Conditions: Breast Cancer
Keywords: Breast Cancer
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 90
Healthy volunteers: no

Brief summary:
This research study is exploring chemotherapy in combination with immunotherapy (a therapy that uses the body's own immune system to control cancer) as a possible treatment for metastatic hormone receptor positive breast cancer.

Detailed description:
This research study is a Phase II clinical trial. Phase II clinical trials test the safety and effectiveness of an investigational intervention to learn whether the intervention works in treating a specific disease. "Investigational" means that the intervention is being studied.

In this research study, The investigators are evaluating the safety and effectiveness of Eribulin mesylate with or without Pembrolizumab in metastatic hormone receptor positive breast cancer.

The FDA (the U.S. Food and Drug Administration) has not approved the combination of Pembrolizumab and Eribulin mesylate for Breast Cancer.

The FDA has not approved Pembrolizumab for this specific type of breast cancer but it has been approved in the United States for other diseases.

The FDA has approved Eribulin mesylate as a treatment option for this type of breast cancer.

Pembrolizumab is a medicine that may treat cancer by working with the immune system. The immune system is the body's natural defense against disease. The immune system sends types of cells called "T cells" throughout the body to detect and fight infections and diseases, including cancer. For some types of cancer, the T cells do not work as they should and are prevented from attacking the tumors. Pembrolizumab is thought to work by blocking a protein in the T cells called PD-1 ("programmed death 1"), which then may allow these cells and other parts of the immune system to attack tumors.

Eribulin mesylate is developed from a natural substance found in a sea sponge. Eribulin mesylate works by preventing cancer cells from multiplying.

The combination of Pembrolizumab and Eribulin mesylate is investigational. The study drugs, when given separately, work in different ways to stop the cancer cells from growing and spreading. However, it is not known if giving the two study drugs at the same time will have a better anti-cancer effect than giving each treatment on its own.

Arms:
arm 1: -Eribulin mesylate will be administered on Days 1 and 8 of each 21 day cycle for 1.4mg/m\^2 intravenously. arm 2: * Pembrolizumab will be administered in clinic once per cycle, given 200mg/m\^2 intravenously prior to Eribulin Mesylate.
* Eribulin mesylate will be administered on Days 1 and 8 of each 21 day cycle for 1.4 mg/m\^2 intravenously.

Interventions: intervention 1: Eribulin Mesylate intervention 2: Pembrolizumab
Intervention descriptions: intervention 1: Eribulin mesylate is developed from a natural substance found in a sea sponge. Eribulin mesylate works by preventing cancer cells from multiplying intervention 2: Pembrolizumab binds to PD-1, an inhibitory signaling receptor expressed on the surface of activated T cells, and blocks the binding to and activation of PD-1 by its ligands, which results in the activation of T-cell-mediated immune responses against tumor cells.

Protocol document:
Prot_SAP_000.pdf: 
 
 
Page 1 
 
NCI Protocol #: N/A  
 
DF/HCC Protocol #: 16-577  
 
TITLE:  A randomized phase II study of eribulin mesylate with or without pembrolizumab for 
metastatic hormone receptor positive breast cancer 
 
 
Coordinating Center: 
 
 
DF/HCC and Dana-Farber/Partners Cancer Care (DF/PCC)  
 
*Principal Investigator (PI): 
Sara Tolaney, MD, MPH 
 
Dana-Farber Cancer Institute  
 
450 Brookline Avenue,  
 
Boston, MA 02215 
 
 
Sara_Tolaney@dfci.harvard.edu 
 
Other Investigators: 
Ines Vaz Luis, MD, MSc 
Dana-Farber Cancer Institute  
Boston, MA 02215 
 
 
Ian Krop, MD, PhD  
Dana-Farber Cancer Institute 
Boston, MA 02215 
  
Eric P Winer, MD 
Dana-Farber Cancer Institute 
Boston, MA 02215 
 
Gerburg Wulf, MD 
Beth Israel Deaconess Medical Center 
Boston, MA 02215 
 
Scott Rodig, MD PhD 
Brigham and Women’s Hospital 
Boston, MA 02215  
 
Evisa Gjini PhD 
Brigham and Women’s Hospital 
Boston, MA 02215 
 
Mariano Severgnini 
Dana-Farber Cancer Institute 
Boston, MA 02215 
 
Laura Spring, MD 
Massachusetts General Hospital 
Boston, MA  02114 
 
Statistician:  
 
 
 
William Barry, PhD 
Dana-Farber Cancer Institute 
Boston, MA 02215 
 
 
 
Hao Guo 
Dana-Farber Cancer Institute  
Boston, MA 02215 
 
IND #:  132715 
IND Sponsor:  Sara Tolaney, MD, MPH 
 
 
Protocol Type / Version # / Version Date: Amendment / Protocol Version 10 / October 18, 
2018 


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SCHEMA 
 
 
Disease:  
Hormone-receptor positive metastatic breast 
cancer 
(0-2 prior lines of chemotherapy for 
metastatic disease) 
Drugs:  
Eribulin mesylate  
Pembrolizumab 
 
R 
A 
N 
D 
O 
M 
I 
Z 
E 
 
TREATMENT 
ARM A: eribulin mesylate 
+pembrolizumab 
 
 
 
 
ARM B: eribulin mesylate         
               Pembrolizumab 
(for patients who wish to cross over) 
 
 
 
 
 
Progressive disease 


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TABLE OF CONTENTS 
 
SCHEMA .........................................................................................................................................3 
1. 
OBJECTIVES ......................................................................................................................7 
1.1 
Study Design ............................................................................................................7 
1.2 
Primary Objectives...................................................................................................7 
1.3 
Secondary Objectives...............................................................................................7 
1.4 
Correlative Objectives .............................................................................................8 
2. 
BACKGROUND .................................................................................................................9 
2.1 
Study Disease(s).......................................................................................................9 
2.2 
The PD-1/PD-L1 pathway in cancer ........................................................................9 
2.3 
Pembrolizumab ......................................................................................................10 
2.4 
Eribulin mesylate ...................................................................................................12 
2.5 
Rationale for the proposed study combination ......................................................16 
2.6 
Correlative Studies Background ............................................................................18 
3. 
PARTICIPANT SELECTION ...........................................................................................20 
3.1 
Eligibility Criteria ..................................................................................................20 
3.2 
Exclusion Criteria ..................................................................................................22 
3.3 
Inclusion of Women and Minorities ......................................................................24 
3.4 
Inclusion Criteria for Crossover Therapy ..............................................................24 
3.5 
Exclusion Criteria for Crossover Therapy .............................................................24 
4. 
REGISTRATION PROCEDURES ...................................................................................25 
4.1 
General Guidelines for DF/HCC Institutions ........................................................25 
4.2 
Registration Process for DF/HCC Institutions .......................................................25 
4.3 
General Guidelines for Other Investigative Sites ..................................................26 
4.4 
Registration Process for Other Investigative Sites.................................................26 
5. 
TREATMENT PLAN ........................................................................................................26 
5.1 
Treatment Regimen ................................................................................................26 
5.2 
Pre-Treatment Criteria, Treatment, end off treatment and follow up visits...........27 
5.3 
Agent Administration.............................................................................................28 
5.4 
Discontinuation of pembrolizumab and Second Course Phase 
(Retreatment Period) ..............................................................................................28 
5.5 
Discontinuation of eribulin mesylate .....................................................................29 
5.6 
General Concomitant Medication and Supportive Care Guidelines ......................29 
5.7 
Criteria for Taking a Participant Off Protocol Therapy .........................................31 
5.8 
Duration of Follow Up ...........................................................................................33 
5.9 
Criteria for Taking a Participant Off Study ...........................................................34 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................34 
6.1 
Management of toxicities attributable to pembrolizumab .....................................35 
6.2 
Management of toxicities attributable to Eribulin mesylate ..................................38 


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7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................41 
7.1 
Adverse Events Lists..............................................................................................41 
7.2 
Adverse Event Characteristics ...............................................................................42 
7.3 
Expedited Adverse Event Reporting ......................................................................42 
7.4 
Expedited Reporting to the Food and Drug Administration (FDA) ......................43 
7.5 
Expedited Reporting to Hospital Risk Management .............................................43 
7.6 
Expedited Reporting to Merck ...............................................................................43 
7.7 
Expedited Reporting to Eisai .................................................................................45 
7.8 
Routine Adverse Event Reporting .........................................................................46 
8. 
PHARMACEUTICAL INFORMATION ..........................................................................46 
8.1 
PEMBROLIZUMAB .............................................................................................46 
8.2 
Eribulin mesylate ...................................................................................................48 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................50 
9.1 
Summary table: research tissue and blood specimen collection ............................51 
9.2 
Fresh Tissue Collection..........................................................................................52 
9.3 
Procedures for obtaining blood specimens for study .............................................52 
9.4 
Sites Performing Correlative Studies .....................................................................53 
9.5 
Blood and tissue banking .......................................................................................54 
9.6 
Hypothesis for correlative objectives.....................................................................54 
9.7 
Planned assays for correlative objectives ..............................................................54 
10. 
STUDY CALENDAR .......................................................................................................57 
11. 
MEASUREMENT OF EFFECT........................................................................................59 
11.1 
Antitumor Effect – Solid Tumors ..........................................................................60 
11.2 
Antitumor Effect – Hematologic Tumors ..............................................................67 
11.3 
Other Response Parameters ...................................................................................67 
12. 
DATA REPORTING / REGULATORY REQUIREMENTS ...........................................69 
12.1 
Data Reporting .......................................................................................................70 
12.2 
Data Safety Monitoring..........................................................................................70 
12.3 
Multicenter Guidelines...........................................................................................70 
13. 
STATISTICAL CONSIDERATIONS...............................................................................71 
13.1 
Study Design/Endpoints.........................................................................................71 
13.2 
Sample Size, Accrual Rate and Study Duration ....................................................71 
13.3 
Interim Monitoring Plan ........................................................................................72 
13.4 
Analysis of Primary Endpoints ..............................................................................72 
13.5 
Analysis of Secondary Endpoints ..........................................................................72 
13.6 
Analysis of Correlative Science Endpoints............................................................73 
13.7 
Reporting and Exclusions ......................................................................................75 
14. 
PUBLICATION PLAN .....................................................................................................75 
REFERENCES ..............................................................................................................................76 


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APPENDIX A 
PERFORMANCE STATUS CRITERIA ..................................................82 
APPENDIX B 
Strong CYP3A Inducers/Inhibitors ............................................................83 
APPENDIX C 
Guidelines for collecting research biopsy tissue ........................................84 
Risks of Research Biopsy and Procedures for Minimizing Risk .......................................84 
Risks of Anesthesia ............................................................................................................85 
APPENDIX D 
Antibodies that can be used for immunohistochemistry in correlative 
studies 
88 
APPENDIX E 
Antibodies that may be used for flow cytometry in correlative studies ....89 
APPENDIX F 
Immunohistochemical staining assays .......................................................91 
APPENDIX G 
TIL isolation from solid tumors .................................................................93 
APPENDIX H 
Flow cytometry procedures ........................................................................95 
APPENDIX I 
Generation of PBMCs ................................................................................96 
 
 
 


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1. OBJECTIVES 
 
1.1 
Study Design 
This is a randomized phase 2 open label study of eribulin mesylate +/- pembrolizumab for 
patients with metastatic hormone receptor (HR) positive breast cancer treated on 0-2 lines of 
chemotherapy. Eighty eight patients will be randomized (1:1) to eribulin mesylate with 
pembrolizumab (Arm A) or eribulin mesylate (Arm B). Patients randomized to eribulin mesylate 
monotherapy will have the option to receive pembrolizumab monotherapy at time of progression. 
Eribulin mesylate will be administered weekly on days 1 and 8 of a 21-day cycle.  
Pembrolizumab will be administered on D1 of each 21 day cycle. PD-L1 tumor positivity will 
not be used for patient selection.  Scientific correlative questions will also be incorporated in this 
study. 
 
1.2 
Primary Objectives 
To compare the progression free survival (PFS) of eribulin mesylate in combination with 
pembrolizumab to eribulin mesylate monotherapy among patients with metastatic HR positive 
breast cancer treated on 0-2 lines of chemotherapy. PFS is defined as the time from study 
randomization to disease progression per RECIST 1.1 or death due to any cause, whichever 
occurred first.  Patients alive without disease progression are censored at the date of last disease 
evaluation.   
 
1.3 
Secondary Objectives 
Efficacy objectives 
1.3.1 To evaluate PFS per immune-related response criteria (irRECIST) among patients treated 
with eribulin mesylate in combination with pembrolizumab and eribulin mesylate 
monotherapy, respectively treated on 0-2 lines of chemotherapy  
1.3.2 To evaluate objective response rate (ORR) among patients treated with eribulin mesylate 
in combination with pembrolizumab and eribulin mesylate monotherapy, respectively. 
ORR will beexamined using RECIST 1.1 and irRECIST criteria. 
1.3.3 To evaluate clinical benefit rate (CBR) among patients treated with eribulin mesylate in 
combination with pembrolizumab and eribulin mesylate monotherapy, respectively. CBR 
will be examined using RECIST 1.1 and irRECIST criteria. 
1.3.4 To evaluate duration of response (DOR) among patients treated with eribulin mesylate in 
combination with pembrolizumab and eribulin mesylate monotherapy, respectively. DOR 
will be examined using RECIST 1.1 and irRECIST criteria. 
1.3.5 To evaluate overall survival (OS) of among patients treated with eribulin mesylate in 
combination with pembrolizumab and eribulin mesylate monotherapy, respectively OS 
will be examined using RECIST 1.1 and irRECIST criteria. 
 
1.3.6 Among patients randomized to Arm B: To explore ORR, CBR, DOR, and PFS in 
patients receiving pembrolizumab after progression on eribulin mesylate monotherapy. 
 
 
Safety objectives  


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1.3.7 To evaluate the safety and tolerability of eribulin mesylate in combination with 
pembrolizumab with eribulin mesylate among patients with metastatic HR positive breast 
cancer whose cancer progressed after at least 2 prior lines of chemotherapy for metastatic 
disease. 
1.3.8 Among patients randomized to Arm B: To explore the safety and tolerability of 
pembrolizumab after progression on eribulin mesylate monotherapy. 
 
1.4 
Correlative Objectives 
1.4.1 To characterize a broad array of immune markers in metastatic HR-positive breast tumors 
(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 vary between patients with and without response (response 
assessed by RECIST 1.1 and immune-related response criteria) 
1.4.3 To characterize serial changes in immune marker profile in peripheral blood mononuclear 
cells (PBMCs) over the course of the trial treatment  
1.4.4 To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in PBMCs vary between patients with and without 
response (response assessed by RECIST 1.1 and irRECIST criteria) 
1.4.5 To investigate whether there is an immune marker in circulating PBMCs that corresponds 
to tumor infiltrating lymphocyte (TIL) percentage in baseline tumor 
1.4.6 In the cohort of patients who have re-biopsy at progressive disease (PD): To characterize 
changes in a broad array of immune markers at time of progression (as characterized in 
aim 1.4.1) to time of progressive disease on trial therapy). 
1.4.7 To collect blood to study cell-free DNA for comparison to tumor specimens. 
1.4.8 To characterize the structure and function of the gut microbiome in patients with breast 
cancer prior to starting this clinical trial. 
1.4.9 To determine whether pre-treatment characteristics of the structure and function of the 
gut microbiome in patients with breast cancer is associated with efficacy of 
pembrolizumab plus eribulin versus eribulin alone. 
1.4.10 To characterize changes in the structure and function of the gut microbiome of patients 
with breast cancer after two cycles of therapy compared to baseline.  
1.4.11 To determine whether changes in the overall diversity of the gut microbiome, estimated 
by the Shannon Index, of patients with breast cancer after two cycles of therapy regimens 
is associated with efficacy of pembrolizumab plus eribulin versus eribulin alone. 
1.4.12 To determine if the abundance and functional profile of specific gut bacteria are 
associated with objective response to pembrolizumab plus eribulin. 
1.4.13 To evaluate the functional pathways that may play a role as a predictive biomarker of 
response to pembrolizumab plus eribulin. 
1.4.14 To explore whether the number and/or type of mutations identified using a next 
generation sequencing (NGS) panel is correlated with patient outcomes (PFS, ORR, 
CBR, and OS). This will be done on DFCI patients only.  
 
 
 


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2. BACKGROUND 
 
2.1 
Study Disease(s) 
 
Globally, breast cancer is the most frequent  female cancer and the leading cause of cancer death 
in women1. In the United States, over ~200,000 new breast cancer cases are expected/year and 
almost 40,000 deaths.2, 3 In women, the lifetime probability of developing invasive breast cancer 
is one in eight overall.4 HR-positive breast cancer (defined as estrogen receptor and/or 
progesterone receptor expressing tumors) accounts for 60-70% of breast cancer. Despite many 
advances in the adjuvant treatment of early-stage disease, 30-40% of women will develop 
systemic relapse in addition to the 6-10% of patients who present with de novo metastatic breast 
cancer,5 and nearly all patients with metastatic breast cancer eventually succumb to their disease. 
Currently the median survival for patients with HR-positive tumors does not exceed 3 years. 6 
Chemotherapy and endocrine therapy remain the backbone of systemic treatment for HR-
positive tumors. More recently, the combination of endocrine therapy plus a molecular-targeted 
agents (such as CDK 4/6 inhibitors, mTOR)  showed to be promising to overcome endocrine 
resistance.7, 8 
 
Nevertheless, remains an urgent need to better understand the biology of human breast cancer, 
and to understand the interaction between tumor and host characteristics, treatment, and clinical 
outcomes and develop new treatment strategies. 
 
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.  Accumulating evidence shows a correlation 
between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in 
various malignancies.  In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector 
T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis and long-term 
survival in many solid tumors. 
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 


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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 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. 
 
The PD-1/PD-L1 pathway in breast cancer 
Accumulating evidence suggests that the immune system can also control breast cancer.   Recent 
data suggests that HR positive breast cancers are positive for PD-L1 expression in about 6% of 
cases, and positive for PD-1 expression in about 43% of cases.9 Additionally, elevated mRNA 
expression of immune markers, including PD-L1, CTLA4, B7H-3, and ID01 was noted in the 
ER+HER2- luminal population. The expression of immune regulatory targets in HR positive 
breast cancer suggests that these patients may benefit from immunotherapeutic approaches.   
A  correlation has been shown between tumor-infiltrating lymphocytes (TILs) in breast cancer 
tissue and improved prognosis.10, 11  Additionally, unsupervised expression profiling of cancer- 
associated stroma revealed a gene signature predictive of good prognosis that was enriched for 
CD8+ T cell responses.12   
 
 
2.3 
Pembrolizumab   
 
Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of the 
IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-
L1 and PD-L2.  KeytrudaTM (pembrolizumab) has recently been approved in the United States 
for the treatment of patients with unresectable or metastatic melanoma and disease progression 
following ipilumumab and, if BRAF V600 mutation positive, a BRAF inhibitor.  Clinical data 
are derived from an ongoing, first-in-human phase I study (PN001, NCT01295827) to evaluate 
the safety and clinical activity of Pembrolizumab as a monotherapy, sponsored by Merck Sharp 
& Dohme.  There are five parts to this study (Parts A-D and F) (Investigator’s Brochure, 2014). 
 
Part A was a 3+3 dose escalation study in subjects with solid tumors to evaluate safety, 
tolerability, pharmacokinetics (PK), and pharmacodynamics, and to determine a maximum 


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tolerated dose (MTD) or preliminary recommended phase 2 doses (RP2Ds).  Doses were 1, 3, 
and 10 mg/kg every 2 weeks (Q2W); doses of either 2 mg/kg or 10 mg/kg were also 
administered every 3 weeks (Q3W).  All 3 dose levels were well tolerated and no dose-limiting 
toxicities (DLTs) were observed; therefore, the MTD was not determined.  The RP2D was 
determined by the sponsor based on safety, PK, and pharmacodynamic measurements, along 
with the strength of antitumor activity signals observed. 
 
