nivolumab (Anti-PD-1; BMS-936558; ONO-4538) in a 
phase I trial in patients with previously treated metastatic renal cell carcinoma 
(mRCC): Long-term patient follow-up.  J Clin Oncol. 31:A4514. 
4. 
Eshleman JR, Lang EZ, Bowerfind GK, et al. Increased mutation rate at the hprt 
locus accompanies microsatellite instability in colon cancer. Oncogene 1995; 10: 
33-7.  
5. 
Investigator Brochure.  (2014). Nivolumab, BMS-936558, MDX1106.  Version 13, 
21 July 2014. Bristol-Myers Squibb.  Lawrenceville, NJ.   
6. 
Karamurzin Y et al. Unusual DNA mistmatch repair-deficient tumors in lynch 
syndrome:  a report of new cases and review of the literature.  Human Pathology 
(2012);43(10): 1677-87. 
7. 
Kobayashi H, Ohno S, Sasaki Y, Matsuura M.  Hereditary breast and ovarian 
cancer susceptibility genes (Review). Oncology Reports (2013)30(3): 1019-29 
8. 
Le DT, Uram JN, Wang H et al. PD-1 Blockade in Tumors with Mismatch-Repair 
Deficiency. Presented as an oral abstract at ASCO Annual Meeting, 2015 May 30, 
2015. Chicago, IL. 
9. 
Timmermann B, Kerick M, Roehr C, et al. Somatic mutation profiles of MSI and 
MSS colorectal cancer identified by whole exome next generation sequencing and 
bioinformatics analysis. PLoS One 2010; 5(12): e15661. 
10. 
Tiwari AK, Roy HK, Lynch HT. Lynch syndrome in the 21st century: clinical 
perspectives.  QJM, An international Journal of medicine 2015: 1-8 
11. 
Yamamoto H, Imai K. Microsatellite instability: an update. Arch Toxicol(2015) 
89:899-921. 
12. 
Topalian, S.L., F.S. Hodi, J.R. Brahmer, et al. (2012). Safety, activity, and immune 
correlates of anti-PD-1 antibody in cancer.  N Engl J Med. 366:2443-2454. 
13. 
Wolchok, J.D., H. Kluger, M.K. Callahan, et al.  (2013). Nivolumab plus ipilimumab 
in advanced melanoma.  N Engl J Med.  369:122-133. 
14. 
Herbst, R.S., et al., Predictive correlates of response to the anti-PD-L1 antibody 
MPDL3280A in cancer patients. Nature, 2014. 515(7528): p. 563-7. 
15. 
Postow, M.A., et al., Immunologic correlates of the abscopal effect in a patient with 
melanoma, in N Engl J Med. 2012. p. 925-31. 
16. 
Schoenfeld, J., et al., Active immunotherapy induces antibody responses that 
target tumor angiogenesis. Cancer Res, 2010. 70(24): p. 10150-60. 
 

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Molecular Analysis for Therapy Choice (MATCH) 
MATCH Treatment Subprotocol Z1D: Nivolumab 
Appendix I 
 
Actionable Mutations for Sub-Protocol EAY131-Z1D 
 
1. Absence of MLH1 by immunohistochemistry or 
2. Absence of MSH2 by immunohistochemistry 
 
 

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MATCH Treatment Subprotocol Z1D: Nivolumab 
Appendix II 
 
Management Algorithms For Endocrinopathy, Gastrointestinal, Hepatic, Neurological, 
Pulmonary, Renal, And Skin Adverse Events 

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Molecular Analysis for Therapy Choice (MATCH) 
MATCH Treatment Subprotocol Z1D: Nivolumab 
Appendix III 
 
EAY131-Z1D Collection and Shipping Kit Order Form 
Kit orders are to be made after patient has consented to participate in EAY131-Z1D. 
Please complete this form correctly, including the valid ECOG-ACRIN case number and 
complete shipping address. If information is missing the kit processing will be delayed. 
FAX to ECOG-ACRIN Central Biorepository and Pathology Facility at (713) 563-6506. 
 
Date:  
EAY131 Patient Case Number:  ____________________ 
 
EAY131-Z1D Kit Requested: Kit will contain three (3) subkits (collection and shipping supplies) 
for (a) Prior to start of treatment, (b) Post-Cycle 2, and (c) End of 
Treatment 
 
Ship Kit to: 
Institution Contact:   
_________________________________ 
Phone Number for Contact:  _________________________________ 
Fax Number for Contact: 
_________________________________ 
Email for Contact: 
_________________________________ 
Institution Address: 
_________________________________ 
 
_________________________________ 
 
_________________________________ 
 
NOTE: 
Questions are to be directed to the ECOG-ACRIN CBPF 
Phone: Toll Free 1-844-744-2420 (713-745-4440 Local or International Sites) 
Fax: 713-563-6506 
Email: eacbpf@mdanderson.org 
Comments: 
 
 

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Molecular Analysis for Therapy Choice (MATCH) 
MATCH Treatment Subprotocol Z1D: Nivolumab 
Appendix IV 
 
