NIMBUS: A Phase II Study of Nivolumab Plus Ipilimumab in Metastatic Hypermutated HER2-negative Breast Cancer
Phase: PHASE2
Conditions: Breast Cancer
Sponsor: Dana-Farber Cancer Institute



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SCHEMA

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TABLE OF CONTENTS
SCHEMA
2
1.
OBJECTIVES5
1.1
Study Design............................................................................................................5
1.2
Primary Objectives...................................................................................................5
1.3
Secondary Objectives...............................................................................................5
1.4
Correlative Objectives .............................................................................................7
1.5
Patient Reported Outcomes Objectives ...................................................................8
2.
BACKGROUND .................................................................................................................8
2.1
Study Disease(s).......................................................................................................8
2.2
The PD-1/PD-L1 pathway in cancer........................................................................8
2.3
Ipilimumab (Yervoy) ...............................................................................................9
2.4
Nivolumab..............................................................................................................14
2.5
Ipilimumab with Nivolumab..................................................................................15
2.6
Rationale ................................................................................................................18
2.7
Correlative Studies Background ............................................................................20
3.
PARTICIPANT SELECTION...........................................................................................23
3.1
Eligibility Criteria..................................................................................................23
3.2
Exclusion Criteria ..................................................................................................25
4.
REGISTRATION PROCEDURES ...................................................................................26
4.1
General Guidelines for DF/HCC Institutions ........................................................26
4.2
Registration Process for DF/HCC Institutions.......................................................27
4.3
General Guidelines for Other Investigative Sites ..................................................27
4.4
Registration Process for Other Investigative Sites.................................................27
5.
TREATMENT PLAN........................................................................................................28
5.1
Treatment Regimen................................................................................................28
5.2
Pre-Treatment Criteria ...........................................................................................28
5.3
Agent administration..............................................................................................29
5.4
General Concomitant Medication and Supportive Care Guidelines......................29
5.5
Criteria for Taking a Participant Off Protocol Therapy.........................................32
5.6
Duration of Follow Up...........................................................................................33
5.7
Criteria for Taking a Participant Off Study ...........................................................33
6.
Dosing Delays/Dose modifications....................................................................................34
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................45
7.1
Expected Toxicities................................................................................................45
7.2
Adverse Event Characteristics...............................................................................46
7.3
Expedited Adverse Event Reporting......................................................................47
7.4
Routine Adverse Event Reporting .........................................................................49

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8.
PHARMACEUTICAL INFORMATION..........................................................................50
8.1
Nivolumab..............................................................................................................50
8.2
Ipilimumab.............................................................................................................52
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................54
9.1
Archival Tissue......................................................................................................55
9.2
Fresh Tissue Biopsy...............................................................................................56
9.3
Blood Collection....................................................................................................62
9.4
Stool Collection .....................................................................................................65
9.5
Central confirmation of the hypermutated status...................................................67
9.6
Additional analysis.................................................................................................67
10.
STUDY CALENDAR .......................................................................................................67
11.
MEASUREMENT OF EFFECT .......................................................................................71
11.1
Antitumor Effect – Solid tumors............................................................................71
11.2
Antitumor Effect – Hematologic Tumors..............................................................78
11.3
Other Response Parameters ...................................................................................78
12.
DATA REPORTING / REGULATORY REQUIREMENTS...........................................80
12.1
Data Reporting.......................................................................................................80
12.2
Data Safety Monitoring..........................................................................................80
12.3
Multicenter Guidelines...........................................................................................81
12.4
Collaborative Research and Future Use of Data and Samples ..............................81
13.
STATISTICAL CONSIDERATIONS...............................................................................82
13.1
Study Design/Endpoints ........................................................................................82
13.2
Sample Size, Accrual Rate and Study Duration ....................................................83
13.3
Analysis of Primary Endpoint................................................................................84
13.4
Analysis of Secondary Endpoints..........................................................................84
13.5
Central confirmation of the tumor mutational burden...........................................85
13.6
Reporting and Exclusions ......................................................................................85
14.
PUBLICATION PLAN .....................................................................................................86
15.
REFERENCES ..................................................................................................................87
APPENDIX A
PERFORMANCE STATUS CRITERIA ..................................................94
APPENDIX B
Guidelines for collecting research biopsy tissue........................................95
Risks of Research Biopsy and Procedures for Minimizing Risk.......................................95
Risks of Anesthesia............................................................................................................96
APPENDIX C
FACT-B Questionnaire..............................................................................99
APPENDIX D
Rotterdam Symptom Checklist................................................................102
Participant ID__________ Cycle#___________.........................................................................102

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APPENDIX E
DF/HCC MULTI-CENTER DATA AND SAFETY MONITORING 
PLAN
104
1.
OBJECTIVES
1.1
Study Design
1.1.1
This is an open-label, single-arm, multicenter, phase 2 study of nivolumab 3 mg/Kg 
intravenously (IV) every 2 weeks (14 days) plus Ipilimumab 1 mg/Kg IV every 6 weeks 
(cycle length= 6 weeks) in subjects with hypermutated metastatic HER-2 negative breast 
cancer previously treated with 0 to 3 chemotherapy regimens in the metastatic setting. 
Mandatory research biopsies will be performed at baseline (if tissue is safely accessible) 
and at day 29 of Cycle 1 (+14 day scheduling window).
1.2
Primary Objectives
1.2.1
To evaluate the efficacy of the nivolumab plus ipilimumab, as defined by objective 
response rate (ORR) according to RECIST 1.1 [Eisenhauer et al., 2009] in patients with 
hypermutated HER2- metastatic breast cancer previously treated with 0 to 3 lines of 
systemic therapy in the metastatic setting.
1.3
Secondary Objectives
Safety objectives
1.3.1
To evaluate the safety and tolerability of the combination of nivolumab plus ipilimumab.
Efficacy objectives:

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1.3.2
To evaluate the objective response rate of the combination according to immune-related 
response criteria (irRC) [Wolchok et al., 2009]. (Section 11). 
1.3.3
To evaluate the Clinical Benefit Rate (CR + PR + SD ≥ 24 weeks) of the combination 
according to RECIST 1.1. 
1.3.4
To evaluate the progression-free survival (PFS) of the combination according to RECIST 
1.1. 
1.3.5
To evaluate the Clinical Benefit Rate (CR + PR + SD ≥ 24 weeks) of the combination 
according to irRC [Wolchok et al., 2009]. (Section 11). 
1.3.6
To evaluate the progression-free survival (PFS) of the combination according to irRC 
[Wolchok et al., 2009]. (Section 11)
1.3.7
To evaluate the objective response rate of the combination according to RECIST 1.1 in 
the population with centrally confirmed (via Foundation Medicine panel) hypermutated 
tumors (≥ 9 mutations/megabase) 
1.3.8
To evaluate the PFS of the combination according to RECIST 1.1 in the population with 
centrally confirmed hypermutated tumors (≥ 9 mutations/megabase) 
1.3.9
To evaluate the overall survival (OS) of the combination.

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1.4
Correlative Objectives
1.4.1
To characterize a broad array of immune markers in metastatic hypermutated HER2-
negative breast cancers. (characterization will be based on histology, protein expression, 
and mRNA expression).
1.4.2
To explore how different immunosuppressive and/or immune-stimulating immune 
marker profiles at baseline correlate with disease response to therapy (PFS, objective 
response assessed by RECIST 1.1 and immune-related response criteria).
1.4.3
To characterize changes in tumor-infiltrating lymphocytes, PD-L1 expression and 
immune gene signatures in the tissue microenvironment (TME) from baseline to after 4 
weeks of the experimental combination.
1.4.4
To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in TME correlates with disease response to therapy 
(response assessed by RECIST 1.1 and immune-related response criteria).
1.4.5
To characterize serial changes in immune marker profile in peripheral blood mononuclear 
cells (PBMCs) and in plasma over the course of the trial treatment.
1.4.6
To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in PBMCs correlates with disease response to therapy 
(response assessed by RECIST 1.1 and immune-related response criteria).
1.4.7
To investigate whether there is an immune marker in circulating PBMCs that corresponds 
to tumor infiltrating lymphocyte (TIL) percentage in baseline tumor.
1.4.8
To collect blood to study cell-free DNA for comparison to tumor specimens before and 
after immunotherapy.
1.4.9
To characterize the structure and function of the gut microbiome in patients with breast 
cancer prior to starting this clinical trial.
1.4.10 To determine whether pre-treatment characteristics of the structure and function of the 
gut microbiome in patients with breast cancer is associated with disease response to 
therapy (response assessed by RECIST 1.1 and irRC, and PFS).
1.4.11 To characterize changes in the structure and function of the gut microbiome of patients 
with breast cancer after 4 weeks of therapy compared to baseline. 
1.4.12 To determine whether changes in the overall diversity of the gut microbiome, estimated 
by the Shannon Index, of patients with breast cancer after after 4 weeks of therapy is 
associated with disease response (response assessed by RECIST 1.1, irRC and PFS).
1.4.13 To determine if the abundance and functional profile of specific gut bacteria are 
associated with objective response therapy (response assessed by RECIST 1.1, irRC and 
PFS).
1.4.14 To evaluate the functional pathways that may play a role as a predictive biomarker of 
disease response to therapy (response assessed by RECIST 1.1, irRC and PFS).
1.4.15 To determine whether pre-treatment characteristics of the structure and function of the 
gut microbiome in patients with breast cancer is associated with therapy-induced grade 
≥2 diarrhea.
1.4.16 To explore whether the cut off of ≥ 14 mutations/megabase is associated with the efficacy 
of this investigational therapy, assessed by ORR per RECIST 1.1. 
1.4.17 To explore whether the cut off of ≥ 14 mutations/megabase is associated with the efficacy 
of this investigational therapy, assessed by PFS per RECIST 1.1
1.4.18 To explore whether the cut off of ≥ 14 mutations/megabase is associated with the efficacy 
of this investigational therapy, assessed by OS per RECIST 1.1

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1.5
Patient Reported Outcomes Objectives
1.5.1
To assess the participant’s quality of life by the FACT-B questionnaire
1.5.2
To assess the participant’s quality of life by the Rotterdam Symptom Checklist
2.
BACKGROUND
2.1
Study Disease(s)
Breast cancer (BC) is the most frequently diagnosed cancer and the second cause of cancer 
death in women in the Unites States[Siegel et al., 2016]. Approximately, 80% of these cancers are 
classified as HER2-negative, comprising those with absent overexpression of the human epidermal 
growth factor receptor 2 (HER2)[O'brien et al., 2010]. Despite progress in the past years regarding 
the biology of breast cancer, metastatic disease is still incurable, independently of the hormonal 
receptor status. Therefore, new treatments are urgently needed.
2.2
The PD-1/PD-L1 pathway in cancer
The importance of intact immune surveillance in controlling outgrowth of neoplastic 
transformation has been known for decades[Schreiber et al., Schreiber, 2012]. Accumulating 
evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and 
favorable prognosis in various malignancies[Mlecnik et al., 2014].  In particular, the presence of 
CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate 
with improved prognosis and long-term survival in many solid tumors[Tosolini et al., 2006, 
Adams et al., 2014, Denkert et al., 2015].
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress 
immune control.  The normal function of PD-1, expressed on the cell surface of activated T-cells 
under healthy conditions, is to down-modulate unwanted or excessive immune responses, 
including autoimmune reactions.  PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member 
related to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor 
signaling upon engagement of its ligands (PD-L1 and/or PD-L2).  The structure of murine PD-1 
has been resolved.  PD-1 and family members are type I transmembrane glycoproteins containing 
an Ig Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail which 
is responsible for the binding of signaling molecules.  The cytoplasmic tail of PD-1 contains 2 
tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif (ITIM) and 
an immunoreceptor tyrosine-based switch motif (ITSM).  Following T-cell stimulation, PD-1 
recruits the tyrosine phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, 
leading to the dephosphorylation of effector molecules such as CD3ζ, PKCθ and ZAP70 which 
are involved in the CD3 T-cell signaling cascade.  The mechanism by which PD-1 down modulates 
T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an 
overlapping set of signaling proteins.  PD-1 was shown to be expressed on activated lymphocytes 
including peripheral CD4+ and CD8+ T-cells, B-cells, T regs and Natural Killer cells.  Expression 
has also been shown during thymic development on CD4-CD8- (double negative) T-cells as well 
as subsets of macrophages and dendritic cells.  The ligands for PD-1 (PD-L1 and PD-L2) are 
constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic 
tissues as well as in various tumors.  Both ligands are type I transmembrane receptors containing 
both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions 

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with no known signaling motifs.  Binding of either PD-1 ligand to PD-1 inhibits T-cell activation 
triggered through the T-cell receptor.  PD-L1 is expressed at low levels on various non-
hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only 
detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic inflammatory 
environments.  PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas 
PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues.  Although healthy 
organs express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant 
levels of this T-cell inhibitor.  PD-1 has been suggested to regulate tumor-specific T-cell expansion 
in subjects with melanoma (MEL).  This suggests that the PD-1/PD-L1 pathway plays a critical 
role in tumor immune evasion and should be considered as an attractive target for therapeutic 
intervention[Intlekofer et al., 2013].
The PD-1/PD-L1 pathway in breast cancer
Unlike melanoma and NSCLC, BC has not been intensively investigated for its 
susceptibility to immunotherapy in clinical settings. However, there is accumulating preclinical 
and clinical evidence suggesting that immune system is critical during natural history of breast 
cancer and the immune system can be modulated to improve outcomes in this disease[Kroemer et 
al., 2015]. It has been recognized that BC is capable of stimulating the immune system, as many 
breast tumors have substantial lymphocyte infiltration [Denkert et al., 2010, Denkert et al., 2015]. 
Additionally, this pathologic feature has prognostic implications, as lymphocyte predominant 
breast cancers are associated with improved prognosis [Denkert et al., 2010, Loi et al., 2013]. 
However, the degree of immune infiltration differs by BC subtype; while a substantial proportion 
of triple negative BC can be richly infiltrated, hormone-receptor positive BC is poorly T-cell 
infiltrated[Dushyanthen et al., 2015]. Recently, it has been demonstrated that the expression of 
PD-1 and PD-L1 differs among breast tumors subtype: HR-positive (30% PD-1; 33% PD-L1), 
triple-negative (70% PD-1; 59% PD-L1) and HER2-positive (60% PD-1; 20% PD-L1)[Gatalica et 
al., 2014].
2.3
Ipilimumab (Yervoy)
Ipilimumab (BMS-734016, MDX010, MDX-CTLA4) is a fully human monoclonal 
immunoglobulin (Ig) G1κ specific for human cytotoxic T-lymphocyte antigen 4 (CTLA-4, cluster 
of differentiation [CD] 152), which is expressed on a subset of activated T cells. CTLA-4 is a 
negative regulator of T-cell activity. Ipilimumab is a monoclonal antibody (mAb) that binds to 
CTLA-4 and blocks the interaction of CTLA-4 with its ligands, CD80/CD86. Blockade of CTLA-4 
has been shown to augment T-cell activation and proliferation, including the activation and 
proliferation of tumor infiltrating T-effector cells. Inhibition of CTLA-4 signaling can also reduce 
T-regulatory cell (Treg) function, which may contribute to a general increase in T-cell 
responsiveness, including the anti-tumor response. 
Ipilimumab has been approved for use in over 47 countries including the United States 
(US, Mar-2011), the European Union (EU, Jul-2011), and Australia (Jul-2011).
2.3.1
Non-Clinical Summary 
Ipilimumab has specificity and a high affinity for human CTLA-4. The calculated 
dissociation constant value from an average of several studies was 5.25 nM. Binding of ipilimumab 
to purified, recombinant human CTLA-4 antigen was also demonstrated by enzyme-linked 

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immunosorbent assay with half-maximal binding at 15 ng/mL, whereas saturation was observed 
at approximately 0.1 μg/mL. No cross-reactivity was observed against human CD28. Ipilimumab 
completely blocked binding of B7.1 and B7.2 to human CTLA-4 at concentrations higher than 6 
and 1 μg/mL, respectively.
Please refer to the ipilimumab investigator’s brochure (IB) for further information on pre-
clinical development.
2.3.2
Clinical Studies 
Bristol-Myers Squibb (BMS) and Medarex, Inc. (MDX, acquired by BMS in Sep-2009) 
have co-sponsored an extensive clinical development program for ipilimumab, encompassing 
more than 19,500 subjects (total number of subjects enrolled in ipilimumab studies) in several 
cancer types in completed and ongoing studies, as well as a compassionate use program. The focus 
of the clinical program is in melanoma, prostate cancer, and lung cancer, with advanced melanoma 
being the most comprehensively studied indication. Ipilimumab is being investigated both as 
monotherapy and in combination with other modalities such as chemotherapy, radiation therapy, 
and other immunotherapies. 
Phase 3 programs are ongoing in melanoma, prostate cancer, and lung cancer. In 
melanoma, 2 completed phase 3 studies have demonstrated a clinically meaningful and statistically 
significant survival benefit in pretreated advanced melanoma and previously untreated advanced 
melanoma, respectively. 
The safety profile of ipilimumab is generally consistent across these trials with a) the 
majority adverse events (AEs) being inflammatory in nature, which is consistent with the proposed 
mechanism of action of ipilimumab; b) the same types of such immune-mediated events in the 
gastrointestinal (GI) tract, skin, liver, and endocrine system being reported; and c) most of these 
events being manageable with immune suppressive therapies. 
In melanoma, 2 BMS-sponsored phase 3 studies are ongoing in subjects with high-risk 
stage III melanoma and pretreated and treatment-naïve advanced melanoma (3 mg/kg versus 10 
mg/kg ipilimumab). 
The completed phase 3 study evaluated ipilimumab in subjects with metastatic castration-
resistant prostate cancer (mCRPC) who had progressed during or following treatment with 
docetaxel. Eligible subjects were randomized to a single dose of bone-directed radiotherapy (RT), 
followed by either ipilimumab 10 mg/kg or placebo (799 randomized: 399 ipilimumab, 400 
placebo). This study did not meet its primary endpoint of overall survival (OS). The hazard ratio 
(HR) of 0.85 (95% confidence interval [CI]: 0.72, 1.00) for survival favored ipilimumab but did 
not reach statistical significance with a p-value of 0.053. Planned sensitivity analyses favored 
ipilimumab, where the greatest benefit appeared to be in subgroups defined by good prognostic 
features and low burden of disease. Additional evidence of ipilimumab activity observed in the 
study included a reduced risk of disease progression relative to placebo (HR = 0.70), superior 
clinical outcomes compared to placebo in tumor regression, and declines in prostate specific 
antigen (PSA). The safety profile in this study was consistent with the previously defined AE 
profile at the same dose. 
A second phase 3 study evaluated ipilimumab 10 mg/kg versus placebo in men with 
asymptomatic or minimally symptomatic, chemotherapy-naïve mCRPC with no visceral 
metastases. A total of 602 subjects were randomized in a 2:1 ratio (400 subjects to 10 mg/kg 
ipilimumab and 202 subjects to placebo). Preliminary data indicate the study did not meet its 
primary endpoint based on intent-to-treat analysis. The HR of 1.11 (95.87% CI: 0.88, 1.39; P value 

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= 0.3667) for OS did not favor ipilimumab. A longer median progression-free survival (PFS) 
interval was observed for the ipilimumab group than for the placebo group, which may be 
indicative of activity of ipilimumab in delaying disease progression. The safety profile in this study 
was generally consistent with the previously defined AE profile at the same dose. 
In addition, a completed, large phase 2 study has investigated the addition of ipilimumab 
to carboplatin and paclitaxel using 2 different schedules (concurrent and phased) in subjects with 
NSCLC or small cell lung cancer (SCLC, a secondary endpoint). Ipilimumab, given in 
combination with paclitaxel/carboplatin in a phased schedule improved immune-related 
progression-free survival (irPFS) compared to the control treatment, but no improvement was seen 
when ipilimumab was given in a concurrent schedule. Phased ipilimumab also improved PFS 
according to modified World Health Organization (mWHO) criteria and showed a trend for 
improved OS. 
The efficacy and safety of ipilimumab in a phased schedule with carboplatin/paclitaxel is 
also being investigated in a phase 3 study in subjects with advanced squamous NSCLC 
(CA184104). The efficacy and safety of ipilimumab in a phased schedule with etoposide/platinum 
in subjects with extensive stage disease SCLC is being investigated in a phase 3 study 
(CA184156). In Study CA184104, the last patient, last visit was achieved in June 2015, and 
database lock occurred on 01-Sep-2015. No final data are currently available, but preliminary data 
indicate that no new safety concerns were identified in the course of standard clinical safety 
monitoring of the study. In Study CA184156, preliminary data indicate the primary endpoint of 
prolonging survival was not achieved, but no new safety signals were identified. 
The unique immune-based mechanism of action is reflected in the clinical patterns of anti-
cancer activity in some patients. Ipilimumab induces an immunologic response, and measurable 
clinical effects emerge after the immunological effects. Tumor infiltration with lymphocytes and 
the associated inflammation (documented by biopsy in some subjects) is likely the cornerstone of 
the effect of ipilimumab and can manifest in various patterns of clinical activity leading to tumor 
control. In some cases, inflammation may not be noted by radiological examination, and objective 
response is observed with the first tumor assessment in a manner seen in patients receiving other 
types of anti-cancer treatments. In other cases, response may be preceded by an apparent increase 
in initial tumor volume and/or the appearance of new lesions, which may be mistaken for tumor 
progression on radiological evaluations. Therefore, in patients who are not experiencing rapid 
clinical deterioration, confirmation of progression is recommended (at the investigator’s 
discretion) to better understand the prognosis, as well as to avoid unnecessarily initiating 
potentially toxic alternative therapies in subjects who might be benefiting from treatment. 
Immune-related response criteria were developed based on these observations to systematically 
categorize novel patterns of clinical activity and are currently being prospectively evaluated in 
clinical studies.
In metastatic diseases, stabilization is more common than response, and in some instances 
is associated with a slow, steady decline in tumor burden over many months, sometimes improving 
to partial and/or complete responses (CRs). Thus, the immune-based mechanism of action of 
ipilimumab results in durable disease control, sometimes with novel patterns of response, which 
contribute to its unique improvement in OS. 
The unique immune-based mechanism of action is also reflected in the safety profile. The 
most common treatment-related AEs are inflammatory in nature, consistent with the mechanism 
of action of the drug and generally medically manageable with topical and/or systemic 
immunosuppressants. Such immunological safety events are described as immune-related adverse 

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events (irAEs) or immune-mediated adverse reactions (imARs). The irAEs are described as AEs 
of unknown etiology, which were consistent with an immune phenomenon and considered causally 
related to drug exposure by investigators. The irAEs primarily involve the GI tract and skin. 
Immune-related AEs in the liver were also observed, particularly in subjects receiving 10 mg/kg. 
Endocrinopathy and neuropathy were important irAEs that were observed less frequently. The 
imARs were adjudicated in a blinded fashion based on Sponsor-physician data review to exclude 
noninflammatory etiologies, such as infection or tumor progression, and to consider available 
evidence of inflammation, such as tumor biopsies or responsiveness to steroids, in an effort to 
determine whether specific AEs or abnormal hepatic laboratory values were likely to be immune 
mediated and associated with ipilimumab treatment. 
The early diagnosis of inflammatory events is important to initiate therapy and minimize 
complications. Inflammatory events are generally manageable using symptomatic or immuno-
suppressive therapy as recommended through detailed diagnosis and management guidelines. 
A program-wide independent Data Monitoring Committee (DMC) reviews data from the 
ipilimumab studies, allowing for an ongoing safety and benefit/risk assessment in subjects 
receiving ipilimumab. The DMC charter includes explicit stopping rules for some studies, allowing 
the DMC to recommend discontinuing further treatment across the ipilimumab program, if 
necessary. 
In summary, ipilimumab offers clinically meaningful and statistically significant survival 
benefit to patients with pretreated advanced melanoma (as 3 mg/kg monotherapy compared to the 
melanoma peptide vaccine gp100) and previously untreated advanced melanoma (at 10 mg/kg in 
combination with dacarbazine [DTIC] compared to DTIC alone) and evidence of clinical activity 
in randomized studies in other tumor types. These findings, together with evidence of a safety 
profile that is manageable with careful monitoring and appropriate intervention for treatment of 
immune-related toxicities, suggest an acceptable benefit to risk ratio. 
Please also refer to the IB for the most comprehensive clinical background information for 
ipilimumab.
2.3.3
Clinical Pharmacokinetics 
The pharmacokinetics (PK) of ipilimumab has been extensively studied in subjects with 
melanoma, at the 3 and 10 mg/kg doses administered as a 1.5-hour IV infusion. The PK of 
ipilimumab was characterized by population PK (PPK) analysis and determined to be linear and 
time invariant in the dose range of 0.3 to 10 mg/kg. The mean CL (±SD) value after IV 
administration of 10 mg/kg was 18.3 ± 5.88 mL/h, and the mean steady-state volume of distribution 
(Vss) [±SD] value was 5.75 ± 1.69 L.
2.3.4
Pharmacodynamics 
CTLA-4 is a key regulator of T-cell activity. Ipilimumab is a CTLA-4 immune checkpoint 
inhibitor that blocks T-cell inhibitory signals induced by the CTLA-4 pathway, increasing the 
number of tumor reactive T-effector cells that mobilize to mount a direct T-cell immune attack 
against tumor cells. Preclinical data indicate that CTLA-4 blockade can also reduce Treg function, 
which may lead to an increase in anti-tumor immune response. Ipilimumab may selectively deplete 
Tregs at the tumor site, leading to an increase in the intratumoral T-effector/Treg cell ratio which 
drives tumor response leading to cell death9. 

