Clinical trial NCT03634800
Conditions: Multiple Myeloma
Sponsor: Weill Medical College of Cornell University
Start date: 2018-10-23; Completion date: 2020-01-16
Enrollment: 6 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Eligible patients have multiple myeloma with measurable disease in the blood and a targetable soft tissue or bony lesion with radiotherapy. All eligible patients will receive immunotherapy (Nivolumab) plus radiotherapy, 6 Gy x 5 fractions, to a targetable lesion. Immunotherapy treatment starts with the first radiotherapy fraction. Nivolumab will be given every 2 weeks. Patients will have specified laboratory values measured bi-monthly and evaluated for response at 12 weeks as defined by International Myeloma Working Group Criteria. Patients will continue to receive their respective immunotherapy until disease progression or dose limiting toxicity is reached.

Detailed description:
All eligible patients will receive immunotherapy (Nivolumab) plus radiotherapy, 6 Gy x 5 fractions, to a targetable lesion. Immunotherapy treatment starts with the first radiotherapy fraction. Nivolumab will be given every 2 weeks. Patients will have specified laboratory values measured bi-monthly and evaluated for response at 12 weeks as defined by International Myeloma Working Group Criteria. Patients will continue to receive their respective immunotherapy until disease progression or dose limiting toxicity is reached.

Arms: arm 1: All eligible patients will receive immunotherapy (Nivolumab) plus radiotherapy (6 Gy x 5 fractions) to a targetable lesion.

* Nivolumab 240 mg IV starts with the first radiotherapy fraction 240 mg IV every 2 weeks from first radiotherapy fraction until disease prograssion or dose limiting toxicity is reached
* Radiotherapy Dose of 6 Gy x 5 days will be given (patients will receive 1 fraction over 5 days for a total of 5 fractions) during the first week of starting Nivolumab
Interventions: intervention 1: Nivolumab 240mg intervention 2: Radiation therapy
Intervention descriptions: intervention 1: Patients with multiple myeloma will receive Nivolumab intravenously at 240 mg every two weeks. Infusions will be given over 60 minutes (not bolus or IV push). Patients will continue to receive infusions every two weeks until disease progression or dose limiting toxicity is reached. intervention 2: Patients will receive radiation for 5 consecutive days. A dose of 6 Gy x 5 days will be administered. Patients may receive radiotherapy to an additional site at 12 weeks if they have stable disease.

Protocol documents:
Prot_SAP_000.pdf: 
Identifiers: NCT03634800 
Unique Protocol ID: 1612017831 
 
Brief Title: Radiotherapy and Immunotherapy 
for Systemic Effect in Myeloma (RISE-M) Trial 
 
 
Version 3.0  
 
Version date: 28JUN2019 
 
IRB approval: 10JUL2019 
 
 
 
Protocol Amendments 
 
Protocol type  
Version number  
Version date  
Amendment 2  
3.0  
06/28/2019  
Amendment 1  
2.0  
03/12/2019  
Initial  
1.0  
09/06/2018  


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Protocol 1612017831 
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TITLE: Radiotherapy and Immunotherapy for Systemic Effect in Myeloma (RISE-M) Trial 
 
 
IRB Protocol #: 1612017831  
Version Number: 3.0 
Version Date : 06/28/2019 
 
Principal Investigators: 
 
 
Dr. Himanshu Nagar – Radiation Oncology Dr. Adriana Rossi – Hematology & Oncology 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Confidentiality Statement 
 
 
This document is confidential and is to be distributed for review only to investigators, potential 
investigators, consultants, study staff, and applicable independent ethics committees or 
institutional review boards. The contents of this document shall not be disclosed to others 
without written authorization from WCM.


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List of Abbreviations 
All abbreviations used throughout the protocol must be defined. 
 
 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CTSC 
Clinical Translational Science Center 
DSMB 
Data Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
HRBFA 
Human Research Billing Analysis Form 
HUD 
Humanitarian Use Device 
ICF 
Informed Consent Form 
IDE 
Investigational Device Exemption 
IMWG 
International Myeloma Working Group 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
PHI 
Protected Health Information 
PI 
Principal Investigator 
REDCap 
Research Electronic Data Capture 
SAE 
Serious Adverse Event 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
UAP 
Unanticipated Problem 
WCM 
Weill Cornell Medicine 
MV  
Megavolts 
Gy 
Gray, derived unit of ionizing radiation dose 
MeV 
Megaelectronvolts 


 
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Protocol Amendments 
 
Protocol type 
Version number 
Version date 
Amendment 2 
3.0 
06/28/2019 
Amendment 1 
2.0 
03/12/2019 
Initial 
1.0 
09/06/2018 
 
 
SUMMARY OF CHANGES: version 3.0 version date : 06/28/2019 
 
1. Adding Dr. Usama Gergis as a co-investigator. 
2. Removing Pragya Yadav as the data manager and adding Drew Ribadeneyra as the data 
manager. 
3. Removing Sheetal Ramnath as the study coordinator. 
4. Adding Ramya Krisha Chunduru as the regulatory coordinator. 
5. Removing Sharanya Chandrasekhar as the study contact.  
 
 
Summary of changes:  
 
1. Changing personnel : Replacing Research Nurse Linda Tegnestam with Kathleen 
Pogonowski 
2. Changing personnel: Data Manager (Myeloma team): Brielle Liotta  
3. Section 3.3 - Exclusion criteria #12 - Prior exposure to immune checkpoint inhibitor 
4. Correcting Nivolumab infusion from 60 mintues to 30 minutes as per BMS and FDA treatment 
prescriptions. 
5. Clarifying stool collection timepoints : Baseline, end of radiation, C4D1 and C7D1 
6. saliva collection added to the calendar for C1D1 (before infusion) 
7. Adding investigator : Dr. Cara Rosenbaum  
8. Updated Appendix 2 : for stool collection timepoints.   
9. Clarifying radiation given as 5 fractions and removing “consecutive days” language.  
10. Removing immune monitoring blood samples from section 8.1, 14.2 and 14.5.  
11. Revising bone marrow aspiration to 20ml. 
 
Informed consent changes :  
 
1. Clarifying that patients will receive 5 fractions of radiation treatment.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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Protocol Summary 
 
Full Title: 
Radiotherapy and Immunotherapy for Systemic Effect in Myeloma 
 
(RISE-M) Trial 
Short Title: 
RISE-M Trial 
 
Clinical Phase: 
 
  II 
Principal Investigator: 
Dr. Himanshu Nagar, MD 
Sample Size: 
N = 30 
Accrual Ceiling: 
This study will enroll 30 subjects and screen up to 50. 
Study Population: 
Eligible patients will have multiple myeloma. 
Accrual Period: 
2 years 
Study Design: 
Eligible patients have multiple myeloma with measurable disease in 
the blood and a targetable soft tissue or bony lesion with radiotherapy. 
All eligible patients will receive immunotherapy (Nivolumab) plus 
radiotherapy, 6 Gy x 5 fractions, to a targetable 
lesion. 
Immunotherapy treatment starts with the first radiotherapy fraction. 
Nivolumab will be given every 2 weeks. Patients will have specified 
laboratory values measured bi-monthly and evaluated for response 
a t  1 2  w e e k s  as defined by International Myeloma Working Group 
Criteria. 
Patients 
will 
continue 
to 
receive 
their 
respective 
immunotherapy until disease progression or dose limiting toxicity is 
reached. 
Study Duration: 
Patients will have approximately 25-30 visits and a follow up for up to 
3 years. 
 
Study Agent/Intervention: 
Patients with multiple myeloma will receive Nivolumab intravenously at   
240 mg every two weeks. Infusions will be given over 30 minutes (not 
bolus or IV push). Patients will continue to receive infusions every two 
weeks until disease progression or dose limiting toxicity is reached. 
Patients will receive 5 fractions of radiation. A  dose of 6 Gy x 5 days will 
be administered. 
Primary Objective: 
The primary aim is to estimate the overall response at 12 weeks using 
IMWG criteria in patients with multiple myeloma when treated with 
immunotherapy and radiotherapy. 
 


 
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Secondary Objectives: 
The secondary aims are: 1) to estimate the median progression free 
survival and the median overall survival for patients treated with 
immunotherapy and radiotherapy, and 2) to assess the toxicity (per 
CTCAE version 4.0) of patients treated with immunotherapy and 
radiotherapy. 
Exploratory Objectives: 
Exploratory analyses will include: 1) investigate the expression of PD- 
L1 and PD-L2 on myeloma plasma cells prior to and at various time 
points after immune checkpoint blockade and radiotherapy, 2) 
investigate the population of T cells (cytotoxic T cells, helper T cells 
and regulatory T cells) and their expression of immune checkpoint 
receptors (PD-1, CTLA-4, LAG3, 2B4, BTLA, TIM3, A2aR) prior to and 
at various time points after checkpoint blockade and radiotherapy, 
3) investigate mutational burden of pre-treatment myeloma samples in 
the bone marrow and target site utilizing FoundationOne Heme 
4) investigate the T cell receptor (TCR) repertoire in the bone marrow 
aspirateprior to and at various time points after immune checkpoint 
blockade and radiotherapy and 5) microbiome analysis prior to and 
after immunoradiotherapy. 
Endpoints: 
The primary endpoint will be the overall response rate using IMWG 
criteria. Other end points include progression free survival and 
overall survival. 


 
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Table of Contents 
Protocol Summary .......................................................................................................................... 7 
SCHEMA ....................................................................................................................................... 12 
1 
Study Objectives .................................................................................................................... 13 
1.1 
Primary Objectives ..................................................................................................................... 13 
1.2 
Secondary Objectives ................................................................................................................. 13 
1.3 
Exploratory Objectives ............................................................................................................... 13 
2 
Background ........................................................................................................................... 13 
2.1 
Disease...................................................................................................................................... 13 
2.2 
Investigational Agent ................................................................................................................. 14 
2.3 
Rationale ................................................................................................................................... 14 
2.3.1 
Overview ........................................................................................................................................................... 14 
2.3.2 
PD1/PDL1 and T-Cell Activation ....................................................................................................................... 14 
2.3.3 
Multiple Myeloma and PD1/PDL1 .................................................................................................................... 15 
2.3.4 
Abscopal Effect of Radiotherapy ...................................................................................................................... 16 
2.3.5 
Radiotherapy and Immunity ............................................................................................................................. 16 
2.3.6 
Harnessing   the   Pro-Immunogenic    Effects    of Radiation in Cancer Treatment: A New Paradigm .......... 17 
2.4 
Risk/Benefit Assessment ............................................................................................................ 18 
2.5 
Correlative Studies Background .................................................................................................. 18 
2.6 
Microbiome Correlative studies .................................................................................................. 21 
2.6.1 
To explore associations of ORR with changes in the microbiome. ................................................................... 21 
3 
Subject Selection ................................................................................................................... 23 
3.1 
Study Population ....................................................................................................................... 23 
3.2 
Inclusion Criteria ........................................................................................................................ 23 
3.3 
Exclusion Criteria ....................................................................................................................... 24 
4 
Registration Procedures......................................................................................................... 24 
4.1 
Patient Registration ................................................................................................................... 24 
5 
Study Calendar ...................................................................................................................... 25 
5.1 
Schedule of Assessment ............................................................................................................. 25 
5.1.1 
Screening Visit – (Day 0  -  day28) ..................................................................................................................... 26 
5.1.2 
Treatment Phase .............................................................................................................................................. 26 
5.2 
Response Assessment at Week 12 .............................................................................................. 26 
5.3 
Duration of Therapy and Criteria for Removal from Study: ........................................................... 26 
5.4 
Duration of Follow Up ................................................................................................................ 26 