Patient Drug Information Handout and Wallet Card 
Information for Patients, Their Caregivers and Non-Study Healthcare Team on Possible 
Interactions with Other Drugs and Herbal Supplements 
The patient ____________________________ is enrolled on a clinical trial using the 
experimental study drug, nivolumab. This clinical trial is sponsored by the National Cancer 
Institute. This form is addressed to the patient, but includes important information for others who 
care for this patient.  
These are the things that you as a healthcare provider need to know: 
• 
This agent can stimulate an autoimmune response, including but not limited no hypophysitis, 
hypothyroiditis, pneumonitis, colitis, hepatitis, and rash. These conditions can be life-
threatening and require emergent intervention. 
To the patient: Take this paper with you to your medical appointments and keep the 
attached information card in your wallet. 
Nivolumab may interact with other drugs which can cause side effects. For this reason, it is very 
important to tell your study doctors of any medicines you are taking before you enroll onto this 
clinical trial.  It is also very important to tell your doctors if you stop taking any regular 
medicines, or if you start taking a new medicine while you take part in this study.  When you talk 
about your current medications with your doctors, include medicine you buy without a 
prescription (over-the-counter remedy), or any herbal supplements such as St. John’s Wort.  It 
is helpful to bring your medication bottles or an updated medication list with you. 
Many health care providers can write prescriptions. You must tell all of your health care 
providers (doctors, physician assistants, nurse practitioners, pharmacists) you are taking part in 
a clinical trial. 
Nivolumab has not been shown to have any effect on cytochrome P450 or other drug 
metabolizing enzymes and is not expected to interact with other medications that are impacted 
by cytochrome P450 or other drug metabolizing enzymes. 
These are the things that you and they need to know: 
Nivolumab must be used very carefully with other medicines. Before you enroll onto the clinical 
trial, your study doctor will work with your regular health care providers  to review any medicines 
and herbal supplements you are taking. 
• 
Please be very careful!  Over-the-counter drugs (including herbal supplements) may contain 
ingredients that could interact with your study drug.  Speak to your doctors or pharmacist to 
determine if there could be any side effects.  
• 
Your regular health care provider should check a frequently updated medical reference or 
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Your study doctor’s name is 
_____________________________________ 
and he or she can be contacted at: 
_____________________________________. 
 
 
 
 
➢ Before you enroll onto the clinical trial, your study 
doctor will work with your regular health care 
providers to review any medicines and herbal 
supplements that you are taking. 
➢ Nivolumab has not been shown to have any effect on 
cytochrome P450 or other drug metabolizing enzymes 
and is not expected to interact with other medications 
that are impacted by cytochrome P450 or other drug 
metabolizing enzymes. 
➢ Your study doctor’s name is 
_____________________________ and can be 
contacted at 
_________________________________.  
STUDY DRUG INFORMATION WALLET CARD   
You are enrolled on a clinical trial using the experimental 
study drug nivolumab.  This clinical trial is sponsored by 
the NCI. Nivolumab may interact with drugs. Because of 
this, it is very important to: 
➢ Tell your doctors if you stop taking any medicines or if 
you start taking any new medicines. 
➢ Tell all of your health care providers (doctors, 
physician assistants, nurse practitioners, or pharmacists) 
that you are taking part in a clinical trial. 
➢ Check with your doctor or pharmacist whenever you 
need to use an over-the-counter medicine or herbal 
supplement. 

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Molecular Analysis for Therapy Choice (MATCH) 
MATCH Treatment Subprotocol Z1D: Nivolumab  
Appendix V 
 
CS-MATCH-0005 
Tissue / blood biomarker analysis of samples collected from patients with mismatch 
repair deficient tumors (non-colorectal cancers) treated with nivolumab (EAY131-Z1D) 
 
Principal Investigators: 
PRINCIPAL INVESTIGATORS: 
Jonathan Schoenfeld, MD MPhil MPH 
CO-INVESTIGATORS: 
Nilofer Azad, MD 
F. Stephen Hodi, MD 
Michael Overman, MD 
Scott Rodig, MD PhD 
Mariano Severgnini, PhD 
BIOSTATISTICS: 
Paul Catalano, ScD 
SUB-PROTOCOL PRINCIPAL INVESTIGATORS: 
Nilofer Azad, MD 
Michael Overman, MD 
Jonathan Schoenfeld, MD MPhil MPH  
Rev. Add16 