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2.3.4.1 Ipilimumab Effect on Circulating T Cells 
CTLA-4 is a negative regulator of T-cell activation. By blocking CTLA-4, ipilimumab 
increases the percentage of peripheral activated T cells and central memory T cells. These changes 
are evidenced by Week 4 and generally remain sustained through Week 1. The sum of these 
changes in immune cell subsets may result in anti-tumor activity, as well as irAEs.
2.3.4.2 Ipilimumab Effect on Absolute Lymphocyte Count 
In clinical studies, ipilimumab increased absolute lymphocyte count (ALC) in peripheral 
blood.
In 214 subjects with advanced melanoma, ipilimumab increased ALC in a dose-dependent 
manner, with the largest increase observed at 10-mg/kg dose. 
In phase 3 studies in advanced melanoma, high baseline ALC was found to be significantly 
associated with longer OS regardless of treatment. However, subjects with low baseline ALC 
treated with ipilimumab demonstrated longer OS relative to subjects not treated with ipilimumab.
2.2.5 Clinical Efficacy 
Ipilimumab prolongs survival in subjects with pretreated and previously untreated 
advanced melanoma and has demonstrated anti-tumor activity in other malignancies, including 
lung, prostate cancer, and renal cell carcinoma. Please refer to the IB for detailed efficacy 
information regarding ipilimumab monotherapy.
2.2.6 Clinical Safety 
Blockade of CTLA-4 by ipilimumab leads to T-cell activation, with the potential for 
clinical inflammatory adverse events (AEs) primarily involving the skin (dermatitis/pruritus), GI 
tract (diarrhea/colitis), liver (hepatitis), endocrine glands (e.g., hypophysitis and adrenal or thyroid 

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abnormalities), and other less frequent organs (e.g., uveitis/episcleritis). The majority of these 
inflammatory AEs initially manifested during treatment; however, a minority occurred weeks to 
months after discontinuation of ipilimumab. The majority of the inflammatory AEs are reversible 
with the guidance issued in Section 6. In rare cases, these inflammatory AEs may be fatal. 
Patients should be assessed for signs and symptoms of enterocolitis, dermatitis, 
neuropathy, and endocrinopathy, and clinical chemistries (including liver function, 
adrenocorticotropic hormone [ACTH] level, and thyroid function tests) should be evaluated. 
During evaluation of a suspected inflammatory AE, all efforts should be made to rule out 
neoplastic, infectious, metabolic, toxin, or other etiologic causes. Serological, immunological, 
imaging, and biopsy with histology (e.g., biopsy-proven lymphocytic) data should be used to 
support the diagnosis of an immune-mediated toxicity or support an alternative cause of the AE. 
In general, for severe inflammatory AEs, ipilimumab should be permanently discontinued, and 
systematic high-dose corticosteroid therapy should be initiated. For moderate immune-mediated 
AEs, ipilimumab should be held or delayed, and moderate-dose corticosteroids should be 
considered. 
Based on limited current clinical experience, corticosteroids do not appear to adversely 
affect the anti-tumor response. For example, disease control was maintained in subjects with 
objective responses who received corticosteroid administration for concomitant serious 
inflammatory AEs.
2.4
Nivolumab
Nivolumab is a fully human, IgG4 (kappa) isotype monoclonal antibody that binds to PD-
1 with nanomolar affinity (KD = 3.06 nM) and a high degree of specificity, thus precluding binding 
to its ligands PD-L1 and PD-L2. Nivolumab does not bind other related family members, such as 
BTLA, CTLA-4, ICOS or CD28. Pre-clinical testing of nivolumab demonstrated that blockade of 
PD-1 results in enhanced T cell proliferation and expression of interferon-gamma (IFN-).
2.4.1
Nivolumab nonclinical toxicology 
Toxicology studies in cynomolgus monkeys revealed that nivolumab was well tolerated at 
doses up to 50 mg/kg given twice weekly for 27 doses. Drug related findings were limited to a 
reversible decrease in triiodothyronine (T3) by 28%, without concomitant abnormalities in other 
markers of thyroid function. Preliminary new non-clinical safety findings of adverse pregnancy 
outcomes and infant losses in the absence of overt maternal toxicity have been reported.66 The 
findings of increased late stage pregnancy loss and early infant deaths/euthanasia in nivolumab 
exposed pregnant monkeys suggest a potential risk to human pregnancy if there is continued 
treatment with nivolumab during pregnancy.
2.4.2
Clinical Experience with nivolumab
 The overall safety experience with nivolumab, as monotherapy or in combination with 
other therapeutics, is based on experience in approximately 1,500 subjects treated to date. For 
monotherapy, the safety profile is similar across tumor types. The one exception is pulmonary 
inflammation AEs which may be numerically greater in subjects with NSCLC because in some 
cases it can be difficult to distinguish between nivolumab related and unrelated causes of 
pulmonary symptoms and radiographic changes. There was no pattern in the incidence, severity, 
or causality of AEs to nivolumab dose level. 
In several ongoing clinical trials, the safety of nivolumab in combination with other 
therapeutics such as ipilimumab, cytotoxic chemotherapy, anti-angiogenics and targeted therapies 

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is being explored. Most studies are ongoing and as such, the safety profile of nivolumab 
combinations continues to evolve. The most advanced combination under development is 
nivolumab and ipilimumab in subjects with MEL. Thus far, the combination of both agents results 
in a safety profile with similar types of AEs as either agent alone, but in some cases with greater 
frequency. 
Overall, the safety profile of nivolumab monotherapy as well as combination therapy is 
manageable and generally consistent across completed and ongoing clinical trials with no MTD 
reached at any dose tested, up to 10 mg/kg. There was no pattern in the incidence, severity, or 
causality of AEs to nivolumab dose level. Most AEs were low grade (grade 1 to grade 2) with 
relatively few related high grade (grade 3 to grade 4) AEs. Most high grade events were 
manageable with the use of corticosteroids or hormone replacement therapy for Endocrinopathies. 
Management algorithms including the use of immunosuppressive agents, such as corticosteroids, 
infliximab, etc., are provided in Section of this protocol. Nivolumab should not be used in subjects 
with active autoimmune disease given the mechanism of action of the antibody.
Nivolumab has demonstrated clinical activity in response evaluable subjects with a variety 
of solid tumor malignancies in the following studies:
1) completed Phase 1 single-dose (MDX1106-01): n = 37 subjects with prostate cancer, 
MEL, NSCLC, RCC, and CRC
2) ongoing Phase 1 multi-dose, dose escalation study with nivolumab monotherapy 
(CA209003/MDX1106-03): NSCLC n = 129, MEL n = 107, RCC n = 34 subjects
3) ongoing Phase 1b study with nivolumab in combination with ipilimumab 
(CA209004/MDX1106-04): MEL n = 82 subjects
4) ongoing Phase 1 study with nivolumab monotherapy or in combination with platinum-
based chemotherapy or erlotinib (CA209012): NSCLC, monotherapy n = 20, combination therapy 
n = 77 subjects
In addition, monotherapy or combination trials with nivolumab are on-going for subjects 
with SCCHN,67 HCC,68 CRC69, GBM70, and NHL.71,72
Updated overall clinical experience for nivolumab is available in the current version of the 
Investigator’s Brochure.
2.5
Ipilimumab with Nivolumab
2.5.1
Clinical Pharmacokinetics 
The pharmacokinetics of nivolumab and ipilimumab were assessed using a PPK approach 
when nivolumab and ipilimumab were administered in combination. The %CV CL, Vss, and t1/2 
of nivolumab were 10.0 mL/h (50.3%), 7.92 L (30.1%), and 24.8 days (94.3%), respectively. When 
ipilimumab 3 mg/kg (registrational dose) was administered in combination with nivolumab, the 
CL of nivolumab was increased by 24%, whereas there was no effect on the CL of ipilimumab. 
However, co-administration with ipilimumab 1 mg/kg did not appear to have an effect on 
nivolumab CL. This is unlikely to be clinically relevant given the observed efficacy with the 
nivolumab and ipilimumab combination regimen. 
When administered in combination, the CL of nivolumab increased by 42% in the presence 
of anti-nivolumab antibodies. There was no effect of anti-ipilimumab antibodies on the CL of 
ipilimumab.

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2.5.2
Immunogenicity 
Of 394 patients who were treated with nivolumab in combination with ipilimumab and 
evaluable for the presence of anti-nivolumab antibodies, 149 patients (37.8%) tested positive for 
treatment-emergent anti-nivolumab antibodies by an ECL assay and 18 patients (4.6%) had 
neutralizing antibodies against nivolumab. Of the 391 patients evaluable for the presence of anti-
ipilimumab antibodies, 33 patients (8.4%) tested positive for treatment-emergent anti-ipilimumab 
antibodies by an ECL assay and 1 patient (0.3%) had neutralizing antibodies against ipilimumab. 
There was no evidence of increased incidence of infusion reactions/hypersensitivity reactions with 
antinivolumab antibody development. There was no evidence of altered toxicity profile associated 
with anti-product antibody development and there was no apparent casual effect of neutralizing 
antibodies on loss of efficacy.
2.5.3
Clinical Efficacy 
In trial CA209012 (NCT01454102; CheckMate 012), an ongoing multi-arm phase 1 safety 
study of nivolumab in chemotherapy-naive NSCLC subjects, 56 subjects were administered 
nivolumab in combination with chemotherapy (gemcitabine / cisplatin, pemetrexed / cisplatin, 
carboplatin / paclitaxel), 21 with nivolumab in combination with erlotinib, and 197 with nivolumab 
in combination with ipilimumab. 
A summary of ORR and PFS rate at 12 months for subjects treated with nivolumab plus 
ipilimumab is provided in the table below. The original nivolumab plus ipilimumab combinations 
using the same dosing regimens as in the melanoma studies (n = 49) were found to be non-tolerable 
for the NSCLC population, and these regimens were not pursued further. The results from newer 
combination dosing regimens (arms N, O, P, Q; n = 148) with lower and less frequent dosing of 
ipilimumab are presented below. The cohorts containing the approved dose of nivolumab (3 
mg/kg) demonstrated improved ORR and PFS compared to the cohorts containing the lower dose 
of nivolumab (1 mg/kg). 
The regimens for these cohorts were: 
Arm N (n=31): nivolumab 1 mg/kg + ipilimumab 1 mg/kg every 3 weeks for 4 cycles, 
followed nivolumab 3 mg/kg every 2 weeks 
Arm O (n=40): nivolumab 1 mg/kg every 2 weeks + ipilimumab 1 mg/kg every 6 
weeks 
Arm P (n= 38): nivolumab 3 mg/kg every 2 weeks + ipilimumab 1 mg/kg every 12 
weeks
Arm Q (n=39): nivolumab 3 mg/kg every 2 weeks + ipilimumab 1 mg/kg every 6 
weeks 

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Hellmann et al published the results of the CheckMate 227, a multicenter randomized phase 
3 trial that examined progression-free survival with nivolumab plus ipilimumab versus 
chemotherapy among patients with a high tumor mutational burden (defined as ≥10 mutations per 
megabase)[Hellmann et al., 2018]. A total of 2877 patients were enrolled in part 1 of the trial from 
August 2015 through November 2016, and 1739 underwent randomization. Of the 1004 patients 
whose tumor mutational burden could be evaluated across all treatment groups, 444 (44.2%) had 
at least 10 mutations per megabase, including 139 patients assigned to nivolumab plus ipilimumab 
and 160 patients assigned to chemotherapy. Patients received nivolumab (3 mg per kilogram of 
body weight every 2 weeks) plus ipilimumab (1 mg per kilogram every 6 weeks) or platinum 
doublet chemotherapy. The progression-free survival among patients with a high tumor mutational 
burden was significantly longer with nivolumab plus ipilimumab than with chemotherapy. The 1-
year progression-free survival rate was 42.6% with nivolumab plus ipilimumab versus 13.2% with 
chemotherapy, and the median progression-free survival was 7.2 months (95% confidence interval 
[CI], 5.5 to 13.2) versus 5.5 months (95% CI, 4.4 to 5.8) (hazard ratio for disease progression or 
death, 0.58; 97.5% CI, 0.41 to 0.81; P<0.001). The objective response rate was 45.3% with 
nivolumab plus ipilimumab and 26.9% with chemotherapy. The benefit of nivolumab plus 
ipilimumab over chemotherapy was broadly consistent within subgroups, including patients with 
a PD-L1 expression level of at least 1% and those with a level of less than 1%. 
More recently, data from the TAPUR trial (ASCO 2019 presentation), demonstrated 
benefit from checkpoint inhibitor monotherapy (pembrolizumab) in patients with metastatic breast 
cancer and tumor mutational burden defined as > 9 mutations per megabase [Alva et al., 2019]. 
2.5.3.1 Clinical Safety 
The original nivolumab plus ipilimumab combinations using the same dosing regimens as 
in the melanoma studies were found to be non-tolerable for the NSCLC population, so 4 additional 

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regimens were examined (arms N, O, P, Q), and the results from these newer cohorts are presented. 
The regimens for these cohorts were: 
Arm N (n=31): nivolumab 1 mg/kg + ipilimumab 1 mg/kg every 3 weeks for 4 
cycles, followed nivolumab 3 mg/kg every 2 weeks 
Arm O (n=40): nivolumab 1 mg/kg every 2 weeks + ipilimumab 1 mg/kg every 6 
weeks 
Arm P (n= 38): nivolumab 3 mg/kg every 2 weeks + ipilimumab 1 mg/kg every 12 
weeks 
Arm Q (n=39): nivolumab 3 mg/kg every 2 weeks + ipilimumab 1 mg/kg every 6 
The most frequently reported drug-related adverse events (AEs) in the newer nivolumab 
plus ipilimumab cohorts were fatigue (29.0%), diarrhea (25.0%), pruritus (23.7%), and fatigue 
(23.1%) in arms N, O, P, Q, respectively. Drug-related serious adverse events (SAEs) reported in 
more than 2 subjects/cohort treated in the newer nivolumab plus ipilimumab cohorts were adrenal 
insufficiency, hypophysitis, pneumonitis, autoimmune hepatitis, diarrhea, colitis, and acute kidney 
injury. 
Drug-related AEs leading to discontinuation reported in more than 1 subject treated in the 
new nivolumab plus ipilimumab arms included ALT increased, AST increased, colitis, myalgia, 
pneumonitis, rash, autoimmune hepatitis, infusion-related reaction, facial nerve disorder, 
esophagitis, and transaminases increased. 
Most deaths were due to disease progression. The only deaths reported due to study drug 
toxicity were in the nivolumab + ipilimumab original treatment groups (respiratory failure 
following Grade 3 colitis, pulmonary hemorrhage, and toxic epidermal necrolysis in a patient with 
history of ulcerative colitis).
In the above mentioned phase 3 trial CheckMate 227 the rate of grade 3 or 4 treatment-
related adverse events was 31.2% with nivolumab plus ipilimumab and 36.1% with 
chemotherapy[Hellmann et al., 2018]. Treatment-related adverse events leading to discontinuation 
were more common with nivolumab plus ipilimumab than with chemotherapy (17.4% vs. 8.9%); 
however, patients continued to receive nivolumab plus ipilimumab longer than chemotherapy. The 
most common treatment-related select adverse events (defined as adverse events of potential 
immunologic causes) with nivolumab plus ipilimumab and nivolumab monotherapy were skin 
reactions (33.9% and 20.7%, respectively); the most common grade 3 or 4 treatment-related select 
adverse events were hepatic events (8.0% and 3.3%, respectively) Overall, treatment-related 
deaths occurred in seven patients (1.2%) treated with nivolumab plus ipilimumab (three died from 
pneumonitis and one each died from myocarditis, acute tubular necrosis, circulatory collapse, and 
cardiac tamponade), six patients (1.1%) treated with chemotherapy (two died from sepsis and one 
each died from multiple brain infarctions, interstitial lung disease, thrombocytopenia, and febrile 
neutropenia with sepsis), and two patients (0.5%) treated with nivolumab (one each died from 
pneumonitis and neutropenia with sepsis).
2.6
Rationale
Breast cancer (BC) is the most frequently diagnosed cancer and the second cause of cancer 
death in women in the Unites States[Siegel et al., 2016]. In the metastatic setting, despite available 
therapies, virtually all patients will die from their disease. Thus, new treatments, such as 

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immunotherapy, are needed. The recognition that overexpression of immune checkpoint molecules 
in the tumor microenvironment has a critical role for antitumor immunity evasion and for cancer 
progression has revolutionized cancer treatment[Pardoll, 2012]. In particular, anti-PD-1/PD-L1 
antibodies have demonstrated clinical activity in more than 15 cancer types and two PD-1 
inhibitors (nivolumab and pembrolizumab) are FDA approved for treatment of advanced mela-
noma, non-small-cell lung carcinoma and renal cell carcinoma[Brahmer et al., 2012, Topalian et 
al., 2012, Topalian et al., 2015]. More than 1,000 clinical trials of checkpoint blockers are currently 
ongoing. In this context, preclinical and clinical evidence suggesting that the immune system can 
recognize and fight against breast cancer [Kroemer et al., 2015, Savas et al., 2015]. It is now 
recognized that a fraction of breast tumors, have substantial lymphocyte infiltration and that this 
pathologic feature has prognostic implications [Savas et al., 2015]. Furthermore, the high rates of 
PD-L1 and PD-1 expression in patients with TNBCs led to clinical trials to address the role of 
PD-1 blockers in this population [Gatalica et al.,  2014].
However, while immunotherapy with PD-1/PD-1 inhibitors has revolutionized treatment 
for many cancers, the majority of patients with metastatic breast cancer are refractory to it. The 
KEYNOTE 086 trial, the largest study evaluating the efficacy of PD-1 inhibitors (Pembrolizumab 
given in monotherapy; flat dose of 200mg every 3 weeks) in patients with mTNBC to date showed 
different outcomes based on the number of previous lines of therapy: cohort A included 170 
patients who had received ≥1 prior chemotherapy for metastatic disease, and showed an objective 
response rate of 4.7% (2% - 9%) [Adams et al., 2015] ; Perhaps, more important is the duration of 
response with the PD-1 inhibitors in patients with mTNBC: 6.3 months; with more than 5 (63%) 
responders without progression of disease at study data cut off; cohort B included 52 patients no 
prior systemic anticancer therapy for metastatic disease, and showed an objective response rate of 
23% (14% - 36%) [Adams et al., 2017]. In patients with HR+ metastatic breast cancer (MBC) has 
been tested, and also only a fraction of patients have shown benefit objective response rates (ORR) 
range from 2.8% to 12%.[Hugo et al., 2015, Dirix et al., 2018] Altogether, these results suggest 
that there must be additional immunosuppressive factors causing resistance to immunotherapy in 
this population, and other agents must be added to anti-PD-1/PD-L1 treatment in order to enhance 
the benefit of immunotherapy in TNBC. In addition, predictive biomarkers are needed to identify 
patients most likely to benefit from immunotherapy. 
High tumor mutational burden (TMB) correlates with high neoantigen burden[Giannakis 
et al., 2016] and high T-cell infiltration. Retrospective studies have been shown that high TMB 
could be predictive of response to immune checkpoint inhibitors (ICI) across different cancer 
types[Snyder et al., 2014, Rizvi et al., 2015, Van Allen et al., 2015]. Moreover, surrogates genomic 
markers of high TMB, including mismatch repair deficiency (dMMR) or micro-satellite instability 
high (MSI-H), or POLE/POLD1 mutations, also have shown to be predictive of response to 
checkpoint blockade[Le et al., 2015, Johanns et al., 2016, Mehnert et al., 2016, Gong et al., 2017]. 
Finally, prospective clinical trials have recently confirmed the importance of high TMB to predict 
benefit of PD-1 inhibition in lung cancer, both as monotherapy[Carbone et al., 2017] and in 
combination with ipilimumab[Hellmann et al.].  
In the United States, both pembrolizumab and nivolumab given as monotherapy are 
approved for the treatment of adult and pediatric  patients with dMMR/MSI-H metastatic 
colorectal cancer with progression after treatment with a fluoropyrimidine, oxaliplatin, and 
irinotecan. Preclinical and clinical data support the idea that nivolumab and ipilimumab act 
synergistically to promote T-cell antitumor activity through complementary mechanisms of 
action[Curran et al., 2010, Larkin et al., 2015, Motzer et al., 2018]. Furthermore, recent data from 

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the CHECKMATE-142 suggests that the combination of a PD-1 inhibitor (Nivolumab) with 
ipilimumab has better efficacy outcomes and is associated with a numerically higher response rate 
(55%; 95% CI, 45 to 64) than nivolumab monotherapy (31%; 95% CI, 21 to 43) in a similar 
population of patients (n = 74) with a comparable median follow-up time[Overman et al., 2017, 
Overman et al., 2018]. Importantly, this study also showed that this combination had a manageable 
safety profile[Overman et al., 2018]. 
High TMB is observed in approximately 2% of all breast cancers, and up to 3.5% in the 
metastatic setting (Barroso-Sousa et al, ASCO Annual Meeting 2018). Given that breast cancers 
are the most common malignancies in woman in the United States, this frequency represents a 
large number of individuals. Importantly, approximately 80% of these hypermutated tumors harbor 
an APOBEC mutational signature, and only approximately 18% have defective DNA mismatch 
repair (MMR) signature. Although pembrolizumab is approved for the treatment of MMR deficient 
tumors independently of cancer type, our data suggests that most hypermutated breast cancers 
would not be identified if using only the MMR status as a biomarker. Therefore, there is a critical 
need to evaluate if hypermutated tumors, independently of MMR status, are more likely to be 
responsive to immune checkpoint inhibitors. 
Thus, we hypothesize that patients with hypermutated HER2-negative metastatic breast 
cancers are more likely to derive an objective response to the combination of nivolumab and 
ipilimumab. We propose to test this hypothesis by evaluating the combination of nivolumab 
3mg/Kg every 2 weeks with ipilimumab 1mg/Kg every 6 weeks (1 cycle = 6 weeks)  in patients 
with hypermutated metastatic HER2- breast cancer in an open-label, single arm phase II study. 
The primary endpoint will be to evaluate the efficacy of the nivolumab plus ipilimumab, as defined 
by objective response rate (ORR) according to RECIST 1.1 in patients with hypermutated HER2- 
metastatic breast cancer previously treated with 0 to 3 lines of systemic therapy in the metastatic 
setting. Additionally, we will perform correlative studies in order to explore the effect of treatment 
on immune-genomic markers in the peripheral blood and in the tumor microenvironment. Research 
biopsies will be performed at baseline (mandatory if the lesion is accessible) and and at day 29 of 
Cycle 1 (+14 day scheduling window). Optional biopsies will be performed at progression in 
patients who experienced an objective response during the study. Moreover, blood samples will 
be collected within 7 days before starting therapy and every 6 weeks for 24 weeks, and then every 
9 weeks. Furthermore, stool samples will be collected within 14 days before starting therapy and 
and at day 29 of Cycle 1 (+14 day scheduling window).  
2.7
Correlative Studies Background
2.7.1
Blood and Tissue Analysis 
The importance of tumor microenvironment and the immunosurveillance in natural history of 
cancer and its outcomes was proved to be true in the last years, with clinical approval of immune 
checkpoint inhibitors[Sharma et al., 2015]. However, less than half of patients with solid tumors 
will derive benefit with these drugs [Hwu et al., 2012, Smith et al., 2012]. Thus, it is crucial to 
elucidate the exact mechanisms of antitumor immunity evasion ongoing in tumor 
microenvironment to successfully develop new cancer immunotherapy and correctly choose the 
best drug for the right patient. This goal can be pursuit through the discovery and validation of 
prognostic and predictive biomarkers.

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A growing body of evidence suggests that patients with advanced solid tumors shows differences 
in tumor microenvironment regarding the presence or absence of a gene expression profile 
indicative of a pre-existing T-cell–inflamed tumor microenvironment[Gajewski, 2015]. Tumors 
classified as T-cell inflamed present a significant infiltration of CD8+ T cells and a type I IFN 
signature. In this group, the main mechanisms of immune evasion are the overexpression of 
immunessupressor molecules acting at the level of the tumor micro- environment, such as immune 
checkpoint molecules (CTLA-4, PD-1/PD-L1, TIM-3, LAG-3), indoleamine-2,3-dioxygenase 
(IDO), and FoxP3. Interestingly, such immunosuppressive molecules seem to be upregulated after 
deflagration of a type I Interferon antitumor response, resulting in T-cell exhaustion, and the so 
called adaptive immune resistance[Gajewski, 2015, Ribas, 2015]. The other group of patients 
presents tumors characterized by a low or absence of intratumoral CD8 T cells and a lack evidence 
of a type I IFN transcriptional signature. This tumor phenotype is called non-T-cell-
inflamed[Gajewski, 2015].
The T-cell inflamed phenotype has positive prognostic value for several types of early stage 
cancer, including breast cancer[Dushyanthen et al., 2015, Perez et al., 2015], suggesting that the 
attempt by the host to generate an anti-tumor immune response reflects a biologic process 
associated with improved patient outcomes[Gajewski, 2015]. In breast oncology, different groups 
have demonstrated that the amount of tumor-infiltrating lymphocytes (TILs) in a tumor specimen, 
commonly assessed simply by histological evaluation of a standard hematoxylin and eosin-stained 
slide by a trained pathologist, is a significant predictor of both response to therapy and overall 
disease outcomes in the neoadjuvant and adjuvant settings [Denkert et al., 2010, Loi et al., 2013, 
Adams et al., 2014, Ali et al., 2014, Salgado et al., 2014, Denkert et al., 2015, Denkert et al., 
2015]. Recently, more in-depth methods of immunologic profiling are being explored in breast 
cancer, for example mRNA expression of immune-activating and immunosuppressive factors, and 
these additional immune profiles also appear to have prognostic significance[Perez et al., 2015]. 
Furthermore, in metastatic setting, the phenotype T-cell-inflamed appears to be associated with 
clinical response to several immunotherapies, including checkpoint blockade[Herbst et al., 2014]. 
Patients with this tumor phenotype seem to be good candidates for immune checkpoint inhibitor 
therapy, alone or in combination. Thus, the bulk of our correlative science in this trial highlights 
our especial interest in characterize a broad array of immune markers in metastatic hypermutated 
HER2-negative breast cancer, investigating whether those markers predict disease response to 
therapy.
Additionally, as a correlative study to this trial, we will characterize the immune marker profile of 
peripheral blood mononuclear cells (PBMCs) in enrolled breast cancer patients.  Furthermore, 
given the demonstrated clinical significance of TILs in breast cancer specimens, we will 
investigate whether there is a peripheral marker whose level corresponds to TIL percentage.  
Lastly, we will evaluate whether there is a correlation between changes in PBMC immune profiles 
and disease response.  Evidence of a correlation would be of significant interest as it would suggest 
the potential presence of a predictive biomarker in the peripheral blood.
These correlative projects are made possible by collaboration with Drs. Elizabeth Mittendorf, 
Catherine Wu, Scott Rodig and Evisa Gjini, and Mariano Severgnini, all of whom are laboratory 
scientists with extensive experience with immune profiling in advanced tumors.  Further details 
can be found in Section 9.