 
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6 
Radiation Therapy Guidelines ................................................................................................ 27 
6.1 
Planning .................................................................................................................................... 27 
6.2 
Treatment ................................................................................................................................. 27 
6.3 
Treatment modifications for Radiation Adverse Events: .............................................................. 28 
6.4 
On Study Evaluations: ................................................................................................................ 28 
7 
Pharmaceutical Information .................................................................................................. 28 
7.1 
Investigational Agent ................................................................................................................. 28 
7.2 
Availability ................................................................................................................................ 28 
7.3 
Preparation and Administration of Nivolumab ............................................................................ 29 
7.4 
Dose Modifications, Delays or Discontinuation of Nivolumab ..................................................... 29 
8 
Correlative Studies ................................................................................................................ 30 
8.1 
Bone Marrow tissue aspirate ..................................................................................................... 30 
8.2 
Tissue biopsies ........................................................................................................................... 30 
8.3 
Other studies : ........................................................................................................................... 30 
8.4 
Microbiome samples .................................................................................................................. 31 
9 
Measurement of Effect .......................................................................................................... 32 
9.1 
Standard IMWG Response Criteria ............................................................................................. 32 
10 
Data Reporting / Regulatory Considerations ...................................................................... 35 
10.1 
Data Collection .......................................................................................................................... 35 
10.1.1 
REDCap ......................................................................................................................................................... 35 
10.2 
Regulatory Considerations ......................................................................................................... 35 
11 
Statistical Considerations ................................................................................................... 35 
11.1 
Sample Size/Accrual Rate ........................................................................................................... 35 
11.2 
Stratification Factors .................................................................................................................. 35 
11.3 
Analysis of Endpoints ................................................................................................................. 35 
11.3.1 
Analysis of Primary Endpoints ...................................................................................................................... 36 
11.3.2 
Analysis of Secondary Endpoints.................................................................................................................. 36 
11.3.3 
Interim Analysis ............................................................................................................................................ 36 
11.3.4 
Reporting and Exclusions .............................................................................................................................. 36 
11.3.5 
Evaluation of toxicity .................................................................................................................................... 36 
11.3.6 
Evaluation of response. ................................................................................................................................ 36 
11.4 
Analysis of Correlative Endpoints ............................................................................................... 37 
11.4.1 
Microbiome .................................................................................................................................................. 37 


 
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11.5 
Statistical analysis of microbiome and metagenome composition ............................................... 37 
12 
Adverse Event Reporting Requirements .............................................................................. 38 
12.1 
Adverse Event Definition ............................................................................................................ 38 
12.1.1 
Collection of Safety Information .................................................................................................................. 38 
12.1.2 
Attribution of the AE: ................................................................................................................................... 38 
12.1.3 
Recording of adverse events ........................................................................................................................ 38 
12.2 
Reporting of Serious Adverse Events (SAEs) ................................................................................ 39 
12.3 
Off Study Criteria ....................................................................................................................... 41 
13 
PROHIBITED AND RESTRICTED THERAPIES DURING THE STUDY ........................................... 42 
13.1 
Prohibited Therapies .................................................................................................................. 42 
13.2 
Data and Safety Monitoring Plan (DSMP) .................................................................................... 42 
13.3 
Informed Consent ...................................................................................................................... 43 
14 
APPENDIX 1: Laboratory Correlate Manual ......................................................................... 43 
14.1 
At Baseline: ............................................................................................................................... 43 
14.2 
Baseline Bone Marrow Aspirate and Core ................................................................................... 44 
14.3 
Baseline Saliva sample ............................................................................................................... 44 
15 
APPENDIX 2. INSTRUCTIONS FOR COLLECTION OF STOOL SAMPLES AT HOME: .................... 45 
References ................................................................................................................................... 47 
 


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SCHEMA 
 
 
Bone marrow aspirate pre-Nivolumab infusion on C2D1, C4D1, C7D1, C10D1 and Q 12 weeks 
thereafter and as needed to confirm CR. 
One course of radiation (6 Gy x 5 fractions), with first radiation AFTER IV Nivolumab. 
All Infusions will have a ± 3 day window.  Nivolumab infusion 240mg q 2 week. 
 


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1 
Study Objectives 
 
1.1 
Primary Objectives 
The primary objective of the study is to estimate the proportion of patients with systemic 
response at 12 weeks using IMWG criteria in patients with multiple myeloma when treated 
with immunotherapy and radiotherapy. Systemic response is defined as partial or complete 
best response within this time 
1.2 
Secondary Objectives 
The secondary objectives of the study are: 1) to estimate the median progression free 
survival and the median overall survival for patients treated with immunotherapy and 
radiotherapy, and 2) to assess the toxicity (per CTCAE version 4.0) of patients treated with 
immunotherapy and radiotherapy. 
1.3 
Exploratory Objectives 
Exploratory analyses will include: 1) investigate the expression of PD-L1 and PD-L2 on 
myeloma plasma cells prior to and at various time points after immune checkpoint blockade 
and radiotherapy, 2) investigate the population of T cells (cytotoxic T cells, helper T cells 
and regulatory T cells) and their expression of immune checkpoint receptors (PD-1, CTLA-
4, LAG3, 2B4, BTLA, TIM3, A2aR) prior to and at various time points after checkpoint 
blockade and radiotherapy, 3) investigate mutational burden of pre-treatment myeloma 
samples in the bone marrow and target site utilizing FoundationOne Heme and 4) 
investigate the T cell receptor (TCR) repertoire in the bone marrow aspirate  prior to and at 
various time points after immune checkpoint blockade and radiotherapy. 
 
 
2 
Background 
2.1 
Disease 
Multiple myeloma remains a disease that requires innovative treatment with an increasing incidence 
of cases in the United States with a 50% mortality rate at 5 years 1. Interest in the role of the immune 
system in attacking malignancies across multiple tumor types has grown tremendously over the past 
few years with the advent of immune checkpoint inhibitors, including multiple myeloma 2. 
The immunomodulatory molecule PD-L1 has higher expression levels on malignant plasma cells and 
myeloma-propagating pre-plasma cell in the bone marrow of patients with multiple myeloma 
compared to healthy donors and patients with MGUS and smoldering multiple myeloma 3. 
Preliminary results from the ongoing KEYNOTE-023 phase  I study  that combined  an anti-PD1 
antibody with Lenalidomide and Dexamethasone in heavily pretreated patients showed a response rate 
of 76% 4. Preliminary results from an ongoing phase II study that combined an anti-PD1 antibody with 
Pomalidomide and Dexamethasone in heavily pretreated patients showed a response rate of 60% 5. 
Adverse events were consistent with previous studies of the drug in other cancers. 
Combining radiotherapy with immunotherapy presents potential therapeutic advantages. Because of its 
localized nature, radiotherapy is devoid of most systemic effects, including interference with systemic 
immunotherapy, commonly encountered with chemotherapy. Additionally, a radiotherapy-focused 
intervention on the tumor may selectively subvert its micro-environment and, in combination with the 
optimal immune intervention, ideally render the cancer an in-situ vaccine. 


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The promising data on the effect of PD1 blockade in multiple myeloma render this an optimal clinical 
setting to test this regimen with radiotherapy to assess the role of radiotherapy in inducing abscopal 
responses. Thus, we are proposing a pilot trial to assess the efficacy of combining PD1 blockade with 
radiotherapy in patients with relapsed/refractory multiple myeloma. 
 
2.2 
Investigational Agent 
Nivolumab (Opdivo®) is a fully human IgG4 PD-1 immune checkpoint inhibiting antibody that 
selectively blocks the interaction between PD-1, which is expressed on activated T cells, and PD-1 
ligand 1 (PD-L1) or 2 (PD-L2), which are expressed on both immune and tumor cells. The interaction 
between PD-1 and PD-L1 or PD-L2 normally results in inhibition of cellular immune responses. 
 
 
2.3 
Rationale 
2.3.1 Overview 
An attractive area of research supports the use of immune manipulations to recover patients’ initial 
antitumor immunity. It is a strategy that has the advantages of being both natural and potentially long-
lasting 6. We propose combining immunotherapy with radiotherapy directed to a target site to establish 
a “hub” for in situ immunization against an irradiated tumor enhancing “tumor rejection” in additional 
areas. The use of radiotherapy has been explored as a viable approach to establish an individualized 
vaccine. In a “proof of principle” clinical trial, radiotherapy was combined with GM- CSF, a dendritic cell 
recruiter and activator, in patients with metastatic solid tumors and was shown to be safe and effective 
at establishing abscopal responses in 27% of treated patients. Those who achieved an abscopal 
response not only possessed a healthier immune system (as demonstrated by a lower neutrophil to 
lymphocyte ratio) at baseline but also demonstrated a longer median survival7. Additionally, our group 
recently completed the accrual of a phase II clinical trial for patients with metastatic non-small cell lung 
cancer treated with ipilimumab, a CTLA-4 inhibitor and T-cell activator, plus radiotherapy to a metastatic 
site. Partial and complete responses were achieved (per RECIST version 1.1) in 18% of treated 
patients (approximately 30% of treated patients had an abscopal response), whereby CTLA-4 
blockade alone historically achieved response rates of only approximately 5 percent 7,8. An example 
of a patient with a complete response to treatment is shown (Figure 1). The patient was treated to 
one liver metastasis, 6 Gy x 5, while receiving CTLA-4 blockade. The post-treatment PET/CT 
(completed in January 2013)  demonstrated a complete response to therapy approximately 4 months 
after the initiation of treatment. The patient remains alive >3 years post treatment 9. 
 
2.3.2 PD1/PDL1 and T-Cell Activation 
The mechanistic rationale for the use of immunotherapy with anti-programmed death 1 (PD-1) 
receptor and anti-programmed death ligand 1 (PD-L1) antibodies are displayed in Figure 1 10. 
Antigen-presenting cells uptake tumor antigens released from cancer cells and present them to naïve 
T cells. This interaction results in activation or tolerization of the T cells, depending on the balance of 
stimulatory and inhibitory signals. Once activated, T cells can recognize and kill cancer cells that 
express and present the antigens in association with major histocompatibility (MHC) molecules, 
leading to immune-mediated tumor elimination. To avoid improper/excessive activation the immune 
system has developed  several “checkpoints”  that hinder T  cell activation  and/or effector function. 
Among them, the PD-1 receptor is expressed on T cells upon activation and inhibits immune responses 
through engagement of PD-L1 and PD-L2 on antigen presenting and cancer cells. While this pathway 
plays an important role in protection of normal tissue from collateral damage during an inflammatory 


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response, it has been shown to be often exploited by tumor cells to evade T cell mediated tumor 
rejection. In fact, monoclonal antibody-mediated blockade of PD-1 or its ligand PD-L1 has been 
successful in the clinic in multiple tumor types 11. Identification of patients who can benefit from 
blockade of PD-1/PDL-1 pathway and of combination treatments that can increase the number of 
responding patients is an area of active investigation, which appears to be related to, but not fully 
explained by PD-1/PD-L1 expression levels. 
 
