Clinical trial NCT03051672
Conditions: Metastatic Breast Cancer
Keywords: Breast Cancer
Sponsor: Dana-Farber Cancer Institute
Start date: 2017-05-22; Completion date: 2019-01-11
Enrollment: 8 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This research study is studying radiation therapy in combination with an immunotherapy as a possible treatment for metastatic hormone receptor (HR) positive, HER2-negative breast cancer.

The interventions involved in this study are:

* Palliative Radiotherapy
* Pembrolizumab

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 palliative radiotherapy ("radiation therapy") in combination with pembrolizumab in HR-positive, HER2-negative breast cancer. This study is designed to test how well radiation therapy in combination with pembrolizumab treats this type of cancer.

The FDA has approved radiation therapy as a treatment option for this type of breast cancer.

The FDA (the U.S. Food and Drug Administration) has not approved pembrolizumab for this specific disease but it has been approved in the United Sates for other types of cancer.

Pembrolizumab is a drug 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 allows these cells and other parts of the immune system to attack tumors.

The combination of pembrolizumab and radiation therapy is investigational. The study drug and radiation therapy, when given separately, work in different waysto help stop the cancer cells from growing and spreading. However, it is not known if giving the study drug and radiation therapy at the same time will have a better anti-cancer effect than giving each treatment on its own.

Arms: arm 1: pembrolizumab : 200 mg intravenously 2 to 7 days prior to radiotherapy (RT) and on day 1 of repeating 21-day cycles. Treatment up to 35 cycles.

Palliative radiation: a total dose of 20 Gy in 5 fractions
Interventions: intervention 1: Pembrolizumab intervention 2: Palliative radiotherapy
Intervention descriptions: intervention 1: Pembrolizumab (formerly MK-3475) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD1 and its ligands, PD-L1 and PD-L2. intervention 2: Palliative radiotherapy aims to shrink cancer, slow down its growth or control symptoms.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Page 1 
 
DF/HCC Protocol #: 16-588 
 
TITLE:   A phase II study of pembrolizumab in combination with palliative radiotherapy 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: 
Romualdo Barroso de Sousa, MD, PhD 
 
Dana-Farber Cancer Institute  
 
450 Brookline Avenue,  
 
Boston, MA 02215 
 
 
 
Ian Krop, MD, PhD  
 
Dana-Farber Cancer Institute 
 
Boston, MA 02215 
  
 
Jonathan D. Schoenfeld, MD, MPhil, MPH 
  
Dana-Farber Cancer Institute 
 
Boston, MA 02215 
 
 
Statistician: 
 
 
William Barry, PhD 
Dana-Farber Cancer Institute 
Boston, MA 02215 
 
Hao Guo 
Dana-Farber Cancer Institute  
Boston, MA 02215 
 
IND #:  132796 
IND Sponsor:  Sara Tolaney, MD, MPH 
 
 
Protocol Type / Version # / Version Date: 
Amendment / April 9, 2019 
 
 


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SCHEMA 
 


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TABLE OF CONTENTS 
 
SCHEMA .........................................................................................................................................2 
1. 
OBJECTIVES ......................................................................................................................5 
1.1 
Study Design ............................................................................................................5 
1.2 
Primary Objectives...................................................................................................5 
1.3 
Secondary Objectives...............................................................................................5 
1.4 
Correlative Objectives .............................................................................................5 
2. 
BACKGROUND .................................................................................................................6 
2.1 
Study Disease(s).......................................................................................................6 
2.2 
The PD-1/PD-L1 pathway in cancer ........................................................................7 
2.3 
Pembrolizumab ........................................................................................................8 
2.4 
Palliative Radiation therapy ...................................................................................10 
2.5 
Rationale ................................................................................................................10 
2.6 
Correlative Studies Background ............................................................................12 
3. 
PARTICIPANT SELECTION ...........................................................................................15 
3.1 
Eligibility Criteria ..................................................................................................15 
3.2 
Exclusion Criteria ..................................................................................................17 
3.3 
Inclusion of Women and Minorities ......................................................................18 
4. 
REGISTRATION PROCEDURES ...................................................................................19 
4.1 
General Guidelines for DF/HCC Institutions ........................................................19 
4.2 
Registration Process for DF/HCC Institutions .......................................................19 
5. 
TREATMENT PLAN ........................................................................................................19 
5.1 
Treatment Regimen ................................................................................................19 
Table 1: Regimen Description .......................................................................................................20 
5.2 
Pre-Treatment Criteria ...........................................................................................20 
5.3 
Treatment period ....................................................................................................21 
5.4 
General Concomitant Medication and Supportive Care Guidelines ......................21 
5.5 
Criteria for Taking a Participant Off Protocol Therapy .........................................24 
5.6 
Duration of Follow Up ...........................................................................................25 
5.7 
Criteria for Taking a Participant Off Study ...........................................................26 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................26 
6.1 
Management of toxicities attributable to pembrolizumab .....................................26 
7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................30 
7.1 
Adverse Events Lists..............................................................................................30 
7.2 
Adverse Event Characteristics ...............................................................................31 
7.3 
Expedited Adverse Event Reporting ......................................................................32 
7.4 
Expedited Reporting to the Food and Drug Administration (FDA) ......................32 


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7.5 
Expedited Reporting to Hospital Risk Management .............................................32 
7.6 
Expedited Reporting of Adverse Events and Events of Clinical Interest to 
Merck .....................................................................................................................32 
7.7 
Routine Adverse Event Reporting .........................................................................35 
8. 
PHARMACEUTICAL INFORMATION ..........................................................................35 
8.1 
PEMBROLIZUMAB .............................................................................................35 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................37 
9.1 
Archival Tissue ......................................................................................................38 
9.2 
Fresh Tissue Biopsy ...............................................................................................38 
9.3 
Blood Collection ....................................................................................................43 
9.4 
Additional analysis.................................................................................................45 
10. 
STUDY CALENDAR .......................................................................................................47 
11. 
MEASUREMENT OF EFFECT........................................................................................51 
11.1 
Antitumor Effect – Solid Tumors ..........................................................................51 
11.2 
Antitumor Effect – Hematologic Tumors ..............................................................58 
11.3 
Other Response Parameters ...................................................................................58 
12. 
DATA REPORTING / REGULATORY REQUIREMENTS ...........................................61 
12.1 
Data Reporting .......................................................................................................61 
12.2 
Data Safety Monitoring..........................................................................................61 
12.3 
Multicenter Guidelines...........................................................................................61 
12.4 
Collaborative Agreements Language .....................................................................61 
13. 
STATISTICAL CONSIDERATIONS...............................................................................62 
13.1 
Study Design/Endpoints.........................................................................................62 
13.2 
Sample Size, Accrual Rate and Study Duration ....................................................63 
13.3 
Stratification Factors ..............................................................................................64 
13.4 
Interim Monitoring Plan ........................................................................................64 
13.5 
Analysis of Primary Endpoint ................................................................................64 
13.6 
Analysis of Secondary Endpoints ..........................................................................64 
13.7 
Reporting and Exclusions ......................................................................................66 
14. 
PUBLICATION PLAN .....................................................................................................66 
REFERENCES ..............................................................................................................................68 
APPENDIX A 
PERFORMANCE STATUS CRITERIA ..................................................74 
APPENDIX B 
Guidelines for collecting research biopsy tissue ........................................75 
Risks of Research Biopsy and Procedures for Minimizing Risk .......................................75 
Risks of Anesthesia ............................................................................................................76 
 
 
 


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1. OBJECTIVES 
 
1.1 
Study Design 
1.1.1 This is a phase II single arm study assessing objective response rate (ORR) according to 
RECIST 1.1 in patients with metastatic hormone receptor (HR)-positive/HER-2 negative 
breast cancer who will receive pembrolizumab in combination with palliative 
radiotherapy (RT). Pembrolizumab will be started 2-7 days before day 1 of RT. Biopsies 
will be performed at baseline (mandatory if tumor tissue is accessible outside the field of 
RT) and the same lesion will be repeated within 7-14 days before the day 1 of cycle 3 of 
pembrolizumab.  Pembrolizumab 200mg will be administered intravenously every 21 
days until disease progression. 
 