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2.7.2
Microbiome Analysis
The gut microbiota has been recognized as a modulator of immune system development 
[Tinchieri, 2015]. Healthy individuals have microbial populations in their intestinal tract that 
vary markedly in composition [Human Microbiome Project, 2012, Qin et al., 2010].  The 
diversity of intestinal microbiota represents a significant challenge to the host’s immune 
defenses, which must balance immune tolerance of beneficial microbes with inflammatory 
responses against pathogens.  Alterations in the gut microbiota and their resulting interactions 
with intestinal epithelieum and the host immune system are associated with many disease, 
including cancer [Roy et al. 2017].  Recently, two preclinical studies provided to ICI, raising the 
possibility that stool microbiota could be used as biomarker predictors of efficacy to 
immunotherapy [Sivan et al., 2015, Vetizou et al., 2015].  Interestingly, postmenopausal women 
with breast cancer have altered composition and low diversity of their gut microbiota compared 
to healthy controls [Goedert et al., 2015].
Identification of biomarkers that predict response to ICI-based therapies can spare de novo 
resistant patients from the unnecessary risks of immune-related adverse events.  In addition, the 
identification of bacterial species associated with response could open new strategies to 
maximize the clinical benefit of cancer immunotherapy through the modulation of gut 
microbiota.
This correlative project is made possible by collaboration with Dr. Elizabeth Mittendorf of Dana-
Farber Cancer Institute and Dr. Andrew Chan of Massachusetts General Hospital. Further details 
can be found in Section 9.  
2.7.3
Tumor Genomic Profile
In addition to the immune microenvironment, intrinsic tumor factors may be associated 
with response to immune checkpoint inhibitors. Although some of the mechanisms related to de 
novo or acquired resistance to ICI have been recently described, including loss of function in beta-
2- microglobulin or defects in the interferon signaling pathway[Gao et al., 2016, Zaretsky et al., 
2016], the knowledge of immune resistance remains largely unknown. Several gene/pathways 
have been described as possible candidates of having an immunosuppressive role in different 
advanced solid tumor, including MYC amplification[Casey et al., 2016], activation in WNT--
catenin pathway[Spranger et al., 2015], activation in MAPK pathway, loss of PTEN[Li et al., 
2016, Peng et al., 2016, George et al., 2017]. On the other hand, few possible biomarkers of 
response to ICI have emerged, including mutational load[Snyder et al., 2014, Rizvi et al., 2015], 
tumor aneuploidy[Davoli et al., 2017], mismatch repair defects[Le et al., 2015], and BRCA2 
mutation[Hugo et al., 2016]. Notably, there is no data on genomic mechanisms of de novo 
resistance to anti-PD-1 therapy in patients with breast cancer.
Therefore, as a correlative study to this trial, we will to explore whether the number and/or 
type of mutations identified using a next generation sequencing (NGS) panel – OncoPanel - is 
correlated with patient outcomes (PFS, ORR, and CBR). This tool is a cancer genomic assay to 
detect somatic mutations, copy number variations and structural variants in tumor DNA extracted 

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from fresh, frozen or formalin-fixed paraffin-embedded samples. The OncoPanel assay surveys 
exonic DNA sequences of 447 cancer genes and 191 regions across 60 genes for rearrangement 
detection. DNA is isolated from tissue containing at least 20% tumor nuclei and analyzed by 
massively parallel sequencing using a solution-phase Agilent SureSelect hybrid capture kit and an 
Illumina HiSeq 2500 sequencer. The targeted NGS assay (OncoPanel) will be performed at the 
Center for Advanced Molecular Diagnostics (Department of Pathology, Brigham and Women's 
Hospital). This assay has been extensively validated and is used as a CLIA-approved clinical 
molecular test in our institution without any additional sequencing assays to validate the findings 
[Wagle et al., 2012]. 
3.
PARTICIPANT SELECTION
Eligibility will be assessed as part of the screening procedures for all patients.
3.1
Eligibility Criteria  
3.1.1 Participants must have histologically or cytologically confirmed invasive breast cancer, 
with metastatic disease. Participants without pathologic or cytologic confirmation of 
metastatic disease should have unequivocal evidence of metastasis from physical 
examination or radiologic evaluation. 
3.1.2 Breast cancer must be HER2-negative by IHC or non-amplified as determined by the 
current ASCO-CAP criteria. If patient has more than one histological result, the most 
recent one will be usedfor inclusion. Participants may be ER/PR positive or negative. 
3.1.3 Patients must harbor tumors with total mutational burden (TMB) of at least 9 mutations 
per megabase assessed by a cancer-gene panel containing more than 300 genes, and 
performed in a CLIA verified laboratory. Tests like Foundation One, Oncopanel (DFCI), 
or IMPACT (MSKCC) are acceptable for including patients on this trial. 
3.1.4 Participants must have measurable disease by RECIST version 1.1.
3.1.5 Participants must agree to undergo a research biopsy, if tumor is safely accessible, at 
baseline and at day 29 cycle 1 (+14 scheduling window). Previously collected archival 
tissue will be obtained on all participants. Participants for whom newly-obtained samples 
cannot be provided (e.g. inaccessible or participant safety concern) may submit an 
archived specimen (block or if not possible, 20 unstained slides). 
3.1.6 Prior chemotherapy: Participants may have received 0-3 prior chemotherapeutic regimens 
for metastatic breast cancer and must have been off treatment with chemotherapy for at 
least 14 days prior to study treatment initiation. 
3.1.7 Patients with hormone receptor positive breast cancer must have progressed on at least one 
prior line of endocrine therapy in the metastatic setting or have disease recurrence while 
on adjuvant endocrine therapy.

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NOTE: Participants should also be adequately recovered from acute toxicities of prior treatment, 
with the exception of alopecia and peripheral sensory neuropathy.
3.1.8 Prior biologic therapy: Patients must have discontinued all biologic therapy at least 14 
days prior to study treatment initiation. 
3.1.9
Prior radiation therapy: Patients may have received prior radiation therapy in either the 
metastatic or early-stage setting. Radiation therapy must be completed 14 days prior to 
study treatment initiation.
NOTE: In all cases, there must be no ongoing complications from prior radiotherapy.
3.1.10The subject is 18 years old. 
3.1.11  ECOG performance status ≤1(Karnofsky ≥70%, see Appendix A).
3.1.12  Participants must have normal organ and marrow function as defined below:
absolute neutrophil count
≥1,000/mcL
platelets
≥100,000/mcL
hemoglobin 
≥ 8 g/dl
total bilirubin
≤1.5 × institutional upper limit of normal (ULN) (or 
≤ 2.0 x ULN in patients with documented Gilbert’s 
Syndrome)
AST(SGOT)/ALT(SGPT)
≤2.5 × institutional ULN or (≤ 3 × institutional 
ULN for participants with documented liver 
metastases)
creatinine
≤1.5 × institutional ULN OR creatinine clearance ≥ 
40 mL/min (using Cockcroft-Gault formula) for 
participants with creatinine levels above 
institutional ULN.
3.1.13  Female subjects of childbearing potential must have a negative pregnancy test (serum or 
urine) at screening.
Note: Childbearing potential is defined as: participants who have not reached a 
postmenopausal state (≥ 12 continuous months of amenorrhea with no identified cause 
other than menopause) and has not undergone surgical sterilization (removal of ovaries 
and/or uterus).

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3.1.14 Women participants of childbearing potential must agree to use an adequate method of 
contraception.  For women, contraception is required starting with the first dose of study 
medication through 150 days (5 months) after the last dose of study medication. Examples 
of contraceptive methods with a failure rate of < 1% per year include bilateral tubal 
ligation, male sterilization, established and proper use of hormonal contraceptives that 
inhibit ovulation, hormone-releasing intrauterine devices, and copper intrauterine devices.  
The reliability of sexual abstinence should be evaluated in relation to the duration of the 
clinical trial and the preferred and usual lifestyle of the patient. Periodic abstinence (e.g., 
calendar, ovulation, symptothermal, or postovulation methods) and withdrawal are not 
acceptable methods of contraception.
3.1.15 Participants on bisphosphonates may continue receiving bisphosphonate therapy during 
study treatment.  Initiation of bisphosphonate or RANKL agent is allowed on study.  
3.1.16 The participant is capable of understanding and complying with the protocol and has 
signed the informed consent document.
3.2
Exclusion Criteria 
3.2.1 Major surgery within 2 weeks before the first dose of study treatment. 
3.2.2 Concurrent administration of other anti-cancer therapy within 14 days of starting protocol 
therapy and during the course of this study.
3.2.3 The participant has received another investigational agent within 14 days of the first dose 
of study drug.
3.2.4 Has received prior therapy with an anti-PD-1, anti-PD-L1, anti-PD-L2, or anti-Cytotoxic 
T-lymphocyte-associated antigen-4 (CTLA-4) antibody.
3.2.5 Known brain metastases that are untreated, symptomatic, or require therapy to control 
symptoms. Participants with a history of treated central nervous system (CNS) metastases 
are eligible. Treated brain metastases are defined as those having no evidence of progression 
for ≥ 2 weeks after treatment, and no ongoing requirement for corticosteroids, as ascertained 
by clinical examination and brain imaging (magnetic resonance imaging or CT scan) 
completed during screening. Subject must be either off corticosteroids, or on a stable or 
decreasing dose of ≤ 10 mg daily (or equivalent) for at least 7 days prior to first study 
treatment. Treatment for brain metastases may include whole brain radiotherapy, 
radiosurgery, or a combination as deemed appropriate by the treating physician. Participants 
with CNS metastases treated by neurosurgical resection or brain biopsy performed within 
28 days before study treatment initiation will be excluded.

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3.2.6 The subject has uncontrolled, significant intercurrent or recent illness. Individuals with a 
history of different malignancy are ineligible except for the following circumstances. 
Individuals with a history of other malignancies are eligible if they have been disease-free 
for at least 3 years or are deemed by the investigator to be at low risk for recurrence of that 
malignancy. 
3.2.7 Participant has an active infection requiring IV antibiotics at initiation of study therapy. 
3.2.8 Patient has a medical condition that requires chronic systemic steroid therapy or on any 
other form of immunosuppressive medication. For example, participants with autoimmune 
disease that requires systemic steroids or immunosuppression agents should be excluded. 
Replacement therapy (eg., thyroxine, insulin, or physiologic corticosteroid replacement 
therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic 
treatment. 
3.2.9 Subjects with current pneumonitis, or requiring supplementary O2 therapy. 
3.2.10 The participant is known to be positive for human immunodeficiency virus (HIV), 
HepBsAg, or HCV RNA. HIV-positive participants on combination antiretroviral therapy 
are ineligible
3.2.11 Participants with any other active malignancy requiring concurrent intervention.
 
3.2.12 Known hypersensitivity to any of the components of ipilimumab or nivolumab. 
3.2.13 The participant has received a live vaccine within 28 days prior to the first dose of trial 
treatment and while participating in the trial. Examples of live vaccines include, but are 
not limited to, the following: measles, mumps, rubella, varicella/zoster, yellow fever, 
rabies, BCG, and typhoid vaccine. The use of the inactivated seasonal influenza vaccine 
(Fluzone®) is allowed.
3.2.14 The participant is pregnant or breastfeeding. 
Inclusion of Women and Minorities
Both men and women of all races and ethnic groups are eligible for this trial. 
4.
REGISTRATION PROCEDURES
4.1
General Guidelines for DF/HCC Institutions
Institutions will register eligible participants in the Clinical Trials Management 
System(CTMS) OnCore. Registrations must occur prior to the initiation of protocol therapy. Any 
participant not registered to the protocol before protocol therapy begins will be considered 
ineligible and registration will be denied.

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An investigator will confirm eligibility criteria and a member of the study team will 
complete the protocol-specific eligibility checklist.
Following registration, participants may begin protocol therapy. Issues that would cause 
treatment delays should be discussed with the Overall Principal Investigator (PI). If a participant 
does not receive protocol therapy following registration, the participant’s registration on the study 
must be canceled. Registration cancellations must be made in OnCore as soon as possible. 
4.2
Registration Process for DF/HCC Institutions
DF/HCC Standard Operating Procedure for Human Subject Research Titled Subject Protocol 
Registration (SOP #: REGIST-101) must be followed.  
4.3
General Guidelines for Other Investigative Sites
Eligible participants will be entered on study centrally at the coordinating center through the 
Study Coordinator or Project Manager.  The list of required forms can be found in section 4.4.
Following registration, participants should begin protocol therapy within 7 days. Issues that 
would cause treatment delays should be discussed with the Overall PI.  If the subject does not 
receive protocol therapy following registration, the subject must be taken off-study in the CTMS 
(OnCore) with an appropriate date and reason entered.  
4.4
Registration Process for Other Investigative Sites
To register a participant, the following documents should be completed by the participating site 
and faxed to 617-632-5152 or e-mailed to ctopm@dfci.harvard.edu to the project manager:

Signed participant consent form

HIPAA authorization form

Eligibility Checklist

Cancer gene panel showing ≥ 9 tumor mutations per megabase

Baseline exam note including medical/surgical history, ECOG, vital signs

Pathology reports including documentation of HER2 status

EKG Report

Laboratory report including hematology, chemistries, PT/PTT, and pregnancy test

Tumor Assessment report (CAP CT and/or MRI)

Brain MRI report (if applicable)
To complete the registration process, the Coordinator or Project manager will follow DF/HCC 
Standard Operating Procedure for Human Subject Research Titled Subject protocol Registration 
(SOP #: REGIST-101) and register the participant on the protocol. The coordinator will fax or e-
mail the participant study number and confirmation of registration to the participating site.
NOTE: Registration can only be conducted during the business hours of 8:00 AM and 5:00 
PM Eastern Standard Time, Monday through Friday. Same day treatment registrations will 
only be accepted with prior notice and discussion with the DF/HCC Lead Institution.

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5.
TREATMENT PLAN
5.1
Treatment Regimen 
This is an open-label, single-arm, multi-center phase 2 study of nivolumab 3 mg/Kg given 
intravenously every 2 weeks, in combination with ipilimumab 1 mg/Kg given intravenously every 
6 weeks, in subjects with hypermutated metastatic HER2- breast cancer previously treated with 0 
to 3 chemotherapy regimens in the metastatic setting. Thirty patients will be enrolled to the study 
to evaluate the efficacy of the nivolumab plus ipilimumab, as defined by objective response rate 
(ORR) according to RECIST 1.1. 
No investigational or commercial agents or therapies other than those described below may 
be administered with the intent to treat the participant’s malignancy. Agents may be administered 
within +/- 3 days of their expected date. 
Table 1: Regimen Description
Agent
Premedication
; Precautions
Dose
Route
Schedule
Cycle 
Length
Nivolumab
Not routinely 
necessary 
unless prior 
infusion 
reaction.
3 mg/kg
IV over 60 
minutes (+/- 5 
minute infusion 
window)
Days 1, 15, 
29
42 days (6 
weeks)
 
Ipilimumab
Not necessary
1 mg/kg 
IV over 60 
minutes (+/- 5 
minute infusion 
window)
Day 1
42 days (6 
weeks) 
5.2
Pre-Treatment Criteria
5.2.1
Cycle 1, Day 1
If screening laboratory values were completed ≤ 96 hours (4 days) before Cycle 1 Day 1, 
laboratory tests do not need to be repeated on Cycle 1 Day 1 and the screening laboratory 
values may be used as the Cycle 1 Day 1 values.
If screening laboratory values were completed > 96 hours (4 days) prior to Cycle 1 Day 
1, laboratory tests must be repeated on Cycle 1 Day 1. Laboratory values drawn on Cycle 
1 Day 1 must re-meet eligibility criteria, exceptions to this are possible following 
discussion with the principal investigator. 

absolute neutrophil count
≥1,000/mcL

platelets
≥100,000/mcL

hemoglobin 
≥ 8 g/dl

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
total bilirubin
≤1.5 × institutional upper limit of normal (ULN) 
(or 2.0 x ULN in patients with documented Gilbert’s 
Syndrome)

AST(SGOT)/ALT(SGPT)
≤2.5 × institutional ULN or ≤ 3 × institutional ULN for 
participants with documented liver metastases

creatinine
>1.5 ×within normal institutional ULN OR
creatinine clearance ≥40 mL/min (using Cockcroft-
Gault formula) for participants with creatinine levels 
above institutional ULN.
5.2.2
Subsequent Cycles, Day 1, and all cycles day 15 and day 29 

absolute neutrophil count
≥1,000/mcL

platelets
≥75,000/mcL

AST(SGOT)/ALT(SGPT)
≤ 2.5 × institutional ULN or ≤ 3 × institutional ULN for 
participants with documented liver metastases

total bilirubin
≤1.5 × institutional ULN (or 2.0 x ULN in patients with 
documented Gilbert’s Syndrome)
5.3
Agent administration
5.3.1
Nivolumab Administration
Nivolumab will be administered as an IV infusion over approximately 60 minutes (+/- 5 minute 
infusion window). Nivolumab will be administered every 2 weeks, on cycle days 1, 15, and 29. 
Weight for dosage calculations should be performed per institutional standards of practice. On 
days where both nivolumab and ipilimumab will be administered, nivolumab should be 
administered first. The ipilimumab infusion will start at least 30 minutes after completion of the 
nivolumab infusion.
5.3.2
Ipilimumab administration
Ipilimumab will be administered as an IV infusion over approximately 60 minutes (+/- 5 minute 
infusion window). Ipilimumab will be administered every 6 weeks, on cycle day 1. Weight for 
dosage calculations should be performed per institutional standards of practice. Re-calculation of 
dosage for fluctuations in body weight should be made in accordance with institutional guidelines. 
On days where both nivolumab and ipilimumab will be administered, nivolumab should be 
administered first. The ipilimumab infusion will start at least 30 minutes after completion of the 
nivolumab infusion.
5.4
General Concomitant Medication and Supportive Care Guidelines
5.4.1
Concomitant Medication Guidelines – 
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed during 
the ongoing trial.  If there is a clinical indication for one of these or other medications or 
vaccinations specifically prohibited during the trial, discontinuation from trial therapy or 

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vaccination may be required.  The investigator should discuss any questions regarding this with 
the overall PI.  
Acceptable Concomitant Medications
All treatments that the investigator considers necessary for a subject’s welfare may be administered 
at the discretion of the investigator in keeping with the community standards of medical care and 
documented in the medical record. All medications being taken by the patient including dosage, 
frequency, route, and dates of administration will be recorded in the electronic medical record. 
Palliative radiotherapy is permitted to areas not followed for response, but presence of new or 
worsening metastases will be considered progression; however, if there is clear evidence of clinical 
benefit, treatment may be continued after completion of palliative radiotherapy. Protocol therapy 
should be interrupted one week before radiotherapy and held until radiotherapy is completed. 
Patients can restart protocol therapy once clinically stable as deemed by the treating investigator, 
but no earlier than one week after completing radiotherapy. 
Prohibited Concomitant Medications
Subjects are prohibited from receiving the following therapies during the Screening and Treatment 
Phase of this trial:

Antineoplastic systemic chemotherapy or biological therapy 

Immunotherapy not specified in this protocol

Investigational agents other than nivolumab and ipilimumab

Radiation therapy (except palliative radiotherapy as noted above)

Live vaccines within 28 days prior to the first dose of trial treatment and while participating 
in the trial.  Examples of live vaccines include, but are not limited to, the following: 
measles, mumps, rubella, varicella/zoster, yellow fever, rabies, BCG, and typhoid vaccine. 
The use of the inactivated seasonal influenza vaccine (Fluzone ®) is allowed.

Systemic glucocorticoids should be avoided for any purpose other than to modulate 
symptoms from radiation or an event of clinical interest of suspected immunologic etiology.  
If corticosteroids are required for this purpose, the minimum effective dose should be used.  
The use of physiologic doses of corticosteroids (10 mg prednisone daily or equivalent) can 
be used without principal investigator (PI) authorization.

Subjects may receive other medications that the investigator deems to be medically 
necessary.
There are no prohibited therapies during the Post-Treatment Follow-up Phase.
5.4.2
Supportive Care Guidelines – General Medications
The following treatments are permitted throughout the duration of the study treatment phase and 
during follow-up:

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
Standard therapies for pre-existing medical conditions unless listed as prohibited therapy. 
Any medication intended solely for supportive care (e.g., analgesics, anti-diarrheal, anti-
depressants) may be used at the investigator’s discretion.  Antiemetics and anti-diarrheal 
medications should not be administered prophylactically before initial treatment with study 
drugs.  At the discretion of the investigator, prophylactic antiemetic and anti-diarrheal 
medication(s) may be used as per standard clinical practice before subsequent doses of 
study drugs or before, during or after radiation treatment.

Bisphosphonate or denosumab therapy to be used in accordance with the approved labeled 
indication and/or nationally recognized treatment guidelines.  Participants already 
receiving bisphosphonate/denosumab at the time of study entry can continue the treatment.  
Participants may initiate treatment with bisphosphonate/denosumab after study entry with 
physician discretion. 

Pain medications administered per standard clinical practice are acceptable while the 
patient is enrolled in the study.
Patients who experience toxicities should be treated symptomatically as clinically indicated.  
Medications that are considered necessary for the subject’s welfare and that are not expected to 
interfere with the evaluation of study treatment or be restricted may be given at the discretion of 
the investigator.  Ancillary treatments will be given as medically indicated.
Potential Drug Interactions: 

Drug-Drug Interaction Potential with Nivolumab
o
Although monoclonal antibodies are not direct inhibitors/inducers of metabolizing 
enzymes, recent literature reports suggest that therapeutic proteins that are 
modulators of cytokines may indirectly affect expression of cytochrome (CYP) 
enzymes. The indirect drug-drug interaction potential of nivolumab was assessed 
using systematic cytokine modulation data for cytokines known to modulate CYP 
enzymes, at single and multiple doses of 0.3 to 10 mg/kg Q3 weeks.
o
There were no meaningful changes in cytokines known to have indirect effects on 
CYP enzymes across all dose levels of nivolumab (0.3, 2 and 10 mg/kg) during the 
course of treatment. This lack of cytokine modulation suggests that nivolumab has 
no or low potential for modulating CYP enzymes, thereby indicating a low risk of 
therapeutic protein-drug interaction.
o
Nivolumab is an IgG4 monoclonal antibody, which is eliminated by mechanisms 
similar to that of other antibodies, namely by non-specific catabolism (mainly by 
enzymes in the reticuloendothelial system). These enzymes are not known to be 
inhibited or induced by drugs, and therefore it is unlikely that other drugs will have 
an impact on the PK of nivolumab. 

Drug-Drug Interaction Potential with Ipilimumab
o
Ipilimumab is a human monoclonal antibody that is not metabolized by cytochrome 
P450 enzymes (CYP2) or other drug metabolizing enzymes. 
o
The use of anticoagulants is known to increase the risk of gastrointestinal 
hemorrhage. Since gastrointestinal hemorrhage is an adverse reaction with 
ipilimumab, patients who require concomitant anticoagulant therapy should be 
monitored closely. 

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5.5
Criteria for Taking a Participant Off Protocol Therapy
Duration of therapy will depend on individual response, evidence of disease progression and 
tolerance. In the absence of treatment delays due to adverse event(s), treatment may continue for 
24 months, or until one of the following criteria applies:

Disease progression by RECIST 1.1 criteria. Please note that although the primary 
endpoint is ORR as defined by RECIST 1.1, patients may remain on protocol therapy 
until the time of disease progression by irRC criteria.  The immune criteria allows 
treatment beyond initial radiographic worsening of disease in order to distinguish 
between pseudoprogression and true disease progression.  

Participants who have attained a confirmed complete response (CR) that have been 
treated for at least 24 weeks on protocol therapy and had at least two treatments with 
nivolumab beyond the date when the initial CR was declared.  Participants who stop 
nivolumab with CR may be eligible for additional nivolumab therapy if they progress 
after stopping study treatment.  This retreatment is termed the Second Course Phase 
Therapy.  See additional details below.

Intercurrent illness that prevents further administration of treatment.

Unacceptable adverse event(s).

Participant demonstrates an inability or unwillingness to comply with the medication 
regimen and/or documentation requirements.

Participant decides to withdraw from the protocol therapy.

General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator.
Participants will be removed from the protocol therapy when any of these criteria apply.  The 
reason for removal from protocol therapy, and the date the participant was removed, must be 
documented in the case report form (CRF) and in the CTMS system (OnCore). Alternative care 
options will be discussed with the participant.
In the event of unusual or life-threatening complications, treating investigators must immediately 
notify the Overall PI, Sara Tolaney, 
Second Course Phase (Retreatment Period) 
Participants may elect to stop nivolumab and ipilimumab with confirmed CR after at least 24 
weeks of treatment.
Subjects who stop nivolumab with CR may be eligible for additional nivolumab therapy if they 
progress after stopping study treatment.  This retreatment is termed the Second Course Phase of 
this study and is only available if the study remains open and the subject meets the following 
conditions:
o
Stopped initial treatment with nivolumab and ipilimumab after attaining an 
investigator-determined confirmed CR according to RECIST 1.1, was treated for at 

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least 24 weeks with nivolumab before discontinuing therapy, and received at least two 
treatments with nivolumab beyond the date when the initial CR was declared
AND
o
experienced an investigator-determined confirmed radiographic disease progression 
after stopping their initial treatment with nivolumab
o
did not receive any anti-cancer treatment other than ipilimumab since the last dose of 
nivolumab
Non-DF/HCC sites will fax or email documentation to the Coordinating Center at 617-632-5152 
or ctopm@dfci.harvard.edu for review. Provided the patient meets criteria per protocol for 
retreatment, the Coordinating Center will process the request and the participating site will be 
notified that the patient may proceed to retreatment.
Subjects who restart treatment will be retreated at the same dose and dose interval as when they 
last received nivolumab.  Visit requirements are as outlined for subjects per the initial treatment 
phase of the trial.  
5.6
Duration of Follow Up
Participants removed from protocol therapy for unacceptable adverse event(s) will be followed 
until resolution or stabilization of the adverse event.
 
Participants who are alive and free of disease progression at the time of removal from protocol 
therapy will be followed for first disease progression event after removal from protocol therapy. 
Participants will be followed for survival until death. This will be performed by checking the 
medical record or reaching out to local provider offices as needed, annually.
Tumor assessments should continue to be performed every 6-12 weeks on these participants until 
radiographic disease progression or initiation of another anti-cancer therapy; however, it will not 
constitute a protocol violation if restaging at this interval is not possible. 
After first disease progression, participants will continue to be followed for survival until death. 
This will be performed by checking the medical record or reaching out to local provider offices as 
needed, annually. 
5.7
Criteria for Taking a Participant Off Study
Participants will be removed from study when any of the following criteria apply:

Lost to follow-up

Withdrawal of consent for data submission

Death
The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the case report form (CRF) and CTMS (OnCore).