Pharmacokinetics 
The half-life (t1/2) of pembrolizumab is approximately 4 weeks and there is no indication of dose 
dependency or half-life in the three dose groups (1, 3, and 10 mg/kg) (Investigator’s Brochure, 
2014).  The long t1/2 supports a dosing interval of every 2 or 3 weeks. 
 
There was a dose-related increase in exposure from 1 to 10 mg/kg.  Serum concentrations of 
pembrolizumab were lower by a factor of approximately 5 in patients receiving 2 mg/kg Q3W 
than in those receiving 10 mg/kg Q3W.  Steady-state trough concentrations were 20% greater in 
the patients receiving 10 mg/kg Q2W than in those receiving the same dose Q3W. 
 
A population pharmacokinetic analysis has been performed using serum concentration time data 
from 476 patients. Within the resulting population PK model, clearance and volume parameters 
of pembrolizumab were found to be dependent on body weight. The relationship between 
clearance and body weight, with an allometric exponent of 0.59, is within the range observed for 
other antibodies and would support both body weight normalized dosing or a fixed dose across 
all body weights.  Pembrolizumab has been found to have a wide therapeutic range based on the 
melanoma indication.  The population PK evaluation revealed that there was no significant 
impact of tumor burden on exposure. In addition, exposure was similar between the NSCLC and 
melanoma indications. Therefore, there are no anticipated changes in exposure between different 
indication settings. 
 
The choice of the 200 mg Q3W as an appropriate dose for the switch to fixed dosing is based on 
simulations performed using the population PK model of pembrolizumab showing that the fixed 
dose of 200 mg every 3 weeks will provide exposures that 1) are optimally consistent with those 
obtained with the 2 mg/kg dose every 3 weeks, 2) will maintain individual patient exposures in the 
exposure range established in melanoma as associated with maximal efficacy response and 3) will 
maintain individual patients exposure in the exposure range established in melanoma that are well 
tolerated and safe.  
A fixed dose regimen will simplify the dosing regimen to be more convenient for physicians and 
to reduce potential for dosing errors.  A fixed dosing scheme will also reduce complexity in the 
logistical chain at treatment facilities and reduce wastage. 
 
Anti-Drug Antibodies (ADA) Data 
The occurrence of ADA has been observed in less than 1% of the patients screened, indicating a 
low potential of pembrolizumab to elicit the formation of ADA.  No impact of ADA on 
pembrolizumab exposure has been observed. 
 
Efficacy 


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When treated with pembrolizumab monotherapy, the overall response rate (ORR) for ipilimumab 
(IPI)-treated patients with melanoma was 25%/27% according to the Response Evaluation 
Criteria in Solid Tumors (RECIST)/investigator-assessed irRECIST, respectively (Investigator’s 
Brochure, 2014).  The ORR for IPI-naïve patients with melanoma was 39%/43% by 
RECIST/investigator-assessed irRC, respectively.  The majority of responses were seen in 
patients with melanoma by 16 weeks of therapy; however, some responses have been reported 
after 24 weeks or more of therapy with pembrolizumab.  Responses can be delayed, and in some 
patients, a RECIST-defined progression followed by response has been observed. 
 
The preliminary ORR for 38 patients with non-small cell lung cancer was 21%/24% by 
RECIST/investigator-assessed irRC, respectively (Investigator’s Brochure, 2014). 
 
Early findings for 27 patients with triple negative breast cancer showed a 18.5% ORR by 
RECIST. 
 
Pharmacodynamics/Biomarkers 
Pharmacodynamic data (IL-2 release assay) has suggested that peripheral target engagement is 
durable (>21 days).   
 
PD-L1 is being investigated as a predictive biomarker for pembrolizumab treatment.  At the 15th 
World Conference on Lung Cancer, Garon et al presented preliminary data on a subset of 
patients suggesting that higher levels of tumor PD-L1 expression are associated with increased 
clinical activity.13  ORR by RECIST 1.1 occurred in 4 out of 7 patients with higher levels of PD-
L1 expression (57%, 95% CI 18-90%) versus 2 out of 22 patients with lower levels of PD-L1 
expression (9%, 95% CI 1-29%).  These data are extremely preliminarily, and PD-L1 is not 
being used for patient selection. 
 
Biomarkers to evaluate immune modulation and markers in the tumor microenvironment, such as 
T-cell infiltration, the baseline expression of markers of T-cell suppression FoxP3 or the 
immunoregulatory enzyme incoleamine 2,3-dioxygenase (IDO) in tumor biopsies, were 
associated with a high response rate in patients with advanced melanoma.14, 15  
 
Safety data 
The most frequent treatment-related adverse events (AEs) were fatigue, nausea, cough, pruritis, 
diarrhea, and rash (Investigator’s Brochure, 2014).  Most AEs were not considered serious.  The 
most commonly-reported immune-related AEs were rash, pruritis, vitiligo, hypothyroidism, 
arthralgia, diarrhea, and pneumonitis. 
 
Important identified risks include: pneumonitis, thyroid disorders (hypothyroidism and 
hyperthyroidism), colitis, diarrhea, hepatitis, nephritis, uveitis, rash/pruritis, and neuropathy. 
 
2.4 
Eribulin mesylate 
 
 
Halichondrin B (Hal B) is a large polyether macrolide isolated from a marine sponge 
(Halichondria okadai).16 It was shown to have anti-cancer activity, based on a microtubule 
destabilizing anti-mitotic mechanism of action.16, 17 Although other cancer drugs, as vinca 


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alkaloids or taxanes, share this mechanism of action, interactions between Hal B and tubulin 
were found to be unique.17-20 Eribulin mesylate induces mitotic block, secondary to tubulin 
polymerization inhibition. It sequesters tubulin into nonproductive aggregates, exerting 
impressive potent anti-cancer effects in vitro and in vivo studies.21 
 
Summary of mechanism of action  
 
 
In vitro studies have demonstrated that analogues of Hal B inhibit cell growth at subnanomolar 
concentrations in a wide variety of cancer cell types, such as breast, ovary, colon and 
melanoma.21 Eribulin mesylate exerts its effects binding to the plus end of microtubules and 
leading to suppression of microtubule growth. It causes tubulin sequestration into nonproductive 
tubulin aggregates. Thereby, it inhibits tubulin polymerization and microtubule dynamics, 
interfering with normal mitotic spindle formation and blocking the prometaphase portion of 
mitosis.22 These results in induction of irreversible cell cycle block at G2/M, point disruption of 
mitotic spindles, and cell death via apoptosis after prolonged mitotic blockage.23 
 
Eribulin mesylate is a substrate for the P-glycoprotein drug efflux pump with reduced potency 
against cells expressing high levels of this pump, however it can retain in vitro efficacy against 
taxane-resistant cell lines due to β-tubulin mutations.24 In vivo, eribulin mesylate has led to tumor 
regression and even eradication against several human cancer xenograft models, including breast 
cancer. 21 
 
Summary of pharmacokinetic (PK) profile and metabolism  
 
Pre-clinical studies 
In animal models (mice, rat and dogs), the pharmacokinetics (PK) of eribulin mesylate after 
intravenous administration is characterized by a rapid distribution phase, a large volume of 
distribution and a prolonged elimination phase (t½:  3.6 -6.9 hours in mice, 15.9- 27.9 hours in 
rats, 21.9-28.2 hours in dogs).   
Eribulin mesylate also has low penetration in brain, likely related to its role as a substrate of the 
P-glycoprotein drug efflux pump. It also has high penetration in tissues such as the lung, bladder, 
renal cortex and medulla, liver, spleen, thyroid, stomach, and salivary gland. 
Unchanged eribulin mesylate is the major circulating compound in plasma following its 
administration. Metabolism is a minor component of eribulin mesylate clearance, with minor 
metabolic changes occurring through cytochrome P450 3A4 (CYP3A4). Eribulin mesylate is 
eliminated primarily unchanged in feces. 
 
Clinical Studies 
In clinical studies, most of these findings were confirmed. Eribulin mesylate’s PK is 
characterized by a rapid distribution phase, with a prolonged elimination phase after intravenous 
infusion. The disposition of eribulin mesylate follows a linear kinetics over the dose range 
studied (0.25mg/m2- 4.0mg/m2). It has low plasma clearance, with a mean clearance=1.16-2.42 
L/hr/m2,  a large mean volume of distribution at steady state (43-114 L/m2) and a half life of 
elimination of 40 hours. The human plasma protein binding of eribulin mesylate occurs at 
concentrations of 100ng/mL to 1.000ng/mL and ranges from 49% to 65%.  
 
 
 


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Eribulin mesylate exposure after multiple doses is comparable to a single dose, with no 
accumulation of eribulin mesylate with weekly administration. As expected based on work in 
pre-clinical models, metabolism is a minor component of eribulin mesylate clearance. 
Metabolites represent less than 0.6% of parent compound in plasma. Renal elimination is also a 
minor route of eribulin mesylate excretion, with less than 10% of drug excreted unchanged in 
urine. Most of excretion of eribulin mesylate is fecal and unchanged. It is unknown if eribulin 
mesylate is excreted into human milk. 
Eribulin mesylate does not induce or inhibit hepatic CYP3A4 activity at clinically relevant 
concentrations. Concomitant administration of ketoconazole, a CYP3A4 inhibitor, or rifampicin, 
a CYP3A4 inducer, had no effect on exposure to eribulin mesylate. Eribulin mesylate does not 
induce or inhibit CYP1A, CYP2C9, CYP2C19, CYP2D6 activity at clinically relevant 
concentrations. 
 
Hepatic impairment may decrease the clearance of eribulin mesylate and prolong the elimination 
half-life, resulting in increased exposure to eribulin mesylate. As such, it is proposed that the 
eribulin mesylate dose in patients with moderate hepatic impairment should be adjusted. No 
studies for patients with severe hepatic impairment have been performed.25 
 
Moderate renal impairment (CrCL 30-50 mL/min) may also impact the clearance of eribulin 
mesylate, resulting in increased exposure. As such, it is proposed that eribulin mesylate dose in 
patients with moderate renal impairment should be adjusted. No studies for patients with severe 
renal impairment have been performed.  
 
Population PK analyses based on Phase 1 and 2 studies showed that eribulin meslyate’s 
clearance is affected by body weight, serum albumin, alkaline phosphatase and bilirubin. The 
effects of age, gender, race and concomitant medications (CYP3A4 inhibitors and inducers) on 
clearance were not found to be significant. 
 
Summary of toxicology data  
 
 
A variety of nonclinical toxicology studies have been conducted to support the use of eribulin 
mesylate in humans. The findings from these studies are summarized below. No significant 
serious adverse events (AE) were observed in any preclinical safety studies with regard to central 
nervous, respiratory, or cardiovascular systems. Eribulin mesylate induced no significant 
reduction in nerve conduction velocity or peak nerve amplitude in caudal and digital nerves, in 
contrast to paclitaxel. The morphological changes in sciatic nerve and dorsal root ganglia were 
less severe in eribulin mesylate than those observed with paclitaxel. In animal models, eribulin 
mesylate induced markedly less neuropathy than paclitaxel. 
 
Bone marrow toxicity appeared to be dose-limiting in both rats and dogs. Intestinal toxicity was 
also present in dogs. Other toxicities that were considered to be drug-related occurred in the 
lymphoid tissue, testes, and skeletal muscle. All observed toxicities (except testicular toxicity) 
were reversible in both dogs and rats. In repeated-dose toxicity studies in rats, testicular toxicity, 
thymic atrophy, bone marrow toxicity, and fiber degeneration of sciatic nerve were found. 


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Although the changes in testes and sciatic nerve were still present after a 14-day recovery period, 
other toxicities were reversible. Repeated-dose toxicity in dogs produced leukopenia, which was 
fully reversible in 14 days with compensatory extramedullary hematopoiesis.  The chronic 
toxicity studies in rats and dogs were conducted over 6 months. In rats, bone marrow and 
testicular toxicity were the most important effects observed.  Hypocellularity of bone marrow 
and a reduction in the weight of testes (correlating with hypocellularity of seminiferous 
epithelium with associated hypospermia/aspermia of the epididymides) were found. Increases in 
alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and cholesterol were 
also observed. In dogs, bone marrow toxicity mainly represented by leukopenia was observed. 
With regard to findings in the testes, this finding was limited to high doses of drugs and included 
decrease of testes weights and microscopic changes with mild to moderate hypocellularity of 
testis with hypospermia/aspermia of the epididymides. In addition, hypercellularity of bone 
marrow, lymphoid depletion in mesenteric lymph nodes and Peyer’s patches, and thymic atrophy 
were noted. These studies suggest that male fertility may be compromised by eribulin mesylate 
treatment. Carcinogenicity studies have not been conducted with eribulin mesylate. Genotoxicity 
was evaluated both in vitro and in vivo. In vitro, results indicated that the effect was due 
primarily to chromosome segregation interference rather than chromosome breakage because 
eribulin mesylate was negative in the Ames test with or without S9 and was weakly positive in 
the L5178Y tk+/- mouse lymphoma mutagenesis assay. In vivo, in rats, dose-related increases of 
micronucleated- polychromatic erythrocytes (MN-PCEs) at either sampling time and dose-
related decreases in the proportion of PCEs were observed, which may indicate some degree of 
bone marrow supression. In the intravenous embryo and fetal developmental studies in rats, 
eribulin mesylate mesylate was given to rats at doses of 0.01, 0.03, 0.10, and 0.15 mg/kg/day on 
gestational days 8, 10, and 12. External and/or soft tissue anomalies were noted at 0.15 
mg/kg/day, indicating that eribulin mesylate has teratogenic potential. 
Summary of safety data for eribulin mesylate (Also refer to Section 6) 
Pivotal Phase III trial 
In the phase III EMBRACE trial, AEs occurred in 99% of patients receiving eribulin mesylate, 
however serious AEs occurred in 25% and AEs leading to therapy discontinuation occurred in 
13% of patients on study.  The primary toxicities with eribulin mesylate were asthenia or fatigue 
(54%) neutropenia (52%). Grade 4 neutropenia lasting more than 1 week occurred in 12% of 
patients and F+N occurred at low incidence (5%).  Patients with elevated ALT/AST or bilirubin 
experienced a higher frequency of grade 4 neutropenia or F+N. Peripheral neuropathy was the 
most common AE leading to discontinuation of therapy, occurring in 5% of patients. Neuropathy 
lasting more than one year occurred in 5% of the patients. Alopecia occurred in 45% of patients. 
Nausea occurred in 35% of patients, but it was grade 3 just in 1% of patients. Diarrhea occurred 
in 18% of cases; in all cases was grade 1 or 2.  Dose interruptions, delays and reductions were 
undertaken in 6%, 49% and 29% in the eribulin mesylate group, respectively.26 
 
Overall Adverse events and serious adverse events 
Overall, in clinical studies, the most common treatment-related toxicities were hematological 
toxicity, mainly neutropenia, asthenia or fatigue, alopecia, nausea and peripheral neuropathy. 
Most AE were grade 1 or 2. 


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Peripheral neuropathy was an important AE leading to discontinuation of therapy. According to 
the investigator’s brochure, the most frequently reported eribulin mesylate related AEs were 
asthenia/fatigue (65%), alopecia (60%), neutropenia (60%), nausea (44%), anemia (28%), 
pyrexia (23%), leucopenia (22%), anorexia (21%), constipation (19%), vomiting (18%), and 
peripheral neuropathy (5.5%; only grade 3). Grade 4 neutropenia occurred in 32% of patients, 
and F+N occurred in 5.5% of patients. The frequency of all other grade 3/4 AEs was less than 
3%.  
 
Summary of efficacy data for eribulin mesylate- Metastatic Breast Cancer 
Phase II studies have observed a significant level of activity in heavily pretreated patients with 
advanced breast cancer. 25, 27, 28 The aim of the Phase II study, E7389-A001-201, was to 
determine the response rate of eribulin mesylate monotherapy in patients with advanced/MBC 
pre-treated with chemotherapy, including an anthracycline and a taxane. Here, an ORR of 11.5% 
(95% confidence interval [CI), 6-20%) and a CBR of 17.2% (95% CI, 10-27%) were achieved. 
The median duration of response was 5.6 months and the median OS was 9.0 months.25, 27 
In addition, the Phase II study E7389-G000-211, determined the response rate of eribulin 
mesylate monotherapy in patients with advanced/MBC pre-treated with chemotherapy, including 
an anthracycline,taxane and capecitabine. ORR by an independent review was 9.3% (95% CI, 
6.1-13.4%), and the CBR was 17.1%. Median duration of response was 4.1 months, and median 
OS was 10.4 months.28 The Phase II study E7389-J081-221 was conducted to determine the 
efficacy and safety of eribulin mesylate in Japanese patients with advanced/MBC pre-treated 
with chemotherapy, including  an anthracycline and a taxane. ORR by an independent review 
was 21.3 % and the CBR was 27.5%.25 These Phase II studies led to the Phase III EMBRACE 
trial (Eisai Inc. MBC Study Assessing Physician's Choice vs. E7389, study E7389-G000-305), a 
multicentre, randomized, open label study. It included 762 locally advanced or MBC patients 
pre-treated with at least 2 and up to 5 chemotherapy regimens.  Women were randomly assigned 
to receive eribulin mesylate (1.4 mg/m2 days 1 and 8 every 21 days) or any other chemotherapy 
by the physician’s and patient’s choice. The study met its primary objective, showing a 
significant increase in OS (Hazard Ratio (HR): 0.81, 95% CI, 0.66-0.99; p=0.041). Treatment 
with eribulin mesylate compared to alternative therapy significantly improved median OS (13.1 
vs. 10.6 months, p=0.041). ORR was also improved in patients treated with eribulin mesylate 
mesylate (12% vs. 5%, p=0.002). CBR was 23% for eribulin mesylate and 17% for provider-
choice therapy. 25-28 
Kaufam et al examined the efficacy of eribulin vs. capecitabine among patients with metastatic 
breast cancer treated up to second line of chemotherapy. In this trial ORRs by independent 
review was 11.0% (95% CI, 8.5% to 13.9%) for the eribulin arm and the median PFS was 4.1 
months (95% CI, 3.5 to 4.3 months) for eribulin.29 
.  
 
2.5 
Rationale for the proposed study combination 
 
Recent evidence suggests that some chemotherapeutic agents rely on the induction of anticancer 
immune responses, and that the innate and adaptive immune systems are critical in determining 
the efficacy of cytotoxic-based regimens.  Based on this recent data, and based on the fact that 
tumor immune infiltrates have been demonstrated to have an impact on survival in breast cancer, 
we propose a study combining chemotherapy with anti-PD1 therapy in metastatic HR+ breast 
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Pembrolizumab is a humanized monoclonal antibody of the IgG4/kappa isotype designed to 
directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2.  Pembrolizumab 
strongly enhances T lymphocyte immune responses in cultured cancer cells, and modulates the 
levels of IL-2 TNF-α, and IFN-ˠ.  The antibody potentiates existing immune responses only in 
the presence of antigen.  The most common adverse events include fatigue, rash, and pruritus.   
 
There is strong rationale that chemotherapy can work synergistically with immune modulation30.   
For example, cyclophosphamide has been shown to deplete T regulatory cells and myeloid-
derived suppressive cells, thereby enhancing antitumor immune response31, 32.  The direct 
cytotoxicity of chemotherapy can also enhance cross presentation of tumor antigens, leading to 
augmented T cell responses directed at the tumor.33  Moreover, cytotoxic chemotherapeutics, 
such as paclitaxel, have been shown to upregulate PD-L1 expression on breast cancer cells.34  
Additionally, administration of taxanes in the preoperative setting results in an increase in the 
levels of tumor-infiltrating lymphocytes (TILs) within the tumor parenchyma.35  In the metastatic 
setting, docetaxel has been shown to increase levels of Th-1 associated cytokines (IL-2, IFN-γ) 
while decreasing negative inflammatory markers, such as tumor necrosis factor beta (TNF-β). 
 