1.2 
Primary Objectives 
1.2.1 To evaluate the efficacy of pembrolizumab in combination with radiotherapy, as defined 
by ORR outside the field of radiation according to RECIST 1.1, in patients with metastatic 
hormone receptor (HR)-positive/HER-2 negative breast cancer. 
 
1.3 
Secondary Objectives 
Safety objectives 
1.3.1 To evaluate the safety and tolerability of pembrolizumab in combination with palliative 
radiotherapy in patients with metastatic hormone receptor (HR)-positive/HER-2 negative 
breast cancer. 
 
Efficacy objectives 
1.3.2 To evaluate the ORR according to immune-related response criteria (irRC). 
1.3.3 To evaluate the abscopal response rate (ARR). 
1.3.4 To evaluate the progression-free survival (PFS) and overall survival (OS) of RT combined 
with pembrolizumab in metastatic hormone-receptor positive, HER2-negative breast 
cancer. 
1.3.5 To evaluate the immune-related progression-free survival (irPFS) of RT combined with 
pembrolizumab in metastatic hormone-receptor positive, HER2-negative breast cancer.  
1.3.6 To evaluate the Clinical Benefit Rate (CR + PR + SD ≥ 24 weeks) of RT combined with 
pembrolizumab in metastatic hormone-receptor positive, HER2-negative breast cancer. 
1.3.7 To evaluate the immune-related Clinical Benefit Rate (irCBR) of RT combined with 
pembrolizumab in metastatic hormone-receptor positive, HER2-negative breast cancer. 
1.3.8 To explore if radiation treatment volume or site of palliative radiation correlates with 
likelihood of achieving clinical benefit.  
 
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 correlate with disease response to therapy (response assessed by 
RECIST 1.1 irRC). 
1.4.3 To characterize changes in tumor-infiltrating lymphocytes, PD-L1 expression and immune 
gene signatures in the tissue microenvironment (TME) from baseline to after 2 cycles of 


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pembrolizumab. 
1.4.4 To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in TME correlates with disease response to therapy 
(response assessed by RECIST 1.1, irRC and ARR). 
1.4.5 To characterize serial changes in immune marker profile in peripheral blood mononuclear 
cells (PBMCs) over the course of the trial treatment. 
1.4.6 To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in PBMCs correlates with disease response to therapy 
(response assessed by RECIST 1.1, irRC and ARR). 
1.4.7 To investigate whether there is an immune marker in circulating PBMCs that corresponds 
to tumor infiltrating lymphocyte (TIL) percentage in baseline tumor. 
1.4.8 To collect blood to study cell-free DNA for comparison to tumor specimens before and 
after immunotherapy. 
1.4.9 To characterize the structure and function of the gut microbiome in patients with breast 
cancer prior to starting this clinical trial. 
1.4.10 To determine whether pre-treatment characteristics of the structure and function of the gut 
microbiome in patients with breast cancer is associated with disease response to therapy 
(response assessed by RECIST 1.1, irRC and ARR). 
1.4.11 To characterize changes in the structure and function of the gut microbiome of patients 
with breast cancer after two cycles of pembrolizumab compared to baseline.  
1.4.12 To determine whether changes in the overall diversity of the gut microbiome, estimated 
by the Shannon Index, of patients with breast cancer after two cycles of therapy is 
associated with disease response (response assessed by RECIST 1.1, irRC and ARR). 
1.4.13 To determine if the abundance or functional profile of specific gut bacteria are associated 
with objective response to therapy (response assessed by RECIST 1.1, irRC and ARR). 
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). 
 
 
2. BACKGROUND 
2.1 
Study Disease(s) 
Breast cancer (BC) is the most frequently diagnosed cancer and the second cause of cancer death 
in American women[Jemal et al., 2011, Siegel et al., 2013]. Approximately, 70% of these cancers 
are HR-positive BC, comprising those expressing estrogen receptor and/or progesterone 
receptor[Burstein et al., 2010]. Despite many advances in the adjuvant treatment of early-stage 
disease, 30-40% of women will develop systemic relapse[Greenberg et al., 2015]. Additionally, 
6-10% of patients present with de novo metastatic breast cancer4. 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 agent (such as CDK 4/6 or mTOR 
inhibitor), are promising strategies to overcome endocrine resistance[Pritchard et al., 2012, Zhang 
et al., 2015]. However, despite all these available systemic therapies, in the advanced setting 
virtually all patients with HR-positive tumors will die of BC and the median survival does not 
exceed 3 years[Nancy et al., 2015]. Thus, new treatment platforms are needed. 
 


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2.2 
The PD-1/PD-L1 pathway in cancer 
The importance of intact immune surveillance in controlling outgrowth of neoplastic 
transformation has been known for decades[Schreiber et al., Schreiber, 2012]. Accumulating 
evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and 
favorable prognosis in various malignancies[Mlecnik et al., 2014].  In particular, the presence of 
CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate 
with improved prognosis and long-term survival in many solid tumors[Tosolini et al., 2006, 
Adams et al., 2014, Denkert et al., 2015]. 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune 
control.  The normal function of PD-1, expressed on the cell surface of activated T-cells under 
healthy conditions, is to down-modulate unwanted or excessive immune responses, including 
autoimmune reactions.  PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related 
to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor signaling 
upon engagement of its ligands (PD-L1 and/or PD-L2).  The structure of murine PD-1 has been 
resolved.  PD-1 and family members are type I transmembrane glycoproteins containing an Ig 
Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail which is 
responsible for the binding of signaling molecules.  The cytoplasmic tail of PD-1 contains 2 
tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif (ITIM) and 
an immunoreceptor tyrosine-based switch motif (ITSM).  Following T-cell stimulation, PD-1 
recruits the tyrosine phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, 
leading to the dephosphorylation of effector molecules such as CD3ζ, PKCθ and ZAP70 which 
are involved in the CD3 T-cell signaling cascade.  The mechanism by which PD-1 down modulates 
T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an 
overlapping set of signaling proteins.  PD-1 was shown to be expressed on activated lymphocytes 
including peripheral CD4+ and CD8+ T-cells, B-cells, T regs and Natural Killer cells.  Expression 
has also been shown during thymic development on CD4-CD8- (double negative) T-cells as well 
as subsets of macrophages and dendritic cells.  The ligands for PD-1 (PD-L1 and PD-L2) are 
constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic 
tissues as well as in various tumors.  Both ligands are type I transmembrane receptors containing 
both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions 
with no known signaling motifs.  Binding of either PD-1 ligand to PD-1 inhibits T-cell activation 
triggered through the T-cell receptor.  PD-L1 is expressed at low levels on various non-
hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only 
detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic inflammatory 
environments.  PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas 
PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues.  Although healthy 
organs express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant 
levels of this T-cell inhibitor.  PD-1 has been suggested to regulate tumor-specific T-cell expansion 
in subjects with melanoma (MEL).  This suggests that the PD-1/PD-L1 pathway plays a critical 
role in tumor immune evasion and should be considered as an attractive target for therapeutic 
intervention[Intlekofer et al., 2013]. 
 