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6.
DOSING DELAYS/DOSE MODIFICATIONS
Dose delays will be made as indicated in the following tables. The descriptions and grading 
scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) 
version 5.0 will be utilized for dose delays and dose modifications. A copy of the CTCAE 
version 5.0 can be downloaded from the CTEP website 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
Given that it may be difficult to tell which agent is causing which toxicity due to overlapping AE 
profiles, it is expected that both agents will be concurrently held or delayed per Table 6.1.
6.1
Toxicity Management
Table 4: Gastrointestinal Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause is identified, treat accordingly and 
continue protocol therapy. Opiates/narcotics may mask symptoms of perforation. Infliximab should not 
be used in cases of perforation or sepsis. 
Grade of Diarrhea / 
Colitis
Management
Follow-Up
Grade 1
Continue protocol therapy. 
Treat symptoms as clinically 
appropriate. 
Monitor patient closely for worsening 
symptoms. 
Educate patient to report worsening 
immediately. 
If worsens: 
Treat as Grade 2 or ≥ Grade 3. 
Grade 2
Delay protocol therapy. 
Treat symptoms as clinically 
appropriate. 
If improves to Grade 1: 
Resume protocol therapy 
If persists > 5-7 days or recurs: 
Administer 0.5-1.0 mg/kg/day 
methylprednisolone or oral equivalent 
When symptoms improve to Grade 1, 
taper steroids over at least 1 month, 
consider prophylactic antibiotics for 
opportunistic infections, and resume 
therapy per protocol. 
If worsens or persists > 3-5 days with 
oral steroids: 
Treat as ≥ Grade 3. 
≥ Grade 3
Permanently discontinue or delay 
protocol therapy *
Administer 1.0 to 2.0 mg/kg/day 
If improves: 
Continue steroids until grade 1, then 
taper over at least 1 month 

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Table 4: Gastrointestinal Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause is identified, treat accordingly and 
continue protocol therapy. Opiates/narcotics may mask symptoms of perforation. Infliximab should not 
be used in cases of perforation or sepsis. 
Grade of Diarrhea / 
Colitis
Management
Follow-Up
methylprednisolone IV or IV 
equivalent 
Add prophylactic antibiotics for 
opportunistic infections 
Consider lower endoscopy as 
clinically appropriate 
If persists > 3-5 days, or recurs after 
improvement: 
Add infliximab 5 mg/kg (if no 
contraindication) 
Consider testing CMV for steroid 
refractory colitis
Note: Infliximab should not be used in 
cases of perforation or sepsis 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
*Discontinue for Grade 4 diarrhea or colitis. For Grade 3 diarrhea or colitis, 1) Nivolumab 
monotherapy: Nivolumab can be delayed. 2) Nivolumab+ Ipilimumab combination: Ipilimumab should 
be discontinuedwhile nivolumab can be delayed. Nivolumab monotherapy can be resumed when 
symptoms improve to Grade 1.
Table 5: Renal Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause is identified, treat accordingly and 
continue protocol therapy. 
Grade of Creatinine 
Elevation
Management
Follow-Up
Grade 1
Continue protocol therapy. 
Monitor creatinine weekly. 
If returns to baseline: 
Resume routine creatinine monitoring 
per protocol 
If worsens: 
Treat as Grade 2-3 or 4 
Grade 2-3
Delay protocol therapy 
Monitor creatinine every 2-3 days 
Administer 0.5 to 1.0 mg/kg/day 
methylprednisolone IV or oral 
equivalent 
Consider renal biopsy with 
nephrology consult as clinically 
appropriate 
If returns to Grade 1: 
Taper steroids over at least 1 month, 
consider prophylactic 
antibiotics for opportunistic infections, 
and resume protocol therapy and routine 
creatinine monitoring per protocol 
If elevations persist > 7 days or 
worsens: 
Treat as Grade 4 

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Table 5: Renal Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause is identified, treat accordingly and 
continue protocol therapy. 
Grade of Creatinine 
Elevation
Management
Follow-Up
Grade 4
Permanently discontinue protocol 
therapy
 
Monitor creatinine daily 
Administer 1.0-2.0 mg/kg/day 
methylprednisolone IV or IV 
equivalent 
Consult nephrologist and consider 
renal biopsy as clinically 
appropriate 
If returns to Grade 1: 
Taper steroids over at least 1 month and 
add prophylactic antibiotics for 
opportunistic infections 
If not improvement or worsening, add 
additional immunosuppression
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
Table 6: Pulmonary Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Evaluate with imaging and pulmonary consultation as clinically appropriate. 
Grade of 
Pneumonitis
Management
Follow-Up
Grade 1
Consider delay of protocol 
therapy 
Monitor for symptoms every 2-3 
days 
Consider Pulmonary and ID 
consults 
Re-image at least every 3 weeks 
If worsens: 
Treat as Grade 2 or ≥ Grade 3. 
Grade 2
Delay protocol therapy 
Obtain Pulmonary and ID 
consults as clinically appropriate
 
Monitor symptoms daily, 
consider hospitalization 
Administer 1.0 mg/kg/day 
methylprednisolone 
IV or oral equivalent 
Consider bronchoscopy, lung 
biopsy as clinically appropriate 
Consider reimaging weekly
If improves: 
Re-imaging every 1-3 weeks, monitoring 
symptoms every 2-3 days.
When symptoms return to near baseline, 
taper steroids over at least 1 month and 
then resume protocol therapy and 
consider prophylactic antibiotics 
If not improving after 2 weeks or 
worsening: 

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Table 6: Pulmonary Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Evaluate with imaging and pulmonary consultation as clinically appropriate. 
Grade of 
Pneumonitis
Management
Follow-Up
Treat as ≥ Grade 3. 
 
≥ Grade 3
Permanently discontinue protocol 
therapy 
Hospitalize as clinically indicated 
Obtain pulmonary and ID 
consults as appropriate 
Administer 2-4 mg/kg/day 
methylprednisolone IV or IV 
equivalent 
Add prophylactic antibiotics for 
opportunistic infections 
Consider bronchoscopy, lung 
biopsy 
If improves to baseline: 
Taper steroids over at least 6 weeks. 
If not improving after 48 hours or 
worsening: 
Add additional immunosuppression. 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
Table 7: Hepatic Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Consider imaging for obstruction. 
Grade of Liver Test 
Function
Management
Follow-Up
Grade 1
Continue protocol therapy 
 
Continue LFT monitoring per protocol 
If worsens: 
Treat as Grade 2 or ≥ Grade 3. 
Grade 2
Delay protocol therapy 
Increase frequency of monitoring 
to every 3 days 
If returns to baseline: 
Resume routine monitoring, resume 
protocol therapy 
If elevations persist > 5-7 days or 
worsen: 
Administer 0.5-1 mg/kg/day 

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Table 7: Hepatic Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Consider imaging for obstruction. 
Grade of Liver Test 
Function
Management
Follow-Up
methylprednisolone or oral equivalent 
and when LFT returns to grade 1 or 
baseline, taper steroids over at least 1 
month, consider prophylactic antibiotics 
for opportunistic infections, and resume 
protocol therapy 
≥ Grade 3
Permanently discontinue protocol 
therapyA 
Increase frequency of monitoring 
to every 1-2 days 
Administer 1.0 to 2.0 mg/kg/day 
methylprednisolone IV or IV 
equivalentB 
Add prophylactic antibiotics for 
opportunistic infections 
Consult gastroenterologist or 
hepatologist as clinically 
appropriate 
If returns to grade 2: 
Taper steroids over at least 1 month 
If does not improve in >3-5 days, 
worsens or rebounds: 
Add mycophenolate mofetil 1 g BID 
If no response within an additional 3-5 
days, consider tacrolimus or other 
immunosuppressants per local guidelines
Note: avoid infliximab due to potential 
risk of liver failure 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
A. Protocol therapy may be delayed rather than discontinued if AST/ALT ≤ 8 × institutional ULN or 
total bilirubin ≤ 5 × institutional ULN. 
B. The recommended starting dose for Grade 4 hepatitis is 2 mg/kg/day 
methylprednisolone IV.
Table 8: Endocrinopathy Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Consider visual field testing, endocrinology consultation, and imaging. 
Event
Management
Follow-Up
Asymptomatic TSH 
Elevation
Or
Hyperglycemia 
requiring no medical 
intervention
Continue protocol therapy. 
If TSH < 0.5 × lower limit of 
normal (LLN), or TSH > 2 × 
ULN, or consistently out of range 
in 2 subsequent measurements: 
include free T4 at subsequent 
cycles as clinically indicated; 
consider endocrinology consult. 
Continue to follow thyroid function per 
protocol.
Monitor blood sugar
If symptomatic or blood sugar requiring 
initiation of treatment or change in 
management, manage as below

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Table 8: Endocrinopathy Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Consider visual field testing, endocrinology consultation, and imaging. 
Symptomatic 
Endocrinopathy 
Or
Hyperglycemia 
requiring initiation of 
treatment or change in 
daily management or 
work up
Evaluate endocrine function 
Consider pituitary scan 
Symptomatic with abnormal 
lab/pituitary scan:
 
Delay protocol therapy 
Administer 1-2 mg/kg/day 
methylprednisolone IV or PO 
equivalent 
Initiate appropriate hormone 
therapy 
No abnormal lab/pituitary MRI 
scan but symptoms persist: 
Continue therapy, Repeat labs in 
1-3 weeks / MRI in 1 month
Hyperglycemia:
Screen for diabetic ketoacidosis 
(DKA). If negative, continue IO 
and treat diabetes. If DKA, hold 
IO, admit, manage DKA. Steroids 
not recommended for 
DKA/hyperglycemia
If improves (with or without 
hormone replacement): 
Taper steroids over at least 1 month and 
consider prophylactic antibiotics for 
opportunistic infections 
Resume protocol therapy 
Patients with adrenal insufficiency may 
need to continue steroids with 
mineralocorticoid component 
Suspicion of adrenal 
crisis (e.g. severe 
dehydration, 
hypotension, shock 
out of proportion to 
current illness)
Delay protocol therapy 
Rule out sepsis 
Stress dose of IV steroids with 
mineralocorticoid activity 
Administer IV fluids 
Consult endocrinologist as 
clinically appropriate 
If adrenal crisis ruled out, then 
treat as above for symptomatic 
endocrinopathy  
Follow until resolution.
If adrenal crisis resolves and patient is 
adequately controlled with hormone 
repclacement, patient can resume therapy 
per protocol.
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 

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Table 9: Skin Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. 
Grade of 
RashA 
Management
Follow-Up
Grade 1 – 2
Continue protocol 
therapy. 
Administer symptomatic 
therapy (e.g. 
antihistamines, topical 
steroids)  
If persists > 1-2 weeks or recurs: 
Consider skin biopsy 
Delay protocol therapy 
Consider 0.5-1.0 mg/kg/day methylprednisolone IV or 
oral equivalent. Once improving, taper steroids over at 
least 1 month, consider prophylactic antibiotics for 
opportunistic infections, and resume protocol therapy 
If worsens: 
Treat as Grade 3-4 
Grade 3-4
Delay or discontinue 
protocol therapy 
Consider skin biopsy 
Dermatology consult as 
clinically appropriate 
Administer 1.0-2.0 
mg/kg/day IV 
methylprednisolone 
IV or IV equivalent 
If returns to Grade 1: 
Taper steroids over at least 1 month and add prophylactic 
antibiotics for opportunistic infections. 
Resume protocol therapy if appropriate. 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
A. If Stevens-Johnson Syndrome (SJS) ,Toxic Epidermal Necrolysis (TEN), Drug 
Reaction with Eosinophilia and Systemic Symptoms (DRESS)is suspected, withhold protocol therapy 
and refer patient for specialized care for assessment and treatment. If SJS or TEN is diagnosed, 
permanently discontinue protocol therapy. 

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Table 10: Neurological Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. 
Grade of Neurological 
Toxicity
Management
Follow-Up
Grade 1
Continue protocol therapy 
Discontinue therapy for select AEs*
Continue to monitor the patient. 
If worsens: 
Treat as Grade 2 or ≥ Grade 3. 
Grade 2
Delay protocol therapy 
Discontinue therapy for select AEs*
Treat symptoms per local guidelines 
Consider neurology consult
Consider 0.5 to 1.0 mg/kg/day 
methylprednisolone IV or oral 
equivalent 
If improves to baseline: 
Resume protocol therapy. 
If worsens: 
Treat as ≥ Grade 3.  
≥ Grade 3
Permanently discontinue protocol 
therapy 
Obtain neurology consult as 
clinically appropriate
 
Treat symptoms per local guidelines 
Administer 1.0-2.0 mg/kg/day 
methylprednisolone IV or IV 
equivalent 
Add prophylactic antibiotics for 
opportunistic infections 
If returns to Grade 2: 
Taper steroids over at least 1 month. 
If worsens or atypical 
presentation:
 
Consider IVIG or other 
immunosuppressive therapies per 
local guidelines. 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
* Discontinue for any grade myasethenia gravis, Guillain-Barre syndrome, treatment-related myelitis, 
or encephalitis
Table 11: Pancreatic Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. 
Grade of Amylase or 
Lipase Elevation, 
Pancreatitis
ManagementB
Follow-Up
Continue protocol therapy. 
Resume routine protocol monitoring. 

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Table 11: Pancreatic Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. 
Grade of Amylase or 
Lipase Elevation, 
Pancreatitis
ManagementB
Follow-Up
Asymptomatic Grade 
1A
Monitor amylase and lipase 
weekly until resolution to 
baseline.  
If worsens: 
Treat as Grade 2-3 or 4. 
Asymptomatic Grade 
2-3A
Delay protocol therapy. 
Monitor amylase and lipase 
weekly until resolution to 
baseline. 
If improves to baseline: 
Resume protocol therapy and routine 
protocol monitoring. 
If worsens: 
Treat as Grade 4. 
Asymptomatic Grade 
4A
Permanently discontinue 
protocol therapy. 
Monitor amylase and lipase 
weekly until resolution. 
Monitor patient until resolution to 
baseline. 
Any Symptomatic 
Elevations in 
Amylase/Lipase, 
Pancreatitis
Permanently discontinue 
protocol therapy. 
Consider GI consult as clinically 
appropriate. 
Administer supportive care. 
Monitor patient until resolution to 
baseline. 
A. Patients should be monitored for signs/symptoms consistent with pancreatitis, including abdominal 
pain and vomiting. 
B. Corticosteroids do not seem to alter the natural course of amylase/lipase elevations. 
Table 12: Ocular Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Work-up for ocular adverse events should also consider pituitary inflammation as a 
potential cause. 
Event Grade
Management
Follow-Up
Grade 1
Continue protocol therapy. 
Monitor patient for worsening 
symptoms. 
Consider ophthalmologist 
consult as clinically appropriate. 
Administer topical 
If worsens: 
Treat as Grade 2 or ≥ Grade 3. 

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Table 12: Ocular Adverse Event Management
Note: Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue 
protocol therapy. Work-up for ocular adverse events should also consider pituitary inflammation as a 
potential cause. 
Event Grade
Management
Follow-Up
corticosteroids as clinically 
indicated. 
Grade 2
Delay protocol therapy. 
Obtain ophthalmologist consult. 
Administer topical 
corticosteroids as clinically 
indicated. 
If improves to baseline: 
Resume protocol therapy. 
If worsens: 
Treat as ≥ Grade 3. 
≥ Grade 3
Permanently discontinue 
protocol therapy. 
Obtain ophthalmologist consult. 
Administer systemic 
corticosteroids as clinically 
indicated. 
Monitor patient until resolution to 
baseline. 
Taper systemic corticosteroids as 
clinically appropriate. 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) 
at start of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of 
oral corticosteroids should be taken into account when switching to the equivalent dose of oral 
corticosteroids. 
Table 13: Infusion-Related Reactions
Event Grade
Management
Follow-Up
Grade 1
Monitor patient until recovery of 
symptoms to baseline. 
Prophylactic premedications are 
recommended for future infusions: 
diphenhydramine 25 – 50 mg (or 
equivalent) and/or acetaminophen 325 to 
1000 mg (or equivalent) at least 30 
minutes before additional infusions. 
Grade 2
Stop infusion. 
Administer diphenhydramine 50 
mg IV (or equivalent) and/or 
acetaminophen 325 – 1000 mg 
PO (or equivalent). 
Corticosteroids or 
bronchodilators may also be 
administered as clinically 
appropriate. 
Monitor patient until symptoms 
Prophylactic premedications are 
recommended for future infusions: 
diphenhydramine 50 mg (or equivalent) 
and/or acetaminophen 325 to 1000 mg (or 
equivalent), administered at least 30 
minutes prior to the infusion. 
If necessary per the judgment of the 
treating investigator, corticosteroids (up 
to 25 mg SoluCortef or equivalent) may 
be used as well. 

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Table 13: Infusion-Related Reactions
Event Grade
Management
Follow-Up
have resolved to baseline. 
After resolution of symptoms, 
the infusion may be restarted at 
50% of the original infusion rate. 
If no further complications ensue 
after 30 minutes, the rate may be 
increased to 100% of the original 
infusion rate. If symptoms recur, 
no further administration of 
nivolumab or ipilimumab should 
occur at that visit. Administer 50 
mg IV diphenhydramine (or 
equivalent) and monitor patient 
until the symptoms resolve to 
baseline. 
≥ Grade 3
Permanently discontinue 
protocol therapy. 
Emergency care should be 
implemented as clinically 
appropriate and per local 
institutional standards of 
practice. 
Monitor patient until resolution to 
baseline. 
6.2
Dose Delays
Dosing of ipilimumab or nivolumab may be held at the treating investigator’s discretion for any 
toxicity of any grade. Additionally, holding of the study agents may be required as indicated in 
the tables in Section 6.1. 
A treatment delay of up to six weeks for nivolumab and up to 12 weeks for ipilimumab is 
allowable. Participants requiring a longer hold should be removed from protocol therapy. 
Exceptions for participants exhibiting clinical benefit may be possible with approval from the 
principal investigator. 
If study agent dosing is held due to toxicity, the counting of cycle days and assessment schedule 
will continue without interruption. For example, a participant who does not receive their Cycle 3 
Day 15 dose of nivolumab due to toxicity will proceed with their next regularly scheduled clinic 
visit (Cycle 3 Day 29) as previously planned. Alternatively, if ipilimumab is held on C3D1 and 
nivolumab is given, the ipilumumab dose would be omitted for that cycle. Additional interim 
clinic visits may be scheduled to manage toxicity, however the cycle will not restart due to 
dosing delays due to adverse events and the assessment schedule (including tumor imaging 
evaluations) will continue as originally planned.

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6.3
Dose Reductions
There will be no dose reductions for nivolumab or ipilimumab allowed. Participants who cannot 
tolerate the assigned doses of nivolumab or ipilimumab should be removed from protocol 
therapy.
6.4
Overdose Management 
There is no available information concerning overdose with nivolumab or ipilimumab. In case of 
overdose, patients must be closely monitored for signs or symptoms of adverse reactions and 
appropriate symptomatic treatment instituted. There are no specific antidotes. Any overdoses of 
either drug should be reported as an SAE to BMS. Please see section 7.3.4 for information on 
reporting. 
 
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The following 
list of reported and/or potential AEs (Section 7.1) and the characteristics of an observed AE 
(Section 7.2) will determine whether the event requires expedited reporting in addition to routine 
reporting.
7.1
Expected Toxicities 
7.1.1
Adverse Events List 
In addition to the list below, please also refer to the investigator’s brochure (IB) for both 
nivolumab and ipilimumab for comprehensive adverse event information.
7.1.1.1.1. Adverse Event List for Nivolumab combined with Ipilimumab 
Common adverse events associated with combination treatment include:

Fatigue 

Fever 

Chills 

Flu-like symptoms 

Infusion-related reactions 

Headache 

Dizziness 

Hyperthyroidism or hypothyroidism 

Cough 

Dyspnea 

Pneumonitis 

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
Hypotension 

Dry mouth 

Anorexia 

Diarrhea 

Nausea 

Vomiting 

Dehydration 

Abdominal pain 

Colitis 

Elevated liver transaminases 

Elevated lipase and/or amylase 

Rash 

Pruritus 

Vitiligo 

Joint pain or stiffness 

Muscle soreness, weakness, stiffness, or spasms 
7.2
Adverse Event Characteristics

CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
5.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 5.0.  A copy of the CTCAE version 5.0 can be 
downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.

For expedited reporting purposes only:  
-
AEs for the agent(s) that are listed above should be reported only if the adverse event 
varies in nature, intensity or frequency from the expected toxicity information which 
is provided.

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.

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7.3
Expedited Adverse Event Reporting 
In the event of an unanticipated problem or life-threatening complications treating 
investigators must immediately notify the Overall PI.
Investigators must report to the Overall PI any serious adverse event (SAE) that occurs 
after the initial dose of study treatment, during treatment, or within 100 days of the last 
dose of Nivolumab and 30 days of the last dose of Ipilimumab. SAEs that occur after 30 
days post-treatment must only be reported to the Overall PI if they are determined to be 
related to Nivolumb or Ipilimumab.  
For Multi-Center Trials where a DF/HCC investigator is serving as the Sponsor, each 
participating institution must abide by the reporting requirements set by the DF/HCC. 
This applies to any medical event equivalent to an unexpected grade 2 or 3 with a 
possible, probable or definite attribution, unexpected grade 4 toxicities, and grade 5 
(death) regardless of study phase or attribution.
7.3.1
Reporting to the Institutional Review Board (IRB)
Investigative sites within DF/HCC will report all serious adverse events directly to the DFCI 
Office for Human Research Studies (OHRS) per the DFCI IRB reporting policy.
Other investigative sites will report to their respective IRBs according to the local IRB’s policies 
and procedures in reporting adverse events. A copy of the submitted institutional AE form should 
be forwarded to the Overall PI within the timeframes detailed in the table below. 
DF/HCC Reportable AEs
Attribution
Gr. 2 & 3 
AE
Expected
Gr. 2 & 3 AE 
Unexpected
Gr. 4 AE 
Expected
Gr. 4 AE
Unexpected
Gr. 5 AE 
Expected or 
Unexpected
Unrelated
Unlikely
Not required
Not required
5 calendar 
days#
5 calendar 
days
24 hours*
Possible
Probable
Definite
Not required
5 calendar 
days
5 calendar 
days#
5 calendar 
days
24 hours*
# If listed in protocol as expected and not requiring expedited reporting, event does not need to 
be reported.
* For participants enrolled and actively participating in the study or for AEs occurring within 
30 days of the last intervention (100 days from last dose of nivolumab), the AE should be 
reported within 1 business day of learning of the event.
The Overall PI will submit AE reports from outside institutions to the DFCI OHRS according to 
DFCI IRB policies and procedures for reporting adverse events. 

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7.3.2
Expedited Reporting to the Food and Drug Administration (FDA)
The Overall PI, as study sponsor, will be responsible for all communications with the FDA. The 
Overall PI will report to the FDA, regardless of the site of occurrence, any serious adverse event 
that meets the FDA/s criteria for expedited reporting following the reporting requirements and 
timelines set by the FDA. 
Any report that meets FDA reporting requirements should also be submitted to BMS per section 
7.3.4. These events are generally those that are both serious, at least possibly related to the 
investigational product(s), and are unexpected.
7.3.3
Expedited Reporting to Hospital Risk Management
Participating investigators will report to their local Risk Management office any participant safety 
reports or sentinel events that require reporting according to institutional policy. 
7.3.4
Expedited Reporting to BMS 
SAEs, whether related or not related to study drug, and pregnancies must be reported to BMS 
within 24 buiness hours of the awareness of the event. SAEs must be recorded on a Medwatch 
Form 3500A.
SAE Email Address:  Worldwide.Safety@BMS.com
SAE Facsimile Number:  609-818-3804
Adverse events generally considered serious are those that occur at any dose during any use of 
BMS product that:

Results in death

Is life-threatening (defined as an event in which the participant was at risk of 
death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe)

Requires inpatient hospitalization or causes prolongation of existing 
hospitalization 

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 subject 
or may require intervention [eg, medical, surgical] to prevent one of the other 
serious outcomes listed in the definition above.) Examples of such events 
include, but are not limited to, intensive treatment in an emergency room or at 
home for allergic bronchospasm; blood dyscrasias or convulsions that do not 
result in hospitalization.) 

Suspected transmission of an infectious agent (eg, pathogenic or 
nonpathogenic) via the study drug is an SAE.

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A planned medical or surgical procedure is not, in itself, an SAE. Generally, any events that meet 
any of the seriousness criteria listed above and that are caused by progression of the disease 
under the study (metastatic breast cancer) are not reportable. Any instance of pregnancy, 
overdose, potential drug-induced liver injury (DILI), and secondary malignancies, though not 
necessarily considered serios per these definitions should be reported as SAEs.  
If only limited information is initially available, a follow-up report is required and should  include 
the same investigator term(s) initially reported.
If an ongoing SAE changes in its intensity or relationship to study drug or if new information 
becomes available, a follow-up SAE report should be sent within 24 hours of awareness to BMS 
(or designee) using the same procedure used for transmitting the initial SAE report. 
All SAEs should be followed to resolution or stabilization.
7.3.5
Reporting of Pregnancy
If, following initiation of the investigational product, it is subsequently discovered that a study 
participant is pregnant or may have been pregnant at the time of investigational product 
exposure, including during at least 5 half-lives after product administration, the investigational 
product will be permanently discontinued in an appropriate manner (eg, dose tapering if 
necessary for participant). 
The investigator must immediately notify Worldwide.Safety@bms.com of this event via either 
the CIOMS, MedWatch or appropriate Pregnancy Surveillance Form in accordance with SAE 
reporting procedures. 
Protocol-required procedures for study discontinuation and follow-up must be performed on the 
participant.
Follow-up information regarding the course of the pregnancy, including perinatal and neonatal 
outcome and, where applicable, offspring information must be reported on the CIOMS, 
MedWatch, BMS Pregnancy Surveillance Form, or approved site SAE form.  A BMS Pregnancy 
Surveillance Form may be provided upon request.
Any pregnancy that occurs in a female partner of a male study participant should be reported to 
BMS. Information on this pregnancy will be collected on the Pregnancy Surveillance Form. In 
order for Sponsor or designee to collect any pregnancy surveillance information from the female 
partner, the female partner must sign an informed consent form for disclosure of this 
information.
7.4
Routine Adverse Event Reporting
All Grade 2 or higher Adverse Events (excluding non-clinically significant laboratory values) 
must be reported in routine study data submissions to the Overall PI on the toxicity case report 
forms.  AEs reported through expedited processes (e.g., reported to the IRB, FDA, etc.) must 
also be reported in routine study data submissions.