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I. 
Introduction 
PD-1 inhibitors have demonstrated impressive response rates and improvements in 
survival across multiple solid tumor types, including melanoma, non-small cell lung 
cancer and renal cell carcinoma [1, 5, 17, 18] and preliminary results demonstrate 
activity across many additional cancers [19]. Response rates to PD-1 inhibitors have 
varied considerably across different disease types, from approximately 40% in 
melanoma patients, to single digit response rates in patients with ovarian cancer [20]. 
And there are few to no responders in other disease types, such as microsatellite stable 
colorectal cancer [9].  Thus, identifying biomarkers that predict response is of significant 
benefit, even more so because patients treated with immune checkpoint blockade may 
have atypical patterns of response that may initially mimic disease progression [21]. 
Additionally, patients that do respond can achieve durable clinical benefit.  
One of the ligands for the PD-1 receptor, PD-L1, is known to be overexpressed in the 
setting of chronic viral infection and, more recently, ubiquitously across many tumor 
types as a mechanism of tumor immune evasion.  Binding of PD-L1 to the PD-1 receptor 
can down regulate T-cell activation by recruiting SHP-2 to the phosphorylated tyrosine 
residue in the cytoplasmic region [22].  In addition to tumor cells, PD-L1 can also be 
expressed on tumor infiltrating lymphocytes, which may also lead to immune 
suppression in the tumor microenvironment.   
PD-L1 overexpression was identified as one of the early potential markers of response 
to inhibitors of the PD-1 pathway [23].  In one of the initial studies testing PD-1 inhibitor 
therapy, none of the 17 patients with tumors that did not express PD-L1 responded to 
treatment with nivolumab (BMS-936558), while 9/25 patients (36%) with PD-L1 
expressing tumors did (p=0.006) [3].  More recently, PD-L1 expression in pretreatment 
tumor samples has demonstrated promise as potential biomarkers in patients treated 
with the anti-PD-L1 antibody MPDL3280A [2].  In this study of 277 patients with a variety 
of tumor types including NSCLC, RCC, melanoma and HNSCC, PD-L1 expression on 
tumor infiltrating lymphocytes was associated with response (p=0.007), although the 
association between PD-L1 expression on tumor cells and response did not reach 
statistical significance (p=0.08).  High PD-L1 expression on tumor cells or increasing 
percentage of infiltrating immune cells has also been found to be a predictive biomarker 
in patients with NSCLC [1, 13].  Membranous PD-L1 was also seen in the majority of 
patients with mismatch repair deficient tumors treated with pembrolizumab in a previous 
study, although expression was not significantly associated with outcome [9].  
In addition to PD-L1 expression, the degree of tumor infiltrating lymphocytes has also 
been associated with benefit following PD-1 pathway blockade.  This has been 
demonstrated for CD8+ T-cells present at the margin between tumor and stroma in 
patients with melanoma treated with the PD-1 inhibitor pembrolizumab [4] as well as in 
patients with mismatch repair deficient tumors, CD8+ T-cells, particularly at the tumor 
invasive fronts, were associated with a trend towards response or stable disease [9]. 
There is an expansive and rapidly growing literature supporting PD-L1, CD3/CD4 and 
other immune marker IHC staining across tumor types.  For example, multiple meta-
analyses of PD-L1 IHC describe IHC staining results in over 16,000 patients across 
almost 100 studies across numerous tumor histologies (Wu, Wu et al. 2015; Pyo, Kang 
et al. 2016). PD-L1 is not an antigen but a conserved mechanism of immune evasion 
whose expression has been demonstrated across multiple tumor types and specifically 
in MSI tumors (Le, Uram et al. 2015) and therefore would be expected to be detectable 
across the various tumor types included in this study.  An additional meta-analysis 

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conducted over 5 years ago identified 52 studies each including over a hundred patients 
that evaluated the prognostic value of tumor infiltrating lymphocytes across multiple 
histologies using IHC for the markers CD3, CD8, CD4, FoxP3 and others (Gooden, de 
Bock et al. 2011). 
In addition to PD-L1 and tumor infiltrating lymphocytes, other immune markers may also 
indicate tumor mediate immune regulation and could also predict response to immune 
therapy either alone or in conjunction with other markers. Additionally, while traditional 
immunohistochemistry generally only allows for the evaluation of one or two proteins at a 
time, multiplex immunofluorescence (IF) simultaneously evaluates expression and 
provides more quantifiable spatial resolution for multiple biomarkers.  Our initial work has 
demonstrated the reproducibility of this technique in identifying various populations of 
tumor infiltrating lymphocytes, as well as PD-L1 expressing tumor cells and PD-L1 and 
PD-1 expressing infiltrating lymphocytes; therefore, we propose to use multiplex IF to 
explore associations between response and expression of PD-1/PD-L1 in the tumor 
versus tumor/stroma interface, as well as co-localized PD-L1 / PD-1 expression.  
In terms of circulating biomarkers, various studies have suggested that patients treated 
with immune checkpoint blockade demonstrate changes that can be detected in the 
peripheral blood.  Herbst et al. found treatment with the PD-L1 inhibitory antibody 
MPDL3280A to be associated with increases in IL-18, ITAC, and CD8+/HLA-DR+/Ki67+ 
T-cells, and a decrease in circulating IL-6 [2]. In patients treated for renal cell carcinoma, 
responders to MPDL3280A with prolonged overall survival demonstrated decreases in 
circulating VEGFA as well as various acute phase proteins [24]. In the previously 
mentioned study evaluating patients with mismatch repair deficient colorectal cancer 
treated with PD-1 inhibitors, decreases in the tumor markers CEA, CA19-9, and CA-125 
were observed in the majority of responding patients depending on their underlying 
tumor type [9].  
In summary, preliminary data demonstrates that tissue markers including PD-L1 
expression and quantification of tumor infiltrating lymphocytes, as well as changes in 
circulating markers such as cytokines and T-cell subsets, may predict activity associated 
with PD-1 pathway inhibitors across tumor types and including patients with mismatch 
repair deficient tumors. Thus, these studies have the potential for clinical utility a method 
of predicting which patients with mismatch repair tumors would be more likely to respond 
to PD-1 directed therapy and then as an early marker of long term benefit in patients 
during the course of therapy.  
II. 
Objectives 
A. 
Primary objectives:  
1. Studies in tissue: The primary objective of this study will be to determine 
associations between PD-L1 expression (quantitatively determined as a 
continuous variable) in tumor tissue at baseline and an objective response to 
protocol treatment.   
2. Peripheral blood studies: To determine association between changes in 
percentages of circulating activated T-cells, specifically CD8+/HLA-DR+/Ki-
67+ T cells and an objective response to protocol treatment.  