The PD-1/PD-L1 pathway in breast cancer 
Unlike melanoma and NSCLC, BC has not been intensively investigated for its susceptibility to 
immunotherapy in clinical settings. However, there are accumulating preclinical and clinical 


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evidence suggesting that immune system is critical during natural history of breast cancer and the 
immune system can be modulated to improve outcomes in this disease[Kroemer et al., 2015]. It 
has been recognized that BC is capable of stimulating the immune system, as many breast tumors 
have substantial lymphocyte infiltration [Denkert et al., 2010, Denkert et al., 2015]. Additionally, 
this pathologic feature has prognostic implications, as lymphocyte predominant breast cancers are 
associated with improved prognosis [Denkert et al., 2010, Loi et al., 2013]. However, the degree 
of immune infiltration differs by BC subtype; while a substantial proportion of triple negative BC 
can be richly infiltrated, hormone-receptor positive BC is poorly T-cell infiltrated[Dushyanthen et 
al., 2015]. Recently, it has been demonstrated that the expression of PD-1 and PD-L1 differs 
among breast tumors subtype: HR-positive (30% PD-1; 33% PD-L1), triple-negative (70% PD-1; 
59% PD-L1) and HER2-positive (60% PD-1; 20% PD-L1)[Gatalica et al., 2014]. 
 
2.3 
Pembrolizumab 
Pembrolizumab (formerly MK-3475) 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 Stated for the treatment of patients with unresectable or metastatic melanoma and 
disease progression following ipilumumab and, if BRAF V600 mutation positive, a BRAF 
inhibitor.  Pembrolizumab will be used in this protocol at the FDA-approved dose and frequency 
(200mg every 3 weeks).  
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 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. 
 


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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 rationale for further exploration of 2 mg/kg and comparable doses of pembrolizumab in solid 
tumors is based on: 1) similar efficacy and safety of pembrolizumab when dosed at either 2 mg/kg 
or 10 mg/kg Q3W in melanoma patients, 2) the flat exposure-response relationships of 
pembrolizumab for both efficacy and safety in the dose ranges of 2 mg/kg Q3W to 10 mg/kg Q3W, 
3) the lack of effect of tumor burden or indication on distribution behavior of pembrolizumab (as 
assessed by the population PK model) and 4) the assumption that the dynamics of pembrolizumab 
target engagement will not vary meaningfully with tumor type. 
 
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 
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 immune-related response criteria (irRECIST), 
respectively (Investigator’s Brochure, 2014).  The ORR for IPI-naïve patients with melanoma was 
39%/43% by RECIST/investigator-assessed irRECIST, 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 irRECIST, respectively (Investigator’s Brochure, 2014). 
 
Early findings for 27 patients with triple negative breast cancer showed an 18.5% ORR by RECIST 
(Nanda R, SABCS 2014). 
 
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 


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suggesting that higher levels of tumor PD-L1 expression are associated with increased clinical 
activity[Leighl et al., 2015].  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. Mutational load, T-cell infiltration and expression of PD-
1/PD-L1 in tumor biopsies, were associated with a high response rate in patients with advanced 
melanoma and non-small cell lung cancer treated with immune checkpoint inhibitors[Herbst et al., 
2014, Snyder et al., 2014, Rizvi et al., 2015].  
 
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 
Palliative Radiation therapy  
Radiation is commonly used throughout the body for patients with metastatic disease for palliative 
purposes. Patients with breast cancer, specifically, may require palliative radiation for bone pain 
or metastatic bony, lymph node or other soft tissue sites that could progress and cause fracture or 
functional compromise. Delivering palliative radiation to a total dose of 20 Gy in 5 fractions is 
commonly used in clinical practice and achieves high rates of pain control and freedom from 
further progression at the irradiated site with less need for retreatment of the irradiated site as 
compared to lower dose regimens[Wu et al., 2003, Hartsell et al., 2005]. Side effects of palliative 
radiation are largely specific to the site being irradiated, but typically include fatigue, skin 
erythema, and long term risks of fibrosis and increased propensity for bone fracture.  
 
2.5 
Rationale 
In the last decade, through the better understanding of tumor immunology, major clinical advances 
have emerged from T-cell based cancer immunotherapy. One of the most important mechanisms 
to evade antitumor immunity is the overexpression of checkpoint immune molecules on the T-cell 
surface, such as CTLA4, LAG3 and PD-1[Mittal et al., 2015].  These molecules, after binding to 
their ligands, initiate inhibitory pathways that negatively modulate T-cell function. It has been 
recognized that BC is capable of stimulating the immune system, as many breast tumors have 
substantial lymphocyte infiltration[Loi et al., 2013]. Additionally, this pathologic feature has 
prognostic implications, as lymphocyte predominant breast cancers are associated with improved 
prognosis. However, the degree of immune infiltration differs by BC subtype; while a substantial 
proportion of triple negative BC can be richly infiltrated, hormone-receptor positive BC is poorly 
T-cell infiltrated[Loi et al., 2013]. Furthermore, only a small proportion of luminal cancers express 
PD-1, PD-L1 or both, respectively 30%, 33% and 15%[Gatalica et al., 2014]. These factors make 
the likelihood of response of hormonal-positive BC to a single anti-PD-1/PD-L1 therapy low. In 
fact, recently, two phase I clinical trials presented the response rate of immune checkpoint 
blockade in patients with HR+ BC. In a non-selected population, the use of Avelumab (an anti-
PD-L1 inhibitor) presented an objective response rate (ORR) of 2.8% while in patients previously 


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selected by PD-L1 expression on tumors, the use of Pembrolizumab resulted in an ORR of 12%. 
Thus, other strategies that elicit an immunogenic tumor microenvironment may be needed in order 
to enhance the activity of PD1/PD-L1 axis inhibitors in patients with hormone-receptor positive 
breast cancer. 
In this context, there is strong preclinical rational to evaluate the combination of radiotherapy (RT) 
with anti-PD1 blockade. Previous studies demonstrated that, in addition to its direct cytoreductive 
effect, RT-induced cell death can be immunogenic, facilitating the recruitment and activation of 
antigen presenting cells (APCs) and priming of tumor antigen-specific T-cells[Shahabi et al., 
2015]. Recently, different groups demonstrated that RT to the tumor bed led to upregulation of 
PD-L1 on tumor cells, dendritic cells, and on myeloid-derived suppressive cells (MDSCs), which 
may contribute to impairment of T-cell function in the tumor[Liang et al., 2013, Deng et al., 2014, 
Sharabi et al., 2014]. Furthermore, these groups also demonstrated that the combination of RT plus 
blockade of the PD-1/PD-L1 axis improved outcomes in different preclinical models compared 
with RT or anti-PD1/PD-L1 alone, including breast cancer models (Figure 1). 
 