A recent study in the 4 T1 breast cancer mouse model demonstrated remarkable synergy between 
therapy with anti-PD-1 antibody and paclitaxel 36. As opposed to paclitaxel monotherapy, the 
combination significantly suppressed tumor growth and 80% of the mice (4 of 5) survived 
tumor-free until day 90. Moreover, theses mice developed memory immune responses as 
demonstrated by their resistance to re-challenge with 4 T1 cells. 
 
 
 
 
 
 
Eribulin mesylate is a synthetic analogue of halichondrin B, a product that is isolated from the 
marine sponge Halichondria okadai.  Eribulin mesylate is designed to inhibit microtubule 
polymerization without affecting depolymerization. This results in apoptosis through an 
Figure 1:  BALB/c mice were 
transplanted s.c. with 5 x 105 4 T1 
cellson day 1 and were pretreated with 
a dose of paclitaxel (10mg/kg) on day 
8, followed by 3 doses of anti-PD-1 
antibody (250μg) on day 9, 12, and 
16.  


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irreversible mitotic block at the G2-M phases. In November 2010, the FDA approved eribulin 
mesylate as a treatment for patients with metastatic breast cancer following the administration of 
at least 2 regimens with an anthracycline and a taxane. This approval was based on data from the 
EMBRACE trial, which demonstrated a statistically significant improvement in overall survival 
for patients receiving eribulin mesylate compared with physician's choice of therapy.26 In a 
second study, eribulin mesylate was compared to capecitabine in patients who had received 0-2 
prior lines of chemotherapy for metastatic disease.    The two studies enrolled 1864 patients, with 
31.5% receiving eribulin mesylate as second-line therapy and 32.7% receiving treatment as third-
line therapy. There was no statistically significant difference in PFS or OS between these two 
treatments.29  Additionally, Dana-Farber has recently completed accrual of a first and second-line 
study of eribulin mesylate for patients with metastatic HR positive breast cancer. 
 
 
This is a randomized phase 2 study of eribulin mesylate with or without pembrolizumab in 
patients with metastatic HR positive breast cancer.   Eribulin mesylate will be administered 
weekly on days 1, 8 of a 21 day cycle.  Pembrolizumab will be administered on D1 of each 21 
day cycle.  Patients who are randomized to eribulin mesylate monotherapy and develop 
progressive disease will be allowed to receive Pembrolizumab at time of progression.  
 
2.6 
Correlative Studies Background 
 
2.6.1    Blood and Tissue Analysis 
There is growing evidence that signatures of tumor-associated immunologic activity are seen in 
breast cancer and are significant prognostic predictors.  Many 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.37-42  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.38 
 
However, the development of immune signatures in breast cancer that go beyond simple 
histology is at a very early stage.  Additionally, much of the work done in this area has been in 
the adjuvant and neoadjuvant settings, with less known about the immunologic profile of 
metastatic disease.  Given the promise of immune-based therapies in other solid malignancies 
such as melanoma and non-small cell lung cancer, with preliminary data demonstrating activity 
of immune checkpoint inhibition in advanced breast cancer patients,43, 44 there is a great need to 
better characterize the immune profile of breast tumors across multiple disease subtypes, and at 
different points in the course of therapy.  The bulk of our correlative science in this trial focuses 
on characterizing a broad array of immune markers in metastatic HR-positive breast cancer, 
investigating whether those markers predict disease response to therapy, and exploring how 
markers change from the start of treatment to the time of development of progressive disease. 
 
In melanoma, the solid malignancy currently at the forefront of understanding the anti-tumor 
immune response, investigation into expression of immune mediators in the peripheral blood is 


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at an early phase.45  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.  
Additionally, 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 (a) changes in 
PBMC immune profiles and disease response to therapy.  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. Scott Rodig and Evisa 
Gjinin, and Mariano Severgnini, all of whom are lab scientists with extensive experience with 
immune profiling in melanoma.  Further details can be found in Section 9. 
 
2.6.2     Microbiome Analysis 
Breast Cancer (BC) is the most frequently diagnosed cancer and the second cause of cancer 
death in American women57.  In the advanced setting, despite multiple available systemic 
therapies, virtually all patients will die from their disease.  Thus, the exploration of new 
treatments, such as immune checkpoint inhibitors (ICI), including pembrolizumab, is imperative. 
 
An increasing body of preclinical and clinical evidence suggests that breast cancer is an 
immunogenic malignancy58.  It is now recognized that a fraction of breast tumors, mainly triple-
negative breast cancer (TNBC), have substantial lymphocyte infiltration, and that this pathologic 
feature has prognostic implications59.  Early clinical trials assessing the efficacy of PD-1/PD-L1 
inhibitors given as monotherapy showed that only a small fraction of patients derive benefit from 
immunotherapy with an approximate 20% objective response rate (ORR) among patients with 
PDL1+ TNBC60,61, and a 12% ORR among those with PDL1+ hormone receptor (HR)-positive 
BC62.  Therefore, new research approaches combining therapeutic agents aiming to boost 
antitumor immunity, as well as developing predictive biomarkers of response, are needed to 
increase the rates of clinical success of immunotherapy in BC. 
 
In this context, the gut microbiota has been recognized as a modulator of immune system 
development63. Healthy individuals have microbial populations in their intestinal tract that vary 
markedly in composition64,65.  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 cancer66.  Recently, two preclinical studies provided to ICI, raising 
the possibility that stool microbiota could be used as biomarker predictors of efficacy to 
immunotherapy67,68.  Interestingly, postmenopausal women with breast cancer have altered 
composition and low diversity of their gut microbiota compared to healthy controls69. 
 
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 
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This correlative project is made possible by collaboration with the BWH/Harvard Cohorts 
Biorepository and an external lab vendor, Microbiome Dx. Further details can be found in 
Section 9.   
 
2.6.3     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-β-
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, CBR, and OS). This tool is a cancer genomic assay 
to detect somatic mutations, copy number variations and structural variants in tumor DNA 
extracted 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 Patients must have histologically or cytologically confirmed Stage IV invasive breast 
cancer. Patients without pathologic or cytologic confirmation of metastatic disease should 
have unequivocal evidence of metastasis from physical examination or radiologic 
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3.1.2 Subjects must have at least one lesion that is not within a previously radiated field that is 
evaluable on computerized tomography (CT) or magnetic resonance imaging (MRI) scan 
per RECIST version 1.1.46 If the subject’s only evaluable disease is within a previously 
radiated field, it must have demonstrated progression since the time of radiation. 
 
3.1.3 Participants must have HR positive, HER2-negative breast cancer (ER>1% and/or, 
PR>1%, HER2-negative per ASCO CAP guidelines, 2013 resulted on the primary tumor 
and/or a metastatic lesion). 
 
3.1.4 Participants must have already received or been intolerant to at least two lines of 
hormonal therapies (including the adjuvant or metastatic setting) or be appropriate 
candidates for chemotherapy  
 
3.1.5 Prior chemotherapy:   
Participants are allowed to have received up to 2 prior lines of chemotherapy in the 
metastatic setting. If a prior chemotherapy was given for less than 1 cycle, it will not be 
counted as a prior line. The last dose of chemotherapy must be ≥14 days prior to 
initiation of study therapy.  Participants should be adequately recovered from acute 
toxicities of prior treatment.  No prior treatment with eribulin mesylate is allowed.  
 
3.1.6 Prior biologic therapy: The last dose of biologic or investigational therapy must be ≥21 
days prior to initiation of study therapy.   
 
3.1.7 Prior hormonal therapy: Hormonal therapy must have been discontinued ≥14 days prior 
to initiation of study therapy. However, continuation of ovarian suppression is allowed. 
 
3.1.8 Prior radiation therapy: Participants may have received prior radiation therapy in either 
the metastatic or early-stage setting.  Radiation therapy must be completed ≥14 days prior 
to initiation of study therapy. 
 
3.1.9 Prior targeted therapy: Targeted therapy must have been discontinued ≥ 14 days prior to 
initiation of study therapy.  
 
3.1.10 Biphosphonates/Denosumab:  Participants on bisphosphonates/denosumab may continue 
receiving bisphosphonate therapy during study treatment.  
 
3.1.11 Participants must have an archival tumor sample available (1 block or 20 unstained 
slides). If no archival tissue is available, participants must be willing to undergo a 
research biopsy of their disease if it is safely accessible.  
 
3.1.12 Age ≥ 18 years of age  
 
3.1.13 ECOG performance status ≤2 (Karnofsky ≥60%, see Appendix A) 
 
 


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3.1.14 Participants must have normal organ and marrow function as defined below: 
 
− 
absolute neutrophil count 
≥1,500/mcL 
− 
platelets 
≥100,000/mcL 
− 
hemoglobin  
≥ 8 g/dl 
− 
total bilirubin 
≤1.5 × institutional upper limit of normal (ULN) 
− 
AST(SGOT)/ALT(SGPT) 
≤2.5 × institutional ULN (≤ 5 × institutional ULN with 
documented liver metastases,  
− 
serum creatinine 
≤1.5mg/dL or calculated GFR ≥60 mL/min 
− 
INR/PT  
 
 
 
≤1.5 times ULN unless participant is receiving 
anticoagulant therapy, as long as PT or PTT is within 
therapeutic range of intended use of anticoagulants 
− 
aPTT/PTT  
 
 
≤1.5 times ULN unless participant is receiving 
anticoagulant therapy, as long as PT or PTT is within 
therapeutic range of intended use of anticoagulants 
 
3.1.15 The effects of eribulin mesylate and pembrolizumab on the developing human fetus are 
unknown.  Pre-clinical data was suggestive of a teratogenic effect of eribulin mesylate. 
For these reasons women of child-bearing potential and men must agree to use adequate 
contraception (hormonal or barrier method of birth control; abstinence) for the duration 
of study participation and 4 months after the last dose of eribulin mesylate and/or 
pembrolizumab. Note: abstinence is acceptable if this is the usual lifestyle and preferred 
contraception for the subject.   
• Should a woman become pregnant or suspect she is pregnant while she or her 
partner is participating in this study, the treating physician and principal 
investigator should be informed immediately.   
• While on the study, women must not breastfeed.   
• Subjects of childbearing potential are defined as those who have not been 
surgically sterilized and/or have had a menstrual period in the past year 
 
3.1.16 Female subjects of childbearing potential, as defined above, must have a either a negative 
urine or a negative serum pregnancy test within seven (7) days of first dose of 
pembrolizumab.  If a urine test is positive or cannot be confirmed as negative, a serum 
pregnancy test will be required. 
 
3.1.17 Ability to understand and willingness to sign a written informed consent document. 
 
3.2 
Exclusion Criteria 
 
3.2.1 Chemotherapy-related or radiation-related toxicities that have not resolved to Grade 1 
severity or lower, except for stable sensory neuropathy (≤ Grade 2) and alopecia. 
 
3.2.2 Participants who are receiving any other investigational agents. 
 


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3.2.3 Previous treatment with eribulin mesylate or any anti-PD-1, PD-L1, or PD-L2 agent or 
participation in any MK-3475 Merck studies. 
 
3.2.4 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to eribulin mesylate or pembrolizumab. 
 
3.2.5 Known brain metastases that are untreated, symptomatic, or require therapy to control 
symptoms. Participants with previously diagnosed brain metastases are eligible if they 
have completed treatment at least 4 weeks prior to registration, are neurologically stable 
and absence of new neurologic symptoms for the last 4 weeks prior to study entry, and 
have recovered from the effects of radiotherapy or surgery.  Any corticosteroid use for 
brain metastases must have been discontinued without the subsequent appearance of 
symptoms for ≥2 weeks before the first study drug.  Treatment for brain metastases may 
have included whole brain radiotherapy, radiosurgery, or a combination as deemed 
appropriate by the treating physician.  
 
3.2.6 Uncontrolled intercurrent illness, including, but not limited to uncontrolled hypertension, 
unstable angina pectoris, uncontrolled cardiac arrhythmia, congestive heart failure-New 
York Heart Association Class III or IV, active ischemic heart disease, myocardial 
infarction within the previous six months, uncontrolled diabetes mellitus, gastric or 
duodenal ulceration diagnosed within the previous 6 months, chronic liver or renal 
disease, severe malnutrition  or psychiatric illness/social situations that would limit 
compliance with study requirements.   
 
3.2.7 Clinically significant electrocardiogram (ECG) abnormality, including a marked baseline 
prolonged QT/QTc ([QT interval/corrected QT interval], eg, a repeated demonstration of 
a QTc interval >500 ms). 
 
3.2.8 Medcial 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 History or evidence of active, noninfectious pneumonitis that required treatment with 
steroids. 
 
3.2.10 History of interstitial lung disease. 
 
3.2.11 Participants known to be positive for the human immunodeficiency virus (HIV), Hepatitis 
B antigen (HepBsAg), or Hepatitis C virus (HCV) RNA. HIV-positive participants on 
combination antiretroviral therapy are ineligible because of the potential for 
pharmacokinetic interactions with Pembrolizumab and/or eribulin mesylate.  In addition, 
these participants are at increased risk of lethal infections.   
 


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3.2.12 Individuals with a history of a second 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 5 years and are deemed by the investigator to be at low risk 
for recurrence of that malignancy. Individuals with the following cancers are eligible if 
diagnosed and treated within the past 5 years: cervical cancer in situ, and non-melanoma 
cancer of the skin.  Patients with other cancers diagnosed within the past 5 years and felt 
to be at low risk of recurrence should be discussed with the study sponsor to determine 
eligibility.  
 
3.2.13 Has received a live vaccine within 28 days of planned start of study therapy. 
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. 
The accrual targets will reflect the expected accrual over the life of the study.  
  
3.4 
Inclusion Criteria for Crossover Therapy 
 
3.4.1 ECOG performance status ≤2 (Karnofsky ≥60%, see Appendix A) 
 
3.4.2 Participants must have normal organ and marrow function as defined below: 
 
− 
absolute neutrophil count 
≥1,000/mcL 
− 
platelets 
≥75,000/mcL 
− 
total bilirubin 
≤1.5 × institutional upper limit of normal (ULN) (≤1.5 
x institutional upper limit of normal (ULN) in patients with well documented 
Gilbert’s Syndrome) 
− 
AST(SGOT)/ALT(SGPT) 
≤3.0 × institutional ULN (≤ 5.0 × institutional ULN 
with documented liver metastases,  
 
3.4.3 Female subjects of childbearing potential, as defined in section 3.1.14, must have either a 
negative urine or a negative serum pregnancy test within seven (7) days of first dose of 
pembrolizumab.  If a urine test is positive or cannot be confirmed as negative, a serum 
pregnancy test will be required. 
 
3.4.4 Participants with accessible disease must be willing to undergo a research biopsy before 
beginning crossover therapy. 
 
 
3.5 
Exclusion Criteria for Crossover Therapy 
 
3.5.1 Clinically significant electrocardiogram (ECG) abnormality, including a marked baseline 
prolonged QT/QTc ([QT interval/corrected QT interval], eg, a repeated demonstration of 
a QTc interval >500 ms). 
 


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3.5.2 History or evidence of active, noninfectious pneumonitis that required treatment with 
steroids. 
 
 
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. 
 
Participants who are randomized, but do not initiate study treatment will be replaced.  
 
The following registration/randomization procedures should be followed: 
 
• An investigator will confirm eligibility criteria and a member of the study team will 
complete the protocol-specific eligibility checklist. 
 
• The eligibility checklist(s) and all pages of the consent form(s) will be faxed to the ODQ 
at 617-632-2295. 
 
• The ODQ Registrar will (a) review the eligibility checklist, (b) register the participant on 
the protocol, and (c) randomize the participant. 
 
• An email confirmation of the registration and/or randomization will be sent to the Overall 
PI, study coordinator(s) from the Lead Site 
 
• Prior to beginning crossover therapy, the crossover eligibility section of the checklist 
should be completed and signed by the treating investigator and re-faxed to the ODQ at 
617-632-2295. The ODQ registrar will review the eligibility checklist, change the 
subject’s treatment arm, and notify the study team. 
 
 
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.   


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4.3 
General Guidelines for Other Investigative Sites 
 
Not applicable  
 
4.4 
Registration Process for Other Investigative Sites 
 
Not applicable 
 
5. TREATMENT PLAN 
 
 
5.1 
Treatment Regimen 
 
This is a phase 2 study of treatment with eribulin mesylate +/- pembrolizumab, in metastatic HR-
positive breast cancer. Eighty-eight participants will be randomized (1:1) to eribulin mesylate 
with pembrolizumab (Arm A) or eribulin mesylate (Arm B). Participants randomized to eribulin 
mesylate monotherapy will have the option to receive pembrolizumab monotherapy at time of 
progression but they must meet the criteria in section 5.2.3 and begin treatment within 2 months 
of progression to crossover. Eribulin mesylate will be administered on days 1 and 8 of each 
21day cycle.  Pembrolizumab will be administered on day 1 of each 21 day cycle. If treatment is 
delayed, all protocol required assessments will be delayed accordingly. 
 
Treatment will be administered on an outpatient basis.  Reported adverse events and potential 
risks are described in Section 7.  Appropriate dose modifications are described in Section 6.  
Details of the regimen are described in Table 1/2.  No investigational or commercial agents other 
than those described below may be administered with the intent to treat the participant’s 
malignancy. 
Treatment regimen is described in Table 1 (Arm A) and in Table 2 (Arm B). 
 
Table 1: Regimen description- ARM A 
Regimen Description 
Agent 
Premedication
; Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
Pembrolizumab 
Not routinely 
necessary unless 
prior infusion 
reaction. 
 
200mg- iv 
 
IV over 
approximately 30 
minutes (range: 
25-40 minutes). 
Infuse prior to 
starting eribulin 
mesylate 
infusion. 
Day 1, q3w 
21 days 
(3 weeks) 
 
 
 


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Eribulin mesylate  
 
No routine 
premedication 
required 
1.4 mg/m2 
D1 and 
D8iv* 
IV, 2-5 minutes. 
May be 
administered 
diluted in 100 mL 
normal saline.  
 
Days 1 and 
8, iv 
 
 
 21 day   
(3 weeks) 
*Dose reductions may be made as per Tables 4-7.  Further details about dose reductions can be found in Section 6. 
 
Table 2: Regimen description- ARM B 
Regimen Description 
Agent 
Premedications
; Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
Eribulin mesylate  
 
No routine 
premedication 
required 
1.4 mg/m2 *  
iv 
IV, 2-5 minutes. 
May be 
administered 
diluted in 100 mL 
normal saline.  
 
Days 1 and 
8, iv * 
 
 
 21 day   
(3 weeks) 
*Dose reductions may be made as per Tables 6-7.  Further details about dose reductions can be found in 
Section 6. 
Note: After progression patients who wish can receive pembrolizumab200mg, at a final concentration of 1 
mg/mL to 10 mg/mL in NS or D5, every 21 days. Pembrolizumab should be administered as described in 
Arm A, pre-treatment criteria will also follow what is recommended in Arm A.    
 
5.2 
Pre-Treatment Criteria, Treatment, end off treatment and follow up visits  
 
5.2.1 Cycle 1, Day 1 
 
Criteria to treat at cycle 1 day 1: 
• Absolute neutrophil count >1500/ mcL 
• Platelets >100,000/ mcL 
• ALT and AST <2.5 x ULN in a patient with no documented liver metastases; ALT 
and AST <5.0 x ULN in a patient with documented liver metastases 
• Total bilirubin <1.5 x ULN (2.0 x ULN in a patient with well documented Gilbert’s 
syndrome) 


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5.2.2 Day 8 and Day 1 of subsequent cycles (including C1D1 and beyond of Crossover) 
 
 
 
5.3 
Agent Administration 
 
5.3.1 Pembrolizumab 
Pembrolizumab will be administered by trained medical personnel at the investigational site.  
Treatment compliance will be monitored through documentation of study treatment 
administration in the subject’s medical record. 
 
Pembrolizumab will be administered in clinic on day 1 (+/- 3 days) of each cycle. It will be 
administered as a 30 minute IV infusion.  Sites should make every effort to target infusion timing 
to be as close to 30 minutes as possible.  However, given the variability of infusion pumps from 
site to site, a window of -5 minutes and +10 minutes is permitted (i.e., infusion time is 25-
40min). 
 