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B. 
Exploratory studies  
1. To associate levels of tumor infiltrating CD8+ T-cells, and PD-L1 expression 
defined categorically, and also in specific cell populations such as tumor cells 
and tumor infiltrating lymphocytes with response 
2. As tissue allows, to explore correlations between response and multiple 
immunologic biomarkers including PD-1, CTLA-4, MHC-1, MHC-2, Lag-3, 
B2M, Na channel and other exploratory multiplex IF to determine expression 
levels of these biomarkers in components of the tumor microenvironment and 
in relation to each other 
3. To evaluate mechanisms of resistance by comparing immunologic markers in 
sequential biopsies and biopsies taken at progression if available  
4. To explore correlations between PD-L1 expression and CD8 + T-cell tumor 
infiltration with overall tumor mutational burden as determined by whole-
exome and targeted sequencing to be performed through the parent NCI 
MATCH study  
5. To explore associations between changes in a panel of circulating immune 
populations and immunologic cytokines with response Methodology 
III. 
Methodology 
Summary:  
Correlative science will involve immune evaluation of archival tissue, serum and PBMCs 
obtained from multiple time points. PD-L1 expression and CD8+ T-cell infiltration will be 
evaluated on cut FFPE slides as below to correlate with response. We will also examine 
changes in these biomarkers over time when sequential biopsy specimens are available. 
Additional tissue, when available will be used for multiplexed IF and assayed for other 
immunologic markers as described in detail below. CyTOF will primarily examine 
changes in circulating activated T-cells over the course of therapy as a predictor of 
response but will also explore other immune subsets and cytokines.   
Patients enrolled on MATCH Z1D have loss of MLH1 or MSH2 as determined by IHC 
performed at the Central Pathologic lab for the NCI MATCH study.  Other MSI testing is 
currently outside of the scope of the current correlative proposal, although additional 
testing can be considered if adequate tissue is remaining. 
No patient identifiers will be provided to investigators with the samples. The trial-specific 
patient ID will be replaced with a unique randomized number and specimens will be 
labeled with a barcode not related to any PHI. The following entities have access to the 
key:  the ECOG-ACRIN biobank, the ECOG-ACRIN Operations Office, and the MATCH 
statisticians. 
Prioritization of tissue:  
Formalin fixed-paraffin embedded (FFPE) tumor slides will be prepared and a slide H&E 
stained from all tumor samples.  Immunohistochemical (IHC) and multiplex 
immunofluorsecent (IF) staining will be performed on FFPE tumor slides using some or 
all of the following antibodies/assays. Since there may not be sufficient tissue to perform 
all the assays in every sample we have prioritized the assays.  In the order of priority 
based on the amount of tissue available, the assays we will perform include:  
- 
IHC to determine intratumoral PD-L1 (CD274) expression 

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- 
IF to determine tumor infiltrating lymphocytes (TILs) using CD8 expression  
- 
Exploratory multiplex IF and IHC to evaluate response and distribution of PD-L1 
staining in conjunction with other markers such as tumor infiltrating lymphocytes and 
PD-1 expressionand, and other immunologic markers will be performed  including 
CD3, CD8, CTLA-4, MHC-1, MHC-2, Lag-3, B2M, Na channel, (these will only be 
performed if there is sufficient tissue)  
Materials and methods for studies in tissue:  
Our Brigham and Women’s Hospital Core Pathology Lab and Immune-oncology Core 
has developed significant expertise for immunohistochemical staining and multiplex 
immunofluorescence under the leadership of Dr. Scott Rodig, Core Director and is now 
part of the CIMAC network. PD-L1, CD3, and CD8 IHC testing, in particular, have been 
established in a CLIA approved setting, and the PD-L1 IHC will be performed in a CLIA-
certified laboratory.  Dr. Rodig’s laboratory has experience with the IF analyses. We plan 
to use positive and negative controls to validate our results.  Extensive validation is not 
feasible for these exploratory biomarkers given the variety of tumor types.  We would 
plan for any hypothesis-generating results to be validated in future studies. 
Regarding our primary endpoint of PD-L1 expression in tumor tissue, mouse monoclonal 
anti-PD-L1 antibody (clone 9A11) was generated in the laboratory of Dr. Gordon 
Freeman (Dana-Farber Cancer Institute) and has been licensed to Cell Signaling 
Technology (Beverly, MA) for commercial use.  This antibody is human gene product 
and can recognize all isoforms.  The specificity of these antibodies has been confirmed 
by western blotting in human cancer cell lines and comparing the results with orthogonal 
methods of analysis (i.e. flow immunophenotyping using a panel of anti-PD-L1 
antibodies).  The specificity of IHC staining was established using genetically defined 
cells lines, and critically, staining was detected in cells with endogenous or engineered 
to express PD-L1 and negative in PD-L1 deficient or knock-down cancer cells. 
Specifically, the 9A11 antibody clone was compared to 4 other antibodies commercially 
available and used in other immunotherapy studies as shown in the figure below 
(Mahoney, Sun et al. 2015) across multiple tumor types such as might be encountered 
on this trial. Additional reproducibility data can be found on the cell signaling technology 
website https://www.cellsignal.com/products/primary-antibodies/pd-l1-405-9a11-mouse-
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Additional data supporting the use of the 9A11 antibody, and/or the techniques to be 
applied in this study, are provided by Howitt et al. JAMA Oncol. 2016 (cervix and vulvar 
cancers), Howitt et al. JAMA Oncol 2015 (MSI high endometrial cancers), Derks et al. 
Oncotarget 2016 (gastric cancer), Roemer et al. JCO 2016 and Ansell et al. NEJM 2015 
(Hodgkin Lymphoma), George et al. Immunity 2017 (leiomyosarcoma), and Strickland et 
al. Oncotarget 2016 (ovarian cancer)   Furthermore, a recently published quantitative 
comparison demonstrates high concordance between PD-L1 staining between 9A11 and 
5 other commercially available antibodies directed against PD-L1 including the PD-L1 
IHC 22C3 PharmDx kit (Dako) approved by the FDA as a companion diagnostic for 
pembrolizumab and the PD-L1 SP142 Ventana test (Ventana medical systems) 
approved by the FDA as a complimentary diagnostic for nivolumab and atezolizumab 
(Gaule et al. JAMA Oncol 2016). This study was performed using automated scoring 
using a PD-L1 index microarray including cell line and tissue controls as well as a 
commercially available, genetically defined PD-L1 engineered cell line array with a range 
of controlled protein expressing cell lines. These data support the feasibility of PD-L1 
IHC, specifically using the 9A11 antibody, in a clinical trial setting across multiple 
disease types, including specifically among patients with MSI tumors and demonstrate 
that the various patterns of PD-L1 expression (tumor, stromal, tumor-stromal interface) 
can be reliably determined.  
Below, we also provide reproducibility data generated in our labs regarding PD-L1 
staining in colorectal and lung cancer. We demonstrate consistency of PD-L1 staining 
with the 9A11 antibody on 2 separate days on 2 sequential tissue slides across 10 