 
Moreover, the effect of localized radiotherapy mediating systemic responses distant from the field 
of radiation (the abscopal effect), has been reported in several types of human malignancies, such 
as melanoma and renal cell carcinoma, after treatment combining RT and immune checkpoint 
inhibitors[Adamow et al., 2012]. Although there is no definitive data confirming the host immune 
response caused the response, the tumor responses observed in these cases were accompanied by 
immunological changes in peripheral blood, including changes in a variety of tumor-associated 
antigens and a decrease in MDSCs. Additionally, the abscopal effect was recently demonstrated 
in mice treated with the combination of RT and anti–PD-L1 therapy, but not in groups receiving 


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either treatment alone, suggesting that this combination may potentiate an abscopal effect on 
distant tumors (Figure 2)[Deng et al., 2014, Dovedi et al., 2015]. 
 
 
 
Due to these data that RT may potentiate the efficacy of PD-1 blockade, we propose evaluating 
the combination of the pembrolizumab with palliative radiotherapy in hormone-positive BC 
patients.  Patients will receive the first dose of pembrolizumab 2-7 days before the beginning of 
RT. Pembrolizumab will be administered every 3 weeks thereafter until disease progression. 
 
2.6 
Correlative Studies Background 
 
2.6.1 Blood and Tissue Analysis  
 
The importance of tumor microenvironment and the immunosurveillance in natural history of 
cancer and its outcomes was proved to be true in the last years, with clinical approval of immune 
checkpoint inhibitors[Sharma et al., 2015]. However, less than half of patients with solid tumors 
will derive benefit with these drugs [Hwu et al., 2012, Smith et al., 2012]. Thus, it is crucial to 
elucidate the exact mechanisms of antitumor immunity evasion ongoing in tumor 
microenvironment to successfully develop new cancer immunotherapy and correctly choose the 
best drug for the right patient. This goal can be pursuit through the discovery and validation of 
prognostic and predictive biomarkers. 
 
A growing body of evidence suggests that patients with advanced solid tumors shows differences 
in tumor microenvironment regarding the presence or absence of a gene expression profile 
indicative of a pre-existing T-cell–inflamed tumor microenvironment[Gajewski, 2015]. Tumors 
classified as T-cell inflamed present a significant infiltration of CD8+ T cells and a type I IFN 
signature. In this group, the main mechanisms of immune evasion are the overexpression of 
immunessupressor molecules acting at the level of the tumor micro- environment, such as immune 
checkpoint molecules (CTLA-4, PD-1/PD-L1, TIM-3, LAG-3), indoleamine-2,3-dioxygenase 
(IDO), and FoxP3. Interestingly, such immunosuppressive molecules seem to be upregulated after 
deflagration of a type I Interferon antitumor response, resulting in T-cell exhaustion, and the so 
called adaptative immune resistance[Gajewski, 2015, Ribas, 2015]. The other group of patients 
presents tumors characterized by a low or absence of intratumoral CD8 T cells and a lack evidence 
of a type I IFN transcriptional signature. This tumor phenotype is called non-T-cell-
inflamed[Gajewski, 2015]. 
 
The T-cell inflamed phenotype has positive prognostic value for several types of early stage 
cancer, including breast cancer[Dushyanthen et al., 2015, Perez et al., 2015], suggesting that the 
attempt by the host to generate an anti-tumor immune response reflects a biologic process 


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associated with improved patient outcomes[Gajewski, 2015]. In breast oncology, different groups 
have demonstrated that the amount of tumor-infiltrating lymphocytes (TILs) in a tumor specimen, 
commonly assessed simply by histological evaluation of a standard hematoxylin and eosin-stained 
slide by a trained pathologist, is a significant predictor of both response to therapy and overall 
disease outcomes in the neoadjuvant and adjuvant settings [Denkert et al., 2010, Loi et al., 2013, 
Adams et al., 2014, Ali et al., 2014, Salgado et al., 2014, Denkert et al., 2015, Denkert et al., 
2015]. Recently, more in-depth methods of immunologic profiling are being explored in breast 
cancer, for example mRNA expression of immune-activating and immunosuppressive factors, and 
these additional immune profiles also appear to have prognostic significance[Perez et al., 2015]. 
Furthermore, in metastatic setting, the phenotype T-cell-inflamed appears to be associated with 
clinical response to several immunotherapies, including checkpoint blockade[Herbst et al., 2014]. 
Patients with this tumor phenotype seem to be good candidates for immune checkpoint inhibitor 
therapy, alone or in combination. Thus, the bulk of our correlative science in this trial highlights 
our especial interest in characterize a broad array of immune markers in metastatic HR-positive 
breast cancer, investigating whether those markers predict disease response to therapy. 
 
Thus, considering the mechanism of action of drugs like anti-PD-1/anti-PD-L1, is the lack of a 
significant T-cell infiltrate and low expression of immune checkpoint molecules may explain the 
reason that certain non inflamed tumor phenotype are associated with de novo resistance to those 
class of drugs. For this group of patients, therapeutic strategies that promote a boost in innate 
immunity, such as a course of radiation therapy, will be crucial to successfully overcoming T-cell 
exclusion and improve the likelihood of benefit of PD-1 blockers.  
 
In breast tumors, particularly the HR-positive subset, the vast majority of patient’s tumors do not 
harbor significant TILs or demonstrate PD-L1 expression and will therefore most likely be 
classified as non-T-cell-inflamed tumors. This explains why ORR recently reported in this 
population ranges from 2.8%-12%[Dirix et al., 2015, Hugo et al., 2015]. Clearly, new approaches 
to boost antitumor immunity are needed in this population.  RT can potentially improve the activity 
of immune checkpoint inhibitors. Because of the preclinical data supporting RT induced immune 
modulation of the tumor microenvironment, we intend to explore how immune biomarkers change 
after the beginning of treatment, including the expression of immune checkpoint molecules, TILs 
and T-cell receptor diversity. 
 
Additionally, as a correlative study to this trial, we will characterize the immune marker profile of 
peripheral blood mononuclear cells (PBMCs) in enrolled breast cancer patients.  Furthermore, 
given the demonstrated clinical significance of TILs in breast cancer specimens, we will 
investigate whether there is a peripheral marker whose level corresponds to TIL percentage.  
Lastly, we will evaluate whether there is a correlation between changes in PBMC immune profiles 
and disease response.  Evidence of a correlation would be of significant interest as it would suggest 
the potential presence of a predictive biomarker in the peripheral blood. 
 