 
 
 
 
 
2.3.3 Multiple Myeloma and PD1/PDL1 
Multiple myeloma is associated with progressive immune dysregulation characterized by decreased 
antigen presenting and effector cell function, loss of myeloma reactive effector T cell populations 
and a bone marrow microenvironment that promotes immune escape 12-14. The role that the PD- 
1/PD-L1 pathway plays in mediating immune escape and the corresponding therapeutic efficacy of PD-
1/PD-L1 blockade in multiple myeloma has emerged as an area of great interest 2. PD-L1 is highly 
expressed on plasma cells isolated from patients with multiple myeloma but not on normal plasma cells 
3. Notably, PD-L1 expression is up-regulated in the setting of relapsed and refractory disease 
suggesting a role in the development of clonal resistance 15. PD-L1 expression is associated with 
increased proliferation and increased resistance to anti-myeloma therapy 16. PD-1 is expressed on 
circulating T cells isolated from patients with advanced multiple myeloma, while expression of PD-1 on 
T cells is reduced in patients who achieve minimal disease state 17. Additionally, increased PD-1 
expression is observed on NK cells derived from myeloma patients associated with loss of effector cell 
function restored via PD-1 blockade 18. These findings support the role of the PD-1/PD-L1 pathway 
in contributing to immune escape in multiple myeloma and suggest that blockade may be an effective 
therapeutic strategy. 
The clinical efficacy of PD-1 blockade is most pronounced in malignancies such as melanoma and 
Figure 1. The mechanistic rationale for the use of immunotherapy with anti-programmed death 1 (PD-1) 
receptor and anti-programmed death ligand 1 (PD-L1) antibodies are displayed. Antigen-presenting cells uptake 
tumor antigens released from cancer cells and present them to naïve T cells. This interaction results in activation 
or tolerization of the T cells, depending on the balance of stimulatory and inhibitory signals. 


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Hodgkin lymphoma characterized by the presence of infiltrating effector cells in the tumor bed. 
Additionally, therapeutic efficacy has been correlated with mutational burden and thought to be 
associated with the presence of neoantigens derived from mutational events producing non-self- 
epitopes targeted by high affinity T cells 19. In contrast, myeloma is characterized by low levels of 
infiltrating effector cells and a modest mutational burden as compared to solid tumors suggesting a more 
restricted neoantigen profile. As such, it is likely that immune checkpoint blockade will be more potent 
when coupled with therapies that stimulate neoantigen presentation. Such approaches, including 
combining immune checkpoint blockade with radiotherapy, immunomodulatory drugs and cellular 
therapies such as tumor vaccines, are currently being studied in clinical trials. 
 
 
2.3.4 Abscopal Effect of Radiotherapy 
Originally described in 1953, the abscopal effect of radiotherapy is a remote effect of ionizing 
radiotherapy on tumors outside of the radiotherapy field 20. The phenomenon was termed the 
abscopal effect, from the Latin ab (position away from) and scopus (mark or target). The abscopal effect 
of radiotherapy is rare and its mechanism remains unexplained, although a variety of biologic events 
have been proposed including the involvement of the immune system 21-23. Investigators have 
reported findings consistent with the abscopal effect, which was the result of recovered anti- tumor 
immunity after radiotherapy 24-26. Notably, Saba et al. reported a case of a patient who originally 
presented with advanced multiple myeloma at the age of 50 who failed multiple therapeutic regimens 
and stem cell transplant. Subsequently, the patient achieved a sustained complete remission after  
receiving palliative radiotherapy to a gastric plasmacytoma and has remained in remission for over 
15 years 27. 
2.3.5 Radiotherapy and Immunity 
Tumor-targeted radiotherapy can generate immune-
stimulating effects and not immune suppression as was 
previously thought 28,29. Moreover, it has become clear 
that radiotherapy can induce profound effects on tumor 
cells and on the tumor microenvironment that can 
enhance or trigger an anticancer immune response. 
First, radiotherapy can induce an immunogenic cell 
death (ICD) of cancer cells 30,31. ICD is a way of cell 
dying that releases tumor antigens and evokes an 
immune response. ICD defines cell death associated 
with the release of immunostimulatory molecular 
signals, collectively termed “danger 
signals” that 
activate the antigen presenting cells, dendritic cells 
(DCs), to uptake the released tumor 
antigens and 
express costimulatory molecules and cytokines that 
together promote the activation 
of T cells and their 
differentiation into anti-tumor effectors. Secondly, 
radiotherapy has been shown to alter the tumor milieu by 
enhancing trafficking of immune cells, inducing inflammatory cytokines, adhesion molecules and NKG2D 
ligands promoting the recognition of cancer cells by cytotoxic T cells (CTL) 9. Thirdly, radiotherapy 
induces upregulation of MHC class I molecules, which are crucial for T cell activation 32. This effect is 
time and dose dependent at least in vitro with a plateau between 48 hours and 72 hours after exposure 
Figure 2. Local radiotherapy in combination with 
immune checkpoint blockade to break immune 
tolerance of established primary tumors has 
been shown to promote effector T cell mediated 
anti-tumor responses. 


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32. Fourthly, radiotherapy upregulates tumor-associated antigens, which are expressed at the surface 
of cells in association with MHC class I antigens 33,34. The upregulation of both MHC-I antigens and 
tumor-associated antigens may evoke the recognition of cancer cells by the immune system. Finally, 
there is evidence that radiotherapy alters the MHC class I associated peptide profile 32. Some of 
these peptides were specifically induced by radiotherapy and may therefore serve as radiotherapy-
specific antigens, which makes the recognition of these cells by the immune system more likely. 
Local radiotherapy in combination with immune checkpoint blockade to break immune tolerance  
of  established  primary  tumors  has  been shown to promote effector T cell mediated anti-tumor 
responses 35,36. The elicited immune response was effective against spontaneous metastases as 
well as the primary tumor. Regressing primary tumors demonstrated an increased infiltration of 
effector T cells and an expanded pool of circulating tumor- specific memory effector T cells was 
observed. These results demonstrate that tumor-directed radiotherapy may induce a therapeutically 
effective anti-tumor response in combination with immune checkpoint blockade, suggesting that the 
irradiated tumor can act as a vaccine and generate anti-tumor T cells that mediate local and systemic 
tumor rejection. 
 
 
2.3.6 Harnessing   the   Pro-Immunogenic    Effects    of Radiation in Cancer Treatment: A New 
Paradigm 
Experimental work done in two syngeneic mouse models (Lewis lung tumors and mammary carcinomas) 
testing radiotherapy with FLT-3 ligand (a growth factor for dendritic cells) demonstrated the induction of 
an immune response that reduced tumor growth outside the field of radiation 37,38. The findings inspired 
a clinical trial testing the combination of subcutaneously injected GM-CSF (a growth factor and activator 
of dendritic cells) with radiotherapy to a metastatic site in patients with solid tumors. GM-CSF increased 
the percentage of dendritic cells and their maturation, facilitating cross-presentation of newly released 
antigens after cell death at the site of radiotherapy. With a standard radiation fractionation of 3.5 Gy x 10 
fractions, abscopal responses were detected in 27% of the patients accrued to the trial 7. Those who 
achieved an abscopal response from therapy presented with a better pretreatment immune system (as 
demonstrated by a baseline neutrophil to lymphocyte ratio < 4) and subsequently demonstrated an 
improved overall survival. Abscopal responses were also detected among 15 patients with low-grade B-
cell lymphoma treated by low-dose radiotherapy to a single tumor site that was injected with a synthetic 
oligodeoxynucleotide (also referred to as CpG) that targets TLR9, express on the surface of dendritic 
cells. These compounds can activate both lymphoma B-cells as well as nearby antigen-presenting cells, 
particularly plasmacytoid dendritic cells, as previously demonstrated in a murine lymphoma model 39. 
Another combination strategy to overcome immune-tolerance consists of the blockade of the 
cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), a negative regulator of T cell activation. 
Prolonged survival and some cures occurred in a syngeneic model of poorly immunogenic mammary 
carcinoma, a process requiring CD8+T cells 35. Postow et al. recently reported a clinical case report 
with the same combination 40. A melanoma patient with disease progression while  receiving 
ipilimumab, a monoclonal antibody that targets CTLA-4, was treated with hypo-fractionated radiotherapy 
to a pleural-based paraspinal metastasis. Several other pre–existing metastases in the spleen and in the 
right lung hilum (outside the radiation field) completely regressed and remained controlled for an 
additional eight months. Importantly, immuno-monitoring of several markers, including antibody 
response to NY-ESO-1 mirrored the clinical course. Seromic analysis detected 10 antigenic targets with 
enhanced antibody responses after radiotherapy. A similar effect was previously reported in a study of 


18 
 
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radiation with a recombinant cancer vaccine to prostate cancer 41. 
Saba et al. describe a case of a patient who originally presented with multiple myeloma in 1996 at the 
age of 50. She failed multiple chemotherapeutic regimens including high-dose melphalan with 
autologous stem cell transplantation. Subsequently, the patient achieved a sustained complete 
remission, after receiving palliative radiotherapy to a symptomatic gastric plasmacytoma. She has 
remained in remission for >15 years 27. These results, although anecdotal, support the concept that 
local radiotherapy and immunotherapy can synergize to produce a therapeutically effective anti-tumor 
immune-response. 
Combining radiotherapy with immunotherapy is promising and presents advantages. Because of its 
localized nature, radiotherapy is devoid of most systemic effects commonly encountered with 
chemotherapy, thereby limiting interference with a systemic immunotherapy. Moreover, a radiotherapy 
focused intervention on the tumor may selectively subvert its micro-environment and in combination with 
the optimal immune intervention, may ideally render the cancer a personalized in situ vaccine. Our group 
recently presented results of a phase II clinical trial of radiotherapy with CTLA-4 blockade in lung cancer 
patients, whereby 18% of treated patients achieved a partial or complete response  RECIST version 1.1 
(30% of treated patients  achieved an abscopal response). Similar to the    trial of radiotherapy in 
combination with GM-CSF for metastatic solid tumors, patients who achieved stable disease or a 
complete/partial response subsequently demonstrated an improved median survival compared to those 
who had disease progression upon completion of their treatment 7. Thus, we are proposing a pilot trial 
that combines PD1 axis blockade and radiotherapy in patients with relapsed/refractory multiple myeloma. 
  
2.4 
Risk/Benefit Assessment 
Patients enrolled on this study have a diagnosis of multiple myeloma. Early results from immune 
checkpoint blockade therapy in multiple myeloma have shown to have a tolerable safety profile 4,5. Based 
on data from two recently halted clinical trials (KEYNOTE-183 and KEYNOTE-185) , the U.S. Food and 
Drug Administration issued a statement to inform the public, health care professionals, and oncology 
clinical investigators about the risks associated with the use of pembrolizumab (a drug in the same class 
of nivolumab) in combination with dexamethasone and an immunomodulatory agent (lenalidomide or 
pomalidomide) for the treatment of patients with multiple myeloma.  The FDA required that all patients in 
these trials be discontinued from further investigation with this drug, because interim results from both 
trials demonstrated an increased risk of death for patients receiving pembrolizumab when it was 
combined with an immunomodulatory agent as compared to the control group. Pembrolizumab is not 
approved for treatment of multiple myeloma. 
The FDA has lifted partial clinical holds placed on the phase I CheckMate-039 and phase II CA204142 
trials exploring nivolumab-based regimens in patients with relapsed/refractory multiple myeloma. The 
agency placed the partial holds on the studies in October 2017, along with a third study, the phase III 
CheckMate-602 trial, also examining a nivolumab combination in myeloma. In this current trial, nivolumab 
will be administed as a single agent and not combined with dexamethasone or immunomodulatory 
agents. A Phase Ib study that included 26 patients with multiple myeloma testing the safety and efficacy 
of nivolumab showed acceptable drug-related adverse events which occurred in 63% of patients where 
most were grade 1 or 2. 
 