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colorectal cases and 7 lung cancer cases. 
 
 
Figure: Analysis of PD-L1 staining in 10 randomly selected colorectal cancers (top) and 7 
randomly selected NSCLC (bottom). IHC was performed on two different days and PD-L1 was 
analyzed in tumor and tumor surrounding stroma. Reproducibility was assessed on the 17 cases 
provided above demonstrating concordance in results obtained on different days.   
Additional data confirming this antibody produced interpretable results on Western blot 
(suggesting specificity) and across a variety of samples including tissues known to be 
positive for PD-L1 such as tonsil, placenta, PD-L1 controls (HDLM-2 and PC-3), and a 
range of tumor tissue can be found here: https://media.cellsignal.com/pdf/29122.pdf.  

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The data presented from Gaule et al. also show specificity given concordance among 
PD-L1 antibodies specifically in the low PD-L1 scoring samples. The data obtained from 
Gaule et al. demonstrates highly concordant and reproducible findings with IHC 
performed using the 9A11 antibody compared to other commercially available and 
validated PD8L1 antibodies FDA approved as companion diagnostics (see figure below): 
 
 
Figures (above): Data demonstrating Signal to Noise ratios and DAB staining with the 9A11 antibody 
compared with other widely used PD-L1 antibodies. Copied from supplementary materials from Gaule 
et al. JAMA Oncol. 2016. 
Beyond PD-L1 IHC, other tissue biomarkers are exploratory.  As above, meta-analyses 
have demonstrated the feasibility of assessing many of these exploratory immune 