These correlative projects are made possible by collaboration with Drs. 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 


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Breast Cancer (BC) is the most frequently diagnosed cancer and the second cause of cancer 
death in American women [DeSantis et al., 2015].  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 malignancy [Kroemer et al., 2015].  It is now recognized that a fraction of breast 
tumors have substantial lymphocyte infiltration, and that this pathologic feature has prognostic 
implications [Stanton, 2016].  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+ TNBC [Dirix et al., 2016, Nanda et al., 2016], and a 12% ORR among those with 
PDL1+ hormone receptor (HR)-positive BC [Rugo et al., 2016].  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 
development [Tinchieri, 2015]. Healthy individuals have microbial populations in their intestinal 
tract that vary markedly in composition [Human Microbiome Project, 2012, Qin et al., 2010].  
The diversity of intestinal microbiota represents a significant challenge to the host’s immune 
defenses, which must balance immune tolerance of beneficial microbes with inflammatory 
responses against pathogens.  Alterations in the gut microbiota and their resulting interactions 
with intestinal epithelieum and the host immune system are associated with many disease, 
including cancer [Roy et al. 2017].  Recently, two preclinical studies provided to ICI, raising the 
possibility that stool microbiota could be used as biomarker predictors of efficacy to 
immunotherapy [Sivan et al., 2015, Vetizou et al., 2015].  Interestingly, postmenopausal women 
with breast cancer have altered composition and low diversity of their gut microbiota compared 
to healthy controls [Goedert et al., 2015]. 
 
Identification of biomarkers that predict response to ICI-based therapies can spare de novo 
resistant patients from the unnecessary risks of immune-related adverse events.  In addition, the 
identification of bacterial species associated with response could open new strategies to 
maximize the clinical benefit of cancer immunotherapy through the modulation of gut 
microbiota. 
 
This correlative project is made possible by collaboration with the BWH/Harvard Cohorts 
Biorepository and Dr. Andrew Chan. 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- 


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microglobulin or defects in the interferon signaling pathway[Gao et al., 2016, Zaretsky et al., 
2016], the knowledge of immune resistance remains largely unknown. Several gene/pathways 
have been described as possible candidates of having an immunosuppressive role in different 
advanced solid tumor, including MYC amplification[Casey et al., 2016], activation in WNT--
catenin pathway[Spranger et al., 2015], activation in MAPK pathway, loss of PTEN[Li et al., 2016, 
Peng et al., 2016, George et al., 2017]. On the other hand, few possible biomarkers of response to 
ICI have emerged, including mutational load[Snyder et al., 2014, Rizvi et al., 2015], tumor 
aneuploidy[Davoli et al., 2017], mismatch repair defects[Le et al., 2015], and BRCA2 
mutation[Hugo et al., 2016]. Notably, there is no data on genomic mechanisms of de novo 
resistance to anti-PD-1 therapy in patients with breast cancer. 
Therefore, as a correlative study to this trial, we will to explore whether the number and/or type of 
mutations identified using a next generation sequencing (NGS) panel – OncoPanel - is correlated 
with patient outcomes (PFS, ORR, 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 Participants must have histologically or cytologically confirmed invasive breast cancer, 
with metastatic disease.  Participants without pathologic or cytologic confirmation of 
metastatic disease should have unequivocal evidence of metastasis from physical 
examination or radiologic evaluation. 
 
3.1.2 Invasive disease must have been tested for ER, PR and HER2.  Participants must have 
hormone-receptor positive, HER2-negative breast cancer defined as: 
 
ER>1% or PR>1% 
 
HER2-negative per ASCO CAP guidelines, 2013 [Wolff et al., 2013] 
 
 
3.1.3 Participant must be a candidate for palliative radiation treatment to at least one bone, 
lymph node, or soft tissue lesion. Radiation of visceral lesions (such as lung or hepatic 
lesions) is not permitted. 
 


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3.1.4 Participant must have measurable disease outside the field of radiation as defined by 
RECIST 1.1. 
 
3.1.5 If tumor is accessible and outside the field of radiation, the participant must be willing to 
undergo a research biopsy at baseline and around their second  cycle of pembrolizumab. 
Participants for whom newly-obtained samples cannot be provided (e.g. inaccessible or 
safety concern) must be willing to submit an archival specimen.  
 
3.1.6 Prior systemic therapy:  
 Participant must be at least 14 days from the last dose of prior chemotherapy, 
endocrine therapy, biological agents (including small molecule targeted therapy) 
or any investigational drug product with adequate recovery of toxicity to baseline, 
or grade 1(with the exception of alopecia and hot flashes) at the time of 
registration.   
 
 There is no limit to the number of prior lines of therapy, including endocrine or 
cytotoxic agents. Systemic treatment naive patients for metastatic disease are also 
eligible.  
 
 Participants may initiate or continue bisphosphonate therapy on study. 
 Continuation of ovarian suppression is allowed.  
 
3.1.7 Prior radiation therapy:  
 Patients must be at least 3 months from prior radiation therapy 
 Re-irradiation of the same field is not allowed  
 
3.1.8 Concurrent administration of other cancer specific therapy during the course of this study 
is not allowed. 
 
3.1.9 The subject is 18 years old 
 
3.1.10 ECOG performance status ≤1 (See Appendix A for details) 
 
3.1.11 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)  
  
 
 
 
(or 2.0 x ULN in patients with documented Gilbert’s 
Syndrome) 
 
AST(SGOT)/ALT(SGPT) 
≤2.5 × institutional ULN or ≤ 5 × institutional ULN for 
participants with documented liver metastases 
 
creatinine 
≤1.5 ×within normal institutional ULN OR creatinine 
clearance ≥60 mL/min/1.73 m2 for participants with 
creatinine levels above institutional ULN. 


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 
International normalized ratio (INR) or Prothrombin Time (PT) <1.5 times the upper 
limit of normal unless subject is receiving anticoagulant therapy, as long as PT or 
PTT is within therapeutic range of intended use of anticoagulants. 
 
Activated Partial Thromboplastin Time (aPTT) <1.5 times the upper limit of normal 
unless subject is receiving anticoagulant therapy, as long as PT or PTT is within 
therapeutic range of intended use of anticoagulants. 
 
3.1.12 Female subjects of childbearing potential must have a negative pregnancy test at 
screening 
 
3.1.13 Female and male subjects of childbearing potential must agree to use an adequate method 
of contraception as outlined in section 5.4.1. Contraception is required starting with the 
first dose of study medication through 120 days after the last dose of study medication    
Note: Abstinence is acceptable if this is the usual lifestyle and preferred contraception for 
the subject. 
 
3.1.14 Resolution of all chemotherapy-related or radiation-related toxicities to Grade 1 severity 
or lower, except for stable sensory neuropathy (≤ Grade 2) and alopecia. 
 
3.1.15 The subject is capable of understanding and complying with the protocol and has signed 
the informed consent document. 
 
3.2 
Exclusion Criteria 
 
3.2.1 Participants who are receiving any other investigational agents. 
 
3.2.2 Previous treatment with any anti-PD-1, PD-L1, or PD-L2 agent. 
 
3.2.3 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to pembrolizumab. 
 
3.2.4 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 (whole brain radiotherapy, radiosurgery, or a 
combination) at least 3 months prior to trial therapy initiation,  
 are neurologically stable, and  
 have recovered from 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 prior to registration.   
 