Pembrolizumab should be administered prior to eribulin mesylate administration.  There should 
be no overlap in timing of the two administrations. 
 
5.3.2 Eribulin mesylate  
Eribulin mesylate will be administered by trained medical personnel at the investigational site.  
Treatment compliance will be monitored through documentation of study treatment 
administration in the subject’s medical record. 
 
Please refer to the FDA-approved package insert for eribulin mesylate for product information 
and a comprehensive list of adverse events. 
 
Eribulin mesylate will be administered on Days 1 and 8 of each 21-day cycle Day 8 Eribulin 
mesylate administration may occur at DFCI Milford, as needed.  
 
Eribulin mesylate will be dosed per institutional guidelines. 
 
5.4 
Discontinuation of pembrolizumab and Second Course Phase (Retreatment Period) 
 
If pembrolizumab is stopped for toxicity, participants are permitted to continue on protocol 
therapy with eribulin mesylate alone.  
Criteria to treat at day 8 and day 1 of subsequent cycles: 
• Absolute neutrophil count >1000/ mcL 
• Platelets >75,000/ mcL 
• ALT and AST <3.0 x ULN in a patient with no documented liver metastases; ALT 
and AST <5.0 x ULN in a patient with documented liver metastases 
• Total bilirubin <1.5 x ULN (2.0 x ULN in a patient with well documented Gilbert’s 
syndrome) 


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Participants may elect to stop pembrolizumab and eribulin mesylate with SD, PR or CR after at 
least 27 weeks of treatment and having had at least two treatments with pembrolizumab after 
documentation of the SD or response. 
 
Subjects who stop pembrolizumab in these conditions may be eligible additional pembrolizumab 
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 pembrolizumab after attaining an investigator-
determined confirmed SD, PR or CR according to RECIST 1.1, and 
1. Was treated for at least 27 weeks with pembrolizumab before discontinuing 
therapy 
2. Received at least two treatments with pembrolizumab beyond the date when the 
initial SD or response was declared 
 
 
OR 
 
o Had SD or response and stopped pembrolizumab treatment after 27 weeks of study 
therapy for reasons other than disease progression or intolerability. 
Subjects who restart treatment will be retreated at the same dose and dose interval as when they 
last received pembrolizumab.  Visit requirements are as outlined for subjects on the initial 
treatment phase of the trial.  
 
 
5.5 
Discontinuation of eribulin mesylate  
 
If eribulin mesylate is stopped for toxicity, participants are permitted to continue on therapy with 
pembrolizumab alone. 
 
 
5.6 
General Concomitant Medication and Supportive Care Guidelines 
 
5.6.1 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 
vaccination may be required.  The investigator should discuss any questions regarding this with 
the Merck Clinical team.  The final decision on any supportive therapy or vaccination rests with 
the investigator and/or the subject's primary physician.  
 
Acceptable Concomitant Medications 


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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.  All concomitant medication will be recorded on the case report form (CRF) 
including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and 
fluids.  If changes occur during the trial period, documentation of drug dosage, frequency, route, 
and date may also be included on the CRF. 
All concomitant medications received within 28 days before the first dose of trial treatment and 
30 days after the last dose of trial treatment should be recorded.  Concomitant medications 
administered after 28 days after the last dose of trial treatment should be recorded for SAEs as 
specified in Section 7.2. 
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 
• Chemotherapy not specified in this protocol 
• Biologic or targeted agents not specified in this protocol 
• Investigational agents other than pembrolizumab  
• Radiation therapy 
• Any systemically active oral, injected, or implanted hormonal method of contraception 
except for progesterone coated intrauterine devices (IUDs) that had been previously 
implanted. 
• Estrogen replacement therapy. 
• 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.  
• Systemic glucocorticoids for any purpose other than to modulate symptoms from an 
event of clinical interest of suspected immunologic etiology.  The use of physiologic 
doses of corticosteroids (10 mg prednisone by mouth daily or equivalent) is allowed.. 
• Care should be taken with concomitant use of strong CYP3A4 inhibitors/inducers (e.g., 
ketoconazole and itraconazole; see Appendix B) and eribulin mesylate. An alternate 
medication with no or minimal potential to inhibit CYP3A4 should be considered. If a 
strong CYP3A4 inhibitor is co-administered with eribulin mesylate, patients should be 
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Subjects who, in the assessment by the investigator, require the use of any of the aforementioned 
treatments for clinical management should be removed from the trial.  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. 
5.6.2 Supportive Care Guidelines – general medications 
 
The following treatments are permitted throughout the duration of the study treatment phase and 
during follow-up: 
 
• Standard therapies for pre-existing medical conditions unless listed as prohibited therapy 
below. 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. 
• Hematopoietic growth factors (e.g., G-CSF, granulocyte macrophage colony stimulating 
factor) may be used at investigator’s discretion for the primary prophylaxis and/or 
management of treatment-emergent neutropenia and/or for secondary prophylaxis as per 
NCCN/European Society for Medical Oncology guidelines 47, 48 or local standard 
practice. However, treatment with granulocyte-colony stimulating factors will not 
permitted in cycle 1 unless the patient has febrile neutropenia and the physician considers 
its use as clinically indicated. It will be left to the treating physician choice from cycle 2.  
• 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. 
• Anticoagulants - Anticoagulation with heparin, heparin derivatives, and/or warfarin may 
be given at the discretion of the treating physician. Coagulation parameters should be 
checked at least once monthly, or more frequently at discretion of treating physician.  
• 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. 
 
 
5.7 
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 
until one of the following criteria applies: 


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• 
Disease progression – in any subject who showed first radiologic evidence of 
progressive disease (PD) by RECIST 1.1 (see Section 11.1.4) and is deemed 
clinically stable, it is at the discretion of the investigator to continue treating the 
subject until progression is confirmed at least 4 weeks from the date of the first 
radiologic evidence of PD.  Further details are described below. 
 
• 
Participants who have attained a confirmed complete response (CR) that have been 
treated for at least 27 weeks with pembrolizumab and had at least two treatments with 
pembrolizumab beyond the date when the initial CR was declared.  Concurrent 
discontinuation of Eribulin mesylate is at the discretion of the investigator.  Subjects 
who then experience radiographic disease progression may be eligible for up to one 
year of additional treatment with pembrolizumab via the Second Course Phase (see 
below) at the discretion of the investigator if no systemic cancer treatment other than 
Eribulin mesylate was administered since the last dose of pembrolizumab, the subject 
meets the safety parameters listed in the Inclusion/Exclusion criteria, and the trial is 
open.  Subjects will resume therapy at the same dose and schedule as at the time of 
initial discontinuation. 
 
• 
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 recorded in the clinical trials management 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, MD MPH at 617-632-3800 or stolaney@partners.org.  
 
Confirmation of Progressive Disease 
 
Pembrolizumab, like other immunotherapeutic agents, may produce antitumor effects by 
potentiating endogenous cancer-specific immune responses. The response patterns seen with 
such an approach may extend beyond the typical time course of image responses seen with 
cytotoxic agents, and can manifest as a clinical response after an initial increase in tumor burden 


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or even the appearance of new lesions. 
 
For any subject who showed first radiologic evidence of progressive disease (PD) by RECIST 
1.1 (see Section 11.1.4) and is deemed clinically stable, it is at the discretion of the investigator 
to continue treating the subject until progression is confirmed at least 4 weeks from the date of 
the first radiologic evidence of PD. If progression is confirmed, the subject will be discontinued 
from study treatment. Otherwise, the subject will continue treatment and radiographic scans. Any 
subject who had initial radiologic progression and is deemed clinically unstable should be 
discontinued from both study drugs and no subsequent scan for confirmation is required. 
 
Further details are as below: 
 
For purposes of PFS assessment on this trial, in addition to radiographic assessment of tumor 
response or progression, the investigator should into account the clinical condition/stability of 
subjects. 
 
• Clinically stable is defined by the following criteria: 
o Absence of signs and symptoms (including worsening of laboratory values) 
indicating disease progression 
o No decline in ECOG performance status 
o Absence of rapid progression of disease 
o Absence of progressive tumor(s) at critical anatomical sites (eg, cord 
compression) requiring urgent alternative medical intervention 
 
Any subject who showed first radiologic evidence of progressive disease and is deemed 
clinically unstable should be discontinued from both study drugs and is not required to have 
repeat imaging for confirmation. 
 
For a clinically stable subject with first radiologic evidence of progressive disease (ie, 
unconfirmed progression of disease), it is at the discretion of the investigator to continue treating 
the subject with the assigned treatment per protocol until progression of disease is confirmed on 
a subsequent scan at least 4 weeks later. If progression is not confirmed on the subsequent scan, 
the subject should continue to receive study treatment and have radiographic scans performed 
every 6 weeks if the patient has been on study for less than 24 weeks, or every 9 weeks for 
patients who have been on study greater than 24 weeks to monitor disease status. If radiologic 
progression is confirmed by subsequent scans, then the subject will be discontinued from study 
treatment. Exceptions may be considered to continue treatment in the presence of clinically 
stable or improved condition only after consultation with the principal investigator. 
 
5.8 
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.   
 
In patients who come off trial for a reason other than progressive disease, if possible during the 
follow-up period, scans should be performed every 9 weeks (if the patients is within 27 weeks of 


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initiation of study treatment) or every 12 weeks (if the patient is greater than 54 weeks from 
initiation of study treatment) to evaluate for disease progression. 
 
All participants will be followed annually by phone or by medical record until death.   
 
 
5.9 
 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). 
 
The research team submits a completed Off Treatment/Off Study form to ODQ when a 
participant comes off study. This form can be found on the ODQ website or obtained from the 
ODQ registration staff.  
 
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
Dose delays and modifications will be made as indicated in the following section. The 
descriptions and grading scales found in the revised NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 4.0 will be utilized for dose delays and dose modifications. A 
copy of the CTCAE version 4.0 can be downloaded from the CTEP website 
 http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
Dosing interruptions are permitted in the case of medical / surgical events or logistical reasons  
not related to study therapy (e.g., elective surgery, unrelated medical events, patient vacation, 
and/or holidays). Subjects should be placed back on study therapy within 3 weeks of the 
scheduled interruption, unless otherwise discussed with the Sponsor. The reason for interruption 
should be documented in the patient's study record. 
 
If there are dosing delays for any reason, all study assessments are to be delayed in the same 
fashion, such that that scans and other assessments occur in conjunction with cycles of treatment. 
 
Eribulin mesylate doses may be delayed and/or reduced.  No re-escalation will be allowed.   
Pembrolizumab may be delayed as a result of toxicities but there are no dose reductions 
allowed.  
 
For toxicities in this Section, which are attributable to pembrolizumab alone, only 
pembrolizumab should be held as directed.  It is permissible to continue eribulin mesylate 
despite discontinuation of pembrolizumab in these select cases. This will be left at physician 
discretion.  


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For toxicities in this Section, which are attributable to eribulin mesylate alone, it is 
permissible to continue pembrolizumab and is left to the treating investigator’s discretion.  
If a decision to stop indefinitely eribulin mesylate is made, it is permissible to continue 
pembrolizumab despite discontinuation of eribulin mesylate  in these select cases. This will 
be left at physician discretion. 
 
  
6.1 
Management of toxicities attributable to pembrolizumab  
  
Adverse events (both non-serious and serious) associated with pembrolizumab exposure may 
represent an immunologic etiology.  These adverse events may occur shortly after the first dose 
or several months after the last dose of treatment.  Pembrolizumab must be withheld for drug-
related toxicities and severe or life-threatening AEs as per Table 4 below. 
 
Table 4: Dose modification guidelines for Pembrolizumab for drug-related adverse events 
 
General instructions: 
1. Corticosteroid taper should be initiated upon AE improving to less than or equal to Grade 1 baseline and continue to 
taper over at least 4 weeks. 
2. For situations where pembrolizumab has been withheld, pembrolizumab can be resumed after AE has been reduced to 
less than or equal to Grade 1 or baseline and corticosteroid has been tapered. Pembrolizumab should be permanently 
discontinued if AE does not resolve within 12 weeks of last dose or corticosteroids cannot be reduced to ≤10 mg 
prednisone or equivalent per day within 12 weeks.  
3. For severe and life-threatening irAEs, IV corticosteroid should be initiated first followed by oral steroid. Other 
immunosuppressive treatment should be initiated if irAEs cannot be controlled by corticosteroids. 
 
Immune-related 
AEs 
Toxicity 
grade or 
conditions 
(CTCAEv4.0) 
Action taken to 
pembrolizumab 
irAE management with 
corticosteroid and/or other 
therapies  
Monitor and follow-up  
Pneumonitis  
Grade 2 
Withhold  
• Administer corticosteroids 
(initial dose of 1-2 mg/kg 
prednisone or equivalent) 
followed by taper 
 
• 
Monitor participants for signs 
and symptoms of pneumonitis 
• 
Evaluate participants with 
suspected pneumonitis with 
radiographic imaging and 
initiate corticosteroid treatment 
• 
Add prophylactic antibiotics for 
opportunistic infections 
Grade 3 or 4, 
or recurrent 
Grade 2 
Permanently 
discontinue 
Diarrhea / Colitis  Grade 2 or 3 
Withhold 
• Administer corticosteroids 
(initial dose of 1-2 mg/kg 
prednisone or equivalent) 
followed by taper 
 
• 
Monitor participants for signs 
and symptoms of enterocolitis 
(ie, diarrhea, abdominal pain, 
blood or mucus in stool with or 
without fever) and of bowel 
perforation (ie, peritoneal signs 
and ileus). 
• 
Participants with ≥ Grade 2 
diarrhea suspecting colitis 
should consider GI consultation 
and performing endoscopy to 
rule out colitis.  
Grade 4 
Permanently 
discontinue 


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• 
Participants with diarrhea/colitis 
should be advised to drink 
liberal quantities of clear fluids.  
If sufficient oral fluid intake is 
not feasible, fluid and 
electrolytes should be 
substituted via IV infusion. 
AST / ALT 
elevation or 
Increased 
bilirubin 
Grade 2 
Continue 
• Consider administering 
corticosteroids (initial dose 
of 0.5 - 1 mg/kg prednisone 
or equivalent) followed by 
taper 
• 
Monitor with liver function tests 
(consider weekly or more 
frequently until liver enzyme 
value returned to baseline or is 
stable 
Grade 3 or 4  
Withhold 
• Administer corticosteroids 
(initial dose of 1-2 mg/kg 
prednisone or equivalent) 
followed by taper 
Type 1 diabetes 
mellitus (T1DM) 
or 
Hyperglycemia 
 
Newly onset 
T1DM or  
Grade 3 or 4 
hyperglycemia 
associated 
with evidence 
of β-cell 
failure 
Withhold 
• Initiate insulin replacement 
therapy for participants 
with T1DM  
• Administer anti-
hyperglycemic in 
participants with 
hyperglycemia  
 
• 
Monitor participants for 
hyperglycemia or other signs 
and symptoms of diabetes. 
Hypophysitis 
Grade 2 
Withhold 
• Administer corticosteroids 
and initiate hormonal 
replacements as clinically 
indicated.   
 
• 
Monitor for signs and symptoms 
of hypophysitis (including 
hypopituitarism and adrenal 
insufficiency)  
Grade 3 or 4  
Withhold or 
permanently 
discontinue1  
Hyperthyroidism  Grade 2 
Continue  
• Treat with non-selective 
beta-blockers (eg, 
propranolol) or 
thionamides as appropriate  
• 
Monitor for signs and symptoms 
of thyroid disorders.  
 
Grade 3 or 4  
Withhold or  
permanently 
discontinue1 
Hypothyroidism 
Grade 2-4 
Continue  
• Initiate thyroid 
replacement hormones (eg, 
levothyroxine or 
liothyroinine) per standard 
of care 
• 
Monitor for signs and symptoms 
of thyroid disorders.  
 
Nephritis and 
Renal 
dysfunction 
Grade 2 
Withhold  
• Administer corticosteroids 
(prednisone 1-2 mg/kg or 
equivalent) followed by 
taper. 
• 
Monitor changes of renal 
function 
 
Grade 3 or 4  
Permanently 
discontinue 
Myocarditis 
Grade 1 or 2 
Withhold 
• Based on severity of AE 
administer corticosteroids 
• 
Ensure adequate evaluation to 
confirm etiology and/or exclude 
other cause 
 
Grade 3 or 4 
Permanently 
discontinue 
Infusion 
Reaction2 
Grade 3 or 4 
Permanently 
discontinue 
• See Table 5 
• 
See Table 5 
All other 
immune-related 
AEs 
Intolerable/ 
persistent 
Grade 2 
Withhold 


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Grade 3 
Withhold or 
discontinue 
based on the type 
of event.  Events 
that require 
discontinuation 
include and not 
limited to:  
Gullain-Barre 
Syndrome, 
encephalitis 
• Based on type and severity 
of AE administer 
corticosteroids  
• 
Ensure adequate evaluation to 
confirm etiology and/or exclude 
other causes 
 
Grade 4 or 
recurrent 
Grade 3  
Permanently 
discontinue 
1. Withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician.   
2. See Table 5 for further guidance on all grades of pembrolizumab infusion reactions.  
NOTE:  
For participants with Grade 3 or 4 immune-related endocrinopathy where withhold of pembrolizumab is required, 
pembrolizumab may be resumed when AE resolves to ≤ Grade 2 and is controlled with hormonal replacement therapy or 
achieved metabolic control (in case of T1DM).   
 
 
Supportive care for pembrolizumab toxicity, particularly suspected immune-mediated toxicity 
Subjects should receive appropriate supportive care measures as deemed necessary by the treating 
investigator.  Suggested supportive care measures for the management of adverse events with 
potential immunologic etiology are outlined below.  
 
Note that several courses of steroid tapering may be necessary as symptoms may worsen when the 
steroid dose is decreased. For each disorder, attempts should be made to rule out other causes such 
as metastatic disease or bacterial or viral infection, which might require additional supportive care. 
The treatment guidelines are intended to be applied when the investigator determines the events to 
be related to pembrolizumab. 
 
Note: if after the evaluation the event is determined not to be related, the investigator is instructed 
to follow the guidance in Section 7 but does not need to follow the treatment guidance provided. 
 
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin 
photography as part of evaluation of the event.  
 
 
• Management of Infusion Reactions: Signs and symptoms usually develop during or 
shortly after drug infusion and generally resolve completely within 24 hours of completion 
of infusion. Table 5 below shows treatment guidelines for subjects who experience an 
infusion reaction associated with administration of pembrolizumab. 
Table 5: Infusion Reaction Treatment Guidelines for pembrolizumab 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
Grade 1 
Mild reaction; infusion interruption 
not indicated; intervention not 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
None 


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NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
indicated 
Grade 2 
Requires infusion interruption but 
responds promptly to symptomatic 
treatment (e.g., antihistamines, 
NSAIDS, narcotics, IV fluids); 
prophylactic medications indicated for 
< =24 hrs 
Stop Infusion and monitor symptoms. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
If symptoms resolve within one hour of stopping 
drug infusion, the infusion may be restarted at 
50% of the original infusion rate (e.g., from 100 
mL/hr to 50 mL/hr).  Otherwise dosing will be 
held until symptoms resolve and the subject 
should be premedicated for the next scheduled 
dose. 
Subjects who develop Grade 2 toxicity despite 
adequate premedication should be 
permanently discontinued from further trial 
treatment administration. 
Subject may be premedicated 1.5h 
(± 30 minutes) prior to infusion of 
pembrolizumab with: 
 
Diphenhydramine 50 mg po (or 
equivalent dose of antihistamine). 
 