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8.
PHARMACEUTICAL INFORMATION
A list of the adverse events and potential risks associated with the investigational agents 
administered in this study can be found in Section 7.1.
8.1
Nivolumab
8.1.1
Description
Nivolumab is also referred to as BMS-936558-01 or BMS-936558. Nivolumab is a soluble 
protein consisting of 4 polypeptide chains, which include 2 identical heavy chains and 2 identical 
light chains. The physical and chemical properties of nivolumab are provided in Table 8.1.1 
below. The geometric mean of terminal T-HALF was 25.6 days and the typical clearance was 8.8 
mL/h, which are consistent with those of full human immunoglobulin antibodies.
Table 14: Nivolumab Physical and Chemical Properties
BMS Number
BMS-936558-01
Other Names
Nivolumab, BMS-936558, MDX1106, ONO-4538, anti-PD-1
Molecular Weight
146,221 daltons
Appearance
Clear to opalescent, colorless to pale yellow liquid, light (few) 
particulates may be present
Solution pH
5.5 to 6.5
8.1.2
Storage and Stability
Vials of Nivolumab injection must be stored at 2°-8°C (36°-46°F) and protected from light, 
freezing and shaking. Shelf-life surveillance of the intact vials is ongoing.
The administration of undiluted and diluted solutions of Nivolumab must be completed within 24 
hours of preparation. If not used immediately, the infusion solution may be stored up to 24 hours 
in a refrigerator at 2°-8°C (36°-46°F) and a maximum of 8 hours of the total 24 hours can be at 
room temperature (20°-25°C, 68°-77°F) and room light. The maximum 8-hour period under room 
temperature and room light conditions includes the product administration period.
CAUTION: The single-use dosage form contains no antibacterial preservative or bacteriostatic 
agent. Therefore, it is advised that the product be discarded 8 hours after initial entry.
8.1.3
Compatibility
No incompatibilities between Nivolumab injection and polyvinyl chloride (PVC), non-
PVC/non-DEHP (di(2-ethylhexyl)phthalate) IV components, or glass bottles have been observed. 
8.1.4
Handling

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Qualified personnel, familiar with procedures that minimize undue exposure to themselves 
and the environment, should undertake the preparation, handling, and safe disposal of the 
chemotherapeutic agent in a self-contained and protective environment. 
8.1.5
Availability
Nivolumab is available as 100 mg vials (10 mg/mL) with a 0.7mL overfill. It is supplied 
in 10 mL type I flint glass vials, with butyl rubber stoppers and aluminum seals.
8.1.6
Preparation
Nivolumab is available as 100 mg vials (10 mg/mL), which include an overfill. It is 
supplied in 10 mL type I flint glass vials, with butyl stoppers and aluminum seals. Nivolumab 
injection can be infused undiluted (10 mg/mL) or diluted with 0.9% Sodium Chloride Injection, 
USP or 5% Dextrose, USP to concentrations no less than 0.35 mg/mL.
8.1.7
Administration
Nivolumab will be delivered in infusion bags with IV infusion lines over 60 minutes (+/- 
5 minutes) using a volumetric pump with 0.2 to 1.2 micron pore size, low-protein binding 
polyethersulfone membrane in-line filter. Weight for dosage calculations should be done per 
institutional standards of practice. Re-calculation of dosage for fluctuations in body weight should 
be made in accordance with institutional guidelines.
8.1.8
Ordering
Nivolumab will be provided by BMS. Each participating institutions is responsible for 
completing the applicable drug supply forms to receive re-supply of Nivolumab for the duration 
of the study. Except in very unusual circumstances, each participating institution will order the 
study agent(s). A participating site may order the agent(s) only after the initial IRB approval for 
the site has been forwarded by the Coordinating Center to the supplier. 
8.1.9
Accountability
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug Accountability 
Record Form (DARF) or another comparable drug accountability form.  (See the NCI 
Investigator’s Handbook for Procedures for Drug Accountability and Storage.)
8.1.10 Destruction and Return
At the end of the study, unused supplies of nivolumab should be destroyed according to 
institutional policies. Destruction will be documented in the Drug Accountability Record Form.

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8.2
Ipilimumab 
8.2.1
Description
Ipilimumab (BMS-734016, MDX-010) is a fully human IgG1κ consisting of 4 polypeptide 
chains; 2 identical heavy chains primarily consisting of 447 amino acids each with 2 identical 
kappa light chains consisting of 215 amino acids each linked through inter-chain disulfide bonds. 
Table 15: IpilimumabPhysical and Chemical Properties
BMS Number
BMS-734016
Other Names
BMS-734016, MDX-010, YERVOY
Molecular Weight
147, 991 Daltons
Appearance
Clear to slightly opalescent, colorless to pale yello liquid, may 
contain particiles
Solution pH
7.0
pI
The isoelectric focusing analysis generates a banding batter in the 
pI range of 8.5 to 8.8, with the major isoform at an approcimate pI 
of 8.7
8.2.2
Form
Ipilimumab injection, 200 mg/40 mL (5 mg/mL), is formulated as a clear to slightly 
opalescent, colorless to pale yellow, sterile, nonpyrogenic, single-use, isotonic aqueous solution 
that may contain particles. Ipilimumab injection, 200 mg/40 mL, is supplied in 50-cc Type I flint 
glass vials, respectively, stoppered with gray butyl stoppers and sealed with aluminum seals. The 
drug product is formulated at a concentration of 5 mg/mL at a pH of 7.0.
8.2.3
Storage and Stability
Ipilimumab Injection, 200 mg/40 mL (5 mg/mL), must be stored refrigerated (2°C to 8°C) and 
protected from light. Ipilimumab injection must not be frozen. 
Partially used vials or empty vials of ipilimumab injection should be discarded at the site according 
to appropriate drug disposal procedures. 
Ipilimumab injection may be stored undiluted (5 mg/mL) or following dilution in 0.9% Sodium 
Chloride Injection, USP or 5% Dextrose Injection in PVC, non-PVC/non-DEHP or glass 
containers for up to 24 hours (at 2°C to 8°C) or room temperature/room light. 
8.2.4
Compatibility
Ipilimumab injection may be diluted in 0.9% Sodium Chloride Injection, United States 
Pharmacopeia (USP) or 5% Dextrose Injection.

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8.2.5
Availability
Ipilimumab will be supplied as an investigational agent by Bristol-Myers Squibb (BMS).
8.2.6
Preparation
Ipilimumab injection (5 mg/mL) can be used for intravenous (IV) administration after transferring 
to a polyvinyl chloride (PVC), non-PVC/non-di-(2-ethylhexyl)phthalate (DEHP), or glass 
container and is stable for 24 hours at 2°C to 8ºC or room temperature/room light (RT/RL). 
Ipilimumab injection may be diluted in 0.9% Sodium Chloride Injection, United States 
Pharmacopeia (USP) or 5% Dextrose Injection. Preparation will follow institutional standard. The 
product may be infused using a volumetric pump at the protocol-specific dose(s) and rate(s) 
through a infusion set with an inline, sterile, nonpyrogenic, low-protein-binding filter (pore size of 
0.2 to 1.2 micrometer). 
Ipilimumab injection must not be administered as an IV push or bolus injection. Care must 
be taken to assure sterility of the prepared solutions since the drug product does not contain any 
antimicrobial preservatives or bacteriostatic agents.
  
8.2.7
Administration
Ipilimumab will be administered as an IV infusion over approximately 60 minutes (± 5 
minute infusion window). Ipilimumab will be administered every 6 weeks, on cycle day 1. 
Calculation of weight should be done per institutional standards of practice. Re-calculation of 
dosage for fluctuations in body weight should be made in accordance with institutional guidelines. 
On days where both nivolumab and ipilimumab will be administered, nivolumab should be 
administered first. The ipilimumab infusion will start at least 30 minutes after completion of the 
nivolumab infusion.
8.2.8
Ordering
Ipilimumab will be ordered by site pharmacy personnel from BMS. Each participating 
institution is responsible for completing the applicable drug supply forms to receive re-supply of 
Ipilimumab for the the duration of the study. Except in very unusual circumstances, each 
participating institution will order the study agent(s). A participating site may order the agent(s) 
only after the initial IRB approval for the site has been forwarded by the Coordinatin Center to the 
supplier. 
8.2.9
Accountability
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug Accountability 
Record Form (DARF) or another comparable drug accountability form. (See the NCI 
Investigator’s Handbook for Procedures for Drug Accountability and Storage.) 

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8.2.10 Destruction and Return
Expired or unused supplies of ipilimumab should be destroyed according to institutional 
policies. Destruction will be documented in the Drug Accountability Record Form. 
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES
In all patients in whom a tumor safely accessible, two biopsies will be required: baseline biopsy 
and on-treatment biopsy and at day 29 of cycle 1 (+14 day scheduling window). Optional biopsies 
will be performed at progression in patients who experienced an objective response during the 
study. We plan to use baseline biopsy tissue to perform a number of immune profiling assays, 
detailed below.  On baseline tumor biopsies, we will perform characterization based on histology 
(TILs), protein expression, and mRNA expression.  Additionally, we will bank specimens for 
possible future DNA analysis, and other further testing.
All participants will be asked to provide a sample of their archival tissue. For participants in whom 
tumor biopsies are not safely accessible, an archival sample will be required in order to enroll. In 
these cases, teams should ensure that an archival sample is available, but it does not need to be at 
the site in order for the patient to start treatment. Though listed as a screening procedure, archival 
tissue may be requested and obtained at any point throughout the trial. 
Serial blood draws for correlative science are required on this trial; blood samples will be collected 
within 7 days before starting therapy; blood draws will be obtained every 6 weeks for 24 weeks, 
and then every 12 weeks, at the end-of-treatment visit in patients who are removed from treatment 
for progressive disease, and all efforts will be made to obtain an additional blood draw at the time 
of progressive disease, in patients who went off treatment for anything other than progressive 
disease.  On each blood draw, we will perform flow cytometry to characterize protein expression 
of immune mediators, detailed below, and additional blood will be banked for future testing. 
All patients will additionally be asked to provide a stool sample at three timepoints: within 14 days 
prior to starting treatment, during treatment at day 29 Cycle 1 (+14 day scheduling window), and 
at the end of treatment. A fourth collection may be requested from patients who experience grade 
≥ 2 diarrhea related to study treatment after discussion with the PI. This collection is not required, 
but is strongly encouraged. These samples will be analyzed for microbiota content. 
Sites may refer to the separate laboratory manual for additional correlative details including 
collection, processing, and shipping instructions. 
Please note that some of the downstream correlative plans for blood and tissue include whole 
exome and single cell sequencing, plus analysis of the data generated, that will be performed by 
Foundation Medicine, Nanostring Technologies, and the Broad Institute of MIT.. All samples 
will be sent in a completely de-identified fashion and staff involved at the Broad Institute, 
Foundation Medicine, and Nanostring Technologies will not have any access to patient health 
information (PHI). The Broad Institute will provide barcode labels for all samples that are 
generated at random by their laboratory software. 

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Summary of Specimens 
Research Sampling
Timepoint
Contents
Destination
Archival Tissue
Anytime on study
1 FFPE Block or 10-
20 5 micron 
unstained slides
Current DFCI CRC
Baseline* (within 28 
days of C1D1, prior 
to treatment start)
5-7 cores
RNAlater Cores – 
Breast Bank
C1D29 (+14 day 
scheduling window)
5-7 cores
Formalin Cores – 
Breast Bank
Fresh Tissue
End of Treatment 
(Optional)
5-7 cores
External Sites Only: 
OCT Core – Breast 
Bank
1 – 10mL Streck 
Tube (cfDNA)
Breast Bank
Blood 
Baseline*, Restaging 
Visits, Time of 
Progression 
5 – 10mL CPT tubes 
(PBMCs)*
Mayer 308 / Mariano 
Severignini
Baseline*
At C1D29 (any time 
between C1D29 and 
C2D1)
End of treatment
Stool
Optional collection at 
the time of grade ≥ 2 
diarrhea
Home Collection Kit 
(DNA Genotek)
Breast Tumor 
Immunology Lab/ 
Elizabeth Mittendorf, 
MD, PhD
Note: The “Breast Bank” and Core Blood and Tissue Bank are the same lab and interchangeable
*Baseline samples preferably completed after registration or on Cycle 1 Day 1 prior to treatment 
start (once a study ID is assigned). Baseline samples completed during screening will not be a 
violation.
9.1
Archival Tissue 
Archival tissue will be obtained on all participants. One block or 10-20 5 micron unstained, 
charged slides will be collected to centrally test for hypermutation status for participants that are 
unable to undergo a baseline biopsy. Local tests will be used for eligibility and if the participant 
had prior Foundation One testing, it will not need to be centrally repeated. Any leftover tissue or 
tissue from participants who had a baseline biopsy will be banked and stored for future research 
as a part of correlatives for this clinical trial or otherwise.

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9.2
Fresh Tissue Biopsy 
9.2.1
Objectives: 

Characterizing immune markers in hypermutated HER2- metastatic breast cancer

To evaluate MET and phospho MET expression in tumor tissue at baseline by 
immunohistochemistry
9.2.2
Collection 
Biopsies will be performed at the below timepoints, if tumor is safely accessible. 
Mandatory:

Baseline (preferably after registration or on Cycle 1 Day 1 prior to dosing): 

Around C1D29 (Any time from C1D29-C2D1 is allowed)
Optional:

At time of progression or end of treatment
For patients proceeding to the retreatment phase, optional biopsies will be performed at the below 
timepoints: 

C1D1 of retreatment, prior to dosing

At the time of progression after retreatment with nivolumab 
Biopsies should not be performed on Friday afternoons, as there may not be time for processing 
of the fresh tissue.  If a biopsy must be performed on Friday morning, the lab of Mariano 
Severgnini and Cathy Wu must be notified ahead of time to ensure that there will be adequate time 
for processing fresh tissue, since fresh tissue cannot be stored over the weekend.  The specimens 
in RNALater, OCT, and formalin may be stored over the weekend and shipped on Monday. 
Specimens in RNA Later and formalin should be stored at room temperature until shipment. 
DFCI Participants:
Ideally five core biopsies will be obtained:

Three cores should be placed in 10% neutral buffered formalin tube supplied by the study.

Two cores should be placed in RNAlater
The order of specimen collection should be:

First core: 10% neutral buffered formalin

Second core: 10% neutral buffered formalin

Third core: RNAlater

Fourth core: RNAlater

Fifth core: 10% neutral buffered formalin

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recommendations from the International TILs Working Group 2014.[Salgado et al., 
2015]
After assessment of the TIL percentage, the pathologists will categorize the specimen 
as lymphocyte predominant breast cancer (LPBC), defined as a tumor that contains 
>60% stromal lymphocytes, or non-LPBC.
Assay 2: Immunohistochemistry
Tissue will be collected and fixed by 10% neutral buffered formalin overnight, 
dehydrated, and paraffin embedded.  Four micrometer-thick sections will be cut.  The 
paraffin blocks and unstained slides will be stored at room temperature.  All 
immunohistochemical staining will be performed in the Center for Immuno-Oncology 
Pathology Core at Dana-Farber/Harvard Cancer Center (DF/HCC) Specialized 
Histopathology Core.
Formalin fixed-paraffin embedded (FFPE) tumor slides will be prepared and H&E 
stained for assessment of TIL in pre- and post-treatment tumor samples.  To identify 
subsets of different immune populations (effector/memory CD8 cells, T regulatory 
cells, dendritic cells, tumor associated macrophages, and Tie-2 expressing monocytes 
(TEM)), immunohistochemical (IHC) staining will be performed on FFPE tumor slices 
using some or all of the following antibodies:
Core set: CD8, PD-1, PD-L1, PD-L2
Others: CD3, CD4, CD25, FoxP3, Indoleamine 2,3 deoxygenase-1 (IDO1), CD11c, 
CD83, CD86, CD56, CD14, CD16, Tie2 (See also Appendix B)
Investigators at our institution have developed IHC staining on paraffin embedded 
tissues for PD-L1, PD-L2, TIM-3, and LAG-3 through our center for Immuno-
Oncology Pathology Core (Scott Rodig MD, PhD Core Director, is a co-investigator 
on this protocol).  PD-L1 IHC has recently been established in a CLIA approved 
laboratory and the remaining assays for CLIA laboratory conduct are being finalized.
These investigators have published the methods, protocols, and data establishing the 
sensitivity and specificity of IHC staining assays using the monoclonal antibodies 
recognizing PD-L1 (CD274, B7-H1, antibody clone 7G11, generated in the lab of 
Gordon Freeman, DFCI) and PD-L2 (CD273, B7-DC, clone 9E5, generated in the 
laboratory of Gordon Freeman, DFCI in two recent manuscripts.[Chen et al., 2013, Shi 
et al., 2014]
Below is a schematic of the workflow for the tissue-based biomarker analysis.

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Tumor will be considered positive if >5% (PD-L1)[Topalian et al., 2012] or >10% (PD-
L2) of the tumor cell population demonstrates unequivocal staining.  PD-1 positivity 
will be defined as >3% positive cells/high power field.[Bachireddy et al., 2014]  All 
IHC stained slides will be evaluated and scored by a pathologist.  A subset of slides 
will be reviewed by a second pathologist to ensure concordance of interpretation.
The semi-quantitative scoring for this study is in accordance with those published 
previously and, as described above, will include scores for both the neoplastic and non-
neoplastic cells within the tumor microenvironment.  Data derived from pathologist 
visual scoring (semi-quantitative, but with increased specificity for delineating 
neoplastic and non-neoplastic cells) and pathologist-assisted, automated scoring 
(quantitative, but without accurately delineating neoplastic and non-neoplastic cells) 
for each marker of interest will be assessed for its clinical value.  As necessary, the data 
from combinations of markers will also be considered (i.e. combined scores from PD-
L1 and PD-L2 expression).  All data will be analyzed in conjunction with the 
biostatistics group.
Assay 3: Flow cytometry
TILs will be isolated from the biopsy specimen as described in the lab manual
Surface staining followed by flow cytometry on the resultant TILs will then be 

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performed as described in the lab manual.  The following antibodies may be used on 
all specimens: (core set)
CD8
PD-1
PD-L1
PD-L2
A selection of the following antibodies may also be used, and additional antibodies 
may be used as well, as deemed appropriate and informative based on the state of the 
immune profiling literature at the time of correlative science performance:
CD4
FOXP3
CD127
(Other antibodies as listed in Appendix B)
Assay 4: RNA analysis
RNA analysis may be performed, and tissue for RNA analysis will be stored, in the 
Core Blood and Tissue Bank (Deborah Dillon, MD).
Messenger RNA (mRNA) expression within tumor biopsy specimens may be assessed 
using the most current and informative methodologies at the time that correlative 
science is performed on all specimens.  NanoString signatures and comprehensive 
RNA sequencing may be used.  Potential genes of interest, based on prior immune 
profiling of breast tumors,[Denkert et al., 2015] include CXCL9, CCL5, CD8ACD80, 
CXCL13, IGKC, CD21, IDO1, PD-1, PD-L1, PD-L2, CTLA4, and FOXP3.
Assay 5: Single cell sequencing
1. Tumor biopsies in DMEM media will be delivered to Dr. Derin Keskin (Immunology 
Lead, TIGL*), Wandi Zhang (Research Technician), or Phuong Le (Research 
Technician) at the Tissue Immunogenomics Lab, TIGL (Dana 520).
2. The tumor will be processed using collagenase, hyalurinidase and DNase to generate 
single cell suspension.
3. These cells will be stained with CD45 antibody. CD45 positive vs negative single cells 
will be sorted into 96 wells plates at the Hematology/Oncology flow cytometry core 
facility (Mayer 584). 
4. Single cell sorted plates will be transferred to Dr. Kenneth Livak (Technology Lead, 
TIGL) and Dr. Shuqiang Li (Senior Scientist) at the Genomics Lab, TIGL (Smith 1048, 
Dana Farber), who will generate single cell RNA-seq libraries. For single-cell TCR 
analysis, the RNA-seq libraries will be sequenced in the TIGL Genomics Lab using next-
generation sequencing (NGS). For single-cell transcriptome analysis, Dr. Li will drop off 
the RNA-seq libraries at the Broad Institute walk-up sequencing station (6th floor, 415 
Main St, Cambridge) for sequencing using NGS.

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*Dr. Catherine Wu is the faculty mentor of TIGL (Translational Immunogenomics 
Laboratory), which was established in 2017. 
Assay 6: Tumor Genomic Profiling
Next generation sequencing with tumor biopsy specimens may be assessed using the 
most current and informative methodologies at the time that correlative science is 
performed on all specimens. This may be at the Center for Advanced Molecular 
Diagnositics (Department of Pathology, Brigham and Women’s Hospital) or 
Foundation Medicine, Inc. 
9.2.5
Sites Performing Correlatives
BWH
DFCI CIO 
DF/HCC Core Blood and Tissue Bank 
Translation Immunogenomics Laboratory (TIGL)
The Broad Institute of MIT
Foundation Medicine, Inc 
Nanostring Technologies, Inc
9.3
Blood Collection 
Research blood collection is mandatory for all patients for flow cytometry and potential DNA 
isolation.  The samples will be banked for these and future research purposes. These specimens 
will become the property of the DF/HCC. 
The following research blood samples are required: 
Baseline (preferably on Cycle 1 Day 1 prior to dosing): 

1-10 mL Streck Tube for cfDNA

5-10mL CPT tubes for PBMCs
Every Restaging Visit (C2D1, C3D1, C4D1, C6D1. C8D1, etc.): 

1-10mL Streck Tube for cfDNA

5-10mL CPT tubes for PBMCs
Off Treatment (if for progressive disease):

1-10 mL Streck Tube for cfDNA

5- 10mL CPT tubes for PBMCs
The following Time of Progression research blood samples are optional for patients who came off 
treatment for a reason other than progressive disease:

1-10 mL Streck Tube for cfDNA

5-10mL CPT tubes for PBMCs
For retreatment patients, research blood samples will be drawn with every restaging visit:

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(Jennifer_Guerriero@dfci.harvard.edu; emittendorf@bwh.harvard.edu)
9.4.2
Analysis of DNA extraction from stool samples
Under the supervision of Dr. Andrew T. Chan, total genomic DNA will be isolated from 0.25g of 
feces using the PowerSoil DNA isolation kit (Mo Bio, USA) at the Clinical and Translation 
Epidemiology Unit at Massachusetts General Hospital. Purified DNA will be separated on a 1% 
agarose gel and quantified by densitometry and spectrophotometry (NanoDrop 1000; Thermo 
Scientific, USA).
9.4.3
Analysis of RNA extraction from stool samples
Under Dr. Chan’s supervision, 16S rRNA analysis will be performed at The V4–V5 region of the 
16S rRNA gene will be amplified and sequenced on an Illumina MiSeq platform at the Broad 
Institute. For each stool sample, replicate PCR reactions will be performed using modified 
universal bacterial primers designed to amplify the V4-V5 16S rRNA region: 563F (59-
nnnnnnnn-NNNNNNNNNNNN-AYTGGGYDTAAAGN G-39) and 926R (59-nnnnnnnn-
NNNNNNNNNNNN-CCGTCAATTYHTTTR AGT-39). 
Using the Illumina TruSeq Sample Preparation procedure, PCR products will be quantified and 
pooled at equimolar amounts before Illumina barcodes and adaptors will be ligated on. The 
completed library will be sequenced on an Ilumina Miseq platform according to the Illumina 
recommended protocol. Sequences will be analysed using mothur version 1.31.115. Sequences 
were aligned using the Silva reference alignment as a template and potentially chimeric 
sequences were eliminated using the UChime algorithm16. Five thousand sequences per patient 
were selected (mean 4,974, s.d. 150) and sequences with a distance-based similarity of Z97% 
were grouped into OTUs using the furthest-neighbour algorithm. OTUs were classified using the 
Greengenes 16S rRNA reference database. OTU-based microbial diversity was estimated by 
calculating two diversity indices, Shannon and Inverse Simpson. OTU-based richness was 
determined by calculating the Chao richness estimate and constructing rarefaction curves. OTUs 
were grouped at different levels of classification (phylum, class, order, family and genus); at 
each level, OTUs that did not have a classification were grouped together by the highest 
available resolution. Feature selection of the intestinal microbia’s composition was performed on 
OTUs with an average abundance 40.01% in either patient group and grouped by phylotype.
9.4.4
Shotgun sequencing and metabolic pathway reconstruction of stool samples
Stool samples from patients included in the trial 2 will be subjected to whole genome shotgun 
sequencing. Libraries will be constructed with Illumina barcodes from the TruSeq DNA Sample 
Prep kit (Illumina) and reagents from KAPA Library Preparation kit (Kapa Biosystems), and 
then sequenced on an Illumina MiSeq platform using 2_250 nucleotide paired-end sequencing, 
according to the manufacturer’s instructions. Sequencing reads will be converted into relative 
abundances of microbial metabolic modules using HUMAnN35, the Human Microbiome Project 
metabolic reconstruction pipeline and mapped to the KEGG36. Relative species abundances will 
be calculated by the MetaPhlAn pipeline37.

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9.4.5
Sites performing correlative analysis
DF/HCC Core Blood and Tissue Bank
MGH Clinical and Translational Epidemiology Unit 
9.5
Central confirmation of the hypermutated status 
All patients with safely accessible tumors need to undergo a research biopsy at baseline, and 
tumor will be centrally sequenced with the FoundationOne medicine panel. The in vitro 
diagnostic (IVD) device, the FoundationOne panel, will be used in accordance with the 
instructions for use (IFU) that are provided in the device’s approved labeling. If participant’s 
previously had testing performed by Foundation One, it will not need to be repeated. Patients in 
which a baseline biopsy was not performed need to provide archival tissue for performing this 
central test.  Eligibility will be determined by local test results.
9.6
Additional analysis
The above-mentioned analyses may be altered based on novel developments in the field of cancer 
immune profiling at the time of correlative science.  Additional markers or alternative technologies 
(based on scientific developments and/or novel technologies) may also be used, to explore 
potential prognostic or predictive candidate markers/panels or markers related to treatment benefit 
and/or safety, to improve diagnostic tests, or to understand breast cancer biology.
10.
STUDY CALENDAR
Screening evaluations are to be conducted within 28 days prior to start of protocol therapy unless 
otherwise specified.  Screening assessments occurring within 4 days prior to initiating study 
treatment do not need to be repeated on Cycle 1 Day 1.  
As detailed in the Study Calendar, laboratory assessments including a negative pregnancy test in 
women of child-bearing potential must be documented within 7 days before the first dose of study 
medication.
In the event that the participant’s condition is deteriorating, laboratory evaluations should be 
repeated within 48 hours prior to initiation of the next cycle of therapy.
Assessments must be performed prior to administration of any study agent. Study assessments and 
agents should be administered within + 3 days of the protocol-specified date, unless otherwise 
noted.