3.2.5 Radiologic or clinical evidence of Spinal Cord Compression. 
 
3.2.6 Spinal Instability Neoplastic Score ≥ 7 unless lesion reviewed by a neurosurgical service 
and considered stable. 
 


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3.2.7 Participants with bone lesions requiring surgical fixation to provide mechanical stability 
are ineligible.  Participants with previously fixed lesions are allowed. 
 
3.2.8 The participant has an 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, severe 
malnutrition  or psychiatric illness/social situations that would limit compliance with 
study requirements. 
 
3.2.9 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). 
 
3.2.10 Participant has a medical condition that requires chronic systemic steroid therapy or on 
any other form of immunosuppressive medication. For example, patients 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. 
 
3.2.11 Has history of (non-infectious) pneumonitis that required steroids or current pneumonitis. 
 
3.2.12 Has a history of interstitial lung disease. 
 
3.2.13 The participant is known to be positive for the human immunodeficiency virus (HIV), 
HepBsAg, or HCV RNA. HIV-positive participants on combination antiretroviral therapy 
are ineligible because of the potential for pharmacokinetic interactions with 
Pembrolizumab.   
 
3.2.14 Individuals with a history of different malignancy are ineligible except for the following 
circumstances. Individuals with a history of other malignancies are eligible if they have 
been disease-free for at least 3 years or are deemed by the investigator to be at low risk 
for recurrence of that malignancy.  
 
3.2.15 Has received a live vaccine within 30 days of planned start of study therapy. 
 
3.2.16 The participant is pregnant or breast-feeding. 
 
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. 
 
 


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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. 
An investigator will confirm eligibility criteria and a member of the study team will 
complete the protocol-specific eligibility checklist. 
Following registration, participants may begin protocol therapy. Issues that would cause 
treatment delays should be discussed with the Overall Principal Investigator (PI). If a participant 
does not receive protocol therapy following registration, the participant’s registration on the study 
must be canceled. Registration cancellations must be made in OnCore as soon as possible.  
 
4.2 
Registration Process for DF/HCC Institutions 
 
DF/HCC Standard Operating Procedure for Human Subject Research Titled Subject Protocol 
Registration (SOP #: REGIST-101) must be followed.   
 
5. TREATMENT PLAN 
 
5.1 
Treatment Regimen 
 
This is a phase II single arm study assessing ORR outside the field of radiation according 
to RECIST 1.1 in patients receiving pembrolizumab in combination with palliative radiotherapy 
in metastatic hormone-positive, HER2-negative breast cancer. 27 patients will be enrolled to 
receive therapy. Pembrolizumab therapy will be started 2-7 days before day 1 of RT. Radiation 
will then be delivered for five treatments of 4Gy (4Gy x 5) over a period of 1 to 2 weeks. The dose 
of pembrolizumab planned to be studied in this trial is 200 mg on day 1 of each 21 day cycle. Each 
cycle will have 21 days (3 weeks – q3w). The dose recently approved in the United States for 
treatment of melanoma subjects is 2 mg/kg Q3W. Although the dose of pembrolizumab studied in 
KN 012, which established efficacy and safety in mTNBC, was 10 mg/kg Q2W, recent studies in 
other tumor types have indicated that 10 mg/kg Q2W and 200 mg Q3W are likely to be similar 
with regard to efficacy and tolerability. 
The choice of the 200 mg Q3W as an appropriate dose for the switch to fixed dosing is 
based on simulations performed using a population pharmacokinetic model of pembrolizumab 
showing that the fixed dose of 200 mg Q3W will provide exposures that 1) are optimally consistent 
with those obtained with the 2 mg/kg dose Q3W, 2) will maintain individual subject exposures in 
the exposure range established in melanoma as associated with maximal efficacy response , and 
3) will maintain individual subjects 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 waste. 
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potential risks are described in Section 7.  Appropriate dose modifications are described in Section 
6.  Details of the regimen are described in Table 1.  No investigational or commercial agents of 
therapies other than those described below may be administered with the intent to treat the 
participant’s malignancy 
 
 
Table 1: Regimen Description 
Agent 
Premedication
; Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
Pembrolizumab 
Not routinely 
necessary unless 
prior infusion 
reaction. 
 
200 mg D1,  
q3w, iv 
 
IV over 
approximately 30 
minutes (range: 
25-40 minutes).  
Please refer to 
Section 8 for 
compatible 
infusion set 
materials 
including in-line 
filter.   
Day 1, q3w 
21 days 
(3 week) 
 
 
 
 
 
5.2 
Pre-Treatment Criteria 
5.2.1 Cycle 1, Day 1 
 
 absolute neutrophil count 
≥1,500/mcL 
 
platelets 
≥100,000/mcL 
 
hemoglobin  
≥ 8 g/dl 
 
total bilirubin 
≤1.5 × institutional upper limit of normal (ULN) 
(or 2.0 x ULN in patients with documented Gilbert’s 
Syndrome) 
 
AST(SGOT)/ALT(SGPT) 
≤2.5 × institutional ULN or ≤ 5 × institutional ULN for 
 participants with documented liver metastases 
 
creatinine  
≤1.5 ×within normal institutional ULN OR creatinine 
clearance ≥60 mL/min/1.73 m2 for participants with 
creatinine levels above institutional ULN. 
 
5.2.2 Subsequent Cycles, Day 1  
 
absolute neutrophil count 
≥1,000/mcL 
 
platelets 
≥75,000/mcL 
 
AST(SGOT)/ALT(SGPT) 
≤ 2.5 × institutional ULN or ≤ 5 × institutional ULN for 
participants with documented liver metastases 
- 
total bilirubin 
≤1.5 × institutional ULN (or 2.0 x ULN in patients with 
documented Gilbert’s Syndrome) 
 


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5.3 
Treatment period 
5.3.1 Pembrolizumab 
Pembrolizumab administration 
Sponsor Merck will provide each investigator with adequate supplies of 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/+10 minutes is permitted. 
 
5.3.2 Other Modality(ies) or Procedures 
 
5.3.2.1 
Palliative Radiation Therapy 
Palliative radiotherapy will be prescribed to a dose of 20 Gy in 5 fractions fractions to be 
delivered to a previously unirradiated area for which palliative radiation is indicated with a dose 
constraint of V20 Gy < 20% for the bilateral lung and V20 Gy < 32% to the bilateral kidney.  All 
patients will undergo CT guided radiation simulation with custom immobilization to be determined 
based on the area being irradiated.  A clinical target volume will be delineated by the treating 
radiation oncologist based on the CT simulation to treat gross disease and account for any 
uncertainty, incorporating information from prior diagnostic imaging and physical exam. 
Irradiation of elective volumes not thought to harbor gross disease is not allowed.  A margin of 
approximately 1 cm and individualized based on the region treated will be added to create the 
planning tumor volume. Conformal treatment planning is encouraged but not mandatory. Intensity 
modulated radiation therapy is permitted. Treatment will be administered on an outpatient basis, 
generally on 5 consecutive business days over a period of one to two weeks. If a treatment is 
missed because of unavoidable events (e.g. weather, machine maintenance, hospitalization, etc.) 
it will be made up for so that there are still 5 total delivered treatments.   
 
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
5.4.1 Contraception 
 
Pembrolizumab may have adverse effects on a fetus in utero.  Furthermore, it is not known if 
pembrolizumab has transient adverse effects on the composition of sperm.   
 