Acetaminophen 500-1000 mg po 
(or equivalent dose of antipyretic). 
Grades 3 or 4 
Grade 3: 
Prolonged (i.e., not rapidly responsive 
to symptomatic medication and/or 
brief interruption of infusion); 
recurrence of symptoms following 
initial improvement; hospitalization 
indicated for other clinical sequelae 
(e.g., renal impairment, pulmonary 
infiltrates) 
Grade 4: 
Life-threatening; pressor or ventilatory 
support indicated 
Stop Infusion. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Epinephrine 
 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
Hospitalization may be indicated. 
Subject is permanently discontinued from 
further trial treatment administration. 
No subsequent dosing 
Appropriate resuscitation equipment should be available in the room and a physician readily available during the period of drug 
administration. 
 
 
 
6.2 
Management of toxicities attributable to Eribulin mesylate 
Subjects should be carefully monitored for toxicity.  If a treatment delay is required for eribulin 
mesylate toxicity, treatment with pembrolizumab is allowed.  If toxicities recur, the dose of 
eribulin mesylate should be reduced.  Once reduced, the dose of eribulin mesylate will not be re-
escalated.   
Dose adjustment recommendations for eribulin mesylate are shown in Tables 6 and delays are 
discussed below in Table 7.  
 If treatment with eribulin mesylate is withheld permanently, participants are allowed to continue 
to receive pembrolizumab therapy.   
 
Table 6. Eribulin mesylate dose modifications  


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Starting dose 
1.4 mg/m2 (1 cycle = 21 days) 
1st dose reduction 
1.1 mg/m2 
2nd dose reduction 
0.7 mg/m2 
 
Table 7. Dose adjustments for Eribulin Mesylate 
Adverse reaction/Toxicity 
Grade/Details 
Eribulin mesylate dose 
modification 
ANC 
< 500 cells/mm3 lasting > 7 days  
with or without use of growth 
factors 
Hold eribulin mesylate until 
recovery to grade < 2 and reduce by 
1 dose level. Prophylactic growth 
factor support should be instituted 
for subsequent cycles. 
< 500 cells/mm3 lasting ≤7 days 
without use of growth factors 
Hold eribulin mesylate until 
recovery to grade < 2. Resume 
eribulin mesylate at the same dose.  
Growth factor support should be 
provided. 
< 500 cells/mm3 lasting ≤ 7 days in 
despite use of growth factors 
Hold eribulin mesylate until 
recovery to grade < 2, and reduce 
by 1 dose level. Continue ongoing 
prophylactic growth factor support 
for subsequent cycles. 
<1000 /mm3 with fever or infection 
without use of growth factors  
Hold eribulin mesylate until 
recovery to grade < 2. Resume 
eribulin mesylate at the same dose.  
Growth factor support should be 
provided. 
<1000 /mm3 with fever or infection 
despite use of growth factors 
Hold eribulin mesylate until 
recovery to grade < 2, and reduce 
by 1 dose level. Growth factor 
support should be provided. 
<1000 /mm3 without fever or 
infection  
First occurrence: Hold eribulin 
mesylate until recovery to grade < 2 
then resume at the same dose.  
Growth factor support should be 
provided .  
 
If uncomplicated neutropenia 
(<1000 /mm3 without fever or 
infection) occurs/recurs despite 
growth factor support, then hold 
eribulin mesylate until recovery to 
grade < 2. Eribulin mesylate may 
be then be resumed at the same 
dose with growth factor support, or 
it may be reduced by one dose 
level.  
 
If a patient misses two consecutive 
Day 8 eribulin mesylate doses due 
to uncomplicated neutropenia, then 
hold eribulin mesylate until 
recovery to grade < 2 , reduce by 1 
dose level, and consider 


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prophylactic growth factor support 
for subsequent cycles.  
Platelets 
Grade 2 
Hold eribulin mesylate until 
recovery to grade < 2  
 Grade 3 
Hold eribulin mesylate until 
recovery to grade < 2 then reduce 
by 1 dose level. 
Grade 4 
Hold eribulin mesylate until 
recovery to grade < 2 then reduce 
by 1 dose level. 
Anemia 
Grade 4 
Hold eribulin mesylate until 
recovery to grade < 2 then reduce 
by 1 dose level. PRBC transfusions 
are permitted to treat anemia on 
protocol. 
 Peripheral neuropathy 
Grade 2 
For intolerable (as determined by 
physician and patient) grade 2, 
decrease eribulin mesylate by one 
dose level.  
 
Grade 3 
Hold eribulin mesylate until 
recovery to grade < 2 then reduce 
by 1 dose level.  
 
Grade 4 
Permanently discontinue eribulin 
mesylate   
Non-hematologic toxicity 
Grade 3, first occurrence 
Hold eribulin mesylate until 
recovery to grade < 2. Maximize 
supportive care measures.  If 
symptom recovery occurs within 1 
week, then eribulin mesylate may 
be resumed at the same dose if 
deemed appropriate by the 
investigator, otherwise, reduce by 1 
dose level. 
Grade 3, despite maximal 
supportive measures 
Hold eribulin mesylate until 
recovery to grade < 2 then reduce 
by 1 dose level. 
Grade 4 
Hold eribulin mesylate until 
recovery to grade < 2 then reduce 
by 1 dose level. 
ANC = absolute neutrophil count  
• Toxicities graded in accordance with National Cancer Institute Common Toxicity Criteria for AEs, version 
4.0 
• Discontinuation should be considered if a dose reduction to <0.7 mg/m2 is required. 
• A minimum of 6 days between D1 and D8 of eribulin mesylate administration will be required and a 
minimum of 10 days between D8 and D22 (i.e. D1 of next cycle) will be required. 
• If a patient does not meet criteria to receive Day 8 of eribulin mesylate, the investigator may decide to make 
up the dose on Day 15 of the cycle. If the Day 15 dose is given, the next cycle should not be initiated until 2 
weeks later. If the Day 15 dose is held again, the next cycle may initiate the following week.   
•  Once a dose is reduced, it should not be re-escalated.   
• Treatment with eribulin mesylate will be discontinued if participants require a delay greater than 3 weeks. 
• Treatment with pembrolizumab is allowed to continue if eribulin mesylate is discontinued.    
 
 


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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 and the characteristics of an observed AE will 
determine whether the event requires expedited reporting in addition to routine reporting. 
 
7.1 
Adverse Events Lists 
 
7.1.1 Adverse Event List(s) for pembrolizumab 
 
In the pembrolizumab monotherapy trials (P001/P002, P012, P013, and P028, plus the P011 
monotherapy arm), the overall incidence of AEs ranged from 83.0% (73 of 88 subjects in P012) 
to 100% (10 of 10 subjects in P011). The most commonly reported AEs included fatigue, 
diarrhea, decreased appetite, nausea, and anemia. The incidence of drug –related AEs (DRAEs) 
ranged from 39.8% (35 of 88 subjects in P013) to 80.0% (8 of 10 subjects in P011). The most 
commonly reported DRAEs across all studies were nausea, fatigue, and diarrhea. The incidence 
of Grade 3-5 DRAEs across studies ranged from 6.8% (6 of 88 in P013) to 12.0% (187 of 1562 
subjects) in P001/P002. The most commonly reported Grade 3-5 DRAEs were anemia, alanine 
aminotransferase increased, and aspartate aminotransferase increased. Most subjects who 
experienced an AE continued in the study, with the incidence of AEs leading to discontinuation 
ranging from 1.9% (8 of 430 subjects in P028) to 12.3% (192 of 1562 subjects in P001/P002). 
The majority of AEs leading to discontinuation were not considered drug related. 
Discontinuations due to a DRAE were infrequent and ranged from 0% (no subjects in P011) to 
4.5% (4 of 88 subjects in P013). The most commonly reported DRAEs leading to discontinuation 
were pneumonitis, alanine aminotransferase increased, and aspartate aminotransferase increased. 
 
List of AEs considered expected:  
• Endorcine disorders: Adrenal insufficiency, Hyperthyroidism, Hypophysitis, 
Hypopituitarism, Hypothyroidism, Secondary adrenal insufficiency, Thyroid disorder 
• Eye disorders: Uveitis 
• Gastrointestinal disorders: Abdominal pain, Colitis, Diarrhoea,Pancreatitis 
• General disorders and administration site conditions: Asthenia, Pyrexia 
• Hepatobiliary disorders: Autoimmune hepatitis, Hepatitis 
• Infusion related reaction 
• Metabolism and nutrition disorders: Diabetic ketoacidosis, Hyponatremia, Type 1 
diabetes mellitus 
• Musculoskeletal and connective tissue disorders: Arthralgia, Back pain,  Myositis 
• Nervous system disorders: Guillain-Barré syndrome 
• Renal and urinary disorders: Nephritis 
• Respiratory, thoracic and mediastinal disorders: Cough,  Pneumonitis 
• Skin and subcutaneous tissue disorders: Pruritis, Rash, Severe skin reaction, Vitiligo 
 
 
 
7.1.2 Adverse Event List(s) eribulin mesylate  
 


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Most AEs on studies with eribulin mesylate are grade 1 or 2.  Peripheral neuropathy was an 
important AE leading to discontinuation of therapy.  
 
The most frequently reported eribulin mesylate related AEs were: 
• asthenia/fatigue  
• alopecia  
• neutropenia  
• nausea  
• anemia  
• pyrexia  
• leucopenia  
• anorexia  
• constipation  
• vomiting  
• and peripheral neuropathy  
 
 
 
 
7.2 
Adverse Event Characteristics 
 
• CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
• For expedited reporting purposes only:   
- 
AEs for the agent(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. 
 
7.3 
Expedited Adverse Event Reporting  
 
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 30 days of the last 
dose of treatment on the local institutional SAE form.  
 


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7.3.1 DF/HCC Expedited Reporting Guidelines 
 
Investigative sites within DF/HCC and DF/PCC will report SAEs directly to the DFCI 
Office for Human Research Studies (OHRS) per the DFCI IRB reporting policy.  
 
 
7.4 
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. 
 
7.5 
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.  
 
7.6 
Expedited Reporting to Merck 
A serious adverse event is any adverse event occurring at any dose or during any use of Merck’s 
product that: 
 
• Results in death; 
• Is life threatening; 
• Results in persistent or significant disability/incapacity; 
• Results in or prolongs an existing inpatient hospitalization; 
• Is a congenital anomaly/birth defect; 
• Is a new cancer (that is not a condition of the study); 
• Is associated with an overdose; 
• Is an other important medical event  
Any serious adverse event, or follow up to a serious adverse event, including death due to any 
cause other than progression of the cancer under study that occurs to any subject from the time 
the consent is signed through 90 days following cessation of treatment, or the initiation of new 
anti-cancer therapy, whichever is earlier, whether or not related to Merck product, must be 
reported within 24 hours to the Sponsor and within 2 working days to Merck Global Safety.  
 
Non-serious Events of Clinical Interest will be forwarded to Merck Global Safety and will be 
handled in the same manner as SAEs.  
 
Additionally, any serious adverse event, considered by an investigator who is a qualified 
physician to be related to Merck product that is brought to the attention of the investigator at any 
time outside of the time period specified in the previous paragraph also must be reported 
immediately to Merck. 
 
SAE reports and any other relevant safety information are to be forwarded to the Merck 


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Global Safety facsimile number:  +1-215-993-1220 
 
A copy of all 15 Day Reports and Annual Progress Reports is submitted as required by FDA, 
European Union (EU), Pharmaceutical and Medical Devices agency (PMDA) or other local 
regulators. Investigators will cross reference this submission according to local regulations to the 
Merck Investigational Compound Number (IND, CSA, etc.) at the time of submission.  
Additionally investigators will submit a copy of these reports to Merck & Co., Inc. (Attn: 
Worldwide Product Safety; FAX 215 993-1220) at the time of submission to FDA. 
 
All subjects with serious adverse events must be followed up for outcome. 
 
7.6.1 
Events of Clinical Interest (ECIs) 
 
Selected non-serious and serious adverse events are also known as Events of Clinical Interest 
(ECI) and must be reported within 24 hours to the Sponsor and within 2 working days to Merck 
Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220). 
 
For the time period beginning when the consent form is signed until treatment 
allocation/randomization, any ECI, or follow up to an ECI, that occurs to any subject must be 
reported within 24 hours to the Sponsor and within 2 working days to Merck Global Safety if it 
causes the subject to be excluded from the trial, or is the result of a protocol-specified 
intervention, including but not limited to washout or discontinuation of usual therapy, diet, 
placebo treatment or a procedure. 
For the time period beginning at treatment allocation/randomization through 90 days following 
cessation of treatment, or 30 days following cessation of treatment if the subject initiates new 
anticancer therapy, whichever is earlier, any ECI, or follow up to an ECI, whether or not related 
to Merck product, must be reported within 24 hours to the Sponsor and within 24 hours to Merck 
Global Safety. 
Events of clinical interest for this trial include: 
 
1. Overdose of Merck product, as defined in Section 7.13.2 - Definition of an Overdose for 
This Protocol and Reporting of Overdose to the Sponsor, that is not associated with 
clinical symptoms or abnormal laboratory results.  
2. An elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of 
normal and an elevated total bilirubin lab value that is greater than or equal to 2X the 
upper limit of normal and, at the same time, an alkaline phosphatase lab value that is less 
than 2X the upper limit of normal, as determined by way of protocol-specified laboratory 
testing or unscheduled laboratory testing.* 
 
*Note:  These criteria are based upon available regulatory guidance documents. The purpose 
of the criteria is to specify a threshold of abnormal hepatic tests that may require an 
additional evaluation for an underlying etiology.  
 
7.6.2 
Definition of an Overdose of Pembrolizumab for This Protocol and Reporting of Overdose to Merck 
 
For purposes of this trial, an overdose of pembrolizumab will be defined as any dose of 1,000 mg 


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or greater.  No specific information is available on the treatment of overdose of pembrolizumab. 
Appropriate supportive treatment should be provided if clinically indicated. In the event of 
overdose, the subject should be observed closely for signs of toxicity.  Appropriate supportive 
treatment should be provided if clinically indicated. 
 
If an adverse event(s) is associated with (“results from”) the overdose of a Merck product, the 
adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria are 
met. 
 
If a dose of Merck’s product meeting the protocol definition of overdose is taken without any 
associated clinical symptoms or abnormal laboratory results, the overdose is reported as a non-
serious Event of Clinical Interest (ECI), using the terminology “accidental or intentional 
overdose without adverse effect.” 
 
All reports of overdose with and without an adverse event must be reported within 24 hours to 
the Sponsor and within 2 working days hours to Merck Global Safety. (Attn: Worldwide Product 
Safety; FAX 215 993-1220) 
 
 
7.6.3 
Reporting of Pregnancy and Lactation to Merck 
 
Although pregnancy and lactation are not considered adverse events, it is the responsibility of 
investigators or their designees to report any pregnancy or lactation in a subject (spontaneously 
reported to them) that occurs during the trial. 
Pregnancies and lactations that occur after the consent form is signed but before treatment 
allocation/randomization must be reported by the investigator if they cause the subject to be 
excluded from the trial, or are the result of a protocol-specified intervention, including but not 
limited to washout or discontinuation of usual therapy, diet, placebo treatment or a procedure.   
Pregnancies and lactations that occur from the time of treatment allocation/randomization 
through 120 days following cessation of Sponsor’s product, or 30 days following cessation of 
treatment if the subject initiates new anticancer therapy, whichever is earlier, must be reported 
by the investigator.  All reported pregnancies must be followed to the completion/termination of 
the pregnancy. Pregnancy outcomes of spontaneous abortion, missed abortion, benign 
hydatidiform mole, blighted ovum, fetal death, intrauterine death, miscarriage and stillbirth must 
be reported as serious events (Important Medical Events). If the pregnancy continues to term, 
the outcome (health of infant) must also be reported. 
Such events must be reported within 24 hours to the Sponsor and within 2 working days to 
Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220) 
 
7.7 
Expedited Reporting to Eisai 
SAEs where the Overall PI considers a relationship to the eribulin therapy to be at least a 
reasonable possibility, will be reported to EISAI on a Medwatch 3500A form within one 
business days of the notification of the event. Serious adverse events (SAEs) that are not related 
to eribulin therapy and non-serious AEs will be provided to Eisai in the final study report and 
any interim reports provided.  


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Events  not considered to be serious adverse events are hospitalizations for:  
• routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition, or for elective procedures  
• elective or pre-planned treatment for a pre-existing condition that did not worsen  
• emergency outpatient treatment for an event not fulfilling the serious criteria outlined 
above and not resulting in inpatient admission  
• respite care  
 
The reports will be sent on MedWatch 3500A form to EISAI at the number/email listed below:  
 
        EISAI safety fax number (732-791-1111) Email: ESI_Safety@eisai.com  
 
 
7.8 
Routine Adverse Event Reporting 
 
All Grade 2 or greater Adverse Events 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. 
 
8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational and other 
agents administered in this study can be found in Section 7.1. 
 
8.1 
PEMBROLIZUMAB 
 
Please refer to the Investigator’s Brochure for detailed agent information, and to the FDA label 
for additional information. 
 
8.1.1 Description 
 
Pembrolizumab is a humanized monoclonal antibody of the IgG4/kappa isotype.  Other 
name: MK-3475, Keytruda.  Pembrolizumab blocks negative immune regulatory 
signaling by binding to the PD-1 receptor, inhibiting the interaction between PD-1 and its 
ligands. 
 
The molecular weight of Pembrolizumab is 148.9-149.5 KDa. 
 
8.1.2 Form 
 
Clinical supplies will be manufactured and provided by Merck as summarized in Table 7. 
 
Table 7:  Product Description 


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Product Name & Potency 
Dosage Form 
Pembrolizumab 100 mg/ 4mL 
Solution for Injection 
 
8.1.3 Storage and Stability 
 
Store intact vials between 2°C-8°C (36°F-46°F).  Do not freeze.  Protect from light by 
storing in the original box. 
 
Stability testing of the intact vials is ongoing. 
 
Administer prepared solutions immediately after preparation.  If not administered 
immediately, prepared solutions may be stored refrigerated for up to 20 hours.  
PEMBROLIZUMAB solutions may be stored at room temperature for a cumulative time 
of up to 4 hours.  This includes room temperature storage of liquid drug product solution 
in vials, room temperature storage of infusion solution in the IV bag, and the duration of 
infusion. 
 
8.1.4 Compatibility 
 
Compatible IV bag materials: PVC plasticized with DEHP, non-PVC (polyolefin), EVA, 
or PE lined polyolefin. 
 
8.1.5 Handling 
 
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.  
 
8.1.6 Availability 
 
Pembrolizumab is an investigational agent and will be supplied free of charge from 
Merck. 
 
8.1.7 Preparation 
 
Pembrolizumab solution for infusion must be diluted prior to administration.  Allow the 
required number of vials to equilibrate to room temperature.  Do not shake the vials.  Do 
not use if opaque or extraneous particulate matter other than translucent to white 
proteinaceous particles is observed.  Do not use if discolored.  To prepare the infusion 
solution add the dose volume of Pembrolizumab to an infusion bag containing 0.9& 
Sodium Chloride Injection, USP of 5% Dextrose Injection, USP.  Gently invert the bag 
10-15 times to mix the solution.  The final concentration must be between 1 mg/mL to 
10 mg/mL. 
 


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8.1.8 Administration 
 
Route of administration: IV infusion only.  Do not administer as an IV push or bolus 
injection. 
 
Method of administration: Infuse over approximately 30 minutes (range: 25-40 minutes) 
using an infusion set containing a low-protein binding 0.2 to 5 m in-line filter make of 
polyethersulfone or polysulfone.  Infusion rate should not exceed 6.7 mL/min.  A central 
line is not required however if a subject has a central venous catheter in place, it is 
recommended that it be used for the infusion.  Do not co-administer drugs through the 
same infusion line.  Following the infusion, flush the IV line with normal saline. 
 
8.1.9 Ordering 
 
Pembrolizumab will be obtained directly from Merck, the study sponsor. 
 
8.1.10 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.) 
 
8.1.11 Destruction and Return 
 
At the end of the study, unused supplies of Pembrolizumab should be destroyed 
according to institutional policies. Destruction will be documented in the Drug 
Accountability Record Form. 
 
8.2 
Eribulin mesylate 
Please refer to the Investigator’s Brochure for detailed agent information, and to the FDA label 
for additional information. 
 