2.5 
Correlative Studies Background 
Aim 1: Investigate the expression of PD-L1 and PD-L2 on myeloma plasma cells prior to and at various 


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time points after checkpoint blockade and radiotherapy. 
Rationale: PD-L1 is highly expressed on plasma cells isolated from patients with multiple myeloma 
but not on normal plasma cells and PD-L1 is not expressed on plasma cells isolated from patients 
with monoclonal gammopathy of unknown significance (MGUS) 3,43-45. It will be important to confirm 
and quantify this finding in our study. Additionally, little is known about the expression of PD-L2 on 
the plasma cells from patients with multiple myeloma, which may affect the immunogenicity of 
patients with multiple myeloma. 
Experimental Design: Expression of these ligands on myeloma plasma cells will be assessed from 
bone marrow biopsies by immunohistochemistry and flow cytometry staining with anti CD45, CD19, 
CD38 and CD138 to identify plasma cells, as well as PD-L1 (CD274) and or PD-L2 (CD273). As 
pre- treatment and serial post-treatment biopsies will be taken, we will compare responses rates 
to expression levels of PD-L1 and PD-L2 on myeloma cells at various time points. 
20 ml bone marrow aspirate and core aspirate will be collected and sent to immunopathology. 
Aim 2: Investigate the population of T cells (cytotoxic T cells, helper T cells and regulatory T cells) and 
their expression of immune checkpoint receptors (PD-1, CTLA-4, LAG3, 2B4, BTLA, TIM3, A2aR) 
prior to and at various time points after immune checkpoint blockade and radiotherapy. 
Rationale: The clinical efficacy of PD-1 blockade is most pronounced in malignancies characterized 
by the presence of infiltrating effector cells in the tumor bed 19. In contrast, myeloma is 
characterized by low levels of infiltrating effector cells, so the role of this biomarker in multiple 
myeloma is unclear. It is also unknown whether bone marrow T cell profiles reflect the T cell profile 
of the tumor itself in multiple myeloma. Therefore, we will quantify and characterize the T cell 
population in the site to be irradiated, bone marrow before immune checkpoint blockade and 
radiotherapy. Additionally, the T cell population will be similarly assayed on serial bone marrow 
biopsies and compared. 
Experimental Design: T cells are broadly categorized into three major groups based on function: 
cytotoxic T cells, helper T cells (Th), and regulatory T cells (Tregs). Differential expression of markers 
on the cell surface, as well as their distinct cytokine secretion profiles, allow different subsets to be 
quantified. For example, CD8+ cytotoxic T cells destroy infected target cells through the release of 
perforin, granzymes, and granulysin, whereas CD4+ T helper cells have little cytotoxic activity and 
secrete cytokines that  act on other leukocytes such as B  cells, macrophages, eosinophils,  or 
neutrophils. Tregs suppress T cell function by several mechanisms including binding to effector T cell 
subsets and preventing secretion of their cytokines. T cell diversity will be examined by flow 
cytometry using the following surface marker antibodies/transcription factors along with the 
expression of immune checkpoint receptors on these cells. 
 
T Cell Subset 
Surface Markers/Transcription Factors 
Cytotoxic 
CD8 


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Th1 
CD4+CXCR3+ 
Th2 
CD4+CCR4+CCR6– 
Th9 
CD4+CCR4– CCR6+ 
Th17 
CD4+CCR4+CCR6+ 
Th22 
CD4+CCR4+CCR6+CCR10+ 
NK/T 
CD3+CD56+ 
Tregs 
CD4+CD25+FoxP3+ 
 
 
Aim 3: Investigate mutational burden of pre-treatment myeloma samples in the bone marrow and 
target site utilizing FoundationOne Heme. 
Rationale: The efficacy of PD-1 blockade 
has been correlated with mutational 
burden and thought to associate with the 
presence of neoantigens derived from 
mutational events producing non-self 
epitopes 19. In contrast, myeloma is 
characterized by a modest mutational 
burden as compared to solid tumors 
suggesting a more restricted neoantigen 
profile. 
Experimental Design: Genomic profiles of 
biopsied  tumors  will  be  performed  
using FoundationOne Heme, a comprehensive   
platform that applies next-generation sequencing to identify all 4 types of genomic alterations (base    
substitutions, insertions and deletions, copy number alterations and rearrangements) across all  
genes known to be drivers of hematologic malignancy. The test sequences the entire coding region 
of 405 cancer-related genes and employs RNA sequencing across 265 genes to capture a broad range 
of gene fusions and provides an interpretive report (Figure), including “tumor mutational burden” 
(TMB), a reproducible estimate of mutation rates across all coding sequences in the genome. 
Spearman rank correlation will be used to explore the significance of the correlation between TMB 
and response. 
Aim 4: Investigate the T cell receptor (TCR) repertoire in the bone marrow aspirate prior to and at 
various time points after immune checkpoint blockade and radiotherapy. 
Rationale: At the center of the process of the immune system distinguishing self from nonself is 
Figure:FoundationOne Heme genomic 


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recognition and binding of a TCR to an antigen displayed in the major histocompatibility complex 
(MHC) on the surface of an antigen-presenting cell (APC). The naïve TCR repertoire is formed by 
somatic rearrangements of non-contiguous genes belonging to the variable (V), diversity (D) and 
joining (J) families (combinatorial diversity). These are adjacent to the constant (C) gene in the 
heterodimeric α–β or γ–δ TCR. Further random insertion and deletion of nucleotides at the 
rearrangement positions create  junctional diversity of the  highly variable  complementarity- 
determining region 3 (CDR3 region). The CDR3 region is unique for every T cell clone and encodes 
the receptor portion that makes the majority of TCR contacts with antigenic peptides bound by 
the MHC. The frequency of a specific CDR3 sequence indicates the abundance of its T cell clone. 
Thus, comprehensive TCR sequencing could provide a representative and quantitative repertoire 
analysis of specific tumor immunity. 46 In a neighboring research project (PI: Demaria), TCR 
repertoire analysis was used to investigate effects of anti-CTLA-4 and/or radiation therapy 
treatment on the TCRβ repertoire of tumor infiltrating lymphocytes (TILs) in a breast carcinoma 
mouse model. The 4T1 tumor model is not sensitive to single treatment using anti-CTLA-4 or 
radiotherapy, but respond well to the combination of both treatments. With TCR repertoire 
analysis, treatment-specific clusters of TILs were identified and to show that highly abundant 
clones rarely were unique to only one animal. Additionally, the data suggest the major role of 
radiotherapy is to increase the frequency of mid-ranked clones whereas anti-CTLA-4 primarily 
expands the top-ranked clones. However, when used together, radiotherapy and anti-CTLA-4 
increased both top- and mid-ranked clones (Rudqvist et al., manuscript in preparation). 
Experimental Design:  TCR sequencing from bone  marrow aspirate will be performed 
using the immunoSEQ assay provided by Adaptive Biotechnologies (AB). DNA from samples 
will be sent for sequencing to AB after which one part of the analysis will be performed utilizing 
AB’s analytical platform the ImmunoAnalyzer 3.0. Furthermore, data will be downloaded and 
processed using various in-house developed tools for TCR repertoire analysis. Since the 
recombination process of the TCR is a random process with large variation between individuals, Dr. 
Rudqvist (postdoc in Radiation Oncology at Weill Cornell Medicine) has developed a method for 
calculating a similarity metric between TCR repertoires (preliminary data not shown). With this 
approach, questions such as when and how a favorable T cell repertoire develops can more easily 
be assessed. 
 
2.6 
Microbiome Correlative studies 
2.6.1 To explore associations of ORR with changes in the microbiome. 
While immune checkpoint blockade results in remarkably prolonged disease control in a subset of 
patients 47,48 modulators of this phenotype are not well understood. Accumulating evidence 
suggests the gut microbiota play a critical role in response to both anti-PD-L1 efficacy and CTLA-4 
blockade: two recently published studies by Vetizou et al. and Sivan et al. demonstrated in mouse 
models that the gut microbiome critically impacts response to immunotherapy 47,48. Remarkably, by 
altering the composition of commensal microbiota, the response to CTLA-4 blockade and anti-PD- 
L1 therapy could be manipulated and therapeutic response markedly enhanced. These pre-clinical 
studies provide strong rationale for our hypothesis that the human gut microbiome composition will 


 
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correlate with response to ICB and RT. Identification of commensal organisms harbored in patients 
with heightened antitumor immune response can ultimately be manipulated to achieve desired 
outcomes in a greater proportion of patients. Fecal transplant is an established clinical technique 
whereby microbiota can be transferred into humans to induce select therapeutic outcomes, providing 
direct clinical applicability of any findings 49. 
The study by Vetizou et al. underscores the dependence of CTLA-4 blockade on commensal 
microbiota 48. First, the authors demonstrate that anti-CTLA-4 therapy is ineffective in mice that are 
housed in germ-free cages or treated with antibiotics. 
By transferring specific gut microbiota back into antibiotic-treated or germ-free mice, the anticancer 
efficacy of CTLA-4 blockade could be recovered. The transfer of T cells specific to Bacteroides fragilis, 
or immunization with dendritic cells exposed to B. fragilis polysaccharides, also  restored the 
antitumor effect of CTLA-4 blockade. Additionally, Vetizou et al. explored human gut microbiome 
changes in 25 malignant melanoma patients treated with CTLA-4 blockade, who at baseline fell into 
three microbial cluster patterns (Clusters A-C). During treatment, the microbial composition altered, 
becoming more similar to cluster pattern “C” and distant from cluster pattern “B”. Subsequent fecal 
transplantation from cluster C patients into mice resulted in a significant response to CTLA-4 
blockade, whereas transplantation from cluster B patients had no response, providing evidence that 
host gut microbial composition has a significant impact on likelihood of ICB success. 
Sivan et al. likewise provides compelling data supporting the importance of gut microbiota in 
determining treatment efficacy to ICB 47. The authors compared growth of melanoma cell lines in 
two sets of mice harboring distinct gut microbiota, with differential tumor growth and response to PD-
L1 therapy, and identified a specific bacterial species, Bifidobacterium, to be associated with 
antitumor immunity. By transferring Bifidobacterium from JAX mice to poorly-responding TAC mice, 
the authors were able to improve baseline anti-tumor immunity, and significantly enhance anti-PD- L1 
therapy response. The therapeutic effect of Bifidobacterium was established to arise from altered 
dendritic cell function and increased priming of tumor-specific CD8+ T cells. Only live bacteria could 
achieve this effect, implying that augmentation of dendritic cell function requires direct interaction of 
host cells with gut microbiota 49. These novel findings signify that the gut microbiome composition 
impacts response to anti-PD-L1 therapy and can be manipulated to enhance treatment response, 
providing impetus for similar analyses in human subjects. 
Taken together, the above preclinical studies lend strong support towards collection of gut 
microbiome samples from patients enrolling on all future immunotherapy studies. We propose to 
collect stool samples from study patients before treatment and at day 21, 3 months and 6 months 
for analysis of microbiome composition and changes after ICB and RT. Both pre-clinical and clinical 
data demonstrate that the addition of RT to ICB creates a robust immune response, eliciting anti- 
tumor immune responses in otherwise non-responding patients. Given that we expect a similar 
heightened response in our proposed study, our patient cohort with its expected dichotomous 
outcomes will be particularly suitable for microbiome analysis. The results will be an important step 
towards understanding, and ultimately harnessing, the interplay between the human gut 
microbiome and anti-tumor immune response. 
Methods Stool collection will occur before treatment, at day 21, at 3 months and 6 months to 


 
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explore changes of the microbiome during ICB. Bacterial diversity and taxonomy will be estimated 
by high throughput sequencing (HTS) of 16S rRNA amplicons, using the MiSeq platform in the 
Genomic Technology Core Lab. Methods have been established in the Blaser Lab and proven to yield 
accurate and reproducible results.50,51 Analysis will be based on the QIIME (Quantitative Insights into 
Microbial Ecology) software package developed by the Knight lab, 52 and machine learning analyses 
developed by NYU Center for Health informatics and Bioinformatics (CHIBI) investigators working 
on NYU Human Microbiome Program studies 53,54. Appendix 2 describes the protocol for stool 
collection. 
 
3 
Subject Selection 
3.1 
Study Population 
Patients with a diagnosis of multiple myeloma who meet the inclusion and exclusion criteria will be 
eligible for participation in this study. 
 