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markers across cancer types.  Our pathology group has published the methods, 
protocols, and data for many of these antibodies including PD-L1 as above and other 
antibodies to be used in this series of studies such as PD-L2. Antibodies for many other 
markers such as CD3 and CD8 are commercial and are currently used in the CLIA-
certified hospital laboratory of Brigham and Women's Hospital for diagnostic purposes. 
As part of the validation assays in a CLIA-certified laboratory, identical cases were 
stained multiple times and under a variety of staining conditions and the results reviewed 
by two certified pathologists.  A positive control sample (such as classical Hodgkin 
Lymphoma for PD-L1 expression; primary mediastinal large B-cell lymphoma for PD-L2 
expression) and negative control sample (benign lymph node) is stained with each 
experimental tissue biopsy sample.  The controls are reviewed by a certified pathologist 
at the time of review of the experimental sample. 
The primary analysis will be performed on PD-L1 expression as a continuous PD-L1 
metric (% positive tumor cells).  Digital, quantitative scoring of stained tissue is 
performed using a slide scanning and analysis platform. Quantitative assessment of 
positive staining uses the commercially provided algorithm for cell identification and 
positive pixels counting within a pre-defined DAB (brown, chromogenic) channel. We 
have shown that this method of analysis shows good correlation with pathologists' 
scoring: Mino-Kenudson M et al., Clin Cancer Res. 2010 Mar 1;16(5):1561-71. 
PMID:20179225. [31]. We have also used this method to score PD-L1 expression in 
tumor cells: Green MR et al., Blood. 2010 Oct 28;116(17):3268-77. PMID: 20628145.[32]  
The quantitative scoring method is summarized in the following steps:  
1. The pathologist takes 3-5 representative pictures for each case at 200x magnification 
2. Tissue classification of the images is performed, where tumor and stroma are 
distinguishably labeled. 
3. The pathologists perform “cell segmentation” where the whole image is segmented 
into individual cells based on hematoxylin as a nuclear stain  
4. The pathologist trains the software to identify PD-L1 (+) and PD-L1 (-) cells on a cell-
to-cell basis, based on expression and comparison to control tissues (tonsil). 
5. Thresholds are established and then applied to the whole image by the pathologist. 
6. A second pathologist checks the threshold, and either approves or disapproves it. In 
case of disproval, the two pathologists reconcile between them. 
7. Scoring for the positive cells is then performed in the tumor compartment.  
Depending on tissue availability, other exploratory IHC analyses will be performed using 
semi quantitative scoring, although multiplex IF may be used as below to analyze 
multiple markers in tandem. Chen et al. [30] describes a semi-quantitative scoring 
method, which is in accordance with typical biomarker scoring in anatomic and surgical 
pathology [30]. Briefly, staining is scored according to intensity (0= no staining, 1= weak 
staining, 2= moderate staining, 3= strong staining), staining pattern (M= predominantly 
cell membrane; C= predominantly cell cytoplasm), and the percentage of cells showing 
positive staining (0-100%). The semi-quantitative scoring is performed for: 1) the 
neoplastic tumor cells and 2) the non-neoplastic infiltrating immune cells. 
In the research setting, all cases are reviewed by two pathologists and any discordant 
results resolved by consensus review. Significantly discordant scoring results have been 
rare during case evaluations [30]. IHC scoring for markers (such as the PD-Ligands) that 
stain macrophages, dendritic cells, and other cells of heterogenous morphology will also 
be semi-quantitative and performed by a pathologist to capture 1) the percentage of 
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membrane or cytoplasmic expression, and 2) the percentage of non-neoplastic cells 
(macrophages, dendritic cells, endothelial cells) positive for biomarker expression, 
intensity of expression, and membrane or cytoplasmic expression. 
Scoring for PD-1+ (and other lymphoid markers) lymphocytes will be accomplished using 
a standard algorithm, developed for quantifying nuclear stains, but found to be 
applicable to quantifying membrane staining of cells with a very high N:C ratio, such as 
lymphocytes (Nuclear algorithm).  The output is number of positive-staining cells per unit 
area (micron2). 
For image analysis: 
IHC stained slides will be digitally scanned using the Aperio ScanScope XT (Leica 
Microsystems, Buffalo Grove, IL). The instrumentation is housed in the Tissue 
Microarray and 
Imaging Core (TMI) facility of the Dana-Farber/ Harvard Cancer Center (DF-/HCC). This 
facility is located adjacent to the office of Dr. Scott Rodig in the Department of 
Pathology. All digital images are stored on servers owned by the TMI core facility and 
accessed via the internet using a password-protected log-in. 
Digital images are viewed using ImageScope Software(version 10.0.35.1800; Leica) on 
standard PCs. Slides are digitally annotated by the pathologist (Dr. S. Rodig) to identify 
the region of interest and analysis. 
Quantitative analysis is performed using analytical software (for PD-Ligands). Intensity of 
staining is also captured automatically using the above algorithms and assigned a score 
(0, 1, 2, or 3) based upon the average optical density of the region or cells. All results 
are exported into an excel spreadsheet. 
Individual scoring data will be compared to clinical parameters to determine if there is an 
association with outcome. Scores using a combination of biomarker data will also be 
considered. 
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The semi-quantitative scoring for this study is in accordance with those published 
previously [30, 33] and will include scores for both the neoplastic and non-neoplastic 
cells within the tumor microenvironment. Data derived from pathologist visual scoring 
(semi-quantitative, but with increased specificity for delineating neoplastic and non-
neoplastic cells) and pathologist-assisted, automated scoring (quantitative, but without 
accurately delineating neoplastic and non-neoplastic cells) for each marker of interest 
will be assessed for its clinical value. As necessary, the data from combinations of 
markers will also be considered.  
All IHC staining will be performed in the Center for Immuno-Oncology Pathology Core at 
Dana-Farber/Harvard Cancer Center Specialized Histopathology Core which is a 
designated Cancer Immune Monitoring and Analaysis Center (CIMAC). Standard 
EnVision two-step (indirect) staining method will be utilized. Four-micrometer-thick 
sections will be cut, deparaffinized, rehydrated and subjected to heat mediated antigen 
retrieval in citrate buffer (pH 6) (Invitrogen) by steaming for 30 minutes. After cooling, 
tissue sections will be incubated with peroxidase block (DAKO, Carpinteria, CA) for five 
minutes, then serum free protein block (DAKO) for 20 minutes. Slides will be incubated 
at room temperature for one hour with a primary antibody. Antibodies will be diluted in 
Da Vinci Green Diluent (Biocare Medical, Concord, CA). EnvisionTM anti-mouse HRP-
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visualization by using the chromogen 3, 3-diaminobenzidine (DAKO). All the sections will 
then be counterstained with haematoxylin, dehydrated, mounted and coverslipped. 
Positive and negative controls shall be included in each staining. Known positive stained 
Hodgkin lymphoma (CD274) will be used as external control (separate slides). Stained 
slides will be stored at room temperature. 
For multiplex immunofluorescence, we use the Opal™ Multiplex Immunohistochemistry 
kit, which is a practical workflow for simultaneous detection of up to 6 tissue biomarkers 
plus nuclear counterstain, within a single image. Antibodies for simultaneous IHC may 
be selected based on performance, rather than species. This kit provides the tools to 
interrogate multiple pathways while retaining context provided by tissue images. 
Methods: Step1. Overnight baked FFPE slides are de-paraffinized and then placed in 
AR6 solution (PerkinElmer) for 15min using a microwave. AR6 solution was developed 
for use in the multicolor immunohistochemistry protocol in microwave treatment, and 
provides antigen retrieval for FFPE tissues, quenches endogenous peroxidase, and 
removes antibodies after epitopes have been labeled with Opal dyes. Step 2. After the 
antigen retrieval step, the slides are cooled in room temperature for 20 min and then 
washed with distilled water and then with Tris buffer. Step 3. Afterwards, the first 
antibody for Protein 1 (example: PD1) is applied for 40 minutes. Step 4. HRP-linked 
secondary antibody (Dako Envision kit) is applied for 30 minutes. Step 5. After a few 
washes in Tris buffer (3x2minutes) the 1st Opal Fluorophore is applied (1:50, diluted in 
Amplification Diluent) for 5 minutes. Step 6. The slides are washed in Tris buffer and 
placed in pre-heated AR6 solution for 15 minutes using a microwave. Step 7. After this, 
the first antibody for Protein 2 (example: CD30) is applied and all the Steps 2-6 are 
repeated. Currently, using the Opal-7 Color Immunohistochemistry Kit, we can stain for 6 
different proteins and Dapi. 
To accurately visualize, analyze and quantify the different biomarkers in tissue sections, 
we use InForm®, which is an automated image analysis software package 
(PerkimElmer). inForm enables accurate per cell quantification of specific biomarkers 
defined within multiple tissue types including tumor and stroma and the phenotyping of 
immune cells and other cells in situ in solid tissue. It also enables the quantitation of 
weakly expressing and overlapping biomarkers within cells, nuclei and membranes, 
which cannot be identified by eye. This sensitive approach provides researchers with the 
confidence to discover indicators of disease and uncover relationships between 
biomarkers, improving results reliability. 
Assay performance 
As above, validation has been performed on retrospective data sets. Protocols have 
been optimized and standardized to minimize staining variance. Positive control and 
negative controls were used and stained separately with each batch of slides for PD-L1 
staining. The IHC staining of PD-L1 has been performed in two different labs by three 
different technicians on whole tissue sections of Hodgkin lymphomas, melanomas, lung 
cancers and renal cell carcinomas. Three readers were involved, confirming the good 
reproducibility of the assay. Consistency of automated PD-L1 scoring has been 
confirmed on 10 colorectal and 7 lung cancer cases.  
Laboratory information 
The IHC and IF staining will be conducted in a research laboratory with GLP standard 
under the direction of Dr. Scott Rodig. Dr. Scott Rodig is the overall supervisor of the lab, 
and is a hematopathologist at the Brigham and Women’s hospital with prior expertise 
evaluating PD1, PDL1 and other immunologic markers in paraffin embedded tumor 