The chemical name for eribulin mesylate is 11,15:18,21:24,28-Triepoxy-7,9ethano-12,15-
methano-9H,15H-furo[3,2-i]furo[2’,3’:5,6]pyrano[4,3-b][1,4]dioxacyclopentacosin-5(4H)-
one,2[(2S)-3-amino-2-hydroxypropyl]hexacosahydro-3-methoxy-26-methyl-20,27-
bis(methylene)-
(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-
methanesulfonate (salt).  It’s molecular weight is: 826.00. 
Structural formula: 
 


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8.2.1 Form 
Eribulin mesylate is a sterile, ready-to-use, clear, colorless aqueous solution for i.v. 
administration.   
 
Eribulin mesylate for i.v. injection will be supplied on an open-label basis by the sponsor in 
single-use vials.  Each single-use vial contains 1 mg/2 mL of clear, colorless solution.  Each 
single-use vial of eribulin mesylate is primarily packaged in a 5 mL nominal volume United 
States Pharmacopeia (USP) Type 1 Flint glass, stoppered with a FluroTec® plug stopper and 
sealed with an aluminum seal and a flip-off cap.  Twelve labeled vials of eribulin mesylate are 
packaged in a labeled carton.  Each of the 6 eribulin mesylate vials within a given labeled 
carton may be assigned individually to 6 different subjects or to the same subject, as required by 
the study site or as clinical supply inventory demands. 
 
8.2.2 Storage Conditions 
Eribulin mesylate must be stored in accordance with labeled storage conditions.  Intact and 
unopened vials must be stored at ambient room temperature.   Do NOT refrigerate or freeze.  
Shelf-life surveillance of the intact vials is ongoing.  The 0.5 mg/mL solution has been shown to 
be stable in syringes at ambient temperature and ambient lighting for up to 4 hours, or under 
refrigeration for up to 24 hours.  The drug is also stable at concentrations ranging from 0.005 
ng/mL to 0.2 mg/mL when diluted in normal saline (0.9% sodium chloride [NaCl]) and kept 
refrigerated in syringes or i.v. bags for up to 48 hours at ambient temperature and ambient 
lighting, or under refrigeration. It is not compatible with solutions with dextrose. 
Product vials that are opened and refrigerated between 2°C to 8°C (36°F – 46°F) must be used 
within 24 hours of being opened, and the remaining drug solution discarded. 
Temperature monitoring for eribulin mesylate is required at the storage location to ensure that 
the study drug is maintained within an established temperature range.  The investigator is 
responsible for ensuring that the temperature is monitored throughout the total duration of the 
trial and that records are maintained; the temperature should be monitored continuously by 
using either an in-house validated data acquisition system, a mechanical recording device such 
as a calibrated chart recorder, or by manual means, such that minimum and maximum 
thermometric values over a specific time period can be recorded and retrieved as required. 
 
 


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8.2.3 Handling 
Qualified personnel, familiar with procedures that minimize undue exposure to themselves and 
the environment, should undertake the preparation, handling, and safe disposal of the eribulin 
mesylate in a self-contained and protective environment. 
 
 
8.2.4 Availability 
Eribulin mesylate is being provided by Eisai for this trial.   
 
8.2.5 Ordering  
Eribulin mesylate will be ordered directly from Eisai. 
 
 
8.2.6 Treatment Compliance 
Eribulin mesylate will be administered by trained medical personnel at the investigational site.  
Treatment compliance will be monitored through drug accountability documentation, as well as 
the recording of study treatment administration in the subject’s medical record.  In addition, all 
details pertaining to the administration of eribulin mesylate, including but not limited to date, 
actual dose, and start and end times of each dose, will be recorded on the Study Drug 
Administration page of the subject’s eCRF. Clinical research associates (CRAs) will review 
treatment compliance during investigational site visits conducted during and at the completion of 
the study. 
 
8.2.7 Drug Supplies and Accountability 
 
Drug supplies and accountability will be as institutional policy.  
 
The investigator, or a responsible party designated by the investigator, will maintain a careful 
record of the inventory and disposition of the agent (investigational or free of charge) using the 
NCI Drug Accountability Record or another comparable drug accountability form. (See the CTEP 
website at http://ctep.cancer.gov/protocolDevelopment  for the “Policy and Guidelines for 
Accountability and Storage of Investigational Agents” or to obtain a copy of the drug 
accountability form.) 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
All patients will be asked to provide archival tumor tissue (either paraffin blocks or 20 unstained 
slides, ideally 4 micron thickness).  However, if archival tissue is not available or not evaluable, 
that will not be a basis to exclude the patient from any portion of the trial or the planned analysis.  
Archival tissue will be used for immune profiling assays to ascertain baseline values in patients 
who choose not to undergo baseline research fresh tissue biopsy or in whom there is insufficient 
tissue or failed testing of any of the planned assays from baseline fresh tissue biopsy. 
 
If obtained, we plan to use baseline biopsy tissue to perform a number of immune profiling 
assays, detailed below, and characterize the samples based on histology (TILs), protein 


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expression, and mRNA expression.  Additionally, specimens we will banked for possible future 
analysis. 
 
Serial blood draws for correlative science are required on this trial; blood draws will be obtained 
every 3 weeks on treatment days prior to the infusion of study drugs, at the end-of-treatment visit 
in patients who go off study for progressive disease, and all efforts will be made to obtain a 
sample at the time of progressive disease from participants who went off study for anything other 
than progressive disease.  We will perform flow cytometry on these samples to characterize 
protein expression of immune mediators as detailed in the lab manual.  Circulating tumor DNA 
will be assessed as well.   
 
An optional research biopsy will be collected for participants who have consented and have 
biopsy accessible tumor on arm B crossing over to pembrolizumab therapy.  
 
An optional  research biopsy at the time of progression will be collected for participants who 
consent and have biopsy accessible tumor.  These biopsies will undergo the same 
characterization testing as described for baseline biopsies.  
 
All patients will additionally be asked to provide a stool sample at three separate timepoints: 
prior to treatment, during treatment, and at the time of disease progression. A fourth collection 
may be requested from patients who experience grade ≥ 2 diarrhea after discussion with the PI. 
This collection is not required, but is strongly encouraged. These samples will be analyzed for 
microbiota content. 
 
Please refer to the separate laboratory manual for additional correlative details including 
collection, processing, and shipping instructions. 
 
 
9.1 
Summary table: research tissue and blood specimen collection 
 
Research 
Sampling 
Time point 
Contents 
Blood  
Cycle 1 Day 1 
1-9 mL Streck Tube 
5- 10mL green top tubes*  
Every Cycle Day 1 
5- 10mL green top tubes * 
Restaging Visits Only 
1-9mL Streck Tube 
Off Treatment for PD 
 
1-9mL Streck Tube 
5- 10mL green top tubes* 
Time of PD, in patients who came off-
study for a reason other than PD (Optional) 
1-9mL Streck Tube 
5- 10mL green top tubes* 
Fresh Tissue  
Pre-treatment (Optional) – within 7 days of 
starting therapy 
5-7cores  
 
Progression** 
5-7cores  
Archival Tissue  
Anytime  
1 block or 20, 4 micron 
unstained slides 


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Stool Sample 
Pre-treatment (within 28 days of starting 
therapy) 
Home Stool Kit (DNA 
Genotek) 
Cycle 3 Day 1 (within 14 days prior) 
Disease Progression 
At the time of grade ≥ 2 diarrhea 
(Optional)*** 
* EDTA (purple top) tubes or CPT tubes may be used interchangeably with green top tubes 
**Biopsy at the time of disease progression is optional for subjects on Arm A. Subjects on Arm 
B will be required to undergo a biopsy if they have accessible disease at the time of progression 
on Eribulin therapy, prior to initiating crossover treatment. Subjects on Arm B Crossover 
Therapy may choose to undergo an optional biopsy at the time of disease progression.  
*** Collection at the time of grade ≥ 2 diarrhea is optional and should be discussed with the 
study PI. 
 
 
9.2 
Fresh Tissue Collection 
 
9.2.1 Collection and handling of biopsy specimens 
 
Biopsies are optional at baseline (within 7 days of starting therapy) and at the time of disease 
progression for subjects on Arm A and subjects on Arm B Crossover Therapy. There will be a 
required biopsy for subjects with accessible disease on Arm B who experience disease 
progression on Eribulin mesylate prior to initiating crossover therapy. Guidelines for biopsies 
from various metastatic sites can be found in Appendix C. Additional collection and processing 
instructions may be found in the separate laboratory manual. 
 
Ideally five core biopsies will be obtained: 
• Two cores should be placed in 10% neutral buffered formalin tube supplied by the study. 
• One core should be placed in RNAlater 
• Two cores should be placed in sterile DMEM 
 
9.3 
Procedures for obtaining blood and stool specimens for study 
 
9.3.1 Blood Collection 
 
Research blood collection is mandatory for all participants for flow cytometry and DNA 
isolation.  The samples will be banked in the DF/HCC Clinical Trials Core Laboratory for these 
and future research purposes. These specimens will become the property of the DF/HCC.  
 
Please refer to the separate laboratory manual for collection and processing instructions 
 
9.3.2 Handling of Blood Specimens 
 
All samples should be de-identified and labeled with the Participant initials, Participant Study ID 
number and date of collection and time point (e.g., “Baseline” or “Cycle 1” or “Progressive 


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Disease”). Please refer to the separate laboratory manual for collection and processing instructions. 
 
• Green Top tubes: 
Five 10mL green top, purple top, or CPT tubes should be filled completely and 
should be processed within 3-4hrs of being drawn.  Will be delivered or shipped, 
ambient overnight to Mariano Severgnini’s lab.  Please contact the lab ahead of time 
by approximately one week to notify of upcoming specimen drop off. 
 
• Streck tubes: 
Fill the Streck tube completely and immediately mix by gentle inversion 8 to 10 times. 
Inadequate or delayed mixing may result in accurate results. 
 
Tube precautions: 
- 
DO NOT FREEZE OR REFRIGERATE TUBES as this could result in cfDNA 
breakage. Blood collected in the Streck tube can be stored for 14 days between 
6-37 degrees Celsius. 
- 
Do not use tubes after expiration date. 
- 
Fill the tube completely; overfilling or underfilling of tubes will result in an 
incorrect blood-to-additive ratio and may lead to incorrect analytical results. 
 
9.3.3     Stool Collection 
 
All stool samples will be collected by each patient at home using a home-based kit with a pre-
paid mailer that provides nearly equivalent metagenomic and metatranscriptomic data to state-of-
the-art fresh-frozen sample-collection protocol.  Patients will be asked to provide samples at the 
following timepoints: 
 
• Baseline 
• After two cycles of therapy 
• At the time of disease progression 
• Optional collection at the time of grade ≥ 2 diarrhea 
 
Most kits will be provided to the patients at their clinic visits. If the study team is unable to 
provide the kits to the patients in clinic, they may be mailed to patients by members of the study 
team. All kits will contain a questionnaire for patients to complete and return with their samples 
regarding timing and conditions surrounding their stool sample.  
 
Please refer to the separate lab manual for collection and processing instructions. 
 
Samples will be stored at the BWH/Harvard Cohorts Repository and will be shipped out to an 
external lab vendor, Microbiome Dx, who will perform the analysis of the samples.  
 
9.4 
Sites Performing Correlative Studies 
 
 
DFCI Center for Immuno-Oncology  
 
DFCI Clinical Trials Core Laboratory 


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BWH/Harvard Cohorts Biorepository 
 
Microbiome Dx – External Lab Vendor 
 
 
 
 
9.5 
Blood, tissue and stool banking 
 
Any leftover blood, tissue, or stool will be banked in the lab of Mariano Severgnini or the 
DF/HCC Clinical Trials Core Laboratory, respectively, as per standard lab protocol, such that it 
can be used for additional or future analyses as needed. 
 
9.6 
Hypothesis for correlative objectives 
 
9.6.1 Hypotheses 
 
• We hypothesize that the immune marker profile in the peripheral blood will change over 
the course of eribulin mesylate  +/- pembrolizumab therapy. 
• We hypothesize that a larger increase in markers of immune activity in the peripheral 
blood will correlate with a better disease response as assessed on concurrent restaging 
scans, and in terms of best radiographic response at any time on trial. 
We hypothesize that an immune marker or composite of markers in the peripheral blood 
at baseline will correspond to TIL percentage in archival tumor tissue. 
• We hypothesize that the immune activity associated with a breast tumor, as assessed both 
in tumor tissue and in circulating blood, will be less at the time of disease progression on 
therapy compared to the time of therapy initiation. 
• We hypothesize that the structure and function of the gut microbiome differs among 
patients with breast cancer. 
• We hypothesize that the structure and function of the gut microbiome before starting 
pembrolizumab and eribulin can be predictive of efficacy of this therapy, with a greater 
microbial diversity, estimated by Shannon index, being predictive of efficacy. 
• We hypothesize that the structure and function of gut microbiome changes in response 
pembrolizumab-based therapy and can predict the likelihood of response to 
pembrolizumab. 
• We hypothesize that the abundance and functional profile of specific gut bacteria is 
associated with response to pembrolizumab. 
 
 
9.7 
Planned assays for correlative objectives 
 
All of the below-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. 
 


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9.7.1 Tumor infiltrating lymphocyte (TIL) percentage and determination of lymphocyte 
predominant breast cancer (LPBC) 
 
Paraffinized, hematoxylin and eosin-stained slides taken from two tissue planes will be derived 
from each biopsy and will be reviewed by certified pathologists.  The extent of lymphocytic 
infiltrate in tumor tissue will be assessed, and stromal TIL percentage will be determined.  More 
detailed guidelines for the quantification of stromal TILs in breast cancer can be found in the 
recommendations from the International TILs Working Group 2014.31 
 
After assessment of the TIL percentage, the specimen may be categorized as lymphocyte 
predominant breast cancer (LPBC), defined as a tumor that contains >60% stromal lymphocytes, 
or non-LPBC. 
 
9.7.2 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 E) 
 
Chen et al37 describe a semi-quantitative scoring method, which is in accordance with typical 
biomarker scoring in anatomic and surgical pathology.  Briefly, staining is scored according to 
intensity (0=no staining, 1=weak staining, 2=moderate staining, 3=strong staining), staining 
patter (M=predominantly cell membrane; C=predominantly cell cytoplasm), and the percentage 
of cells showing positive staining (0-100%).  The semi-quantitative scoring is performed for: 1) 
the neoplastic tumor cells and 2) the non-neoplastic infiltrating immune cells.  Significant 
discordant results have been rare during case evaluations.37 
 
Further details of the immunohistochemical assay and assessment are described in the lab 
manual for this protocol. 
 
It should be noted that the above staining protocols are based on standard methods used at the 
time of protocol writing.  It is possible that at the time protein expression assays are conducted, 
novel and improved methods for staining will exist.  In this case, we plan to use the best 
available, best validated experimental method available at the time. 
 


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9.7.3 Flow cytometry, genomic analysis of biopsy tissue 
 
TILs will be isolated from the biopsy specimen and assessed by surface staining as described in 
the lab manual for this protocol.  
 
Messenger RNA (mRNA) expression within tumor biopsy specimens will 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,27 include CXCL9, 
CCL5, CD8ACD80, CXCL13, IGKC, CD21, IDO1, PD-1, PD-L1, PD-L2, CTLA4, and FOXP3.  
Additional DNA analysis, for example to assess mutational load and neoantigen burden, may 
also be performed. 
 
9.7.4 Analysis of PBMCs 
 
PBMCs will be generated as described in the lab manual for this protocol, and used to assess 
immune cell populations by flow cytometry. 
 
9.7.5 Analysis of cell-free DNA 
 
Blood will be collected at baseline, restaging visits and at time of progression for evaluation of 
cell-free DNA (cfDNA).  The cfDNA will be banked in the DF/HCC Clinical Trials Core 
laboratory for future research purposes.  The banked samples will be used to analyze DNA, RNA 
and protein in future studies.   
 
9.7.6 Analysis of 16S rRNA gene sequencing 
 
Microbial DNA is extracted using the Mag-Bind Universal Pathogen DNA Kit (Omega Bio-
Tek). Briefly, 250 mg of the specimen is transferred to a deep-well plate for bead beating 
followed by DNA precipitation and purification following the manufacturer’s instructions. 
Finally, DNA is eluted in 100 uls of Elution Buffer and stored at -80°C until further use. 16S 
sequencing libraries are generated by amplifying the v3-v4 hypervariable regions of the 16S 
gene in a polymerase chain reaction using primers F341and R785. Resulting amplicons are 
tagged with unique molecular barcodes that are later used to demultiplex sequencing reads into 
individual sample buckets. Libraries are loaded on a MiSeq flowcell and sequenced following 
Illumina’s loading instructions. Sequence data are retrieved from the instrument by converting 
base call format files into fastq files for data processing purposes.  
 
MicrobiomeDX uses BacPro™, a proprietary algorithm, to inspect and validate sequencing files 
by employing demultiplexing, trimming, merging, and quality filtering steps. Paired sequencing 
reads are merged using an overlap of 25 bp allowing for 10 base mismatches. Merged sequences 
are dereplicated and clustered in a de-novo fashion using VSEARCH, while filtering out 
sequence chimeras and singletons. Representative sequences from each cluster are mapped 
against the SILVA database at 99% sequence identity to obtain accurate taxonomic 
classifications and relative abundances. In parallel, feature tables are constructed to derive alpha 
diversity indices, and distance matrices are built to derive beta diversity indices. The BacProTM 


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pipeline generates a comprehensive report that includes alpha diversity scores describing 
community richness and evenness, taxonomic composition with relative abundances, and beta 
diversity metrics to determine the in-between sample differences based on the bacterial 
communities identified.  
 
 
 
 
9.7.7 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. 
 
 
 
10. STUDY CALENDAR 
 
Consent may be obtained 28 days prior to the start of protocol therapy. Baseline evaluations are 
to be conducted within 21 days prior to start of protocol therapy (unless otherwise specified).  If 
these screening assessments occur within 3 days before start of study treatment, they do not need 
to be repeated on Cycle 1 Day 1.  Scans must be done within 28 days prior to the start of 
protocol therapy. 
 
As detailed in the Study Calendar, a negative pregnancy test in women of child-bearing potential 
(as defined in the eligibility criteria) must be documented within 7 days prior to the start of 
protocol therapy. 
 
If participants agree to a baseline tumor biopsy, this biopsy should be obtained within 7 days 
before starting protocol therapy. 
 
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. 
 
Day 8 assessments may be performed at DFCI Milford, as needed; however, due to correlative 
requirements, the Day 1 visits, scans, and/or biopsies may not occur at this location.  
 