68
Cycle 1
Cycle 2-4 
Cycle 5+ 
Screeninga
Day 1
Day 15
Day 29
Day 1
Day 15
Day 29 
Day 1
Day 15
Day 29 
End of
Treatmentq
Follow-Up 
Medical Historyb
X
Physical examc
X
X
X
X
X
X
X
Concurrent medicationsd
X
X
X
X
X
ECOG 
X
X
X
X
X
Adverse event evaluation
X
X
X
X
X
X
X
X
X
Xs
Vital signse
X
X
X
X
X
X
X
X
X
X
X
Hematology panelf
X
X
X
X
X
X
X
X
X
X
X
Chemistry panelg
X
X
X
X
X
X
X
X
X
X
X
Urine Protein Creatinine Ratio
X
X
X
X
TSH/fT4
X
X
X
Xh
X
ACTH/Cortisol
X
X
X
Xh
Amylase and Lipase
X
X
X
Xh
Coagulation panel (PT/PTT)
X
Pregnancy testi
Xi
Xi
Xi
Xi
Xi
Xi
Xi
Xi
Xi
12- lead EKG
X
Tumor Assessmentj
X
X
 
X
Xk
Xk
Research Bloodl
X
X
X
X
X
Research Biopsym
X
X 
X
Archival Tumor Retrievaln
X
Quality of Lifer
X
X
X
Research Stool Collection & 
Questionnaireo, p (Section 9.4)
X
X
X
Survival (Section 5.6) 
X
a. Screening assessments are to be conducted within 28 days prior to start of protocol therapy unless otherwise specified.  If these screening assessments occur 
within 4 days before start of study treatment, then they may serve as the baseline Cycle 1 Day 1 values.
b. Medical history includes clinically significant diseases, surgeries, and cancer history (including prior cancer therapies and procedures).
c.  A complete physical examination will be performed at baseline.  A limited physical exam will be completed prior to therapy on Days 1,15 and 29 for Cycles 1 
and on Day 1 of every cycle beginning with Cycle 2.
d. All medications taken from the time informed consent is signed through 30 days after the last dose of study therapy will be captured in the participant’s electronic 
medical record. 

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e. Vital sign assessments include measurements of heart rate, systolic and diastolic blood pressures, respiratory rate, temperature and weight. 
f. Hematology includes: hemoglobin, hematocrit, platelet count, RBC count, WBC count, and percent and absolute differential count.  Results must be available 
prior to the administration of study drug.
g. Chemistry testing includes: sodium, potassium, chloride, bicarbonate, glucose, BUN, creatinine, calcium, total bilirubin, total protein, albumin, ALT, AST, and 
alkaline phosphatase. LDH will be drawn at baseline only (during screening or on C1D1). Results must be available prior to the administration of study drug.
h. TSH, free T4, ACTH, cortisol, amylase, and lipase will be performed during screening, on Day 1 of Cycles 1-4 and then every other cycle on Day 1. These lab 
tests should be drawn prior to treatment, however, treatment can be administered prior to the results of these tests. 
i. In female subjects of child-bearing potential as defined in the eligibility criteria, the screening pregnancy test (urine preferred, serum acceptable) must be 
performed within 7 days before the first dose of study medication. A urine pregnancy test should be repeated within 24 hours of the first dose of study treatment 
on C1D1. Urine pregnancy tests should be repeated every 4 weeks (+/- 1 week) while receiving study treatment (this is approximately every other Nivolumab 
infusion). Any urine pregnancy test that comes back as positive should be confirmed with a serum hCG.      
j. Tumor assessments should consist of 1) CT and/or MRI of the chest/abdomen/pelvis, 3) any other imaging studies (CT neck, plain films, etc.) as clinically 
indicated by the treating physician. The same radiographic procedures and technique must be used throughout the study for each patient (e.g., if the patient had CT 
chest/abdomen/pelvis performed during screening, then she should subsequently undergo CT performed using the same radiologic protocol throughout the 
remainder of the study). Tumor assessments will be performed at baseline, at day 1 (7 day scheduling window) of Cycles 2, 3, and 4 and then every 12 week   at 
day 1 (7 day scheduling window) of every odd cycle: C6, C8, C10, etc..  Additional scans are permitted as clinically indicated. All known sites of disease 
documented at screening should be re-assessed at each subsequent tumor evaluation. 
k. For those taken off the study for reasons other than progressive disease, tumor measurements should continue to be repeated every 6-12 weeks until first disease 
progression or start of a new anti-cancer therapy. Every effort should be made to maintain this scheduled, but it is understood that it may not be possible in all  
cases. Failure to complete scans at this interval will not be deemed protocol violations. 
l. Collected at screening, every restaging, end of treatment (if removed for progressive disease) and optionally at time of progression. See section 9.0 
m. Baseline tumor biopsy should be obtained within 28 days prior to initiating protocol therapy. On treatment biopsies should be obtained at C1D29 (+14 day 
scheduling window). A tumor biopsy may be performed at the end of treatment. See Section 9.2.
n. Archival tumor sample should be collected (block or if not possible, 10-20 5mincron, unstained positively charged slides). This sample may be collected at any 
time throughout the lifetime of the study.
o. Baseline stool collection should be obtained within 14 days before starting protocol therapy. On treatment stool collection should be obtained at C1D29 (anytime 
from C1D29-C2D1). A stool collection should be obtained at the end of treatment. As these collections are for exploratory correlative purposes, failure to provide 
a sample at these timepoints will not constitute a protocol violation. See section 9 and/or lab manual for stool collection and processing instructions.
p.  An optional stool sample may be collected at the time of grade ≥ 2 diarrhea after discussion with the PI. As these collections are for exploratory correlative 
purposes, failure to provide a sample or complete the stool questionnaires at these timepoints will not constitute a protocol violation. See section 9 and/or lab 
manual for stool collection and processing instructions.
q. End of treatment visit is to occur within 30 days of final administration of study treatment.  End of treatment assessments do not have to be repeated if the same 
assessments were performed within 7 days (28 days for tumor assessments) prior to the visit.

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r. FACT B and the Rotterdam Symptom Checklist should be completed baseline or C1D1 (any time prior to first dose), D1 of C2-C4, and then on D1 of every other 
cycle (C6D1, C8D1, etc.) These questionnaires may be found in Appendices C and D. 
s. An adverse event assessment is performed 100 days (-15/+30 days) after the last dose of nivolumab. This may be conducted in person or by phone by the 
investigator or suitably trained and qualified delegate (i.e. MD, RN, NP, etc). 

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11.
MEASUREMENT OF EFFECT
For the purposes of this study, participants should be re-evaluated for response 
approximately every 6 weeks for the first 24 weeks and then every 12 weeks thereafter. If cycles 
are delayed for any reason, the scan schedule should be moved to align with cycles of treatment 
received.
11.1
Antitumor Effect – Solid tumors 
Response and progression in sites of metastases will be evaluated in this study using the 
international criteria proposed by the RECIST 1.1 criteria [Eisenhauer et al., 2009].  Changes in 
the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter 
in the case of malignant lymph nodes are used in the RECIST criteria. 
11.1.1 RECIST 1.1 Definitions
Evaluable for Target Disease response.  Only those participants who have measurable 
disease outside the field of radiation present at baseline, have received at least one cycle of 
therapy, and have had their disease re-evaluated will be considered evaluable for target 
disease response.  These participants will have their response classified according to the 
definitions stated below.  (Note:  Participants who exhibit objective disease progression 
prior to the end of cycle 1 will also be considered evaluable.)
Evaluable Non-Target Disease Response.  Participants who have lesions present at baseline 
that are evaluable but do not meet the definitions of measurable disease, have received at 
least one cycle of therapy, and have had their disease re-evaluated will be considered 
evaluable for non-target disease.  The response assessment is based on the presence, 
absence, or unequivocal progression of the lesions. 
11.1.2 Disease Parameters
Measurable disease.  Measurable lesions are defined as those that can be accurately 
measured in at least one dimension (longest diametB er  to be recorded) as ≥ 20 mm by 
chest x-ray or ≥10 mm with CT scan, MRI, or calipers by clinical exam.  All tumor 
measurements must be recorded in millimeters (or decimal fractions of centimeters).
Note: Tumor lesions that are situated in a previously irradiated area are not considered 
measurable.
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a 
lymph node must be ≥15 mm in short axis when assessed by CT scan (CT scan slice 
thickness recommended to be no greater than 5 mm).  At baseline and in follow-up, only 
the short axis will be measured and followed.
Non-measurable disease.  All other lesions (or sites of disease), including small lesions 
(longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), 

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

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superficial (e.g., skin nodules and palpable lymph nodes) and ≥10 mm in diameter as 
assessed using calipers (e.g., skin nodules).  In the case of skin lesions, documentation by 
color photography, including a ruler to estimate the size of the lesion, is recommended. 
Chest x-ray.  Lesions on chest x-ray are acceptable as measurable lesions when they are 
clearly defined and surrounded by aerated lung; however, CT is preferable. 
Conventional CT and MRI. This guideline has defined measurability of lesions on CT scan 
based on the assumption that CT thickness is 5mm or less. If CT scans have slice thickness 
greater than 5 mm, the minimum size of a measurable lesion should be twice the slice 
thickness. MRI is also acceptable in certain situations (e.g. for body scans).  
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which 
greatly impact image quality, lesion conspicuity, and measurement.  Furthermore, the 
availability of MRI is variable globally.  As with CT, if an MRI is performed, the technical 
specifications of the scanning sequences used should be optimized for the evaluation of the 
type and site of disease.  Furthermore, as with CT, the modality used at follow-up should 
be the same as was used at baseline and the lesions should be measured/assessed on the 
same pulse sequence.  It is beyond the scope of the RECIST guidelines to prescribe specific 
MRI pulse sequence parameters for all scanners, body parts, and diseases.  Ideally, the 
same type of scanner should be used and the image acquisition protocol should be followed 
as closely as possible to prior scans.  Body scans should be performed with breath-hold 
scanning techniques, if possible.
FDG-PET. While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis 
of FDG-PET imaging can be identified according to the following algorithm: 
(a) Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of 
PD based on a new lesion.
(b)  No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive 
FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is 
PD.  If the positive FDG-PET at follow-up is not confirmed as a new site of disease on 
CT, additional follow-up CT  scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not 
PD.
(c)  FDG-PET may be used to upgrade a response to a CR in a manner similar to a 
biopsy in cases where a residual radiographic abnormality is thought to represent 
fibrosis or scarring.  The use of FDG-PET in this circumstance should be prospectively 
described in the protocol and supported by disease-specific medical literature for the 
indication.  However, it must be acknowledged that both approaches may lead to false 
positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity.

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Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake 
greater than twice that of the surrounding tissue on the attenuation corrected image.
PET-CT. At present, the low dose or attenuation correction CT portion of a combined PET-
CT is not always of optimal diagnostic CT quality for use with RECIST measurements.  
However, if the site can document that the CT performed as part of a PET-CT is of identical 
diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of 
the PET-CT can be used for RECIST measurements and can be used interchangeably with 
conventional CT in accurately measuring cancer lesions over time.  Note, however, that 
the PET portion of the CT introduces additional data which may bias an investigator if it 
is not routinely or serially performed.  
MIBG (meta-iodobenzylguanidine).The following is recommended, to assure high 
quality images are obtained. 
Patient preparation: Iodides, usually SSKI (saturated solution of potassium iodide), are 
administered to reduce thyroidal accumulation of free radioiodine, preferably 
beginning the day prior to injection and continuing for 3 additional days (4 days total). 
For infants and children, one drop t.i.d. is sufficient, for adolescents 2 drops t.i.d., and 
for adults 3 drops t.i.d. Participants and/or parents are asked about exposure to potential 
interfering agents. If none is noted, an indwelling intravenous line is established. The 
dose of MIBG is administered by slow intravenous injection over 90 seconds. 
Images from the head to the distal lower extremities should be obtained. 
I-123MIBG scintigraphy is performed to obtain both planar and tomographic images. 
Planar: Anterior and posterior views from the top of the head to the proximal lower 
extremities are obtained for 10 minutes at 24 hours and occasionally at 48 hours 
following injection of 10 mCi/1.7 square meters of body surface area (~150 μCi/kg, 
maximum 10 mCi). Anterior views of the distal lower extremities are adequate. A large 
field of view dual head gamma camera with low energy collimators is preferred. 
SPECT: Most participants receiving I-123 MIBG also undergo SPECT at 24 hours, 
using a single or multi-headed camera with a low energy collimator. The camera is 
rotated through 360 degrees, 120 projections at 25 seconds per stop. Data are 
reconstructed using filtered back projections with a Butterworth filter and a cut off 
frequency of 0.2-0.5. SPECT/CT may be performed at institutions with this capacity.
Ultrasound. Ultrasound is not useful in assessment of lesion size and should not be used as 
a method of measurement. Ultrasound examinations cannot be reproduced in their entirety 
for independent review at a later data and, because they are operator dependent, it cannot 
be guaranteed that the same technique and measurements will be taken from one 
assessment to the next. If new lesions are identified by ultrasound in the course of the study, 
confirmation by CT or MRI is advised. If there is concern about radiation exposure from 

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CT, MRI may be used instead of CT in selected instances. 
Endoscopy, Laparoscopy. The utilization of these techniques for objective tumor 
evaluation is not advised. However, such techniques may be useful to confirm complete 
pathological response when biopsies are obtained or to determine relapse in trials where 
recurrence following complete response (CR) or surgical resection is an endpoint.
Tumor markers. Tumor markers alone cannot be used to assess response. 
Cytology, Histology. These techniques can be used to differentiate between partial 
responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor 
types, such as germ cell tumors, where known residual benign tumors can remain).
The cytological confirmation of the neoplastic origin of any effusion that appears or 
worsens during treatment when the measurable tumor has met criteria for response or stable 
disease is mandatory to differentiate between response or stable disease (an effusion may 
be a side effect of the treatment) and progressive disease.
11.1.3.1
Evaluation of Target Lesions
Complete Response (CR):  Disappearance of all target lesions.  Any pathological lymph 
nodes (whether target or non-target) must have reduction in short axis to <10 mm.
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of target 
lesions, taking as reference the smallest sum on study (this includes the baseline sum if 
that is the smallest on study).  In addition to the relative increase of 20%, the sum must 
also demonstrate an absolute increase of at least 5 mm.  (Note:  the appearance of one 
or more new lesions is also considered progressions).
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 
study.
11.1.3.2
Evaluation of Non-Target Lesions
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
short axis).
Note:  If tumor markers are initially above the upper normal limit, they must normalize 
for a patient to be considered in complete clinical response.
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or maintenance 

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of tumor marker level above the normal limits.
Progressive Disease (PD):  Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions.  Unequivocal progression should not 
normally trump target lesion status.  It must be representative of overall disease status 
change, not a single lesion increase.    
Although a clear progression of “non-target” lesions only is exceptional, the opinion of 
the treating physician should prevail in such circumstances, and the progression status 
should be confirmed at a later time by the review panel (or Principal Investigator).
11.1.3.3
Evaluation of New Lesions
The finding of a new lesion should be unequivocal (i.e. not due to difference in 
scanning technique, imaging modality, or findings thought to represent something other 
than tumor (for example, some ‘new’ bone lesions may be simply healing or flare of 
pre-existing lesions). However, a lesion identified on a follow-up scan in an anatomical 
location that was not scanned at baseline is considered new and will indicate PD. If a 
new lesion is equivocal (because of small size etc.), follow-up evaluation will clarify if 
it truly represents new disease and if PD is confirmed, progression should be declared 
using the date of the initial scan on which the lesion was discovered.
11.1.3.4
Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).  The patient's best 
response assignment will depend on the achievement of both measurement and 
confirmation criteria.
For Participants with Measurable Disease (i.e., Target Disease)
Target 
Lesions
Non-Target 
Lesions
New 
Lesions
Overall 
Response
Best Overall Response when 
Confirmation is Required*
CR
CR
No
CR
4 wks Confirmation**
CR
Non-CR/Non-
PD
No
PR
CR
Not evaluated
No
PR
PR
Non-CR/Non-
PD/not 
evaluated
No
PR
4 wks Confirmation**
SD
Non-CR/Non-
PD/not 
evaluated
No
SD
PD
Any
Yes or No
PD
Any
PD***
Yes or No
PD
Any
Any
Yes
PD
no prior SD, PR or CR

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
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion.
**        Only for non-randomized trials with response as primary endpoint.
***      In exceptional circumstances, unequivocal progression in non-target lesions may be
            accepted as disease progression.
Note:  Participants with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.”  Every effort should be made to document the 
objective progression even after discontinuation of treatment.
For Participants with Non-Measurable Disease (i.e., Non-Target Disease)
Non-Target Lesions
New Lesions
Overall Response
CR
No
CR
Non-CR/non-PD
No
Non-CR/non-PD*
Not all evaluated
No
not evaluated
Unequivocal PD
Yes or No
PD
Any
Yes
PD

‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign 
this category when no lesions can be measured is not advised
11.1.4 Duration of Response
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date 
that recurrent or progressive disease is objectively documented (taking as reference for 
progressive disease the smallest measurements recorded since the treatment started, or 
death due to any cause. Participants without events reported are censored at the last disease 
evaluation).
Duration of overall complete response: The duration of overall CR is measured from the 
time measurement criteria are first met for CR until the first date that progressive disease 
is objectively documented, or death due to any cause. Participants without events reported 
are censored at the last disease evaluation.
Duration of stable disease:  Stable disease is measured from the start of the treatment until 
the criteria for progression are met, taking as reference the smallest measurements recorded 
since the treatment started, including the baseline measurements. 
11.1.5 Clinical Benefit rate 
Clinical benefit rate: defined as CR, PR and stable disease (SD) ≥  24 weeks.

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11.2
Antitumor Effect – Hematologic Tumors
N/A
11.3
Other Response Parameters
11.3.1 Definition of Tumor Response Using Immune-Related Response Criteria (irRC)
The sum of the products of the largest perpendicular diameters (SPD) at tumor assessment 
using the immune-related response criteria (irRC) for progressive disease incorporate the 
contribution of new measurable lesions.  Each net Percentage Change in Tumor Burden 
per assessment using irRC criteria accounts for the size and growth kinetics of both old and 
new lesions as they appear.
11.3.1.1
Impact of New Lesions on irRC
New lesions in and of themselves do not qualify as progressive disease.  However, their 
contribution to total tumor burden is included in the SPD which in turn feeds into the irRC 
criteria for tumor response.  Therefore, new non-measurable lesions will not discontinue 
any subject from the study.
11.3.1.2
Definition of Target Lesions Response Using irRC

irComplete Response (irCR): Complete disappearance of all target lesions.  This category 
encompasses exactly the same subjects as “CR” by the mWHO criteria.

irPartial Response (irPR): Decrease, relative to baseline, or 50% or greater in the sum of 
the products of the two largest perpendicular diameters of all target and all new measurable 
target lesions (i.e., Percentage Change in Tumor Burden).  Note: the appearance of new 
measurable lesions is factored into the overall tumor burden, but does not automatically 
qualify as progressive disease until the SBD increases by >25% when compared to SPD at 
nadir.

irStable Disease (irSD): Does not meet criteria for irRC or irPR, in the absence of 
progressive disease.

irProgressive Disease (irPD): At least 25% increase Percentage Change in Tumor Burden 
(i.e. taking SPD of all target lesions and any new lesions) when compared to SPD at nadir.
11.3.1.3
Definition of Non-Target Lesions Response Using irRC

irComplete Response (irCR): Complete disappearance of all non-target lesions.  This 
category encompasses exactly the same subjects as “CR” by the mWHO criteria.

irPartial Response (irPR) or irStable Disease (irSD): Non-target lesion(s) are not 
considered in the definition of PR; these terms do not apply.

irProgressive Disease (irPD): Increases in number or size of non-target lesion(s) does not 
constitute progressive disease unless/until the Percentage Change in Tumor Burden 
increases by 25% (i.e. the SPD at nadir of the target lesions increases by the required 
amount).

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11.3.1.4
Definition of Overall Response Using irRC
Overall response using irRC will be based on these criteria:

Immune-Related Complete Response (irCR): Complete disappearance of all tumor 
lesions (target an non-target) together with no new measurable/unmeasurable lesions for at 
least 4 weeks from the date of documentation of complete response.

Immune-Related Partial Response (irPR): The sum of the products of the two largest 
perpendicular diameters of all target lesions is measured and captured as the SPD baseline.  
At each subsequent tumor assessment, the SPD of the two largest perpendicular diameters 
of all target lesions and of new measurable lesions are added together to provide the 
Immune Response Sum of Product Diameters (irSPD).  A decrease, relative to baseline, of 
the irSPD compared to the previously SPD baseline  of 50% or greater is considered an 
irPR.

Immune-Related Stable Disease (irSD): irSD is defined as the failure to meet criteria for 
immune complete response or immune partial response, in the absence of progressive 
disease

Immune-Related Progressive Disease (irPD): It is recommended in difficult cases to 
confirm PD by serial imaging.  Any of the following will constitute PD:

At least 25% increase in the SPD of all target lesions over baseline SPD calculated for 
the target lesions.

At least 25% increase in the SPD of all target lesions and new measurable lesions 
(irSPD) over the baseline SPD calculated for the target lesions.
Criteria for determining overall response by irRC are summarized as follows:

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11.3.1.5
Immune-Related Best Overall Response Using irRC (irBOR)
irBOR is the best confirmed overall response over the study as a whole, recorded between 
the date of first dose until the last tumor assessment before subsequent therapy (except for 
local palliative radiotherapy for painful bone lesions) for the individual subject in the study.  
For the assessment of irBOR, all available assessments per subject are considered.
irCR or irPR determinations included in the irBOR assessment must be confirmed by a 
second (confirmatory) evaluation meeting the criteria for response and performed no less 
than 4 weeks after the criteria for response are first met.
11.3.1.6
Patient Reported Outcome Assessments
The PRO outcome measure for this study is as follows: Scores from the FACT-B 
assessment (Appendix C) and Rotterdam Symptom Checklist (Appendix D).
12.
DATA REPORTING / REGULATORY REQUIREMENTS
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements).
12.1
Data Reporting
12.1.1 Method
The Office of Data Quality (ODQ) will collect, manage, and perform quality checks on the 
data for this study.
12.1.2 Responsibility for Data Submission
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the  Office of Data Quality in accordance with DF/HCC SOPs.
12.2
Data Safety Monitoring
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity 
and accrual data from this study. The committee is composed of clinical specialists with experience 
in oncology and who have no direct relationship with the study. Information that raises any 
questions about participant safety will be addressed with the Overall PI and study team.
The DSMC will review each protocol up to four times a year or more often if required to review 
toxicity and accrual data. Information to be provided to the committee may include: up-to-date 
participant accrual; current dose level information; DLT information; all grade 2 or higher 
unexpected adverse events that have been reported; summary of all deaths occurring with 30 days 

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of intervention for Phase I or II protocols; for gene therapy protocols, summary of all deaths while 
being treated and during active follow-up; any response information; audit results, and a summary 
provided by the study team. Other information (e.g. scans, laboratory values) will be provided 
upon request. 
12.3
Multicenter Guidelines
This protocol will adhere to the policies and requirements of the DF/HCC Multi-Center Data and 
Safety Monitoring Plan. The specific responsibilities of the Overall PI, Coordinating Center, and 
Participating Institutions and the procedures for auditing are presented in Appendix E.

The Overall PI/Coordinating Center is responsible for distributing all IND Action Letters 
or Safety Reports to all participating institutions for submission to their individual IRBs 
for action as required.

Mechanisms will be in place to ensure quality assurance, protocol compliance, and adverse 
event reporting at each site.

Except in very unusual circumstances, each participating institution will order the study 
agent(s) directly from supplier. A participating site may order the agent(s) only after the 
initial IRB approval for the site has been forwarded to the Coordinating Center.
12.4
Collaborative Research and Future Use of Data and Samples
Tissue, blood, stool, bodily fluids, and other materials derived from these will be collected in this 
study to analyze genes, DNA, RNA, proteins and cells for the study’s correlative endpoints and 
potential future research, utilizing new types of biomarker testing as it becomes available. 
These samples and any data generated as a part of these clinical trials may be used for future 
research studies and may be provided to collaborating investigators both within and outside of the 
DF/HCC for either correlative endpoints or secondary use. Samples and data may be shared with 
outside non-profit academic investigators, as well as with for-profit pharmaceutical investigators 
or commercial entitites, with whom we collaborate. When samples or data are sent to collaborators 
and when any research is performed on them, all information will be identified with a code, and 
will not contain any PHI, such as name, birthday, or MRNs. 
In order toe allow the greatest amount of research to be performed on the specimens and 
information generated as a part of this trial, researchers in this study may share results of genetic 
sequencing with other scientists. De-identified specimen or genetic data may be placed into one of 
more publicly-accessible scientific databases, such as the National Institutes of Health’s Database 
for Genotypes and Phenotypes (dbGaP). The results from the correlative research on this study 
will be shared with these public databases. Through such databases, researchers from around the 
world will have access to de-identified samples or data for future research. More detailed 
information, beyond the public database, may only be accessed by scientists at other research 
centers who have received special permission to review de-identified data.