3.2 
Inclusion Criteria 
1. Subject is, in the investigator’s opinion, willing and able to comply with the protocol 
requirements. 
2. Subject has given voluntary written informed consent before performance of any study- 
related procedure not part of normal medical care, with the understanding that consent may be 
withdrawn by the subject at any time without prejudice to their future medical care. 
3. Must have received 2 consecutive cycles of systemic myeloma therapy. 
4. Documented refractory or relapsed and refractory (R/R) multiple myeloma 
a. patients had less than minimal response, or had achieved at least a minimal response to 
previous treatment, but progressed within 6 months 
b. patients who have failed treatment with, are intolerant to, or ane not candidates for all 
available therapies known to be active for treatment of relapsed or refractory myeloma 
c. patients must have failed, be intolerant or are ineligible to treatment with at least 3 lines 
of therapy, including an IMiD, proteasome inhibitor and anti-CD38 agent 
5. Targetable plasmacytoma, either intra-or extramedullary that is visualized on imaging 
(PET/CT or MRI) and is causing symptoms (eg. pain, neurological compromise) or potential 
to cause symptom as per clinician’s judgement; and measurable disease at screening, 
defined as one or more of the following: 
a. Serum IgG, IgA, or IgM M-protein ≥ 0.5 g/dL 
b. Urine M-Protein ≥ 200 mg excreted in a 24-hour collection sample 
c. Involved serum free light chain (sFLC) > 100 mg/L provided the FLC ratio is abnormal 
6. Males and Females at least 18 years or legal age of consent per local regulations. 
7. Women of childbearing potential (WOCBP) must have two negative serum or urine 
pregnancy tests (minimum sensitivity 25 mIU/mL or equivalent units of HCG). One 10-14 days 
prior to start of the study drug and one within 24 hours prior to the start of study drug. 
8. Investigators shall counsel WOCBP and male subjects who are sexually active with WOCBP 
on the importance of pregnancy prevention and the implications of an unexpected 
pregnancy Investigators shall advise WOCBP and male subjects who are sexually active with 
WOCBP on the use of highly effective methods of contraception. Highly effective methods of 
contraception have a failure rate of < 1% when used consistently and correctly. 
9. No condition which would cause unacceptable risk. 


 
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3.3 
Exclusion Criteria 
1. Subjects with solitary bone or extramedullary plasmacytoma as the only evidence of plasm 
cell dyscrasia. 
2. Subjects with monoclonal gammopathy of undetermined significance (MGUS), smoldering 
multiple myeloma (SMM), Waldenstrom’s macroglobulinemia, or POEMS syndrome (plasma 
cell dyscrasia with poly neuropathy, organomegaly, endocrinopathy, monoclonal protein, 
and skin changes). 
3. Subjects with active plasma cell leukemia (defined as either 20% of peripheral blood white 
blood cell count comprised of plasma/CD138+ cells or an absolute plasma cell count of 2 x 
109/L). 
4. Subjects within 100 days of stem cell transplantation. 
5. Subjects within 4 weeks of surgery, unless cleared by surgeon. 
6. Women who are of childbearing potential not complying to the above described 
contraceptive measures or are breastfeeding, and sexually active fertile men whose partners 
are WOCBP if they are not complying to the above described contraceptive measures. 
7. Any uncontrolled or severe cardiovascular or pulmonary disease determined by the 
investigator, including: 
a. NYHA functional classification III or IV, congestive heart failure, unstable or poorly 
controlled angina, uncontrolled hypertension, arrhythmia, or myocardial infarction in 
the past 12 months 
b. Subjects with interstitial lung disease that is symptomatic or may interfere with the 
detection or management of suspected drug-related pulmonary toxicity 
8. Active infection or know HBV/HCV/HIV. 
9. Subjects with an active, known or suspected autoimmune disease. Subjects with type I 
diabetes mellitus, hypothyroidism only requiring hormone replacement, skin disorders (such 
as vitiligo, psoriasis, or alopecia) not requiring systemic treatment, or conditions not expected 
to recur in the absence of an external trigger are permitted to enroll. 
10. Subjects with a condition requiring systemic treatment with either corticosteroids (> 10 mg 
daily prednisone equivalent) or other immunosuppressive medications within 14 days of 
initiation of study drug. Inhaled or topical steroids, and adrenal replacement steroid doses > 
10 mg daily prednisone equivalent, are permitted in the absence of active autoimmune 
disease. 
11. Previous radiotherapy to the area of the target area. 
12. Prior exposure to immune checkpoint inhibitor. 
13. Persistent toxicities of ≥ Grade 1 from prior treatment (including chemotherapy, targeted 
therapy, experimental agents, radiation, or surgery). 
 
4 
Registration Procedures 
4.1 
Patient Registration 
Patients will be centrally registered with the Office of Billing Compliance. To register a 
patient, submit the following documents via the JIRA Registration Process: 
• 
Legible copy of the HRBAF 
• 
First and last page of signed informed consent form 
Registration must be completed within 24 hours of the signing of informed consent. 


 
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5 
Study Calendar 
5.1 
Schedule of Assessment 
 
Procedure 
Screeni
ng 
(Day0
C1D1 
(±3 
days) 
C1D15 
(±3 
days) 
Cycles 2-4 Cycles 5+ Cycles 5+  
End of 
Study 
D1 
D15 
D1 
D15 
Informed Consent 
X 
Medical History 
X 
X 
X 
X 
X 
X 
Physical Exam 
X 
X 
X 
X 
X 
X 
Vital Signs + weight 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status (ECOG) 
X 
X 
X 
X 
X 
X 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
Second Primary Malignancy 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events Assessment 
X 
X 
X 
X 
X 
X 
X 
X 
Bone Marrow Aspiration/Biopsya 
Xa 
Xa 
Xa 
 
Xa 
Biopsy of Targeted Lesion 
X 
Radiologic bone imaging: 
PET/CT, MRI 
X 
X 
 
X 
CBC w/ plts & diff 
X 
X 
X 
X 
X 
X 
X 
X 
CMP, Mg, Uric Acid, LDH 
X 
X 
X 
X 
X 
X 
X 
X 
Serum or urine pregnancy test 
X 
X 
X 
X 
X 
X 
X 
X 
TSH 
X 
X 
X 
Beta 2 microglobulin 
X 
X 
Response Assessment 
Serum quantitative immunoglobulins 
X 
X 
X 
X 
 
X 
free light chains, SPEP & SIFE 
X 
X 
X 
X 
 
X 
24-Hour urine for urine total protein 
X 
X 
X 
X 
 
X 
UPEP & UIFE 
X 
X 
X 
X 
 
X 
TREATMENT 
Nivolumab Infusionc 
Xc 
Xc 
Xc 
Xc 
Xc 
Xc 
Radiation to Targeted Lesionb 
Xb 
RESEARCH PROCEDURES 
 
Stool Samples (before nivo infusion)d 
Xd 
Xd  
Xd  
Saliva Sample collection (before nivo) 
X 
 
 
 
 
Foot notes: 
a. Bone marrow aspirate pre-nivolumab infusion on C2D1, C4D1, C7D1, C10D1 and Q 12 weeks thereafter and as needed to 
confirm CR 
b. One course of radiation (6GY x 5 fractions), with first radiation on D1 AFTER IV nivolumab 
c. 
All Infusions will have a ± 3 day window.  
d. Stool samples will be collected on Day1, end of Radiation, C4D1 and C7D1.  


 
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5.1.1 Screening Visit – (Day 0  -  day28) 
After proper documentation of the informed consent process, patients will undergo screening 
procedures which will include standard of care labs, specified laboratory values as defined 
by International Myeloma Working Group (IMWG) Criteria, PET/CT –MRI, baseline imaging, 
bone marrow aspiration biopsy, and target lesion biopsy as outlined in section 5.1 schedule 
of assessments. 
 
5.1.2 Treatment Phase 
Once eligible, patients will receive immunotherapy, nivolumab, intravenously at 240 mg 
every two weeks. Infusions will be given over 30 minutes (not bolus or IV push).  Patients 
will also receive radiation of 6 Gy x 5 fractions the first week of starting immunotherapy. 
Immunotherapy treatment starts with the first radiotherapy fraction. Nivolumab will be 
given every 2 weeks. Patients will have specified laboratory values measured monthly and 
evaluated for response as defined by International Myeloma Working Group Criteria. 
Patients will continue to receive their respective immunotherapy for up to three years or 
until disease progression or dose limiting toxicity is reached. Patients will remain on therapy 
for up to 6 cycles in the setting of stable disease and for up to a year in the setting of a PR 
or better by 6 cycles. 
5.2 
Response Assessment at Week 12 
Patients will have specified laboratory values measured monthly and evaluated for response 
as defined by International Myeloma Working Group Criteria. Patients without progression 
of disease as defined by IMWG criteria including assessment of plasmacytoma(s) by PET/CT will 
continue to receive nivolumab every 2 weeks. 
 
5.3 
Duration of Therapy and Criteria for Removal from Study: 
In the absence of treatment delays due to adverse event(s), treatment may continue until one 
of the following criteria applies: 
1. Progression of disease as defined by IMWG critera, 
2. Intercurrent illness that prevents further administration of treatment, 
3. Unacceptable adverse event(s), 
4. Patient decides to withdraw from the study, or 
5. General  or   specific  changes   in  the  patient’s  condition  render  the  patient 
unacceptable for further treatment in the judgment of the investigator. 
 
5.4 
Duration of Follow Up 
Patients will be followed for 3 years after removal from study or until death, whichever 
occurs first. Patients removed from treatment  for unacceptable adverse events will be 
followed until resolution or stabilization of the adverse event


 
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6 
Radiation Therapy Guidelines 
6.1 
Planning 
After informed consent is obtained, a targetable plasmacytoma lesion that is accessible for biopsy 
is selected by the treating physician as the site for local radiotherapy under Standard of Care 
practices to establish a diagnosis. The area of interest is imaged at CT planning for conformal 
treatment. There is no contrast media used in the CT planning. There is an exposure to small 
amounts of radiation with the use of CT scan. CT scan thickness should be ≤0.5 cm through the 
tumor region. These images will be used in 3D treatment planning in accordance with the dose 
specification constraints. 
The CTV is defined as the plasmacytoma lesion of interest with the expected motion changes, while 
the PTV is the CTV plus a margin ≤ 1 cm, dependent on the anatomical location, to account for 
setup uncertainty. While gating is not part of the current conformal setting at Stich Radiation 
Center, WCM, efforts are made to consistently treat chest and abdominal lesions with the patient 
maintaining shallow breathing, to limit the movement of the volume treated during respiration. 
6.2 
Treatment 
Radiotherapy is delivered by external beam using linear accelerators capable of delivering ≥ 
4megavoltage  x- rays. The PTV will encompass all of the biopsied plasmacytoma lesion, and will 
be defined by the treating physician. The PTV will be treated daily, Monday-Friday. A dose of 30 
Gy in 5 fractions of 6 Gy each is delivered, daily, to an isodose surface encompassing ≥90% of the 
PTV. 
Radiation Dose specification: The planning target volume receives a minimum of 90% of the 
prescription dose. 
Treatment Machine: A linear accelerator with ≥ 4 MV x-rays is required. 
Immobilization Technique: Patients will be set-up for treatment and CT scanning, and planned for 
treatment. The specific immobilization technique will be determined at the discretion of the 
treating physician. 
Target Positioning Verification: Digitally acquired radiographic images will be used to verify the 
position of the target with respect to the treatment machine’s isocenter using digitally 
reconstructed radiographs (DRRs) as a reference image set. Both kV and MV images may be used 
to verify setup. 
IGRT Target Localization: In addition to the portal imaging, cone-beam CT (CBCT) images will be 
acquired prior to treatment for each fraction. By using IGRT to image the plasmacytoma in “real- 
time”, the operator may automatically align the plasmacytoma with the treatment machine on 
each day of treatment. If shifts are made based upon the CBCT images, the portal images will be 
repeated. 
Treatment Planning: 3D-Conformal or IMRT treatment planning is allowed. This includes “field-in- 
field” beams as well as the use of dynamic multi-leaf collimator (MLC) derived using inverse


 
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planning or electronic compensator techniques. Field arrangements and technique should be 
chosen that satisfy the PTV dose coverage constraints and normal tissue dose constraints using 
Dose-Volume Histogram (DVH) analysis. By carefully selecting the gantry and table angle 
combinations that do not enter or exit through other organs of the body, the dose can be 
confined to the traditional treatment volumes. 
Non-coplanar beam arrangements are encouraged, but not required. 
 