Male subjects will be considered to be of non-reproductive potential if they have azoospermia 
(whether due to having had a vasectomy or due to an underlying medical condition).   
 
Female subjects will be considered of non-reproductive potential if they are either:  
 postmenopausal (defined as at least 12 months with no menses without an alternative 
medical cause; in women < 45 years of age a high follicle stimulating hormone (FSH) 
level in the postmenopausal range may be used to confirm a post-menopausal state in 
women not using hormonal contraception or hormonal replacement therapy. In the 


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absence of 12 months of amenorrhea, a single FSH measurement is insufficient.);  
OR  
 have had a hysterectomy and/or bilateral oophorectomy, bilateral salpingectomy or 
bilateral tubal ligation/occlusion, at least 6 weeks prior to screening;  
OR  
 has a congenital or acquired condition that prevents childbearing. 
 
Female and male subjects of reproductive potential must agree to avoid becoming pregnant or 
impregnating a partner, respectively, while receiving study drug and for 120 days after the last 
dose of study drug by complying with one of the following:   
 practice abstinence† from heterosexual activity;  
OR  
 use (or have their partner use) acceptable contraception during heterosexual activity.   
 
Acceptable methods of contraception are‡: 
  
Single method (one of the following is acceptable): 
• 
intrauterine device (IUD) 
• 
vasectomy of a female subject’s male partner  
• 
contraceptive rod implanted into the skin 
 
Combination method (requires use of two of the following): 
• 
diaphragm with spermicide (cannot be used in conjunction with cervical cap/spermicide) 
• 
cervical cap with spermicide (nulliparous women only)   
• 
contraceptive sponge (nulliparous women only)  
• 
male condom or female condom (cannot be used together) 
• 
hormonal contraceptive: oral contraceptive pill (estrogen/progestin pill or progestin-only 
pill), contraceptive skin patch, vaginal contraceptive ring, or subcutaneous contraceptive 
injection 
  
†Abstinence (relative to heterosexual activity) can be used as the sole method of 
contraception if it is consistently employed as the subject’s preferred and usual lifestyle 
and if considered acceptable by local regulatory agencies and ERCs/IRBs.  Periodic 
abstinence (e.g., calendar, ovulation, sympto-thermal, post-ovulation methods, etc.) and 
withdrawal are not acceptable methods of contraception.  
  
‡If a contraceptive method listed above is restricted by local regulations/guidelines, then 
it does not qualify as an acceptable method of contraception for subjects participating at 
sites in this country/region. 
 
Subjects should be informed that taking the study medication may involve unknown risks to the 
fetus (unborn baby) if pregnancy were to occur during the study.  In order to participate in the 
study subjects of childbearing potential must adhere to the contraception requirement (described 
above) from the day of study medication initiation (or 14 days prior to the initiation of study 
medication for oral contraception) throughout the study period up to 120 days after the last dose 


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of trial therapy.  If there is any question that a subject of childbearing potential will not reliably 
comply with the requirements for contraception, that subject should not be entered into the study. 
 
5.4.2 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 overall PI.   
 
Acceptable Concomitant Medications 
All treatments that the investigator considers necessary for a subject’s welfare may be administered 
at the discretion of the investigator in keeping with the community standards of medical care.  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.   
Prohibited Concomitant Medications 
Subjects are prohibited from receiving the following therapies during the Screening and Treatment 
Phase of this trial: 
 Antineoplastic systemic chemotherapy or biological therapy  
 Immunotherapy not specified in this protocol 
 Investigational agents other than pembrolizumab  
 Radiation therapy not specified in this protocol 
 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 30 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 radiation 
or an event of clinical interest of suspected immunologic etiology.  The use of physiologic 
doses of corticosteroids (10 mg prednisone daily or equivalent) can be used without 
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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. 
5.4.3 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. 
Any medication intended solely for supportive care (e.g., analgesics, anti-diarrheal, anti-
depressants) may be used at the investigator’s discretion.  Antiemetics and anti-diarrheal 
medications should not be administered prophylactically before initial treatment with study 
drugs.  At the discretion of the investigator, prophylactic antiemetic and anti-diarrheal 
medication(s) may be used as per standard clinical practice before subsequent doses of 
study drugs or before, during or after radiation treatment. 
 Bisphosphonate or denosumab therapy to be used in accordance with the approved labeled 
indication and/or nationally recognized treatment guidelines.  Participants already 
receiving bisphosphonate/denosumab at the time of study entry can continue the treatment. 
 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. 
 
 
5.5 
Criteria for Taking a Participant Off Protocol Therapy 
 
Duration of therapy will depend on individual response, evidence of disease progression and 
tolerance. In the absence of treatment delays due to adverse event(s), treatment may continue for 
an indefinite number of cycles, or until one of the following criteria applies: 
 
 
Disease progression 
 
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 
 
Completed 35 treatments with pembrolizumab 


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 
General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator 
 
Participants will be removed from the protocol therapy when any of these criteria apply.  The 
reason for removal from protocol therapy, and the date the participant was removed, must be 
documented in the case report form (CRF) and in the CTMS system (OnCore). Alternative care 
options will be discussed with the participant. 
 
For sites using Centralized  Subject Registrations with the office of ODQ, 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.  
 
In the event of unusual or life-threatening complications, treating investigators must immediately 
notify the Overall PI, Sara Tolaney, MD at 617-632-5743 or stolaney@partners.org. 
 
Participants may elect to stop pembrolizumab with CR after at least 24 weeks of treatment and 
having had at least two treatments with pembrolizumab after documentation of the CR, or if they 
have been on pembrolizumab after 35 cycles..  
 
These patients may be eligible for up to 17 additional study treatments if they progress after 
stopping study treatment provided they meet the requirements detailed in Sections 3.1 and 3.2. 
This retreatment is termed the Second Course Phase of this study and is only available if the study 
remains open and the subject meets all the eligibility criteria included in section 3 and the following 
conditions: 
 
o Stopped initial treatment with pembrolizumab after attaining an investigator-
determined a confirmed CR according to RECIST 1.1, was treated for at least 24 weeks 
with pembrolizumab before discontinuing therapy, and received at least two treatments 
with pembrolizumab beyond the date when the initial CR was declared 
 
 
OR 
 
o Had a CR and stopped pembrolizumab treatment after 24 months of study therapy for 
reasons other than disease progression or intolerability. 
OR 
o Received 35 cycles of study therapy without disease progression 
 
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.  Patients must meet cycle 1 day 1 pre-treatment criteria to initiate 
therapy. 
 
5.6 
Duration of Follow Up 
 


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Participants removed from protocol therapy for unacceptable adverse event(s) will be followed 
until resolution or stabilization of the adverse event.   
 
For participants who are alive and free of disease progression at the time of removal from protocol 
therapy, tumor assessments should continue to be performed every 6-12 weeks until progression 
or death, whichever occurs first.  
 
All participants will be followed annually by medical record or phone until death. 
 
5.7 
Criteria for Taking a Participant Off Study 
 
Participants will be removed from study when any of the following criteria apply: 
 Lost to follow-up 
 Withdrawal of consent for data submission 
 Death 
 
The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the case report form (CRF) and CTMS (OnCore). 
 