 


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Screening 
C1 
C2 
C3 
Cycle 4 and 
subsequent cycles 
Crossovera 
Off-
Treatmentm  
Follow-
Up 
D1 
D8s 
D1 
D8s 
D1 
D8s 
D1 
D8s 
Arm A- Drug 
administration 
 
 
 
 
 
 
 
 
 
 
 
 
Pembrolizumab  
 
X 
 
X 
 
X 
 
X 
 
 
 
 
Eribulin mesylate  
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Arm B- Drug 
administration  
 
 
 
 
 
 
 
 
 
 
 
 
Eribulin mesylate  
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Pembrolizumab  
 
 
 
 
 
 
 
 
 
X 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
X 
 
 
Physical exam 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Weight 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
Performance status 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
Concurrent medications 
X 
X----------------------------------------------------------------------------------------------------------X 
 
Adverse event evaluation 
 
X----------------------------------------------------------------------------------------------------------X 
 
Hematology panelb  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Chemistry panelc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Cortisolt 
X 
 
 
X 
 
X 
 
X 
 
 
 
 
TSHd 
X 
 
 
X 
 
 
 
X 
 
X 
 
 
Coagulation panel 
(PT/PTT) 
X 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy teste 
X 
 
 
 
 
 
 
 
 
X 
 
 
EKG 
X 
 
 
 
 
 
 
 
 
X 
 
 
Tumor Assessmentsf, h 
X 
 
 
 
 
 
 
Xg 
 
 
 
X i 
Tumor biopsy 
Xj 
 
 
 
 
 
 
 
 
Xk  
Xk 
 
Archival tumor samplesl 
X 
 
 
 
 
 
 
 
 
 
 
 
Research Blood 
Collection  
 
X 
 
X 
 
 
X 
 
X 
 
X 
Xn 
Xo 
Research Stool 
Collectionq 
X 
 
 
 
 
X 
 
 
 
 
X 
 
Stool Questionnairer 
X 
 
 
 
 
X 
 
 
 
 
X 
 
Survival 
 
 
 
 
 
 
 
 
 
 
 
Xp 
1 cycle = 21 Days 
a. Participants on Arm B crossing over to Pembrolizumab monotherapy must meet the criteria provided in Section 3.4 and 3.5 to begin therapy with 
pembrolizumab. 
 
b. Hematology to include: CBCA  
 
c. Chemistries to include: Chloride, potassium, sodium, BUN, serum creatinine, phosphorus, calcium, albumin, total protein, alkaline phosphatase, 
ALT, AST, total bilirubin (NOTE: the frequency of checking magnesium levels is left up to the treating provider) 


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d. TSH collected at screening, C2D1, and every other cycle for participants receiving pembrolizumab.  TSH is required at time of crossover. 
 
e. In female participants of child-bearing potential (as defined in the eligibility criteria), a urine or serum pregnancy test must be performed within 7 
days prior to the first dose of study medication.  If the urine test is positive or cannot be confirmed as negative, then a serum test is required.  For 
female participants of child-bearing potential crossing over, a negative pregnancy test is also required within 7 days of starting crossover therapy. 
 
f. Baseline tumor assessments must be done within 28 days prior to the start of therapy. Tumor assessments will be performed every 3 cycles (9 
weeks) for the first 18 cycles (1 year).  If after Cycle 18 scans (1 year), a participant has SD or better by RECIST the frequency of assessments 
may be reduced to every 4 cycles (12 weeks). If treatment is delayed, scans should continue to align with cycles of treatment, rather than weeks 
on treatment. Effort should be made to use the same type of imaging to assess measurable lesions at baseline and in follow-up. Confirmatory 
scans 4 weeks after documented response should be obtained 
 
g. For any subject who showed first radiologic evidence of progressive disease (PD) by RECIST 1.1 (see Section 11.1.4) and is deemed clinically 
stable (see section 5.6), it is at the discretion of the investigator to continue treating the subject until progression is confirmed at least 4 weeks 
from the date of the first radiologic evidence of PD. If progression is confirmed, the subject will be discontinued from study treatment. 
Otherwise, the subject will continue treatment and radiographic scans. Any subject who had initial radiologic progression and is deemed 
clinically unstable should be discontinued from both study drugs and no subsequent scan for confirmation is required.   
 
h. Tumor assessments should continue as scheduled for participants on Arm B who crossover to receive pembrolizumab.   
 
i. During follow-up of patients who went off study for reasons other than PD, tumor assessments should continue every 9 to 12 weeks from the date 
of the last assessment until progressive disease is documented 
 
j. Optional biopsy should be obtained within 7 days before starting protocol therapy.  See Section 9, appendices, and lab manual for biopsy 
handling and processing instructions.  
 
k. Optional tumor biopsy should be offered only in patients who go off study for progressive disease.  See Section 9, appendices, and lab manual for 
biopsy handling and processing instructions. For patients randomized for the eribulin mesylate arm who wish to cross over to pembrolizumab 
arm, a biopsy at progression is mandatory for those whose disease is amenable for biopsy. 
 
l. 1 block or 20 unstained slides should be collected.  If archival tissue is not available, patients with safely accessible disease should have a baseline 
biopsy. 
 
m. Off 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 prior to the visit. 
 
n. Research blood should only be collected on patients who come off treatment for progressive disease.   
 
o. Optional blood collection for immune cells/cfDNA may be offered to patients who develop progressive disease during follow-up.   
 
p. Survival status should be collected annually, either by review of the medical record or by phone call (see Section 5.8). 
 
q. Baseline stool collection should be obtained within 28 days before starting protocol therapy. The C3D1 stool collection should be performed as 
close to C3D1 as possible, but may be collected up to 14 days prior. A sample will additionally be collected at the time of disease progression. 
An optional stool sample may be collected at the time of grade ≥ 2 diarrhea after discussion with the PI. Stool collections will not occur on 
Pembrolizumab Crossover therapy for patients randomized to Arm B. 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. 
 
r. Each stool collection kit will contain a questionnaire for the patients to complete regarding the conditions surrounding their collection. These will 
be a part of the kit and are not to be administered in clinic. Failure to complete these questionnaires are the required or optional timepoints will 
not constitute a protocol violation.  
 
s. D8 assessments may occur at DFCI Milford, as needed. Additionally, if a subject is on Arm A and discontinues Eribulin, D8 visits will not be 
required. D8 visits are also not required when Arm B subjects are receiving Crossover Pembrolizumab monotherapy.  
 
t. For Arm A subjects, Cortisol blood draws will be required at baseline and every D1. For Arm B subjects, Cortisol blood draws will be drawn at 
baseline, as treatment arm is likely unknown, and then not again until all D1s of Crossover treatment. This will not be required when on Eribulin 
alone.  
 
 
 
 
11. MEASUREMENT OF EFFECT 
 


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11.1 
Antitumor Effect – Solid Tumors 
 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) 
[Eur J Ca 45:228-247, 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.  
 
Pembrolizumab, like other immunotherapeutic agents, may produce antitumor effects by 
potentiating endogenous cancer-specific immune responses. The response patterns seen with 
such an approach may extend beyond the typical time course of image responses seen with 
cytotoxic agents, and can manifest as a clinical response after an initial increase in tumor burden 
or even the appearance of new lesions. 
 
For any subject who showed first radiologic evidence of progressive disease (PD) by RECIST 
1.1 (see Section 11.1) and is deemed clinically stable, it is at the discretion of the investigator to 
continue treating the subject until progression is confirmed at the next scheduled restaging (or 
with a confirmatory completed at least 4 weeks from the initial date of PD).  If progression is not 
confirmed on the subsequent scan, the subject should continue to receive treatment and have 
radiographic scans performed according to the study calendar [approximately every 3 cycles (9 
weeks) for the first 12 months and then every 4 cycles (12 weeks)]. If radiologic progression is 
confirmed, then the subject should be discontinued from all study treatment.  If the treating 
investigator feels that the participant is clinically stable, demonstrates improved condition, or is 
clearly continuing to benefit from the treatment; the PI may approve the participant to continue 
to receive study treatment.  In all participants, the date of progression will be documented as the 
first date progression was observed.  
 
11.1.1 Definitions 
 
Evaluable for Target Disease response.  Only those participants who have measurable 
disease 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 diameter  to be recorded) as ≥ 20 mm by 


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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), 
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.  


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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 
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 


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FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is 
PD.  If the positive FDG-PET at follow-up is not confirmed as a new site of disease 
on CT, additional follow-up CT  scans are needed to determine if there is truly 
progression occurring at that site (if so, the date of PD will be the date of the initial 
abnormal FDG-PET scan).  If the positive FDG-PET at follow-up corresponds to a 
pre-existing site of disease on CT that is not progressing on the basis of the anatomic 
images, this is not PD. 
(c)  FDG-PET may be used to upgrade a response to a CR in a manner similar to a 
biopsy in cases where a residual radiographic abnormality is thought to represent 
fibrosis or scarring.  The use of FDG-PET in this circumstance should be 
prospectively described in the protocol and supported by disease-specific medical 
literature for the indication.  However, it must be acknowledged that both approaches 
may lead to false positive CR due to limitations of FDG-PET and biopsy 
resolution/sensitivity. 
 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake 
greater than twice that of the surrounding tissue on the attenuation corrected image. 
 
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 


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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 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. If markers are 
initially above the upper normal limit, they must normalize for a participant to be 
considered in complete clinical response.  Specific guidelines for both CA-125 response 
(in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been 
published [JNCI 96:487-488, 2004; J Clin Oncol 17, 3461-3467, 1999; J Clin Oncol 
26:1148-1159, 2008].  In addition, the Gynecologic Cancer Intergroup has developed 
CA-125 progression criteria which are to be integrated with objective tumor assessment 
for use in first-line trials in ovarian cancer [JNCI 92:1534-1535, 2000]. 
 
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. 


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Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters. 
 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of 
target lesions, taking as reference the smallest sum on study (this includes the 
baseline sum if that is the smallest on study).  In addition to the relative increase of 
20%, the sum must also demonstrate an absolute increase of at least 5 mm.  (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 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 


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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 
4 wks Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-CR/Non-
PD/not 
evaluated 
No 
SD 
 
PD 
Any 
Yes or No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
∗ 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
**        Only for non-randomized trials with response as primary endpoint. 
***      In exceptional circumstances, unequivocal progression in non-target lesions may be 
            accepted as disease progression. 
 
Note:  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 


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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) ≥  27 weeks. 
 
 
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 (irRECIST) 
 
The sum of the product of the longest diameter of lesions (SPD) at tumor assessment 
using the immune-related response criteria (irRECIST) 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 irRECIST 
 
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 
irRECIST criteria for tumor response.  Therefore, new non-measurable lesions will not 


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discontinue any subject from the study. 
 
11.3.1.2 
Definition of Target Lesions Response Using irRECIST 
 
• irRECIST Complete Response (irRECIST CR): Complete disappearance of all target 
lesions.  This category encompasses exactly the same subjects as “CR” by the mWHO 
criteria. 
• irRECIST Partial Response (irRECIST PR): 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. 
• irRECIST Stable Disease (irRECIST SD): Does not meet criteria for irRECIST RC or 
irPR, in the absence of progressive disease. 
• irRECIST Progressive Disease (irRECIST PD): 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 irRECIST 
 
• irRECIST Complete Response (irRECIST CR): Complete disappearance of all non-
target lesions.  This category encompasses exactly the same subjects as “CR” by the 
mWHO criteria. 
• irRECIST Partial Response (irRECIST PR) or irRECIST Stable Disease 
(irRECIST SD): Non-target lesion(s) are not considered in the definition of PR; these 
terms do not apply. 
• irRECIST Progressive Disease (irRECIST PD): 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). 
 
11.3.1.4 
Definition of Overall Response Using irRECIST 
 
Overall response using irRECIST will be based on these criteria: 
 
• Immune-Related Complete Response (irRECIST CR): 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 (irRECIST PR): 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 (irRECIST SPD).  
A decrease, relative to baseline, of the irRECIST SPD compared to the previously SPD 
baseline  of 50% or greater is considered an irRECIST PR. 


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• Immune-Related Stable Disease (irRECIST SD): irRECIST SD 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 (irRECIST PD): 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 nadir SPD calculated for 
the target lesions. 
 At least 25% increase in the SPD of all target lesions and new measurable lesions 
(irRECIST SPD) over the baseline SPD calculated for the target lesions. 
 
Criteria for determining overall response by irRECIST are summarized as follows: 
 
 
11.3.1.5 
Immune-Related Best Overall Response Using  irRECIST (irRECIST BOR) 
 
irRECIST BOR 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. 
 
irRECIST CR or irRECIST PR determinations included in the irRECIST BOR 
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. 
 
12. DATA REPORTING / REGULATORY REQUIREMENTS 
 


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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 
 
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  ODQ according to the schedule set by ODQ. 
 
12.2 
Data Safety Monitoring 
 
The DF/HCC Data and Safety Monitoring Board (DSMB) will review and monitor study 
progress, toxicity, safety and other data from this study. The board is chaired by a medical 
oncologist from outside of DF/HCC and has external and internal representation. Information 
that raises any questions about participant safety or protocol performance will be addressed by 
the Overall PI, statistician and study team. Should any major concerns arise, the DSMB will 
offer recommendations regarding whether or not to suspend the study. 
 
The DSMB will meet twice a year to review accrual, toxicity, response and reporting 
information. Information to be provided to the DSMB may include: participant accrual; 
treatment regimen information; adverse events and serious adverse events reported by category; 
summary of any deaths on study; 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 
 
N/A 
 
12.4 
Collaborative Research and Future Use of Data and Biospecimens 
 
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.  


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In order to 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. 
 
 
13. STATISTICAL CONSIDERATIONS 
 
 
13.1 
Study Design/Endpoints 
 
This is a randomized phase II open label study of eribulin mesylate +/- Pembrolizumab for 
patients with metastatic HR positive breast cancer treated with 0-2 line of chemotherapy. Eighty-
eight patients will be randomized (1:1) to eribulin mesylate with pembrolizumab or eribulin 
mesylate. Patients randomized to eribulin mesylate monotherapy will have the option to receive 
pembrolizumab monotherapy at time of progression. 
 
The primary endpoint is PFS,  defined as the time from study randomization to disease 
progression per RESICT 1.1 or death due to any cause, whichever occurred first.  Patients alive 
without disease progression are censored at the date of last disease evaluation.  The primary 
objective is to compare PFS of patients randomized to receive eribulin mesylate in combination 
with pembrolizumab (Arm A) versus those randomized to receive  eribulin mesylate 
monotherapy (Arm B). 
 
Secondary endpoints include PFS per irRECIST criteria, ORR per RECIST 1.1 and irRECIST, 
DOR, CBR, OS, safety and tolerability.  For the patients randomized to the eribulin monotherapy 
arm who elect to receive the combination after progression, we will also  perform an exploratory 
analysis of all clinical endpoints for patients using measurements at progression on monotherapy 
as the baseline tumor assessment. 
 
13.2 
Sample Size, Accrual Rate and Study Duration 
 
The target accrual is 88 patients (44 in eribulin mestlate alone arm and 44 in eribulin 
mesylate+pembrolizumab combination arm). The study has 83% power to distinguish a 3 month 
improvement in PFS from 4.1 to 7.1 months (HR = 0.58) with a one-sided alpha of 0.1. This 
statement of power assumes a final analysis when 70 PFS events are observed, and would be 
anticipated to occur after a constant accrual over 18 months with 6 months of additional follow-
up. Further assumptions are a constant hazard of PFS and dropout such that 5% of patients are 
lost-to-follow-up at 1 year. This allows for one futility analysis at 50% information (35 PFS 


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events) and would stop early if the Z-statistics from the logrank test is less than 0 (i.e. observed 
HR from a Cox model > 1.0).  
 
The expected accrual rate is 3-5 patients per month, and the accrual is expected to complete 
within 18-24 months. 
 
13.3 
Interim Monitoring Plan 
 
A single interim analysis for futility is planned to minimize the likelihood of exposing study 
patients to an inactive regimen. The interim analysis will be performed at 50% information, 
when the 44th patient is evaluable for response.  Stopping criteria were set considering Lan-
Demets (O'Brien Fleming) beta-spending function, and shifting the boundary slightly to Z = 0 
(from Z = 0.116) to correspond to no observed difference in the proportion of response or worse 
outcomes with the combination of Eribulin/Pembro. This boundary corresponds to a Bayesian 
predicted probability of 3.5% of concluding superiority at final analysis with a non-informative 
prior of treatment effects.   Futility criteria are non-binding, and enrollment will not be 
suspended for patient follow-up and interim analysis. No early stopping for efficacy will be 
considered. 
 
13.4 
Analysis of Primary Endpoints 
 
The primary analysis of the study will be the comparison of PFS per RECIST 1.1 between Arm 
A and Arm B using logrank test and group sequential methods to control the overall one-sided 
Type I error rate at 0.1.  
 
The null and alternative hypotheses are defined using the hazard ratio (HR) of the Arm A to Arm 
B as: 
H0: HR ≤ 1  
H1: HR > 1 
 
All inferences will be conducted in the intent-to-treat manner, and the survival function will be 
summarized using the Kaplan-Meier methods according to randomized treatment assignment. 
Final analysis will be performed after 70 PFS events are observed or at least 12 months after the 
last patient is enrolled on study, whichever occurs first.  
 
13.5 
Analysis of Secondary Endpoints 
 
Efficacy Endpoints  
All patients who initiated protocol therapy will be evaluated for PFS per irRECIST criteria, 
ORR, CBR, and OS. ORR and CBR will be evaluated using RECIST 1.1 and irRECIST criteria 
(as defined in Section 11.3). DOR will be evaluated among patients who had CR or PR. All these 
endpoints will be estimated separately in the two treatment arms. 
 
The objective response rate (CR + PR) and immune-related response rate (irCR + irPR) will be 
reported by arm with 90% exact confidence intervals.  Objective response rate and immune-
related response rate will be compared between Arm A and Arm B, respectively using a Pearson 


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chi-squared test for difference in proportions with a one-sided α of 0.1.   
 
Clinical benefit is defined as CR, PR or SD ≥ 27weeks.  Clinical benefit will be calculated using 
RECIST 1.1 and irRECIST criteria, respectively.  Clinical benefit rate (CR+PR+SD≥27 wks) per 
RECIST 1.1 and irRECISTcriteria will be reported respectively with 95% exact confidence 
intervals.  CBR will be compared between two treatment arms using a Pearson chi-squared test 
for difference in proportions with a one-sided α of 0.1.   
 
PFS per irRECIST and OS will be also analyzed using Kaplan–Meier product-limit estimatesand 
95% confidence bands. PFS per irRECIST is defined as the time from study randomization to 
disease progression per irRECIST or death due to any cause, whichever occurred first.  Patients 
alive without disease progression per irRECIST are censored at the date of last disease 
evaluation.  OS is defined as the time from study randomization to death due to any cause.  
Patients will be censored at the date they were last known alive. 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.   
 
DOR is defined as the time from CR or PR achieved until renewed disease progression is 
detected.  DOR will be calculated per RECIST 1.1 and irRECIST criteria, respectively, and 
descriptive statistics will be used to summarize the intervals observed by arm.  
 
In the group of patients treated with Pembrolizumab after  progression on eribulin mesylate, 
CBR, PFS, OS, and DOR after they received Pembrolizumab will be estimated using binomial, 
Kaplan-Meier and descriptive statistics, respectively with 95% confidence intervals.   For this 
group, PFS and OS will start from the time of initiating Pembrolizumab. Under an assumption 
that approximately two-thirds of patients elect to cross over (n=30), the maximum width to an 
exact confidence interval for a proportion will be 0.374.  
 
 
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 4.0. 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 using a Pearson chi-squared test for difference in proportions with a 
one-sided α of 0.1.. 
 
 
13.6 
Analysis of Correlative Science Endpoints 
 
All analyses of correlative scientific endpoints are exploratory and hypothesis-generating. Any 
promising findings will be tested in future studies.  
 
13.6.1  Blood and tissue correlative science endpoints: 
• We will describe the presence and abundance of multiple immune markers in metastatic 
HR-positive breast tumors (characterization will be based on histology, protein 


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expression, and mRNA expression; see Section 9 for further details on characterization) 
using frequency tables and descriptive statistics (mean, standard deviation, median , and 
inter-quartile range). 
• We will explore the correlation of immunosuppressive and/or immune-stimulating 
immune marker profiles at baseline to disease response to therapy (response assessed by 
RECIST 1.1 and irRECISTcriteria) using odds ratios and 95% confidence intervals. 
• We will summarize serial changes in immune marker profile in peripheral blood 
mononuclear cells (PBMCs) from baseline (pre-trial therapy) using mean, standard 
deviation, median , and inter-quartile range. 
• We will plot serial changes in the immunosuppressive and/or immune-stimulating 
immune marker profile in PBMCs by disease response to therapy (response assessed by 
RECIST 1.1 and irRECISTcriteria) 
• We will explore an immune marker or set of markers in circulating PBMCs that 
corresponds to tumor infiltrating lymphocyte (TIL) percentage in baseline tumor 
• In the cohort of patients who have re-biopsy at progressive disease (PD): We will 
summarize changes in a broad array of immune markers from baseline to time of 
progressive disease on trial therapy using mean, standard deviation, median , and inter-
quartile range. 
 