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13.
STATISTICAL CONSIDERATIONS
13.1
Study Design/Endpoints
This is an open-label, single-arm, multicenter, phase 2 study of nivolumab 3 mg/Kg intravenously 
(IV) on day 1 of each 14 days plus Ipilimumab 1 mg/Kg IV every 6 weeks in subjects with 
hypermutated metastatic HER2- breast cancer previously treated with 0 to 3 chemotherapy 
regimens in the metastatic setting. The target enrollment is 30 evaluable patients. Patients that do 
not start protocol therapy will be considered unevaluable and will be replaced, therefore total 
enrollment may exceed 30 pateints.
Primary Endpoint
The primary endpoint is ORR of nivolumab in combination with ipilimumab, according to 
RECIST 1.1 (Section 11), in patients with with hypermutated HER2- metastatic breast cancer 
previously treated with 0 to 3 lines of chemotherapy in the metastatic setting.
Secondary endpoints include:
Secondary endpoints include the ORR of the combination according to immune-related response 
criteria (irRC) [Wolchok et al., 2009] (Section 11), the clinical benefit rate according to RECIST 
1.1. and irRC, PFS according to RECIST 1.1. and irRC, the OS, the ORR, PFS and OS of the 
combination according to RECIST 1.1 in the population with centrally confirmed hypermutated 
tumors (≥ 9 mutations/megabase); and whether the cut off of ≥ 14 mutations/megabase is 
associated with the efficacy of this investigational therapy, assessed by ORR, PFS and OS. 
Blood and Tissue correlative science objectives include: 
To characterize a broad array of immune markers in metastatic hypermutated HER2-negative 
breast cancers. (characterization will be based on histology, protein expression, and mRNA 
expression).
To explore how different immunosuppressive and/or immune-stimulating immune marker 
profiles at baseline correlate with disease response to therapy (PFS, objective response 
assessed by RECIST 1.1 and immune-related response criteria).
To characterize changes in tumor-infiltrating lymphocytes, PD-L1 expression and immune 
gene signatures in the tissue microenvironment (TME) from baseline to after 4 weeks of the 
experimental combination.
To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in TME correlates with disease response to therapy 
(response assessed by RECIST 1.1 and immune-related response criteria).
To characterize serial changes in immune marker profile in peripheral blood mononuclear 
cells (PBMCs) and in plasma over the course of the trial treatment.
To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in PBMCs correlates with disease response to therapy 
(response assessed by RECIST 1.1 and immune-related response criteria).
To investigate whether there is an immune marker in circulating PBMCs that corresponds to 

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tumor infiltrating lymphocyte (TIL) percentage in baseline tumor.
To collect blood to study cell-free DNA for comparison to tumor specimens before and after 
immunotherapy.
To explore whether the cut off of ≥ 14 mutations/megabase is associated with the efficacy of 
this investigational therapy, assessed by ORR per RECIST 1.1. 
To explore whether the cut off of ≥ 14 mutations/megabase is associated with the efficacy of 
this investigational therapy, assessed by PFS per RECIST 1.1
To explore whether the cut off of ≥ 14 mutations/megabase is associated with the efficacy of 
this investigational therapy, assessed by OS per RECIST 1.1
Stool and microbiome correlative science endpoints:
Overall, we plan to describe the landscape of gut microbiota in patients with BC who will receive 
the combination of ipilimumab plus nivolumab, and the changes in their gut microbiota after 4 
weeks of the combination. Statistical analyses of intestinal microbiota samples will be performed 
using R Statistical Language (v3.1.1) and GraphPad Prism (version 6.0e) software packages. 
Unpaired Mann–Whitney rank sum test (two-tailed) will be used for comparisons of continuous 
variables between two groups. Bar plots will be used to represent the data’s mean at the center 
values, with error bars to indicate standard deviation.  In order to explore the association of 
response (objective response according RECIST 1.1 and PFS) to baseline microbiota diversity, 
and changes from baseline in microbiota, inference will be based on Wilcoxon rank sum tests and 
estimates of predictive value along the continuous scales will be visualized using receiver 
operating characteristic (ROC) curves and reported with c-index and confidence intervals derived 
from variance estimates of Somers rank correlation. Unadjusted P-values will be considered  
significant for the Mann–Whitney rank sum test. 
We will quantify microbiome features from amplicon, metagenome, metatranscriptome using 
established pipelines to identify strain-level taxonomic, functional gene, transcriptional, and 
microbially-mediated metabolite profiles associated with BC patients with and without 
immunotherapy70-76. We will use modified multivariate linear modeling to identify statistically 
significant features associated with outcomes. Statistical tests for association with these outcomes 
and covariates will be performed using the sparse generalized linear model MaAsLin, which 
provides random effects models for both log-Gaussian and zero-inflated negative binomial link 
functions. Computational workflows for these steps are implemented as AnADAMA2 
(http://huttenhower.sph.harvard.edu/anadama) 
workflows, 
a 
reproducible 
data 
handling 
environment that captures all provenance during the analysis process.
13.2
Sample Size, Accrual Rate and Study Duration
Based on data of recent trials with a population of individuals with metastatic breast 
cancer treated with anti-PD-1/PD-1 agents in monotherapy, and considering that our population 
will not be previously selected by PD-L1 expression status a true rate of 5% or less would not be 
of clinical interest. The study will follow a two-stage design in the first stage 14 patients will be 
enrolled. If in this first stage there is at least 1 patient with objective response (complete or particl 
response), accrual will continue to the second stage where an additional 16 patients will be 

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enrolled. If there are at least 4 patients with ORR among the 30 evaluable patients, the regimen 
will be considered worthy of further study. If the true response rate is 5%, the chance the regimen 
is declared worthy of further study is less than 5%. If the true response rate is 25%, the chance that 
the regimen is declared worthy of further study is >90%. The design has been evaluated under 
departures from the assumption that 100% of the enrollments will be confirmed as hypermutated 
tumors (>9mut/Mb). The rational for this evaluation is represented by multiple platforms to 
identify hypermutated tumors and validation of the hypermutation status after  enrollment. We 
considered power based on exact conditional testing (type I error <0.05), with frequencies of non 
validated hypermutation status equal to 5%, 10%, 15% or 20% in the enrolled population, and 
analyses 
restricted 
to 
patients 
with 
validated 
hypermutation 
status. 
Under 
these 
parameters,  restricting continuation to the second stage with OR for one or more patients with 
validated hypermutation status, power  is  equal to 0.90, 0.90, 0.88, 0.86.
13.3
Analysis of Primary Endpoint
The primary endpoint is ORR of the experimental combination, which will be assessed among all 
patients who initiated protocol therapy. Radiographic response will be assessed using RECIST 1.1 
criteria as defined in section 11. Objective response will require confirmatory scans as indicated.  
The ORR (CR + PR) will be reported with 90% exact confidence intervals.   (per RECIST 1.1 
criteria; Section 11). 
13.4
Analysis of Secondary Endpoints
Efficacy Endpoints 
All patients who initiated protocol therapy will also be evaluated for ORR according irRC, and for 
CBR and PFS, according RECIST. Clinical benefit is defined as CR, PR or SD ≥ 24 weeks 
according RECIST 1.1. ORR according to irRC will be reported with 90% exact confidence 
intervals. CBR will be reported respectively with 95% exact confidence intervals. PFS will be 
analyzed using Kaplan–Meier product-limit estimates and will be plotted using Kaplan-Meier 
plots. PFS is defined as the time from study randomization to disease progression according 
RECIST 1.1, medical judgment or death due to any cause, whichever occurred first.  Patients alive 
without disease progression are censored at the date of last disease evaluation.  The hazard ratio 
for each time-to-event endpoint will be estimated with 95% confidence intervals derived from the 
Cox proportional hazard model, but no hypothesis testing will be conducted.  
Safety and tolerability 
All patients will be evaluable for toxicity from the time of their first treatment with any study 
agent. Toxicity will be graded according to NCI CTCAE, Version 5.0. Toxicities will be 
summarized by maximum grade and by treatment arm.  Incidence rate of each toxicity will be 
reported with 95% exact CI.  The incidence rates of any grade 3+ toxicity will be compared 
between two arms using Fisher’s exact test.
Correlative endpoints
Recently, Herbst et al have demonstrated that patients who presented an increase of at least 5% in 
expression of PD-L1 in tumor microenvironment experienced a bigger likelihood to respond to 
treatment with the anti-PD-L1 Atezolizumab[Herbst et al., 2014]. Also, modifications in molecular 

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signature of tumor microenvironment also correlated with response rate to this drug. Because of 
this rationale, we plan to perform two research biopsies in tumor lesion: one at baseline and the 
other one at day 29 cycle 1 of the experimental treatment. 
With a sample size of 30 and assuming that there will be patients without accessible lesions, the 
table below indicates the power available to detect 20%, 30%, 40%, or 50% increases of PD-L1 
positivity rate. The power calculation is based on McNemar’s test with 1-sided alpha of 0.05 and 
assuming 2% of patients unexpectedly show PD-L1 positivity only in the baseline assessment. The 
calculation was done using East v6.3 (Cytel Inc).
 
Increase of PD-L1 positivity 
rate post treatment
# of paired biopsies available
Power
20%
15
54%
 
20
64%
30%
15
75%
 
20
85%
40%
15
90%
 
20
96%
50%
15
97%
 
20
> 99%
PD-L1 positivity seen at baseline and C3D1 samples will be summarized using contingency tables. 
An exploratory analysis is planned to evaluate PD-L1 change in continuous scale.
13.5
Central confirmation of the tumor mutational burden
All patients who initiated protocol therapy will need to agree to undergo a research biopsy, if tumor 
is safely accessible, at baseline.  Participants for whom newly-obtained samples cannot be 
provided (e.g. inaccessible or participant safety concern) may submit an archived specimen (block 
or if not possible, 20 unstained slides). The tumor mutational burden will be accessed by 
Foundation One panel that will be used as a central confirmation test for the cutoff of ≥ 9 Mut/Mb. 
13.6
Reporting and Exclusions
13.6.1 Evaluation of Efficacy
For this Phase II trial, the efficacy evaluable population is a modified intent-to-treat (ITT) 
population.  The modified ITT population consists of all patients who initiate protocol 
therapy, even if there are major protocol therapy deviations.  Patients that do not start 
protocol therapy will be considered unevaluable and will be replaced. 
Subanalyses may then be performed on the basis of a subset of participants, excluding those 
for whom major protocol deviations have been identified (e.g., early death due to other 
reasons, early discontinuation of treatment, major protocol violations, etc.).  However, 
these subanalyses may not serve as the basis for drawing conclusions concerning treatment 
efficacy, and the reasons for excluding participants from the analysis should be clearly 

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reported.  If applicable to the endpoint, the 95% confidence intervals should also be 
provided. 
13.6.2 Evaluation of Safety
The safety population will be used in the safety data summaries.  The safety population 
consists of all patients who took at least one dose of any randomized treatment and who 
have at least one post-baseline safety assessment. Note that a patient who had no adverse 
events constitutes a safety assessment.  Patients who have received at least one dose of 
study drug but have no post-treatment safety data of any kind would be excluded. 
14.
PUBLICATION PLAN
The results should be made public within 1 year of reaching the end of the study. The end of the 
study is the time point at which the last data items are to be reported, or after the outcome data are 
sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate and Study 
Duration. If a report is planned to be published in a peer-reviewed journal, then that initial release 
may be an abstract that meets the requirements of the International Committee of Medical Journal 
Editors. A full report of the outcomes should be made public no later than three (3) years after the 
end of the study. 

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Programmed Cell Death 1 Ligand 2 (Pd-L2) Is a Distinguishing Feature of Primary Mediastinal 
(Thymic) Large B-Cell Lymphoma and Associated with Pdcd1lg2 Copy Gain. Am J Surg Pathol 
38: 1715-1723.
Siegel, R.L., Miller, K.D., and Jemal, A. (2016) Cancer Statistics, 2016. CA Cancer J Clin 66: 7-
30.
Smith, D.C., Mcdermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B. et al. 
(2012) New England Journal. 2443-2454.
Snyder, A., Makarov, V., Merghoub, T., Yuan, J., Zaretsky, J.M., Desrichard, A. et al. (2014) 
Genetic Basis for Clinical Response to Ctla-4 Blockade in Melanoma. The New England journal 
of medicine: 2189-2199.
Spranger, S., Bao, R., and Gajewski, T.F. (2015) Melanoma-Intrinsic Beta-Catenin Signalling 
Prevents Anti-Tumour Immunity. Nature 523: 231-235.
Topalian, S.L., Drake, C.G., and Pardoll, D.M. (2015) Immune Checkpoint Blockade: A 
Common Denominator Approach to Cancer Therapy. Cancer Cell 27: 450-461.
Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., Mcdermott, D.F. et al. 
(2012) Safety, Activity, and Immune Correlates of Anti-Pd-1 Antibody in Cancer. N Engl J Med 
366: 2443-2454.
Tosolini, M., Camus, M., Berger, A., Wind, P., and Lagorce-Page, C. (2006) References and 
Notes 1. 313: 1960-1965.
Van Allen, E.M., Miao, D., Schilling, B., Shukla, S.A., Blank, C., Zimmer, L. et al. (2015) 
Genomic Correlates of Response to Ctla-4 Blockade in Metastatic Melanoma. Science 350: 207-
211.
Wagle, N., Berger, M.F., Davis, M.J., Blumenstiel, B., Defelice, M., Pochanard, P. et al. (2012) 
High-Throughput Detection of Actionable Genomic Alterations in Clinical Tumor Samples by 
Targeted, Massively Parallel Sequencing. Cancer Discov 2: 82-93.
Wolchok, J.D., Hoos, A., O'day, S., Weber, J.S., Hamid, O., Lebbe, C. et al. (2009) Guidelines 
for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response 
Criteria. Clin Cancer Res 15: 7412-7420.
Zaretsky, J.M., Garcia-Diaz, A., Shin, D.S., Escuin-Ordinas, H., Hugo, W., Hu-Lieskovan, S. et 
al. (2016) Mutations Associated with Acquired Resistance to Pd-1 Blockade in Melanoma. N 
Engl J Med: 

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Zaretsky, J.M., Garcia-Diaz, A., Shin, D.S., Escuin-Ordinas, H., Hugo, W., Hu-Lieskovan, S. et 
al. (2016) Mutations Associated with Acquired Resistance to Pd-1 Blockade in Melanoma. N 
Engl J Med: 

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APPENDIX A
PERFORMANCE STATUS CRITERIA
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, no complaints, no evidence 
of disease.
0
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction.
90
Able to carry on normal activity; 
minor signs or symptoms of disease.
80
Normal activity with effort; some 
signs or symptoms of disease.
1
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work).
70
Cares for self, unable to carry on 
normal activity or to do active work.
60
Requires occasional assistance, but 
is able to care for most of his/her 
needs.
2
In bed <50% of the time.  
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities.  Up and about 
more than 50% of waking hours.
50
Requires considerable assistance and 
frequent medical care.
40
Disabled, requires special care and 
assistance.
3
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours.
30
Severely disabled, hospitalization 
indicated.  Death not imminent.
20
Very sick, hospitalization indicated. 
Death not imminent.
4
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair.
10
Moribund, fatal processes 
progressing rapidly.
5
Dead.
0
Dead.

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APPENDIX B
GUIDELINES FOR COLLECTING RESEARCH BIOPSY TISSUE 
Tissue specimens will be collected from metastatic lesions using standard institutional 
procedures. The amount of tissue collected may follow the guidelines listed below:
Skin/chest wall: A goal of 2 4-mm punch biopsies will be obtained.
Lymph node: A goal of 3-6 core biopsy specimens will be obtained using an 18-gauge needle.
Liver: A goal of 3-6 core biopsy specimens will be obtained using an 18-gauge needle.
Lung: Because of the risk of pneumothorax associated with core needle biopsies of lung nodules,
no core biopsies of lung nodules are mandated on this protocol, unless they are clinically
indicated.
Bone: Because the yield of malignant tissue from bone biopsies tends to be relatively low, if a
patient has another accessible site of disease (i.e. skin, lymph node, liver), that site should be
biopsied preferentially. If bone is the only biopsy-accessible site, then a goal of 3-6 core biopsy
specimens will be obtained using an 11-13 gauge needle.
Please note that the above are guidelines for the amount of tissue to be obtained, and are
not meant to replace clinical judgment at the time the procedure is performed. Less than the
goal quantity of tissue is accepted for each type of biopsy, and will be left to the clinical
judgment of the physician performing the procedure.
Coded laboratory specimens will be stored in the Tumor Bank of the DFCI. These specimens 
will become the property of DFCI. Patients will be informed that their specimens may be used 
for research by investigators at DF/HCC and other approved collaborators. Shared specimens 
will be identified with a sample ID number; all patient identifying material will be removed.
Risks of Research Biopsy and Procedures for Minimizing Risk
Potential risks according to site are:
Skin/chest wall (punch biopsy):

Likely: local discomfort and minor bleeding

Less likely: moderate or major bleeding, or infection
Lymph node, liver, or bone (core needle biopsy):

Likely: local discomfort and minor bleeding

Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due
to bleeding or other complications, infection, damage to adjacent organs. Additional risks may be 
present if intravenous conscious sedation is required
Breast (core biopsy):

Likely: local discomfort and minor bleeding.

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
Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due 
to bleeding or other complications, infection, pneumothorax, damage to adjacent organs.
Pleural fluid (thoracentesis):

Likely: local discomfort and minor bleeding

Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due to bleeding or other complications, infection, pneumothorax, damage to adjacent  
organs
Ascites fluid (paracentesis):

Likely: local discomfort and minor bleeding

Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due to bleeding or other complications, infection, bowel perforation or damage to 
adjacent organs. In order to minimize the risk of a biopsy, only qualified personnel 
will perform these procedures. 
Prior to the procedure, the physician performing the procedure will discuss the risks with each 
study participant, answer any questions, and obtain separate procedure consent. Patients will be 
evaluated for comorbidities or concomitant medications that may increase the risk of potential 
complications. For biopsies of lesions that are not superficial and clearly palpable, imaging 
studies such as CT or ultrasound will be used to guide the biopsy in order to minimize the risk of 
damage to adjacent structures. After lymph node biopsies, patients will be observed a minimum 
of 2 hours (range 2-4 hours) after the procedure, or according to standard institutional guidelines. 
After liver biopsies, patients will be observed a minimum of 4 hours (range 4-6 hours) after the 
procedure, or according to standard institutional guidelines. Less than the goal quantity of tissue 
is accepted for each type of biopsy, and will be left to the clinical judgment of the physician 
performing the procedure.
Risks of Anesthesia
Local Anesthesia
All biopsy procedures require local anesthesia using lidocaine, xylocaine, or related
compounds. There is a small risk of an allergic reaction associated with these drugs.
In order to minimize the risk of local anesthesia, only qualified personnel will perform
the biopsy procedure. Patients will be queried if they have had previous allergic
reactions to local anesthetics.
Intravenous Conscious Sedation
Certain biopsy procedures, such as lymph node, liver, or bone biopsies, may require
intravenous conscious sedation (IVCS). IVCS is a minimally depressed level of consciousness 
that retains the patient’s ability to maintain a patent airway independently and continuously and 
respond appropriately to physical stimulation and verbal commands.
The risks of intravenous conscious sedation include: inhibition of the gag reflex and concomitant 
risk of aspiration, cardiopulmonary complications (myocardial infarction, cardiac arrhythmias, 
hypoxemia), and allergic reactions to the sedative or analgesic medications. These risks are small 

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but real; for example, in a prospective study of 14,149 patients undergoing IVCS during upper 
gastrointestinal endoscopies, the rate of immediate cardiopulmonary events was 2 in 
1000.[Quine et al., 1995] The 30-day mortality was 1 per 2,000 cases. In this study, there was a 
strong association between lack of monitoring and use of high-dose benzodiazepines with 
adverse outcomes. There was also an association between the use of local anesthetic sprays to the 
oropharynx and the development of pneumonia. In order to minimize the risk of intravenous 
conscious sedation, only qualified personnel will be responsible for conscious sedation. A 
minimum of two individuals will be involved in the care of patients undergoing conscious 
sedation—the physician performing the biopsy procedure, and the individual (M.D. or R.N.) who 
monitors the patients and his/her response to both the sedation and the procedure, and who is 
capable of assisting with any supportive or resuscitative measures. The room where the 
procedure utilizing IVCS takes place will have adequate equipment to provide supplemental 
oxygen, monitor vital signs, and maintain an airway should this be necessary. An emergency cart 
will also be immediately accessible to the room where the procedure is to take place, and 
emergency support services will be available on page. Patients will be screened and evaluated for 
their fitness to undergo conscious sedation by a trained physician. Patients with active cardiac 
disease are excluded from this study. No local anesthetic spray to the oropharynx will be 
necessary, given that endoscopy is not a planned procedure. Following the procedure, patients 
will be observed closely in the recovery room for a minimum of 2 hours.
General Anesthesia
Because of the higher risk of general anesthesia compared with local anesthesia or intravenous 
conscious sedation, biopsies that would require general anesthesia in order to be performed are 
not permitted on this protocol.

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For Biopsies of Soft Tissue, Liver, Bone, Breast, Etc:
1. After biopsy is performed, the tissue mass is placed on a sterile gauze
2. Using forceps, separate the tumor tissue
3. Place 2 pieces (cores) of tumor tissue in each cassette (typically end up with 3 cassettes
per biopsy); the last cassette will contain many small pieces of tumor tissue
4. Fill cassettes with OCT
a. Completely cover tissue
b. Limit the amount of bubbles
5. Place cassettes on dry ice and prepare for transport by limiting OCT leakage
6. Return samples to the lab and complete freezing of samples in OCT with dry ice (about
10 minutes freezing time)
7. Once samples are frozen, place in plastic bag; label bag with date, protocol number,
patient number, and number of initials included
8. Store in –80C freezer
For Effusions and Ascites
1. Fluid sample should be split into two equal aliquots
2. One aliquot should be spun down into a pellet and snap frozen in an ETOH/dry ice bath
or in liquid N2
3. One aliquot should be fixed and processed as a standard cell block.
Note: if the sample preparation is done by a clinical cytopathology laboratory, it is important to
explain that the sample is for research purposes only and that no thin prep should be performed
as this uses up a significant portion of the sample.
For Fine Needle Aspiration Samples
A goal of 3 passes:
1. One pass should be evacuated and rinsed directly into 2mL of room temperature Trizol for
RNA analysis.
2. One pass should be evacuated and rinsed directly into 2mL of room temperature Trizol for
DNA analysis.
3. One pass should be evacuated and rinsed directly into 10-20mL of RPMI to prepare a cell 
block.

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APPENDIX C
FACT-B Questionnaire
Participant ID__________ Cycle#___________
Below is a list of statements that other people with your illness have said are important. Please 
circle or mark one number per line to indicate your response as it applies to the past 7 days.
PHYSICAL WELL-BEING
Not at 
all
A little 
bit
Some-
what
Quite a 
bit
Very 
much
GP1
I have a lack of energy
................................................................................................................
0
1
2
3
4
GP2
I have nausea
................................................................................................................
0
1
2
3
4
GP3
Because of my physical condition, I have trouble meeting the needs 
of my family
................................................................................................................
0
1
2
3
4
GP4
I have pain
................................................................................................................
0
1
2
3
4
GP5
I am bothered by side effects of treatment
................................................................................................................
0
1
2
3
4
GP6
I feel ill
................................................................................................................
0
1
2
3
4
GP7
I am forced to spend time in bed
0
1
2
3
4
SOCIAL/FAMILY WELL-BEING
Not at 
all
A little 
bit
Some-
what
Quite a 
bit
Very 
much
GS1
I feel close to my friends
................................................................................................................
0
1
2
3
4
GS2
I get emotional support from my family
................................................................................................................
0
1
2
3
4
GS3
I get support from my friends
................................................................................................................
0
1
2
3
4
GS4
My family has accepted my illness
................................................................................................................
0
1
2
3
4
GS5
I am satisfied with family communication about my illness
................................................................................................................
0
1
2
3
4
GS6
I feel close to my partner (or the person who is my main support)
................................................................................................................
0
1
2
3
4

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EMOTIONAL WELL-BEING
Not 
at all
A little 
bit
Some-
what
Quite
a bit
Very 
much
GE1
I feel sad
.............................................................................................
0
1
2
3
4
GE2
I am satisfied with how I am coping with my illness
.............................................................................................
0
1
2
3
4
GE3
I am losing hope in the fight against my illness
.............................................................................................
0
1
2
3
4
GE4
I feel nervous
.............................................................................................
0
1
2
3
4
GE5
I worry about dying
.............................................................................................
0
1
2
3
4
GE6
I worry that my condition will get worse
0
1
2
3
4
FUNCTIONAL WELL-BEING
Not 
at all
A little 
bit
Some-
what
Quite
a bit
Very 
much
GF1
I am able to work (include work at home)
.............................................................................................
0
1
2
3
4
GF2
My work (include work at home) is fulfilling
.............................................................................................
0
1
2
3
4
GF3
I am able to enjoy life
.............................................................................................
0
1
2
3
4
GF4
I have accepted my illness
.............................................................................................
0
1
2
3
4
GF5
I am sleeping well
.............................................................................................
0
1
2
3
4
Q1
Regardless of your current level of sexual activity, please answer the 
following question. If you prefer not to answer it, please mark this 
box           and go to the next section.
GS7
I am satisfied with my sex life
0
1
2
3
4

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GF6
I am enjoying the things I usually do for fun
.............................................................................................
0
1
2
3
4
GF7
I am content with the quality of my life right now
0
1
2
3
4
ADDITIONAL CONCERNS
Not at 
all
A little 
bit
Some-
what
Quite
a bit
Very 
much
B1
I have been short of breath
.............................................................................................
0
1
2
3
4
B2
I am self-conscious about the way I dress
.............................................................................................
0
1
2
3
4
B3
One or both of my arms are swollen or tender
.............................................................................................
0
1
2
3
4
B4
I feel sexually attractive
.............................................................................................
0
1
2
3
4
B5
I am bothered by hair loss
.............................................................................................
0
1
2
3
4
B6
I worry that other members of my family might 
someday get the same illness I have
.............................................................................................
0
1
2
3
4
B7
I worry about the effect of stress on my illness
.............................................................................................
0
1
2
3
4
B8
I am bothered by a change in weight
.............................................................................................
0
1
2
3
4
B9
I am able to feel like a woman
0
1
2
3
4
P2
I have certain parts of my body where I experience pain
.............................................................................................
0
1
2
3
4

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104
APPENDIX E
DF/HCC MULTI-CENTER DATA AND SAFETY MONITORING 
PLAN
DFCI IRB Protocol #: 18-561

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TABLE OF CONTENTS
SCHEMA
2
1.
OBJECTIVES5
1.1
Study Design............................................................................................................5
1.2
Primary Objectives...................................................................................................5
1.3
Secondary Objectives...............................................................................................5
1.4
Correlative Objectives .............................................................................................7
1.5
Patient Reported Outcomes Objectives ...................................................................8
2.
BACKGROUND .................................................................................................................8
2.1
Study Disease(s).......................................................................................................8
2.2
The PD-1/PD-L1 pathway in cancer........................................................................8
2.3
Ipilimumab (Yervoy) ...............................................................................................9
2.4
Nivolumab..............................................................................................................14
2.5
Ipilimumab with Nivolumab..................................................................................15
2.6
Rationale ................................................................................................................18
2.7
Correlative Studies Background ............................................................................20
3.
PARTICIPANT SELECTION...........................................................................................23
3.1
Eligibility Criteria..................................................................................................23
3.2
Exclusion Criteria ..................................................................................................25
4.
REGISTRATION PROCEDURES ...................................................................................26
4.1
General Guidelines for DF/HCC Institutions ........................................................26
4.2
Registration Process for DF/HCC Institutions.......................................................27
4.3
General Guidelines for Other Investigative Sites ..................................................27
4.4
Registration Process for Other Investigative Sites.................................................27
5.
TREATMENT PLAN........................................................................................................28
5.1
Treatment Regimen................................................................................................28
5.2
Pre-Treatment Criteria ...........................................................................................28
5.3
Agent administration..............................................................................................29
5.4
General Concomitant Medication and Supportive Care Guidelines......................29
5.5
Criteria for Taking a Participant Off Protocol Therapy.........................................32
5.6
Duration of Follow Up...........................................................................................33
5.7
Criteria for Taking a Participant Off Study ...........................................................33
6.
Dosing Delays/Dose modifications....................................................................................34
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................45
7.1
Expected Toxicities................................................................................................45
7.2
Adverse Event Characteristics...............................................................................46
7.3
Expedited Adverse Event Reporting......................................................................47
7.4
Routine Adverse Event Reporting .........................................................................49