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samples [30]. PD-L1 IHC and the multiplex IF panel examining CD8+ T-cell infiltration 
will be performed under the auspices of the CIMAC. Other exploratory IF panels, 
specifically those exploring hypothesized mechanisms of resistance (CTLA-4, MHC-1, 
MHC-2, Lag-3, B2M, Na channel) will be performed in the same laboratory but are not 
currently planned to be incorporated as core CIMAC assays.    
Laboratory methods for evaluating serum and PBMCs:   
Peripheral blood mononuclear cells (PBMC’s) will be analyzed using CyTOF via the 
CIMAC. DFCI CIMAC reference document is in development and validation data will be 
sent to NCI under the CIMAC vetting process. Existing analytical validation data is 
available from Klensteuber et al.: Standardization and Quality Control for High-
Dimensional Mass Cytometry Studies of Human Samples Cytometry A. 2016 Oct; 
89(10): 903–913.  As an example of the capabilities of  the CyTOF assay in general to 
be offered through the CIMAC, please see attached reference document provided by 
Human Immune Monitoring Ceter at Mt. Sinai. As described in this reference document, 
“CyTOF offers single-cell level detection of 40+ markers with inter-channel cross talk 
levels <4% and very low background signals (<2 integrated dual counts ion per cell). The 
accuracy of individual antibodies is expected to be comparable to the analogous 
antibody clones used for conventional flow cytometry, and the accuracy of defined cell 
population identification is comparable to, if not better than flow cytometry.”    
Cytokine / serum analysis:  
Cytokines will be analyzed using Olink via the CIMAC (Mt. Sinai). Please see attached 
reference document provided by Human Immune Monitoring Center at Mt. Sinai. As 
described in this reference document, O-link is “a 92-parameter targeted soluble factor 
immunoassay” that allows for “quantification and correlation of multiplexed cytokine 
levels.”   Precision, analytical sensitivity, and analytical specificity are described in more 
detail in this document.    
A. Facilities & personnel 
PD-L1 IHC and multiplex IF including CD8+ T-cell analysis will be performed in the 
laboratory of Dr. Scott Rodig at the DFCI as part of the DFCI CIMAC.  Other 
multiplex IF panels described above examining mechanisms of resistance will be 
performed in the same laboratory, but are not currently CIMAC assays.   
CyTOF will be performed via the DFCI CIMAC under the direction of Dr. Mariano 
Severgnini.  
Olink will be performed via the Mt. Sinai CIMAC - Human Monitoring Core / Dr. 
Miriam Merad MD PhD, Director. 
IV. 
Statistical Considerstions 
A. 
Endpoints 
1. 
Primary 
a) PD-L1 positivity 
b) Percent of circulating, activated CD8+/HLA-DR+/Ki-67+ T-cells  
2. 
Secondary 
a) 
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b) 
Infiltration of lymphocytes per high powered field  
c) 
Exploratory analyses of expression levels of various serum 
cytokine and tumor immune biomarkers 
B. 
Case selection and Sample Size 
All consenting cases with adequate biospecimen available from the Z1D subarm.   
Sample size estimate: 44 patients for tissue analyses, 37 patients for blood 
analyses 
C. 
Analysis plan:  
Regarding the primary endpoint of PD-L1 expression in tumor tissue, an unpaired 
t test will be used to compare the percent positive tumor cells in responders vs. 
non-responders. Responders will be determined using the primary endpoint of 
the trial – objective tumor response. Using the data provided in the 
supplementary appendix to Le et al. as a basis for the power calculations (Le DT, 
Uram JN, Wang H, et al. PD-1 blockade in tumors with mismatch-repair 
deficiency. N Engl J Med 2015;372:2509-20) it is assumed that PD-L1 
expression, measured as a percentage of positive cells, will have a mean 
percentage of roughly 20% and a standard deviation of about 10%. Using these 
figures and assuming also that roughly 24% of all patients in this subarm will be 
responders, with approximately 44 patients with tissue available (roughly 11 
responders and 33 non-responders) there is at least 80% power to detect a 
mean difference of 10% (e.g., 29% versus 19%) in PD-L1 expression between 
responders and non-responders using a two-sided 0.05 level t-test. The 24% 
figure used for the calculations was chosen based on the results of this arm 
recently presented at SITC.  
Given the small sample size of this subarm, a categorical analysis to correlate 
objective response with PD-L1 positivity (for example via Fisher’s exact test 
using a 5% cutpoint for PD-L1 positivity) will not have meaningful power, hence 
the proposed analyses will be done on the continuous PD-L1 metric and any 
categorical analyses will be considered exploratory. Following the logic of the 
power analyses above, there is 80% power to detect a 1.00 standard deviation 
difference in the means of any quantitative biomarker between responders and 
non-responders (assuming the overall response rate above) using a two-sided 
0.05 level t-test.  
The primary objective is to examine the association between a single biomarker 
(PD-L1 expression) and response.  Thus, no statistical adjustments are planned 
for this analysis.  Other planned IF and IHC testing is exploratory and largely 
based on mechanistic hypotheses (e.g. exploring for treatment resistance as a 
consequence of defects in the MHC I or MHC II expression and antigen 
presentation pathways).  Therefore, exploratory analyses will be descriptive in 
nature with no planned adjustment for multiple testing. 
When genomics data are available from the NCI MATCH study we may perform 
exploratory analyses to examine associations with protein expression across the 
markers tested. Specifically, there are immediate plans to attempt to look at 
numbers of MOIs based on the NCI MATCH assay as a proxy for high mutational 
burden for arm Z1D and correlate this with response.  We will correlate this 
measure with our primary endpoint of PD-L1 expression as well as with our other 
exploratory endpoints.  