Dose calculations with tissue inhomogeneity correction must be used. 
After completion of the first course of radiotherapy + nivolumab on Day 1 (or within 24 hours or 
RT), the patient will start on day 15 the regimen of nivolumab 240 mg q2 weeks. 
Response assessment will  be performed every 2 weeks as standard of care. 
 
6.3 
Treatment modifications for Radiation Adverse Events: 
Dosing delay: The patient should have resolution or return to pre-treatment baseline of all grade 
3-4 toxicities prior to start of the next immunotherapy treatment. 
 
6.4 
On Study Evaluations: 
As summarized in the Study Calendar, patients are evaluated pre-treatment for measurable disease 
in the blood and a targetable soft tissue or bony lesion with radiotherapy. Patients will have 
specified laboratory values measured bi-monthly and evaluated for response as defined by 
International Myeloma Working Group Criteria. Response assessments will be performed every 2 
weeks. 
 
7 
Pharmaceutical Information 
7.1 
Investigational Agent 
Nivolumab is a human monoclonal antibody that blocks the interaction between PD-1 and its 
ligands, PD-L1 and PD-L2. Nivolumab is an IgG4 kappa immunoglobulin that has a calculated 
molecular mass of 146 kDa. Binding of the PD-1 ligands, PD-L1 and PD-L2, to the PD-1 receptor 
found on T cells, inhibits T-cell proliferation and cytokine production. Upregulation of PD-1 ligands 
occurs in some tumors and signaling through this pathway can contribute to inhibition of active T- 
cell immune surveillance of tumors. Nivolumab is a human immunoglobulin G4 (IgG4) monoclonal 
antibody that binds to the PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, releasing 
PD-1 pathway-mediated inhibition of the immune response, including the anti-tumor immune 
response. In syngeneic mouse tumor models, blocking PD-1 activity resulted in decreased tumor 
growth. 
7.2 
Availability 
Nivolumab is an investigational agent supplied to investigators by Bristol-Myers Squib


 
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7.3 
Preparation and Administration of Nivolumab 
Dose Calculations 
For Multiple myeloma: Nivolumab is given every 2 weeks. The recommended dose of nivolumab 
is a flat dose of 240mg administered as an intravenous infusion over 30 minutes every 2 weeks 
until disease progression or unacceptable toxicity. 
Visually inspect drug product solution for particulate matter and discoloration prior to 
administration. Nivolumab is a clear to opalescent, colorless to pale-yellow solution. Discard 
the vial if the solution is cloudy, discolored, or contains extraneous particulate matter other than 
a few translucent-to-white, proteinaceous particles. Do not shake the vial. 
 
7.4 
Dose Modifications, Delays or Discontinuation of Nivolumab 
 
Recommendations for OPDIVO modifications are provided in Table 1. There are no 
recommended dose modifications for hypothyroidism or hyperthyroidism. Interrupt or slow the 
rate of infusion in patients with mild or moderate infusion reactions. Discontinue OPDIVO in 
patients with severe or life-threatening infusion reactions.  
 
 


 
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8 
Correlative Studies 
 
8.1 
Bone Marrow tissue aspirate 
In addition to this, 20 ml sample of bone marrow tissue will be collected at the same intervals. 
 
8.2 
Tissue biopsies 
4 core pre treatment tissue biopsies will be taken for diagnostic confirmation and correlative 
studies. 
 
8.3 
Other studies : 
A baseline sputum (saliva) sample will be obtained from all patients for gene expression profiles. 
 


 
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8.4 
Microbiome samples 
Stool collection for microbiome analysis will be collected at Baseline, end of radiation, C4D1 and 
C7D1. to explore changes of the microbiome during ICB. Appendix 2 describes the protocol for 
stool collection.


 
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9 
Measurement of Effect 
9.1 
Standard IMWG Response Criteria 
Response to multiple myeloma will be assessed based on the International Myeloma Working Group Criteria (IMWG) as described in the table 
below. 
 
Standard IMWG response criteria* 
Stringent complete 
response 
Complete response as defined below plus normal FLC ratio** and absence of clonal cells in bone marrow biopsy 
by immunohistochemistry (κ/λ ratio ≤4:1 or ≥1:2 for κ and λ patients, respectively, after counting ≥100 plasma 
cells)++ 
Complete response 
Negative immunofixation on the serum and urine and disappearance of any soft tissue plasmacytomas and <5% 
plasma cells in bone marrow aspirates 
Very good partial 
response 
Serum and urine M-protein detectable by immunofixation but not on electrophoresis or ≥90% reduction in 
serum M-protein plus urine M-protein level < 100mg per 24 hours. 
Partial response 
≥50% reduction of serum M-protein plus reduction in 24 h urinary M-protein by ≥90% or to <200 mg per 24h; If 
the serum and urine M-protein are unmeasurable, a ≥ 50% decrease in the difference between involved and 
uninvolved FLC levels is required in place of the M-protein criteria; If serum and urine M-protein are 
unmeasurable, and serum-free light assay is also unmeasurable, ≥ 50% reduction in plasma cells is required in 
place of M-protein, provided baseline bone marrow plasma-cell percentage was ≥ 30%. In addition to these 
criteria, if present at baseline, a ≥50% reduction in the size (SPD)§§ of soft tissue plasmacytomas is also 
required. 
Minimal response 
≥25% but ≤49% reduction of serum M-protein and reduction in 24-h urine M-protein by 50–89%. In addition to 
the above listed criteria, if present at baseline, a ≥50% reduction in the size (SPD)§§ of soft tissue 
plasmacytomas is also required. 
 
 


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Stable disease 
Not recommended for use as an indicator of response; stability of disease is best described by providing the 
time-to-progression estimates. Not meeting criteria for complete response, very good partial response, partial 
response, minimal response, or progressive disease 
Progressive 
disease‡‡,Ω 
Any one or more of the following criteria: 
Increase of 25% from lowest confirmed response value in one or more of the following criteria: 
Serum M-protein (absolute increase must be ≥0·5 g/dL); 
Serum M-protein increase ≥1 g/dL, if the lowest M component was ≥5 g/dL; 
Urine M-protein (absolute increase must be ≥200 mg/24 h); 
In patients without measurable serum and urine M-protein levels, the difference between involved and 
uninvolved FLC levels (absolute increase must be >10 mg/dL); 
In patients without measurable serum and urine M-protein levels and without measurable involved FLC 
levels, bone marrow plasma-cell percentage irrespective of baseline status (absolute increase must be 
≥10%); Appearance of a new lesion(s), ≥50% increase from nadir in SPD§§ of >1 lesion, or ≥50% increase in 
the longest diameter of a previous lesion >1 cm in short axis; 
≥50% increase in circulating plasma cells (minimum of 200 cells per μL) if this is the only measure of disease 
Clinical relapse 
Clinical relapse requires one or more of the following criteria: Direct indicators of increasing disease and/or end 
organ dysfunction (CRAB features) related to the underlying clonal plasma-cell proliferative disorder. It is not used 
in calculation of time to progression or progression-free survival but is listed as something that can be reported 
optionally or for use in clinical practice; Development of new soft tissue plasmacytomas or bone lesions 
(osteoporotic fractures do not constitute progression); 
Definite increase in the size of existing plasmacytomas or bone lesions. A definite increase is defined as a 50% 
(and ≥1 cm) increase as measured serially by the SPD§§ of the measurable lesion; 
Hypercalcaemia (>11 mg/dL); 
Decrease in haemoglobin of ≥2 g/dL not related to therapy or other non-myeloma-related conditions; Rise 
in serum creatinine by 2 mg/dL or more from the start of the therapy and attributable to myeloma; 
Hyperviscosity related to serum paraprotein 


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Relapse from 
complete response 
(to be used only if the 
end point is disease- 
free survival) 
Any one or more of the following criteria: 
Loss of MRD negative state (evidence of clonal plasma cells on NGF or NGS, or positive imaging study 
for recurrence of myeloma); 
Reappearance of serum or urine M-protein by immunofixation or electrophoresis; 
Development of ≥5% plasma cells in the bone marrow; 
Appearance of any other sign of progression (ie, new plasmacytoma, lytic bone lesion, or hypercalcaemia) 
*Derived from international uniform response criteria for multiple myeloma. Minor response definition and clarifications derived from 
Rajkumar and colleagues. When the only method to measure disease is by serum FLC levels: complete response can be defined as a 
normal FLC ratio of 0·26 to 1·65 in addition to the complete response criteria listed previously. Very good partial response in such 
patients requires a ≥90% decrease in the difference between involved and uninvolved FLC levels. All response categories require two 
consecutive assessments made at any time before the institution of any new therapy; all categories also require no known evidence of 
progressive or new bone lesions or extramedullary plasmacytomas if radiographic studies were performed. Radiographic studies are not 
required to satisfy these response requirements. Bone marrow assessments do not need to be confirmed. Each category, except for 
stable disease, will be considered unconfirmed until the confirmatory test is performed. The date of the initial test is considered as the 
date of response for evaluation of time dependent outcomes such as duration of response. 
**All recommendations regarding clinical uses relating to serum FLC levels or FLC ratio are based on results obtained with the validated 
Freelite test (Binding Site, Birmingham, UK). 
++Presence/absence of clonal cells on immunohistochemistry is based upon the κ/λ/L ratio. An abnormal κ/λ ratio by 
immunohistochemistry requires a minimum of 100 plasma cells for analysis. An abnormal ratio reflecting presence of an abnormal clone 
is κ/λ of >4:1 or <1:2. ‡‡Special attention should be given to the emergence of a different monoclonal protein following treatment, 
especially in the setting of patients having achieved a conventional complete response, often related to oligoclonal reconstitution of the 
immune system. These bands typically disappear over time and in some studies have been associated with a better outcome. Also, 
appearance of monoclonal IgG κ in patients receiving monoclonal antibodies should be differentiated from the therapeutic antibody. 
§§Plasmacytoma measurements should be taken from the CT portion of the PET/CT, or MRI scans, or dedicated CT scans where 
applicable. For patients with only skin involvement, skin lesions should be measured with a ruler. Measurement of tumor size will be 
determined by the SPD. 
‡‡Positive immunofixation alone in a patient previously classified as achieving a complete response will not be considered progression. 
For purposes of calculating time to progression and progression-free survival, patients who have achieved a complete response and are 
MRD-negative should be evaluated using criteria listed for progressive disease. Criteria for relapse from a complete response or relapse 
from MRD should be used only when calculating disease-free survival. 
Ω In the case where a value is felt to be a spurious result per physician discretion (eg, a possible laboratory error), that value will not be 
considered when determining the lowest value. 
 