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8.
PHARMACEUTICAL INFORMATION..........................................................................50
8.1
Nivolumab..............................................................................................................50
8.2
Ipilimumab.............................................................................................................52
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................54
9.1
Archival Tissue......................................................................................................55
9.2
Fresh Tissue Biopsy...............................................................................................56
9.3
Blood Collection....................................................................................................62
9.4
Stool Collection .....................................................................................................65
9.5
Central confirmation of the hypermutated status...................................................67
9.6
Additional analysis.................................................................................................67
10.
STUDY CALENDAR .......................................................................................................67
11.
MEASUREMENT OF EFFECT .......................................................................................71
11.1
Antitumor Effect – Solid tumors............................................................................71
11.2
Antitumor Effect – Hematologic Tumors..............................................................78
11.3
Other Response Parameters ...................................................................................78
12.
DATA REPORTING / REGULATORY REQUIREMENTS...........................................80
12.1
Data Reporting.......................................................................................................80
12.2
Data Safety Monitoring..........................................................................................80
12.3
Multicenter Guidelines...........................................................................................81
12.4
Collaborative Research and Future Use of Data and Samples ..............................81
13.
STATISTICAL CONSIDERATIONS...............................................................................82
13.1
Study Design/Endpoints ........................................................................................82
13.2
Sample Size, Accrual Rate and Study Duration ....................................................83
13.3
Analysis of Primary Endpoint................................................................................84
13.4
Analysis of Secondary Endpoints..........................................................................84
13.5
Central confirmation of the tumor mutational burden...........................................85
13.6
Reporting and Exclusions ......................................................................................85
14.
PUBLICATION PLAN .....................................................................................................86
15.
REFERENCES ..................................................................................................................87
APPENDIX A
PERFORMANCE STATUS CRITERIA ..................................................94
APPENDIX B
Guidelines for collecting research biopsy tissue........................................95
Risks of Research Biopsy and Procedures for Minimizing Risk.......................................95
Risks of Anesthesia............................................................................................................96
APPENDIX C
FACT-B Questionnaire..............................................................................99
APPENDIX D
Rotterdam Symptom Checklist................................................................102
Participant ID__________ Cycle#___________.........................................................................102

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APPENDIX E
DF/HCC MULTI-CENTER DATA AND SAFETY MONITORING 
PLAN
104
1. INTRODUCTION
1.1
Purpose
1.2
Multi-Center Data and Safety Monitoring Plan Definitions
2. GENERAL ROLES AND RESPONSIBILITIES
2.1
DF/HCC Sponsor
2.2
Coordinating Center 
2.3
Participating Institution
3. DF/HCC REQUIREMENTS FOR MULTI-CENTER PROTOCOLS
3.1
Protocol Distribution
3.2
Protocol Revisions and Closures
3.3
Informed Consent Requirements
3.4
IRB Documentation
3.5
IRB Re-Approval
3.6
Participant Confidentiality and Authorization Statement 
3.7
DF/HCC Multi-Center Protocol Registration Policy
3.8
DF/HCC Protocol Case Number
3.9
Safety Assessments and Toxicity Monitoring
3.10
Data Management
4. REQUISITIONING INVESTIGATIONAL DRUG
5. MONITORING: QUALITY CONTROL
5.1
Ongoing Monitoring of Protocol Compliance
5.2
Monitoring Reports
5.3
Accrual Monitoring
6. AUDITING: QUALITY ASSURANCE
6.1
DF/HCC Internal Audits
6.2
Audit Notifications
6.3
Audit Reports 
6.4
Participating Institution Performance

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1.
INTRODUCTION
The Dana-Farber/Harvard Cancer Center Multi-Center Data and Safety Monitoring Plan 
(DF/HCC DSMP) outlines the procedures for conducting a DF/HCC Multi-Center research 
protocol. The DF/HCC DSMP serves as a reference for any sites external to DF/HCC that are 
participating in a DF/HCC clinical trial. 
1.1
Purpose
To establish standards that will ensure that a Dana-Farber/Harvard Cancer Center Multi-Center 
protocol will comply with Federal Regulations, Health Insurance Portability and Accountability 
Act (HIPAA) requirements and applicable DF/HCC Standard Operating Procedures.       
1.2
Multi-Center Data and Safety Monitoring Plan Definitions
DF/HCC Multi-Center Protocol:  A research protocol in which one or more outside institutions 
are collaborating with Dana-Farber/Harvard Cancer Center where a DF/HCC investigator is the 
sponsor. DF/HCC includes Dana-Farber/Partners Cancer Care (DF/PCC) Network Clinical Trial 
Affiliates. 
Lead Institution:  One of the Dana-Farber/Harvard Cancer Center consortium members (Dana-
Farber Cancer Institute (DFCI), Massachusetts General Hospital (MGH), Beth Israel Deaconess 
Medical Center (BIDMC), Boston Children’s Hospital (BCH), Brigham and Women’s Hospital 
(BWH) responsible for the coordination, development, submission, and approval of a protocol as 
well as its subsequent amendments per the DFCI IRB and applicable regulatory guidelines 
(CTEP, Food and Drug Administration (FDA), Office of Biotechnology Activities (OBA) etc.).  
The Lead Institution is typically the home of the DF/HCC Sponsor. The Lead Institution also 
typically serves as the Coordinating Center for the DF/HCC Multi-Center Protocol.  
DF/HCC Sponsor:  The person sponsoring the submitted Multi-Center protocol who takes 
responsibility for initiation, management and conduct of the protocol at all research locations. In 
applicable protocols, the DF/HCC Sponsor will serve as the single liaison with any regulatory 
agencies (i.e. FDA). The DF/HCC Sponsor has ultimate authority over the protocol and is 
responsible for the conduct of the study at DF/HCC and all Participating Institutions. In most 
cases the DF/HCC Sponsor is the same person as the DF/HCC Overall Principal Investigator; 
however, both roles can be filled by two different people.
Participating Institution:  An institution that is outside the DF/HCC and DF/PCC consortium 
that is collaborating with DF/HCC on a protocol where the sponsor is a DF/HCC Investigator.  
The Participating Institution acknowledges the DF/HCC Sponsor as having the ultimate authority 
and responsibility for the overall conduct of the study.
   
Coordinating Center: The entity (i.e. Lead Institution, Medical Monitor, Contract Research 
Organization (CRO), etc) that provides administrative support to the DF/HCC Sponsor in order 
that he/she may fulfill the responsibilities outlined in the protocol document and DSMP, and as 
specified in applicable regulatory guidelines (i.e. CTEP Multi-Center Guidelines). In general, the 

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Lead Institution is the Coordinating Center for the DF/HCC Multi-Center Protocol. 
DF/HCC Office of Data Quality (ODQ): A group within DF/HCC responsible ensuring high-
quality standards are used for data collection and the ongoing management of clinical trials, 
auditing, and data and safety monitoring. ODQ also coordinates quality assurance efforts related 
to multi-center clinical research.
DF/HCC Research Informatics Office (RIO): A group within DF/HCC responsible for 
providing a comprehensive data management platform for managing clinical trial data.
2
GENERAL ROLES AND RESPONSIBILITIES
For DF/HCC Multi-Center Protocols, the DF/HCC Sponsor, the Coordinating Center, and the 
Participating Institutions are expected to adhere to the following general responsibilities: 
2.1
DF/HCC Sponsor
The DF/HCC Sponsor, Sara Tolaney, MD, MPH will accept responsibility for all aspects of 
conducting a DF/HCC Multi-Center protocol which includes but is not limited to: 

Oversee the coordination, development, submission, and approval of the protocol as well 
as subsequent amendments. 

Ensure that the investigators, study team members, and Participating Institutions are 
qualified and appropriately resourced to conduct the protocol.  

Include the Multi-Center Data and Safety Monitoring Plan as an appendix to the protocol.

Ensure all Participating Institutions are using the correct version of the protocol.

Ensure that each participating investigator and study team member receives adequate 
protocol training (and/or a Site Initiation Visit prior to enrolling participants) and 
throughout trial’s conduct as needed.

Ensure the protocol will be provided to each participating site in a language 
understandable to all applicable site personnel when English is not the primary language. 

Monitor progress and overall conduct of the study at all Participating Institutions. 

Ensure all DFCI Institutional Review Board (IRB), DF/HCC and other applicable (i.e. 
FDA) reporting requirements are met. 

Review data and maintain timely submission of data for study analysis. 

Act as the single liaison with FDA (investigator-held IND trials) as applicable.

Ensure compliance with all requirements as set forth in the Code of Federal Regulations, 
applicable DF/HCC requirements, HIPAA requirements, and the approved protocol.

Commit to the provision that the protocol will not be rewritten or modified by anyone 
other than the DF/HCC Sponsor.

Identify and qualify Participating Institutions and obtain accrual commitments prior to 
extending the protocol to that site.

Monitor accrual and address Participating Institutions that are not meeting their accrual 
requirements. 

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2.2
Coordinating Center 
The general responsibilities of the Coordinating Center may include but are not limited to:

Assist in protocol development. 

Maintain FDA as applicable.

Review registration materials for eligibility and register participants from Participating 
Institutions in the DF/HCC clinical trial management system (CTMS).

Distribute protocol and informed consent document updates to Participating Institutions 
as needed.

Oversee the data collection process from Participating Institutions.

Maintain documentation of Serious Adverse Event (SAE) reports and 
deviations/violation submitted by Participating Institutions and provide to the DF/HCC 
Sponsor for timely review and submission to the DFCI IRB, as necessary. 

Distribute serious adverse events reported to the DF/HCC Sponsor that fall under the 
DFCI IRB Adverse Event Reporting Policy to all Participating Institutions.

Provide Participating Institutions with information regarding DF/HCC requirements that 
they will be expected to comply with.

Carry out plan to monitor Participating Institutions either by on-site or remote 
monitoring. 

Maintain Regulatory documents of all Participating Institutions which includes but is not 
limited to the following: local IRB approvals/notifications from all Participating 
Institutions, confirmation of Federalwide Assurances (FWAs) for all sites, all SAE 
submissions, Screening Logs for all sites, IRB approved consents for all sites

Conduct regular communications with all Participating Institutions (conference calls, 
emails, etc) and maintain documentation all relevant communications.
2.3
Participating Institution
Each Participating Institution is expected to comply with all applicable federal regulations and 
DF/HCC requirements, the protocol and HIPAA requirements. 
The general responsibilities for each Participating Institution may include but are not limited to:

Document the delegation of research specific activities to study personnel.

Commit to the accrual of participants to the protocol.

Submit protocol and/or amendments to their local IRB.

Maintain regulatory files as per sponsor requirements.

Provide the Coordinating Center with regulatory documents or source documents as 
requested.

Participate in protocol training prior to enrolling participants and throughout the trial as 
required (i.e. teleconferences).

Update Coordinating Center with research staff changes on a timely basis.

Register participants through the Coordinating Center prior to beginning research related 
activities. 

Submit Serious Adverse Event (SAE) reports to local IRB per institutional requirements 
and to the Coordinating Center, in accordance with DF/HCC requirements. 

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
Submit protocol deviations and violations to local IRB per institutional requirements and 
to the DF/HCC Sponsor in accordance with DF/HCC requirements.

Order, store and dispense investigational agents and/or other protocol mandated drugs per 
federal guidelines and protocol requirements.

Have office space, office equipment, and internet access that meet HIPAA standards.

Participate in any quality assurance activities and meet with monitors or auditors at the 
conclusion of a visit to review findings.

Promptly provide follow-up and/or corrective action plans for any monitoring queries or 
audit findings.
3
DF/HCC REQUIREMENTS FOR MULTI-CENTER PROTOCOLS
The following section will clarify DF/HCC Requirements and further detail the expectations for 
participating in a DF/HCC Multi-Center protocol. 
3.1
Protocol Distribution
The Coordinating Center will distribute the final DFCI IRB approved protocol and any 
subsequent amended protocols to all Participating Institutions.   
3.2
Protocol Revisions and Closures
The Participating Institutions will receive notification of protocol revisions and closures from the 
Coordinating Center.  It is the individual Participating Institution’s responsibility to notify its 
IRB of these revisions.

Non life-threatening revisions: Participating Institutions will receive written notification 
of protocol revisions regarding non life-threatening events from the Coordinating Center. 
Non-life-threatening protocol revisions must be IRB approved and implemented within 
90 days from receipt of the notification.

Revisions for life-threatening causes: Participating Institutions will receive immediate 
notification from the Coordinating Center concerning protocol revisions required to 
protect lives with follow-up by fax, mail, e-mail, etc.  Life-threatening protocol revisions 
will be implemented immediately followed by IRB request for approval.

Protocol closures and temporary holds: Participating Institutions will receive 
notification of protocol closures and temporary holds from the Coordinating Center. 
Closures and holds will be effective immediately.  In addition, the Coordinating Center, 
will update the Participating Institutions on an ongoing basis about protocol accrual data 
so that they will be aware of imminent protocol closures.
3.3
Informed Consent Requirements
The DF/HCC approved informed consent document will serve as a template for the informed 
consent for Participating Institutions. The Participating Institution consent form must follow the 

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consent template as closely as possible and should adhere to specifications outlined in the 
DF/HCC Guidance Document on Model Consent Language for PI-Initiated Multi-Center 
Protocols.  This document will be provided separately to each Participating Institution upon 
request.
Participating Institutions are to send their version of the informed consent document and HIPAA 
authorization, if a separate document, to the Coordinating Center for review and approval prior 
to submission to their local IRB. The approved consent form must also be submitted to the 
Coordinating Center after approval by the local IRB for all consent versions.
The Principal Investigator (PI) at each Participating Institution will identify the physician 
members of the study team who will be obtaining consent and signing the consent form for 
therapeutic protocols. Participating institutions must follow the DF/HCC requirement that only 
attending physicians obtain informed consent and re-consent for all interventional drug, biologic, 
or device research.  
3.4
IRB Documentation
The following must be on file with the Coordinating Center:

Initial approval letter of the Participating Institution's IRB. 

Copy of the Informed Consent Form(s) approved by the Participating Institution’s IRB. 

Participating Institution’s IRB approval for all amendments.

Annual approval letters by the Participating Institution's IRB.
3.5
IRB Re-Approval
Verification of IRB re-approval from the Participating Institutions is required in order to 
continue research activities.  There is no grace period for continuing approvals.
The Coordinating Center will not register participants if a re-approval letter is not received from 
the Participating Institution on or before the anniversary of the previous approval date.  
3.6
Participant Confidentiality and Authorization Statement
In 1996, congress passed the first federal law covering the privacy of health information known 
as the Health Insurance Portability and Accountability Act (HIPPA). Any information, related to 
the physical or mental health of an individual is called Protected Health Information (PHI). 
HIPAA outlines how and under what circumstances PHI can be used or disclosed. 
In order for covered entities to use or disclose protected health information during the course of a 
study, the study participant must sign an authorization statement.  This authorization statement 
may or may not be separate from the informed consent document.  The Coordinating Center, 
with the approval from the DFCI IRB will provide a consent template, with information 
regarding authorization for the disclosure of protected health information. 
The DF/HCC Sponsor will use all efforts to limit its use of protected health information in its 

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trials. However, because of the nature of these trials, certain protected health information must 
be collected. DF/HCC has chosen to use authorizations, signed by the participant in the trial, 
rather than limited data sets with data use agreements.
   3.6.1   
DF/HCC Multi-Center Protocol Confidentiality
All documents, investigative reports, or information relating to the participant are 
strictly confidential. Whenever reasonably feasible, any participant specific reports 
(i.e. Pathology Reports, MRI Reports, Operative Reports, etc.) submitted to the 
Coordinating Center should be de-identified. It is recommended that the assigned 
protocol case number (as described below) be used for all participant specific 
documents. Participant initials may be included or retained for cross verification of 
identification. 
3.7
DF/HCC Multi-Center Protocol Registration Policy
   3.7.1 
Participant Registration and Randomization 
Please refer to Protocol Section 4.3 and 4.4 for participant registration information. 
Treatment cannot begin until site has received confirmation that participant has been 
registered with DF/HCC CTMS. 
   3.7.2
Initiation of Therapy
Participants must be registered with the DF/HCC CTMS before the initiation of 
treatment or other protocol-specific interventions. Treatment and other protocol-
specific interventions may not be initiated until the Participating Institution receives 
confirmation of the participant’s registration from the Coordinating Center. The 
DF/HCC Sponsor and DFCI IRB must be notified of any violations to this policy.
   3.7.3
Eligibility Exceptions
No exceptions to the eligibility requirements for a protocol without DFCI IRB approval will be 
permitted. All Participating Institutions are required to fully comply with this requirement. The 
process for requesting an eligibility exception is defined below.
3.8
DF/HCC Protocol Case Number
At the time of registration, the following identifiers are required for all subjects: initials, date of 
birth, gender, race and ethnicity. Once eligibility has been established and the participant 
successfully registered, the participant is assigned a unique protocol case number.  Participating 
Institutions should submit all de-identified subsequent communication and documents to the 
Coordinating Center, using this case number to identify the subject.  
   3.8.1
Protocol Deviations, Exceptions and Violations
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to ensure that researchers do not initiate changes in approved research without IRB 
review and approval, except when necessary to eliminate apparent immediate hazards 
to the participant. DF/HCC requires all departures from the defined procedures set 
forth in the IRB approved protocol to be reported to the DF/HCC Sponsor, who in 
turn is responsible for reporting to the DFCI IRB.
For reporting purposes, DF/HCC uses the terms “violation”, “deviation” and 
“exception” to describe departures from a protocol. All Participating Institutions must 
adhere to these requirements for reporting to the DF/HCC Sponsor and will follow 
their institutional policy for reporting to their local IRB.
   3.8.2
Definitions
Protocol Deviation: Any departure from the defined procedures set forth in the IRB-
approved protocol which is prospectively approved prior to its implementation.
Protocol Exception:  Any protocol deviation that relates to the eligibility criteria, e.g.
enrollment of a participant who does not meet all inclusion/exclusion criteria.
Protocol Violation: Any protocol departure that was not prospectively approved by 
the IRB prior to its initiation or implementation. 
   3.8.3
Reporting Procedures
DF/HCC Sponsor: is responsible for ensuring that clear documentation is available in 
the medical record and/or regulatory documents to describe all protocol exceptions, 
deviations and violations. The DF/HCC Sponsor will also be responsible for ensuring 
that all protocol violations/deviations are promptly reported per DFCI IRB guidelines. 
Participating Institutions: Protocol deviations require prospective approval from the 
DFCI IRB. The Participating Institution must submit the deviation request to the 
Coordinating Center who will then submit the deviation request to the DFCI IRB. 
Upon DFCI IRB approval the deviation is submitted to the Participating Institution 
IRB, per institutional policy. A copy of the Participating Institution’s IRB report and 
determination will be forwarded to the Coordinating Center within 10 business days 
after the original submission.  The deviation may not be implemented without all 
required approvals.
All protocol violations must be sent to the Coordinating Center in a timely manner. 
The Coordinating Center will provide training for the requirements for the reporting 
of violations. 
Coordinating Center:  Upon receipt of the violation/deviation report from the 
Participating Institution, the Coordinating Center will submit the report to the 
DF/HCC Sponsor for review. Subsequently, the Participating Institution’s IRB 
violation/deviation report will be submitted to the DFCI IRB for review per DFCI 

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IRB reporting guidelines. 
3.9
Safety Assessments and Toxicity Monitoring
The study teams at all participating institutions are responsible for protecting the safety, rights 
and well-being of study participants. Recording and reporting of adverse events that occur during 
the course of a study help ensure the continuing safety of study participants. 
All participants receiving investigational agents and/or other protocol mandated therapy will be 
evaluated for safety. The safety parameters include all laboratory tests and hematological 
abnormalities, physical examination findings, and spontaneous reports of adverse events reported 
by participants.  All toxicities encountered during the study will be evaluated according to the 
NCI criteria specified in the protocol. Life-threatening toxicities must be reported immediately to 
the DF/HCC Sponsor via the Coordinating Center. 
Additional safety assessments and toxicity monitoring will be outlined in the protocol.
   3.9.1
Guidelines for Reporting Serious Adverse Events 
Guidelines for reporting Adverse Events (AEs) and Serious Adverse Events (SAEs) 
are detailed in protocol section 7.
Participating Institutions must report the SAEs to the DF/HCC Sponsor and the 
Coordinating Center following the DFCI IRB Adverse Event Reporting Policy.
The Coordinating Center will maintain documentation of all Participating Institution 
Adverse Event reports and be responsible for communicating to all participating 
investigators, any observations reportable under the DFCI IRB Reporting 
Requirements.  Participating Institutions will review and submit to their IRB 
according to their institutional policies and procedures
   3.9.2
Guidelines for Processing IND Safety Reports 
The DF/HCC Sponsor will review all IND Safety Reports and ensure that all IND 
Safety Reports are distributed to the Participating Institutions. Participating 
Institutions will review/submit to their IRB according to their institutional policies 
and procedures.
3.10
Data Management
DF/HCC RIO develops case report forms (CRF/eCRFs), for use with the protocol.  These forms 
are designed to collect data for each study. DF/HCC RIO provides a web based training for all 
eCRF users.
   3.10.1
Data Forms Review

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Data submissions are monitored for timeliness and completeness of submission. If 
study forms are received with missing or questionable data, the submitting institution 
will receive a written or electronic query from the DF/HCC Office of Data Quality, 
Coordinating Center, or designee. 
Responses to all queries should be completed and submitted within 14 calendar days.  
Responses may be returned on the written query or on an amended paper case report 
form, or in the case of electronic queries, within the electronic data capture (eDC) 
system. In the case of a written query for data submitted on a paper case report form, 
the query must be attached to the specific data being re-submitted in response.  
If study forms are not submitted on schedule, the Participating Institution will 
periodically receive a Missing Form Report from the Coordinating Center noting the 
missing forms. 
4
REQUISITIONING INVESTIGATIONAL DRUG
The ordering of investigational agent is specified in the protocol section 8. 
Participating Institutions should order their own agent regardless of the supplier.
If the agent is commercially available, check with the local Director of Pharmacy and/or the 
Research Pharmacy to ensure that the agent is in stock. If the agent is not stocked, ensure that the 
agent can be ordered once the protocol is approved by the local IRB. 
If the agent is investigational, ensure that the pharmacy will be able to receive and store the agent 
according to state and federal requirements. The local IRB should be kept informed of who will 
supply the agent (i.e., NCI or a pharmaceutical company) so that any regulatory responsibilities 
can be met in a timely fashion. 
5
MONITORING: QUALITY CONTROL
The quality control process for a clinical trial requires verification of protocol compliance and 
data accuracy. The Coordinating Center, with the aid of the DF/HCC Office of Data Quality, 
provides quality control oversight for the protocol.
5.1
Ongoing Monitoring of Protocol Compliance
The Participating Institutions may be required to submit participant source documents to the 
Coordinating Center for monitoring. Participating Institution may also be subject to on-site 
monitoring conducted by the Coordinating Center. 
The Coordinating Center will implement ongoing monitoring activities to ensure that 
Participating Institutions are complying with regulatory and protocol requirements, data quality, 
and participant safety. Monitoring practices may include but are not limited to source data 

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verification, and review and analysis of eligibility requirements, informed consent procedures, 
adverse events and all associated documentation, review of study drug administration/treatment, 
regulatory files, protocol departures reporting, pharmacy records, response assessments, and data 
management. 
Additionally, a plan will be formulated to provide regular and ongoing communication to
Participating Institutions about study related information which will include participation
in regular Lead Institution initiated teleconferences. Teleconferences will occur every 2
weeks and will continue regularly until completion of accrual. Upon completion of
accrual, teleconferences will occur monthly until all patients complete protocol therapy.
Upon completion of protocol therapy, teleconferences will occur every 3 months until study 
completion. Additional communication may be distributed via “Newsletter” or email as deemed 
appropriate by DF/HCC Sponsor.
On-Site Monitoring: On-site monitoring will occur on an as-needed basis. Participating 
Institutions will be required to provide access to participants’ complete medical record and source 
documents for source documentation verification during the visit. In addition, Participating 
Institutions should provide access to regulatory documents, pharmacy records, local policies 
related to the conduct of research, and any other trial-related documentation maintained by the 
Participating Site. On-site monitoring visits can be substituted with remote (virtual) monitoring 
visits at the discretion of the Principal Investigator. 
Remote Monitoring: Remote monitoring will be performed on an as-needed basis by the Clinical 
Trial Monitor. Sites will be asked to provide source documentation via fax, email, or mail as 
specified by the Clinical Trial Monitor for virtual monitoring.
5.2
Monitoring Reports
The DF/HCC Sponsor will review all monitoring reports to ensure protocol compliance. The 
DF/HCC Sponsor may increase the monitoring activities at Participating Institutions that are 
unable to comply with the protocol, DF/HCC Sponsor requirements or federal and local 
regulations. 
5.3
Accrual Monitoring
Prior to extending a protocol to an external site, the DF/HCC Sponsor will establish accrual 
requirements for each participating institution. Accrual will be monitored for each participating 
institution by the DF/HCC Sponsor or designee. Sites that are not meeting their accrual 
expectations may be subject to termination. Sites are expected to accrue at least 3 patients per 
year.
6
AUDITING: QUALITY ASSURANCE
Auditing is a method of Quality Assurance and involves the systematic and independent 
examination of all trial related activities and documents.  Audits determine if evaluated activities 
were appropriately conducted and whether data was generated, recorded and analyzed, and 

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accurately reported per the protocol, applicable Standard Operating Procedures (SOPs), and the 
Code of Federal Regulations (CFR).
6.1
DF/HCC Internal Audits
 
All Participating Institutions are subject to audit by the DF/HCC Office of Data Quality (ODQ). 
Typically, approximately 3-4 participants would be audited at the site over a 2-day period. If 
violations which impact participant safety or the integrity of the study are found, more 
participant records may be audited. 
6.2
Audit Notifications
It is the Participating Institution’s responsibility to notify the Coordinating Center of all external 
audits or inspections (e.g., FDA, EMA, NCI) that involve this protocol. All institutions will 
forward a copy of final audit and/or re-audit reports and corrective action plans (if applicable) to 
the Coordinating Center, within 12 weeks after the audit date. 
6.3
Audit Reports 
The DF/HCC Sponsor will review all final audit reports and corrective action plans, if 
applicable. The Coordinating Center, must forward any reports to the DF/HCC ODQ per 
DF/HCC policy for review by the DF/HCC Audit Committee. For unacceptable audits, the 
DF/HCC Audit Committee would forward the final audit report and corrective action plan to the 
DFCI IRB as applicable.
6.4
Participating Institution Performance
The DF/HCC Sponsor and the DFCI IRB are charged with considering the totality of an 
institution’s performance in considering institutional participation in the protocol.
Participating Institutions that fail to meet the performance goals of accrual, submission of timely 
and accurate data, adherence to protocol requirements, and compliance with state and federal 
regulations, may be recommended for a six-month probation period. Such institutions must 
respond with a corrective action plan and must demonstrate during the probation period that 
deficiencies have been corrected, as evidenced by the improved performance measures. 
Participating Institutions that fail to demonstrate significant improvement will be considered by 
the DF/HCC Sponsor for revocation of participation. A DF/HCC Sponsor and/or the DFCI IRB 
may terminate a site’s participation if it is determined that a site is not fulfilling its 
responsibilities as described above.