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The primary endpoint of studies to be conducted on peripheral blood will evaluate 
changes in percentage of circulating CD8+/HLA-DR+/Ki-67+ T cells and 
response. We will compare the change in percentage of this cell population 
between responder and non-responder patients. To give a sense of power for 
studies to be done on paired tissue and blood samples (baseline versus 
progression), and blood samples (paired within patient comparing baseline vs 
progression) for analyses of various quantitative biomarkers being proposed 
here, differences will be taken for all measures and the hypotheses is that 
biomarkers will change (increase or decrease) post therapy. PD-L1 expression 
on tumor cells, infiltrating and circulating T-cell populations, myeloid derived 
suppressor cells, and immunologic cytokines and other immunologic markers.  
Assuming that within this cohort we will obtain paired samples on conservatively 
40 patients, using a two-sided 0.05 level paired t-test there is at least 80% power 
to detect a 0.46 SD change in mean of a quantitative correlative parameter using 
a two-sided test with 5% type I error. Quantitative measure will be transformed to 
attain normality or appropriate non-parametric tests will be conducted. With this 
level of consent for these studies in this cohort there is sufficient power for 
meaningful analyses. Power for these tests will be further improved if a greater 
percentage of the cohort of 47 patients consent and provide post-baseline 
(progression) samples for analysis.  
D. 
Rationale for the sample size estimate: 
As described above, assuming that roughly 24% of all patients in this subarm will 
be responders, with 44 patients (expected distribution to be roughly 11 
responders and 33 non-responders) there is at least 80% power to detect a 
mean difference of 10% (e.g., 29% versus 19%) in PD-L1 expression between 
responders and non-responders using a two-sided 0.05 level t-test.  
For the blood studies, assuming approximately 40 patients have usable 
specimens pre and post treatment, using a two-sided 0.05 level paired t-test 
there is at least 80% power to detect a 0.46 SD change in mean of a quantitative 
correlative parameter using a two-sided test with 5% type I error. 
We propose to evaluate associations with PD-L1 expression measured as a 
continuous metric of positivity (as described above) as opposed to a single cutoff.  
Exploratory analyses will assess specific cutpoints of PD-L1 positivity (e.g. 1%, 
5%, 10%, etc.) as potential biomarkers, as these cutpoints have been described 
previously in the literature. To our knowledge there are unfortunately no on-
treatment pathologic specimens available and therefore magnitude of change on 
treatment cannot be assessed.  As mentioned in the above statistical analysis 
plan, assuming that within this cohort we will obtain paired samples on 
conservatively 40 patients that yield evaluable data for our flow-cytometric 
biomarkers, using a two-sided 0.05 level paired t-test there is at least 80% power 
to detect a 0.46 SD change in mean of a quantitative correlative parameter using 
a two-sided test with 5% type I error. Quantitative measures will be transformed 
to attain normality or appropriate non-parametric tests will be conducted as 
appropriate.  We will also compare changes between responder and non-
responder groups for the purpose of our analyses. 
E. 
Statistical considerations for secondary objectives (if applicable) 
Our secondary objectives for testing multiple biomarkers in tissue and serum are 
exploratory. However, these objectives are based on strong biologic rationale 

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patients with mismatch repair deficiency or other tumors.  
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