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10 Data Reporting / Regulatory Considerations 
10.1 Data Collection 
The data collection plan for this study is to utilize REDCap to capture all treatment, 
toxicity, efficacy, and adverse event data for all enrolled patients. 
10.1.1 REDCap 
REDCap (Research Electronic Data Capture) is a free data management software system 
that is fully supported by the Weill-Cornell Medical Center CTSC. It is a tool for the 
creation of customized, secure data management systems that include Web-based 
data-entry forms, reporting tools, and a full array of security features including user and 
group based privileges, authentication using institution LDAP system, with a full audit 
trail of data manipulation and export procedures. REDCap is maintained on CTSC- 
owned servers that are backed up nightly and support encrypted (SSL-based) 
connections.  Nationally, the software is developed, enhanced and supported through a 
multi-institutional consortium led by the Vanderbilt University CTSA. 
10.2 Regulatory Considerations 
All protocol amendments and consent form modifications will be made by the Principal 
Investigator. Bristol-Myers Squibb will have the opportunity to review and approve the 
changes prior to submission of these changes to the local IRB. 
11 Statistical Considerations 
The primary endpoint of this study is response rate. The trial design is a single arm,  
single-center, Simon optimal two-stage design. Patients will be evaluated for response at 12 
weeks (84 days ± 7 days). Patients will be enrolled in two stages with 13 patients in the first 
stage, and potentially an additional 13 patients in the second stage. The decision to enroll 
patients into the second stage will depend on the number of responses observed in the first 
stage, as described below. 
11.1 Sample Size/Accrual Rate 
11.2 Stratification Factors 
None 
11.3 Analysis of Endpoints 
As mentioned, a Simon’s two-stage optimal design [Simon, 1989] will be used. The null 
hypothesis that the true response rate is 20% will be tested against a one-sided alternative. 
If there are 3 or fewer responses in these 13 patients, the study will be stopped. Otherwise 
13 additional patients will be accrued for a total of 26 patients. The null hypothesis will be 
rejected if 10 or more responses are observed in 26 patients. This design yields a one-
sided type I error of 0.025 and power of 60% when the true response rate is 40%. The 
target sample size is 26 evaluable patients and up to 30 patients (4 extra patients) will be 
accrued to account for non-evaluable patients or patients who withdraw prior to evaluation.


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11.3.1 Analysis of Primary Endpoints 
The primary analysis will be an evaluation of the response rate. The trial decision rules are to 
conclude the treatment strategy is not worth further evaluation if in the first evaluable 13 
patients, there are 3 or fewer patients with a response. If in the first 13 evaluable patients, 
there is at least 4 patients with a response, an additional 13 evaluable patients will be enrolled. 
If in the first 26 evaluable patients, there are 10 or more responses are seen, it will be 
concluded that this treatment strategy is worth further evaluation (the null hypothesis will be 
rejected). If in the first 26 evaluable patients, 9 or fewer responses are observed, the treatment 
will not be recommended for further evaluation in this patient population. The response rate 
will be estimated with a one-sided 95% exact binomial procedure (66). Analyses will be 
performed in R (R Core Team, 2017). 
11.3.2 Analysis of Secondary Endpoints 
With adequate follow-up time, secondary endpoints of progression-free survival (PFS) and 
overall survival (OS) will be assessed by Kaplan-Meier survival analysis and 95% confidence 
intervals for median PFS/OS will be calculated using Greenwood’s formula. PFS will be 
defined as the time from first treatment day until objective or symptomatic progression. OS will 
be defined as the time from first treatment day until death. The frequency of subjects 
experiencing toxicities will be tabulated. Toxicities will be assessed and graded according to 
CTCAE v. 4.0 terminology. Exact 95% confidence intervals around the toxicity proportions will 
be calculated to assess the precision of the obtained estimates. 
11.3.3 Interim Analysis 
None Planned. 
11.3.4 Reporting and Exclusions 
11.3.5 Evaluation of toxicity 
11.3.6 Evaluation of response. 
All patients included in the study will be assessed for response to treatment if they have received 
at least 1 nivolumab infusion. 
All patients will be evaluable for toxicity from the time of their first treatment with 
Investigational agent. A DLT will be defined as any grade 4 non-hematological toxicity or 
any toxicity that interrupts treatment for more than 6 weeks,  treatment-related deaths, or 
grade 3 treatment-related non-hematological toxicities. Once 8 patients have been 
enrolled, we will use a continuous Bayesian toxicity stopping rule that will pause the study 
for review if the posterior probability that the DLT event rate is greater than 20% is > 
80%, using an uninformative prior of Beta(1,1). The trial will reviewed if 3 or more of the 
first 8 patients experience DLTs, or 4 or more of the first 9-12, or 5 or more of the first 
13-16, 6 or more of the first 17-21, 7 or more of the first 22-25.


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11.4 Analysis of Correlative Endpoints 
 
11.4.1 Microbiome 
 
11.5 Statistical analysis of microbiome and metagenome composition 
Statistical analysis of microbiome and  metagenome composition will be performed in the R 
statistical programming environment 56 using package phyloseq 57, which incorporates and builds 
upon community ecology packages such as ade4 and vegan and employs the flexible graphic 
system ggplot2, to easily visualize complex data relationships. For 16S data, we will evaluate the 
adequacy of sequencing efforts using rarefaction plots. Alpha diversity index for each will be 
characterized through dominance, equitability, richness, evenness. The diversity metrics will be 
calculated at Operational Taxonomic Unit (OTU) and higher taxonomical levels to best characterize 
the community structure. We will test for associations of each of these alpha diversity metrics with 
the time relative to radiation 
exposure, using one-way ANOVA after even-sampling the 
observations to a depth cut-off maximizing the number of samples and depth. In addition, rank-
abundance plots will be used to visualize differences in abundance of dominant taxa in the clinical 
and phenotypic groups. We will utilize skyline plots to visualize the patterns of community structure 
in terms of relative abundances in the collected samples between before and after the radiation 
treatment or between case and control samples. Similarly, for metagenomic data, skyline plots 
will be used to reveal functional compositions of the samples. Heat-maps will be plotted to visualize 
clustering patterns in the data. 
 
Univariate analyses. To circumvent instabilities associated with rare species, which are difficult to 
detect uniformly in all samples, we will focus our primary univariate analyses only on highly 
abundant taxa, i.e. those present at 1% or more relative abundance across all specimens. This 
approach will also help to reduce multiple comparison-related Type I-error inflation, which will be 
formally controlled by false discovery rate (FDR) 58. The differences in presence or absence of 
specific taxa and functional categories will be assessed by χ2-test at all taxonomical levels. Paired- 
Sample Wilcoxon Signed Rank Test will be used to establish the differences in relative abundance 
of taxa between before and after the radiation treatment or between paired samples. Depending 
on the dynamics observed, subsequent analyses may focus on less abundant taxa, and at higher 
depth, as indicated above. 
 
Multivariate analyses. Multivariate association of the entire microbiome/metagenome with 
clinical and phenotypic factors will be examined with Principal Coordinate Analysis (PCoA) on 
Jensen-Shannon, weighted and unweighted Unifrac, and χ2  (correspondence analysis) distances. 
Likewise, these distances will be used to build non-parametric multivariate Analysis of variance 
(ANOVA) models (ADONIS) 59  to allow for simultaneous measurement of univariate and 
interaction effects of the clinico-phenotypic variables on the microbiome. Starting with the 
microbiota and metagenomic features significantly different in univariate analyses, we will use the 
dimension-reduction method, canonical correlation analysis (CCA) to identify the bacteria taxa 
which are related radiation treatment, according to benchmarked methodologies of our prior 
studies 54,60. We will utilize a phylogenetic structure-constrained penalty function to impose 
phylogenetic relationships among bacteria on the model selection 61. 
 
Longitudinal analysis: We will study the evolution of microbiome over time and how that evolution 
is associated with the radiation treatment. The relative abundances at each taxonomical level will 


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be first normalized by log-ratio transformation 62. Then the transformed relative abundance of each 
individual taxa at multiple time points will be fitted by the linear mixed model along with the time 
effect and all subject-specific characteristics as the independent covariates. For the nonlinear 
trend, we will combine the nature splines with linear mixed model in the data analysis. The same 
model will be applied on the indices calculated in the ecology microbial analysis.  For the joint 
analysis of more than one taxon. we will use MCMCglmm R package to implement the multivariate 
generalized linear mixed model. This package gives a large flexibility 
in analyzing correlated 
multiple longitudinal response by allowing different types of covariance structure 63. 
 
12 Adverse Event Reporting Requirements 
CTCAE Version 4.0 will be used for tracking the Adverse Events in this study. 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. The 
investigator will be required to provide appropriate information concerning any findings that 
suggest significant hazards, contraindications, side effects, or precautions pertinent to the safe 
use of the drug or device under investigation. 
Safety will be monitored by evaluation of adverse events reported by patients or observed by 
investigators or research staff, as well as by other investigations such as clinical laboratory tests, 
x-rays, electrocardiographs, etc. 
 
12.1 Adverse Event Definition 
12.1.1 Collection of Safety Information 
CTCAE term (AE description) and grade: The descriptions and grading scales found in the revised 
NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE 
reporting. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov). 
12.1.2 Attribution of the AE: 
Definite – The AE is clearly related to the study treatment. 
Probable – The AE is likely related to the study treatment. 
Possible – The AE may be related to the study treatment.  
Unlikely – The AE is doubtfully related to the study treatment. 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
12.1.3 Recording of adverse events 
At every clinical visit, the patients will be assessed for adverse events. Adverse events will be 
documented in patient’s charts (EPIC). All adverse events will be recorded on a patient specific AE 
log. The AE log will be maintained by the research staff and kept in the patient’s research chart. 
AEs will also be updated in REDCAP. 
 
 
A serious AE or reaction is any untoward medical occurrence that at any dose: 
• 
results in death, 
• 
is life-threatening (defined as an event in which the patient or subject was at risk of 
death at the time of the event; it does not refer to an event which hypothetically 


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might have caused  death if it were more severe), 
• 
requires inpatient hospitalization or prolongation of existing hospitalization, (refer to 
note for  exceptions), 
• 
results in persistent or significant disability/incapacity, 
• 
is a congenital anomaly/birth defect, 
• 
is an important medical event (defined as a medical event(s) that may not be 
immediately life-threatening or result in death or hospitalization but, based upon 
appropriate medical and scientific judgment, may jeopardize  the  patient/subject  
or may require intervention (e.g., medical, surgical) to prevent one of the other 
serious outcomes listed in the definition above). Potential drug induced liver injury ( 
DILI) is also considered an important medical event. 
NOTE: 
Pregnancy:  Incidence  of  pregnancy  is  not  considered  a  SAE;  pregnancy  must,  however,  be  
reported immediately to investigator. Cancer/Overdose: An overdose is defined as the accidental 
or intentional ingestion of any dose of a product that is considered both excessive and medically 
important, and must be immediately reported. 
Hospitalizations (exceptions): 
Criteria for hospitalizations not reported as SAEs include admissions for: 
Planned as per protocol medical/surgical procedure 
Routine health assessment requiring admission for baseline/trending of health status documentation 
(e.g., routine colonoscopy) 
Medical/surgical admission for purpose other than remedying ill health state (planned prior to entry 
into study trial; appropriate documentation required) 
Admission encountered for other life circumstance that carries no bearing on health status and 
requires no medical/surgical intervention (e.g., lack of housing, economic inadequacy, care-giver 
respite, family circumstances, administrative) 
An SAE report should be completed for any event where doubt exists regarding its status of 
seriousness. 
 