13.6.2  Stool and microbiome correlative science endpoints: 
 
Overall, we plan to describe the landscape of gut microbiota in patients with BC who will 
receive pembrolizumab, and the changes in their gut microbiota after two cycles of 
pembrolizumab. 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 progression-free survival) 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. 
 


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13.6.3  Tumor Genomic Profile correlative science endpoints: 
 
All analyses of Oncopanel in correlation with patient outcomes are exploratory and hypothesis-
generating. Any promising findings will be explored in future studies.  
 
13.7 
Reporting and Exclusions 
 
13.7.1 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 randomized patients who initiate 
protocol therapy, even if there are major protocol therapy deviations.   
 
 
 
13.7.2 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 24 months 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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Has a Critical Role in Antiviral Innate Immunity. Nat Immunol 17: 241-249. 
 
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Promotes Resistance to T Cell-Mediated Immunotherapy. Cancer Discov 6: 202-216. 
 
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. 
 
Rizvi, N.A., Hellmann, M.D., Snyder, A., Kvistborg, P., Makarov, V., Havel, J.J. et al. (2015) 
Cancer Immunology. Mutational Landscape Determines Sensitivity to Pd-1 Blockade in Non-
Small Cell Lung Cancer. Science 348: 124-128. 
 
Davoli, T., Uno, H., Wooten, E.C., and Elledge, S.J. (2017) Tumor Aneuploidy Correlates with 
Markers of Immune Evasion and with Reduced Response to Immunotherapy. Science 355:  
 
 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. 
 
 
 
 


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


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APPENDIX B 
STRONG CYP3A INDUCERS/INHIBITORS 
 
The list provided below is not exhaustive.  For a more comprehensive, frequently updated list, 
please visit: http://medicine.iupui.edu/clinpharm/ddis/table.aspx. 
 
Medications that strongly inhibit CYP3A: 
Amprenavir 
Atazanavir 
Boceprevir 
Clarithromycin 
Conivaptan 
Delavirdine 
Diltiazem 
Erythromycin 
Fosamprenavir 
Indinavir 
Itraconazole 
Ketoconazole 
Lopinavir 
Mibefradil 
Miconazole 
Nefazodone 
Nelfinavir 
Posaconazole 
Ritonavir 
Saquinavir 
Telaprevir 
Telithromycin 
Verapamil 
Voriconazole 
Grapefruit, grapefruit juice, or any product containing grapefruit 
 
Medications that strongly induce CYP3A: 
Carbamazepine 
Felbamate 
Nevirapine 
Phenobarbital 
Phenytoin 
Primidone 
Rifabutin 
Rifampin 
Rifapentin 
St. John’s wort 
 
 


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APPENDIX C 
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 5-7 core biopsy specimens will be obtained using an 18-gauge needle. 
 
Liver: A goal of 5-7core 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. 
 
If a patient is undergoing resection of a lesion for clinical reasons (i.e. wedge resection of a 
new lung lesion for confirmation of diagnosis or re-testing of hormone receptor or HER2 status; 
or, resection of a chest wall lesion; or, resection of a lymph node), then the patient may opt to 
have a portion of that tissue (roughly equivalent to the goal amount of tissue listed in the 
guidelines above, i.e. the equivalent of two 5-mm punch biopsies of the skin, or 3-6 18-gauge 
core biopsies) stored for research at the time of the procedure (provided that the tissue is 
processed as specified), in which case, the patient would not be required to undergo a separate 
research biopsy at baseline on this protocol. 
 
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 
 


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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. 
• 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 


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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 
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 1000.56 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, unless they are being done for clinical reasons, and excess tissue 
that otherwise would have been discarded is then banked for the purpose of 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 D 
ANTIBODIES THAT CAN BE USED FOR IMMUNOHISTOCHEMISTRY IN 
CORRELATIVE STUDIES 
 
 
 


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APPENDIX E 
ANTIBODIES THAT MAY BE USED FOR FLOW CYTOMETRY IN 
CORRELATIVE STUDIES 
 
Cell Type 
Antibody 
Color 
Clone 
T effector 
CD4 
FITC 
SK3 
  
  
PC7 
SK3 
  
CD62L 
APC 
DREG-56 
  
CD69 
PE 
FN50 
T regs 
CD4 
FITC 
SK3 
  
  
PC7 
SK3 
  
CD25 
PE 
Bc96 
  
  
PC5 
B1.49.9 
  
FOXP3 
PE 
PCH101 
  
  
FITC 
PCH101 
  
CD127  
APC 
eBioRDR5 
NK 
CD3 
FITC 
UCHT1 
  
  
PC7 
UCHT1 
  
CD56 
PE 
NCAM16.2 
  
CD57 
PE 
TB01 
NKT 
CD3 
FITC 
UCHT1 
  
  
PC7 
UCHT1 
  
CD56 
PE 
NCAM16.2 
  
TCR a/b 
APC 
BW242/412 
  
 CD314 (NKG2D) 
PE 
ON72 
MDSC 
HLA-DR 
PC7 
L243 
  
  
FITC 
L243 
  
CD11b 
FITC 
Bear1 
  
CD14 
APC 
61D3 
  
CD33 
PE 
WM53 
Cytotoxic 
CD8 
APC 
BW135/80 
  
  
PE 
BW135/80 
  
CD3 
FITC 
UCHT1 
  
  
PC7 
UCHT1 
Memory T 
CD197 (CCR7) 
PE 
3D12 
  
CD45RO 
FITC 
UCHL1 
  
CD45RA 
PC7 
HI100 
  
CD4 
FITC 
SK3 
  
  
PC7 
SK3 
  
CD8 
APC 
BW135/80 
  
  
PE 
BW135/80 
B cells 
CD5 
BV421 
UCHT2 
  
  
FITC 
UCHT2 
  
CD19 
PC7 
SJ25C1 
  
  
PE 
SJ25C1 
  
  
APC 
SJ25C1 


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CD20 
FITC 
2H7 
Classic Monocytes 
CD14 
APC 
61D3 
  
CD16 
FITC 
eBioCB16 
Dendritic 
CD123 
APC 
6H6 
  
CD303a 
FITC 
201A 
  
CD11c 
FITC 
11-0116 
  
CD141 
APC 
M80 
  
CD1c 
PE 
L161 
Macrophages 
CD40 
APC 
5C3 
Progenitors 
CD34 
PE 
4H11 
Intracellular Cytokines 
IL-10 
PE 
JES3-9D7 
  
IL-17a 
PercP Cy5 
eBio64DEC1
7 
  
INFg 
APC 
B27 
  
TNFa 
FITC 
Mab11 
Co-stimulatory and inhibitory markers 
CD134 (OX40) 
APC 
ACT-35 
  
CD137 (4-1BB) 
FITC 
4B4 
  
CD154 (CD40L) 
PercP 710 
24-31 
  
CD223 (LAG3) 
PercP 710 
3DS223H 
  
CD252 (OX40L) 
PE 
11C3.1 
  
CD278 (ICOS) 
FITC 
ISA-3 
  
Tim-3 
BV421 
F38-2E2 
  
CD274 (PD-L1) 
PE 
MIH1 
  
CD279 (PD-1) 
FITC 
MIH4 
  
  
PE 
MIH4 
  
  
APC 
MIH4 
  
CD357 (GITR) 
APC 
621 
  
CD152 (CTLA-4) 
efluor 660 
14D3 
Proliferation 
Ki-67 
FITC 
20Raj1 
 


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APPENDIX F 
IMMUNOHISTOCHEMICAL STAINING ASSAYS 
 
Design of immunohistochemical assay 
The immunohistochemical assay for PD-L1 and PD-L2 is semi-quantitative while PD-1 stained 
slides will be scanned by an automated scanning microscope and quantitatively analyzed by 
Aperio image analysis system (Lecia Biosystems) after they are evaluated and positive cells are 
manually counted by a pathologist. 
 
Standard EnVision two-step (indirect) staining method will be utilized.  Four micrometer-thick 
sections will be cut, deparaffinized, rehydrated, and subjected to heat modified antigen retrieval 
in citrate buffer (pH 6) (Invitrogen) by steaming for 30 minutes.  After cooling, tissue sections 
will be incubated with peroxidase block (DAKO, Carpinteria, CA) for five minutes, then serum 
free protein block (DAKO) for 20 minutes.  Slides will be incubated at room temperature for one 
hour with a primary antibody.  Antibodies will be diluted in Da Vinci Green Diluent (Biocare 
Medical, Concord, CA).  EnVision™ anti-mouse HRP-labeled polymer (DAKO) will be applied 
to the sections for 30 minutes, followed by visualization using the chromogen 3,3-
diaminobenzidine (DAKO).  All the sections will then be counterstained with hematoxylin, 
dehydrated, mounted, and coverslipped.  Positive and negative controls shall be included in each 
staining.  Known positive stained Hodgkin Lymphoma (PD-L1), tonsil (PD-1), and melanoma 
(PD-L2) slides will be used as external control (separate slides).  Stained slides will be stored at 
room temperature. 
 
In a pilot study performed by our correlative scientists, immunoreactivity for PD-L1 was 
detected in the cytoplasm and membrane while PD-L2 and PD-1 expression was observed in the 
cytoplasm.  Scoring for PD-L1 and PD-L2 will be semi-quantitative/ordered categorical.  The 
percentage of the tumor cells staining positive for PD-L1 or PD-L2 and the intensity of the 
staining will be recorded (using the scale 0=no staining, 1=weak staining, 2=moderate staining, 
3=intense staining).  Absolute PD-1 positive cells will be counted under microscope lens x20 
power field.  Five representative areas will be chosen to count.  The average number from 5 
areas will be recorded and compared with data from image analysis. 
 
For PD-1 staining, slides will be scanned by an automated scanning microscope and analyzed by 
Aperio image analysis system (Lecia Biosystems).  Tumor areas will be marked by a pathologist 
to exclude non-neoplastic areas, such as stroma, normal epithelial, and necrotic regions. The 
software will be used to count the number of positive cells in each tissue.  The percentage of PD-
1 positive cells will be calculated.  Data will be compared with that of manual counting by a 
pathologist to exclude tissue artifacts that cannot be recognized by computer image software. 
 
Assay performance 
Protocols of these three antibodies have been optimized and standardized to minimize staining 
variance.  Positive control and negative controls were used and stained separately with each 
batch of slides.  The IHC staining of three markers (PD-1, PD-L1, PD-L2) has been performed in 
two different labs by three different technicians on whole tissue sections of Hodgkin lymphomas, 
melanomas, lung cancers, and renal cell carcinomas.  Three readers were involved, confirming 
the good reproducibility of the assay. 
 
Thresholds of positivity 


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Tumor will be considered positive if >5% (PD-L1)53 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.54  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. 
 
 


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APPENDIX G 
TIL ISOLATION FROM SOLID TUMORS 
 
1. Prepare an enzyme solution of collagenase, hyaluronidase and deoxyribonuclease in 
advance: 
a. Dissolve collagenase (collagenase type I, cat#17100-017, Invitrogen) in DMEM 
at a concentration of 1 mg/ml 
b. Add hyaluronidase (hyaluronidase type V, cat#H6254, Sigma-Aldrich) to a final 
concentration of 1mg/ml (1,500 units/ml) and deoxyribonuclease 
(deoxyribonuclease I, type IV, cat#5025, Sigma-Aldrich) to a final concentration 
of 50 micrograms/ml (100 units/ml) 
c. Filter the solution with a 10 ml sterile syringe, a sterile 23G needle, and a sterile 
0.2 µm filter.  
2. Record the date and time of the start of TIL isolation.* 
3. Dissect patient tumor sample into pieces as small as possible with sterile scissor or 
scalpel. Note: Mincing of tumor may be facilitated by lining up two scalpels in parallel. 
4. Submerge the pieces of tumor in 5 - 10 ml prepared enzyme solution in a 50 ml conical 
tube. 
5. Enzymatically digest tumor pieces in 37°C waterbath for one to two hours; every 15 
min., vigorously shake the tube. 
6. Put a sterile cell strainer (100 µm, 352360, BD Falcon) on a 50 ml conical tube, and pass 
the digested tumor solution through the strainer; the flowthrough will be collected in the 
50 ml conical tube. Rinse the strainer and undigested tumor once with PBS. 
7. Add 2-5 ml complete DMEM medium (with 10% FBS + 50 µg/ml gentamycin) into 
flowthrough to stop digestion. 
8. Spin the tube at 1500 rpm for 5 min in a centrifuge at room temperature. 
9. In the meantime, put undigested tumor tissue into a sterile 50 ml conical tube, and add 5-
10 ml enzyme solution and continue with digestion from step 4.  
10. Repeat step 3 to step 8, based on tissue digestion  
 
NOTE: For samples with lot of red blood cells and/or undigested debris that has passed through 
the cell strainer, the following is recommended before proceeding to step 11: 
A. Resuspend the cell pellet in 10 ml complete DMEM medium 
B. Add 10 ml Ficoll Paque Plus (Cat# 17-1440-03; GE Healthcare) in a 50 ml conical tube. 
C. Slowly and gently layer the digested tumor suspension onto the Ficoll Paque Plus. 
D. Centrifuge the tube at room temperature at 1500 - 2000 rpm (1000 g) with slow acceleration 
and deceleration for 20 - 30 min. 
E. Pipette off the interface between complete DMEM and the Ficoll Paque Plus (lower part), and 


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transfer the layer into a 50 ml conical tube.  The bottom pellet will be composed of red blood 
cells and undigested debris. 
F. Add 2 - 3X bed volume of PBS to dilute Ficoll. 
G. Spin the tube at 1500 rpm for 5 min in a centrifuge at room temperature. 
H. Aspirate supernatant and proceed to step 11.   
 
11. Resuspend cell pellets in complete DMEM medium plus gentamycin 50ug/ml and 
combine cells-TIL into one sterile 50 ml conical tube.. 
12. Centrifuge at 1500 rpm for 5 min in a centrifuge at room temperature. 
13. Aspirate off the supernatant and remove a small aliquot to record the cell count and 
viability, then place the tube on ice. 
14. For each timepoint, collect the following parameters: 
a. Cell viability (%) before freezing* 
b. Total yield of TIL (x 106 cells/mL/vial) isolated prior to freezing* 
15. Resuspend the cell suspension in pre-chilled PBMC freezing media (CTL-cryoABS kit, 
CTL cellular Technology) 
a. Transfer 1 ml aliquots of the cell suspension to a cryovial labeled with the 
supplied Quintiles labels.  A minimum of one (1) cryovial should be obtained 
with a minimum concentration of cells at 1x106 cells/mL/vial.   
b. For each cryovial prepared, please record the total # of cells in the cryovial*.  If 
there are more than 2x106 cells, then aliquot cells into as many cryovials as 
possible at a concentration of 1x106 cells/ml/vial. 
16. Store in 1 ml aliquots in cryovials at -80⁰C in a slow freeze container.  Leave undisturbed 
overnight or for a minimum of 12 hrs and a maximum of 24 hrs. 
17. Transfer into liquid nitrogen for long-term storage.  Record the time, date, and location 
that the samples* were placed in liquid nitrogen storage. 
18. At the sponsor’s request, samples should be batch shipped at the end of study.  Please, 
follow the shipping instructions provided.  Samples will be shipped on liquid nitrogen. 


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APPENDIX H 
FLOW CYTOMETRY PROCEDURES 
 
Prep without Permeabilization  
 
KEEP EVERYTHING ON ICE 
1. Thaw cell vial in 37 degree water bath until completely thawed. 
2. Resuspend cells in 50 ml of RPMI medium (Gibco, 11A75-093) + 10% FBS + 1X final  
Anti-Anti (Gibco, 15240-062) in a 50 ml conical tube (Corning, 430290). 
3. Culture cells in 2 T-150 culture flasks (Corning, 431465) overnight (25 ml per flask) 
• In one flask, activate cells by adding 0.4 ml (whole vial) of Dynabeads Human T- 
Activator (Gibco, 1161D).  Before adding beads to flask, wash beads according to 
manufacturer’s protocol. 
4. Incubate cells for 24 hours at 37 degrees with 5% CO2 
5. Remove cells and media from flask and filter through 70 micron cell filter (Biologix, 15-
1070) into 50 ml conical tube.  
6. Spin conical tubes for 5 min at 1800 rpm in a Sorvall Legend XTR centrifuge. 
7. Make wash/blocking media: PBS +2.5% FBS (Gibco, 14040). 
8. Vacuum media off pellet, resuspend pellet in calculated volume of wash/blocking media 
according to calculations from cell density + number of wells and tubes for 700,000 
cells/tube in 100 µl. 
9. Pipet 100 µl/ well of cells + wash/blocking media containing FcR Blocking Reagent 
(Milteny Biotec, 130-059-901) into v-bottomed plate (Costar, 3894) according to well map 
(let sit for 20-30 minutes on ice). 
10. Spin plate at 1800 rpm for 5 minutes at 4C in Sorvall Legend XTR centrifuge 
11. Mix antibody cocktails in flat bottomed plate (amount according to manufacturer 
specifications or from previously developed assays) 
12.  After plate with cells is finished spinning, aspirate liquid off pellet by carefully tilting the 
plate.  Add appropriate antibody cocktails from flat bottomed plate according to well map 
after pipetting up and down to mix at least three times. 
13. Let plate with cells + antibodies sit for 45 minutes on ice in the dark. 
14. Spin plate as previously described in step 11 
15.  Aspirate off liquid by tilting plate and wash with 150 µl/well of wash/blocking media, 
pipetting up at down to mix at least 3 times (described in step 6) 
16. Resuspend cells in 150 µl/well in wash/blocking media  
17. Keep plate and single tubes (single color controls) on ice,  in the dark or covered with 
aluminum foil until read by Fortessa LTS II (Beckton- Dickinson). 
 


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APPENDIX I 
GENERATION OF PBMCS 
 
1. Pour blood from green-cap tubes (heparin treated tubes) into two 50 ml conical tubes 
(Corning, 430290). 
2. Spin tubes at 1500 rpm for 10 min (Sorvall Legend XTR centrifuge). 
3. Aspirate 2 ml plasma/tube and aliquot into 4 tubes mircocentrifuge tubes (Fisherbrand, 
05-408-138)  
4. Spin plasma at 3000 RPM for 5 minutes (Sorvall Legend Micro 21R centrifuge) 
5. Aspirate plasma into Cryogenic tubes 2 ml plasma/ tube (Corning, 430488). 
6. Dilute blood 1:1 with PBS. (Blood amount should not exceed 25 ml per tube.) 
7. Take 2 new 50 ml conical tubes and add 12 ml ficoll-paque (Cat# 17144003; GE 
Healthcare) per tube. 
8. Slowly and gently layer the diluted blood on top of the ficoll-paque of the tube with a 
maximum volume of 35 ml. 
9. Centrifuge the tube at 1900 rpm for 20 min at room temperature with slow acceleration 
(#7) and deceleration (#7) (Sorvall Legend XTR centrifuge). 
10. Remove the PBMC layer from between the upper layer (diluted plasma) and middle layer 
(ficoll-paque) and transfer into a 50 ml conical tube. The lower layer is composed of red 
blood cells. 
11. Completely fill conical tube containing isolated PBMC with PBS, mixing well. 
12. Count viable cells by mixing 10 µl Trypan Blue with 10µl PBMC/PBS dilution in a 
microcentrifuge tube. Load 10 µl of mixture onto Countess Cell Counting Chamber Slide 
(Invitrogen, C10283) and read with Countess Automated Cell Counter (Invitrogen).  
13. Centrifuge the tubes containing PBMC/PBS mixture at 1500 rpm for 5 min at room 
temperature (Sorvall Legend XTR centrifuge).  
14. Remove PBS, and resuspend PBMC pellet in appropriate amount of freezing solution so 
that there are approx 5 × 106 cells/cryo vial in 300-500 µl of Fetal Bovine Serum (heat 
inactivated) plus 15% DMSO.    
15. Put vials in CoolCell container (Biocision Inc.) and transfer to -80C freezer overnight.  
16. Transfer cells to liquid nitrogen tank