12.2 Reporting of Serious Adverse Events (SAEs) 
Following the subject’s written consent to participate in the study, all SAEs should be collected and 
reported, including those thought to be associated with clinical trial procedures. SAE terminology 
and severity grading will be based on CTCAEv4. 
This is a Weill Cornell Medicine investigator-sponsored study in which the study drug will be 
provided by BMS. The principal investigators are responsible for reporting SAEs to the IRB, 
Data Safety Monitoring Board (DSMB)  and the FDA or other applicable regulatory authority. 
The principal investigator is responsible for submitting follow-up reports for all SAEs regarding the 
patient’s subsequent course until the SAE has resolved or until the patient’s condition stabilizes (in 
the case of persistent impairment), or the patient dies. Reports of SAEs will be submitted to the 
WCM IRB and to DSMB.  
The causal relationship to study drug is determined by a physician and should be used to assess 
all AEs. The causal relationship can be one of the following: 


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Related: There is a reasonable causal relationship between the study drug and the AE. The event 
responds to withdrawal of study drug (dechallenge), and recurs with rechallenge when  clinically 
feasible. 
Not related: There is not a reasonable causal relationship between  s t u d y drug 
administration and the AE. 
• 
Adverse events classified as "serious" require expeditious handling and reporting to 
WCM to  comply with regulatory requirements. 
• 
All SAEs whether related or unrelated to nivolumab, must be immediately reported 
to WCM and BMS (by the investigator or designee) within 24 hours of becoming 
aware of the event. If only limited information is initially available, follow-up reports 
are required. The original SAE form must be kept on file at the study site. All 
SAEs should be reported to WCM and BMS via confirmed facsimile (fax) 
transmission, or scanned and reported via electronic mail to: 
SAE Email Address: Worldwide.Safety@BMS.com 
SAE Fax Number: 609-818-3804 
• 
Collection of complete information concerning SAEs is extremely important. Full 
descriptions of each event will be followed by WCM. Thus, follow-up information 
which 
becomes available as the SAE evolves, as well as supporting 
documentation (e.g., hospital discharge summaries and autopsy reports), should 
be collected subsequently, if not available at the time of the initial report, and  
immediately sent using  the same procedure as the initial SAE report. 
• 
An overdose is defined as the accidental or intentional ingestion of any dose of a 
product that is considered both excessive and medically important. For reporting 
purposes, WCM considers an overdose, regardless of adverse outcome, as an 
important medical event. 
• 
AEs should be followed to resolution or stabilization, and reported as SAEs if  they 
become serious. This also applies to subjects experiencing AEs that cause 
interruption or discontinuation of nivolumab, or those experiencing AEs that are 
present at the end of their participation in the study; such subjects should receive 
post-treatment follow-up as appropriate. 
• 
All SAEs must be collected which occur within 100 days of discontinuation of 
dosing or completion of the patient’s participation in the study if the last scheduled 
visit occurs at a later time. In addition, the Investigator should notify WCM of any 
SAE that may occur after this time period which they believe to be certainly, 
probably, or possibly related to nivolumab. 
 
Pregnancy 
 
Sexually active women of childbearing potential must use an effective method of birth control during 
the course of the study, in a manner such that risk of failure is minimized. 
Before enrolling women of childbearing potential in this clinical trial, Investigators must review the 
guideline 
about study participation for WOCBP which can be found in the GCP Manual for 
Investigators. The topics include the following: 
• 
General Information 


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• 
Informed Consent Form 
• 
Pregnancy Prevention Information Sheet 
• 
Drug Interactions with Hormonal Contraceptives 
• 
Contraceptives in Current Use 
• 
Guidelines for the Follow-up of a Reported Pregnancy 
Prior to study enrollment, WOCBP must be advised of the importance of avoiding pregnancy 
during trial participation and the potential risk factors for an unintentional pregnancy. The subject  
must sign an informed consent form documenting this discussion. 
All WOCBP MUST have a negative pregnancy test within 72 hours prior to receiving 
nivolumab. The minimum sensitivity of the pregnancy test must be 25 IU/L or equivalent units of  
hCG. If the pregnancy test is positive, the subject must not receive nivolumab and must not be  
enrolled in the study. 
In addition, all WOCBP should  be instructed  to contact the  Investigator  immediately if  they 
suspect they might be pregnant (e.g., missed or late menstrual period) at any time during study 
participation. Additionally, all pregnancies must be reported to BMS via confirmed facsimile (fax) 
transmission, or scanned and reported via electronic mail to: 
SAE Email Address: Worldwide.Safety@BMS.com 
SAE Fax Number: 609-818-3804 
If following initiation of study treatment, it is subsequently discovered that a trial subject is 
pregnant or may have been pregnant at the time of nivolumab exposure, including during at 
least 6 half-lives after product administration, nivolumab will be permanently discontinued in an 
appropriate manner (e.g., dose tapering  if necessary  for  subject  safety).  Exceptions  to  
nivolumab  discontinuation  may be considered  for  life-threatening  conditions only after  
consultation with the Principal Investigator or as otherwise specified in this protocol. Protocol- 
required procedures for study discontinuation and follow-up must be performed on the subject 
unless contraindicated by pregnancy (e.g., x-ray studies). Other appropriate pregnancy follow- 
up procedures should  be  considered  if  indicated.  In  addition, the course of the  pregnancy,  
including perinatal and  neonatal outcome. Infants should be followed for a minimum of eight  
weeks. 
Follow-up information regarding the course of the pregnancy, including perinatal and neonatal 
outcome and, where applicable, offspring information can be reported on a Pregnancy 
Surveillance Form provided by BMS. Any pregnancy that occurs in a female partner of a male 
study participant should be reported to BMS. 
Adverse events that are considered non-serious events, including lab abnormalities will be 
documented in REDCap database and will be reported to BMS on a regular basis. In addition to 
that, WCMC Datasafety monitoring committee will be reviewing the study on an annual basis and 
will review the adverse events collected on the study. 
 
 
12.3 Off Study Criteria 
1. Intercurrent illness that prevents further administration of treatment, 
2. Unacceptable toxicity (defined in Section 15), 


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3. Patient decides to withdraw from the study, or 
4. General  or  specific  changes  in  the  patient's  condition  render  the  patient 
unacceptable for further treatment in the judgment of the investigator. 
5.   Authorized Physicians must notify the data manager and Principle Investigator when 
a patient is taken off study. 
 
 
13 PROHIBITED AND RESTRICTED THERAPIES DURING THE STUDY 
13.1 Prohibited Therapies 
Patients in this study may not use vaccines for the treatment of cancer for up to one month before 
the first dose of nivolumab. Concomitant systemic or local anti-cancer medications or treatments 
are prohibited while receiving study treatments. 
• 
Patients may not use any of the following therapies during the study: 
• 
Any non-study anti-cancer agent (investigational or non-investigational); 
• 
Any other investigational agents; 
• 
Any other CTLA-4 or anti PD-1 or PD-L1 inhibitors or agonists; 
• 
Immunosuppressive agents; 
• 
Chronic systemic corticosteroids. 
 
 
13.2 Data and Safety Monitoring Plan (DSMP) 
Study monitoring 
There will be monthly reports generated for study accrual, observed AEs, and data timeliness. 
The study team will review these reports. In addition, the study will undergo periodic review by the 
Weill Cornell data safety and monitoring board (DSMB). 
DSMB Safety Review 
The protocol will be reviewed by the Data Safety Monitoring Board (DSMB) on a semi-annual basis. 
Safety reports will be submitted to the DSMB every six months. 
 
 
ADMINISTRATIVE SECTION 
 
 
Compliance with the Protocol and Protocol Revisions 
 
The study will be conducted as described in the final approved protocol. Documentation of 
approval signed by the chairperson or designee of the IRB(s) will be sent to the WCM protocol  
manager. 
All revisions (protocol amendments, administrative letters, and changes to the informed consent) 
will be submitted to the WCM protocol manager. The Investigator will not implement any deviation 
or change to the protocol without prior review and documented approval/favorable opinion from 
the IRB of an Amendment, except where necessary to eliminate an immediate  hazard(s) to study 


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patients. 
 
13.3 Informed Consent 
The Investigator will ensure that patients are clearly and fully informed about the purpose, potential 
risks, and other critical issues regarding clinical trials in which they volunteer to participate. 
Preparation of the consent form is the responsibility of the Investigator and will include all elements 
required by the Code of Federal Regulations 21 Part 50.25 and the local IRB. Written informed 
consent will be obtained by IRB approved physicians listed on the title page of this protocol. The 
informed consent form will be signed by the subject and the registering physician. Once signed, a 
copy will be given to the subject and one will be maintained with the subject’s medical record. Once 
eligibility is confirmed and informed consent is documented, the patient will be registered by the 
study coordinator/data manager. The study samples obtained will be for the purpose of immune-
monitoring study when funds become available. 
 
14 APPENDIX 1: Laboratory Correlate Manual 
 
14.1 At Baseline: 
1. 
Tissue Biopsies (4 core biopsies) 
4 core biopsies will be collected by interventional radiology form the same lesion and placed in 
one container as described below. 
 
• Immediately after tumor exeresis, put the specimen into a appropriately labeled 
container (i.e. 50ml Falcon, specialized OR containers etc.) into sterile saline 
(ideally including Penicillin and Streptomycin) 
• Keep the container 4oC on wet ice (if possible) 
• Bring as soon as possible the tissue sample from the operating theatre to the 
pathology triage area: THIS needs to be coordinated with Dr. Inghirami and 
pathology. 
• Identify the sample using only the trial ID# of the patient and date for all subsequent 
steps 
• On arrival, place the tissue sample in a Petri dish, maintaining it in sterile 
conditions (use sterile tweezers etc.) 
Place the tissue in a sterile Petri dish containing 1-2 ml of tissue culture medium supplemented 
with 10% FCS and antibiotics 
 
TRIAGE: 
 
Priority list, depending on amount of viable tissue: 
 
1. Place one portion in RNA later (for RNA Seq), which will be stored frozen [Needs to be in 
emergency power-line freezer for clinical samples in Rad Onc] 
 
2. PDX: Cut viable tissue into in strip of 2-4 x2mm long with a maximum width of 1 mm 
(alternative prepare cubes of 1 mm3) 
• 
Fresh sample can be used immediate for animals implants 
• 
Please use 2-4 1mm3 cube tissue cubes for each s.c. implantation site 


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3. Histology: One core (or a portion of a core) and be processed for paraffin embedding. The 
blocks will be used for 1 H&E, and stored for multiplexing IHC 
 
Freeze dry samples (for protein and DNA extraction etc.), ~3-5x1mm portion 
 
5. Biobank (unlikely that sufficient tissue will be available, but should be considered): Put 4 
tissue fragments into 1 ml freezing medium (RPMI 1640, 20% FCS, 10% DMSO 
 
Place printed bar coded identification vials and place them to cryovials. Prepare at least 3-5 bar 
coded vials for multiple experiments/implants (biobank). Place the tissue vials into an appropriate 
container with isopropyl alcohol at – 80°C for least 12 hours. 
Transfer and store the cryovials into a liquid nitrogen tank. Record into the data base sample, 
vials (RNA, DNA, protein and viable) and location. 
 
 
14.2 Baseline Bone Marrow Aspirate and Core 
20ml bone marrow aspirate will be obtained at baseline from all patients. The samples will be 
sent to immunopathology 
 
14.3 Baseline Saliva sample 
Approximately 5ml saliva (sputum) will be obtained from patients for genetic profiling. 
 


45 
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46 
 
Protocol 1612017831 
Version Date 06/28/2019 
 
 
 
 
Label the collection bag with your: 
• 
Name 
• 
Date 
• 
Time of collection 
 
Place the specimens into the collection bag labeled urine and stool. Store the samples in the 
refrigerator until you leave for your appointment (drop off site). 
 
STEP 6: When you are ready to transport the sample to the drop off site, remove the frozen 
gel packs from your freezer. Place one pack in the bottom of the provided lunch bag. Place 
the stool specimen container. 


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Protocol 1612017831 
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