For Centralized  Subject Registrations, 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 table(s). 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). Participants held for these reasons require prior approval from the PI and should resume 
therapy within 3 weeks of the scheduled interruption. The reason for interruption should be 
documented in the participant's study record. 
 
No dose reductions are allowed for pembrolizumab.  
 
If there are dosing delays for any reason, all study assessments are to be delayed in the same 
fashion, such that scans and other assessments occur in conjunction with cycles of treatment. 
 
6.1 
Management of toxicities attributable to pembrolizumab  
  
Adverse events (both non-serious and serious) associated with pembrolizumab exposure may 


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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 2 below. 
 
Table 2: 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 or 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.  
 
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. 
Grade 4 
Permanently 
discontinue 
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 


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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 -
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 
 Based on type and 
severity of AE administer 
corticosteroids  
 
Ensure adequate evaluation to 
confirm etiology and/or 
exclude other causes 
 
Grade 3 
Withhold or 
discontinue 
based on the type 
of event.  Events 
that require 
discontinuation 
include and not 
limited to:  
Gullain-Barre 
Syndrome, 


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encephalitis 
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 in Table 2 and Table 3. 
 
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 events of clinical interest (ECI) reporting 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 3 below shows treatment guidelines for subjects who experience an 
infusion reaction associated with administration of pembrolizumab. 
Table 3: Infusion Reaction Treatment Guidelines for pembrolizumab 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
Grade 1 
Mild reaction; infusion interruption 
not indicated; intervention not 
indicated 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
None 
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 
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 


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NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
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. 
(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. 
 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The following 
list of reported and/or potential AEs (Section 7.1) and the characteristics of an observed AE 
(Section 7.2) will determine whether the event requires expedited reporting in addition to routine 
reporting. 
 
7.1 
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 


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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) for Radiation Therapy 
The risks of radiation depend on the area of the body that will be treated with radiation.  In 
general, the risks of radiation to an area of the soft tissue, lymph or bone are listed below:   
 
 Tiredness 
 Skin reddening & irritation 
 thickening of skin in the treatment field 
 Blistering, ulceration or tissue death 
 Delayed wound healing 
 Alopecia  
 Swelling 
 Pain 
 Destruction of bone or cartilage and increased likelihood of fracture 
 Damage to muscles 
 
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 


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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  
 
7.3.1 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.  
 
7.3.2 DF/HCC Expedited Reporting Guidelines 
 
Investigative sites within DF/HCC will report AEs 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 of Adverse Events and Events of Clinical Interest to Merck 
 
7.6.1 Serious Adverse Events 
 
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; 


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 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 30 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 to 
the Overall PI within 24hrs of being notified of the event and 2 working days 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 Global 
Safety facsimile number:  +1-215-993-1220 
 
All subjects with serious adverse events must be followed up for outcome. 
 
7.6.2 Events of Clinical Interest (ECIs) 
 
Selected non-serious and serious adverse events are also known as Events of Clinical Interest (ECI) 
and must be recorded as such on the Adverse Event case report forms/worksheets and reported to 
the Overall PI within 24hrs of being notified of the event and within 2 working days to Merck 
Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220).  Events of clinical interest 
for this trial include: 
 
1. Overdose 
For purposes of this trial, an overdose of pembrolizumab will be defined as any 
dose of 1,000 mg 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 
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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.” 
 
 
2. Elevated AST or Lab value  
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.  The trial site 
guidance for assessment and follow up of these criteria can be found in the 
Investigator Trial File Binder (or equivalent). 
 
Additional adverse events: 
 
  
   
 
ECIs (both non-serious and serious adverse events) from the date of first dose through 30 days 
following cessation of treatment or the initiation of a new anticancer therapy, whichever is 
earlier, need to be reported to the overall PI within 24hrs of being notified of the event  and 
within 2 working days to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 
993-1220), regardless of attribution to study treatment, consistent with standard SAE reporting 
guidelines. 
 
 
Subjects should be assessed for possible ECIs prior to each dose.  Lab results should be 
evaluated and subjects should be asked for signs and symptoms suggestive of an immune-
related event.  Subjects who develop an ECI thought to be immune-related should have 
additional testing to rule out other etiologic causes.  If lab results or symptoms indicate a 
possible immune-related ECI, then additional testing should be performed to rule out other 
etiologic causes.  If no other cause is found, then it is assumed to be immune-related. 
 
 
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), including the pregnancy of a male subject's female partner that occurs during 
the trial or within 120 days of completing the trial, or 30 days following cessation of treatment if 


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the subject initiates new anticancer therapy, whichever is earlier.  All subjects and female partners 
of male subjects who become pregnant 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 to the Overall PI within 24hrs of being notified of the event and 2 
working days to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220) 
 
7.7 
Routine Adverse Event Reporting 
 
All Grade 2 or higher 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 4. 
 
Table 4:  Product Description 
Product Name & Potency 
Dosage Form 
Pembrolizumab 100 mg/ 4mL 
Solution for Injection 
 
8.1.3 Storage and Stability 
 


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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 a total cumulative storage 
time at room temperature and refrigeration of 24 hours.  PEMBROLIZUMAB solutions 
may be stored at room temperature for a cumulative time of up to 6 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. 
 
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 


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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. 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
 
In all patients in whom a tumor outside the field of radiation is accessible, a baseline tumor biopsy 
is required. We plan to use baseline biopsy tissue to perform a number of immune profiling assays, 
detailed below.  On baseline tumor biopsies, we will perform characterization based on histology 
(TILs), protein expression, and mRNA expression.  Additionally, we will bank specimens for 
possible future DNA analysis, and other further testing. 
 
Serial blood draws for correlative science are required on this trial; blood draws will be obtained 
every 3 weeks 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 an additional blood draw 
at the time of progressive disease, in patients who went off study for anything other than 
progressive disease.  On each blood draw, we will perform flow cytometry to characterize protein 
expression of immune mediators, detailed below, and additional blood will be banked for future 
testing. 
 
All patients will additionally be asked to provide a stool sample at three 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. 
 


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Please refer to the separate laboratory manual for additional correlative details including 
collection, processing, and shipping instructions. 
 
 
9.1 
Archival Tissue 
 
1 block or 10-20 5 micron unstained, charged slides will be collected for future research. 
  
9.2 
Fresh Tissue Biopsy  
 
9.2.1 Objectives:  
 
 
Characterizing immune markers in metastatic HR-positive breast tumors 
 
 
9.2.2 Collection 
 
Biopsies are required at screening and C2D1 (up to 3 days before and 7 days after C2D1). 
 
Biopsies should not be performed on Friday afternoons, as there may not be time for processing 
of the fresh tissue.  If a biopsy must be performed on Friday morning, the lab of Mariano 
Severgnini must be notified ahead of time to ensure that there will be adequate time for processing 
fresh tissue, since fresh tissue cannot be stored over the weekend.  The specimens in RNALater 
and formalin may be stored over the weekend and shipped on Monday. Specimens in RNA Later 
and formalin should be stored at room temperature until shipment.  
 
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 
 
The order of specimen collection should be: 
 First core: 10% neutral buffered formalin 
 Second core: Sterile DMEM 
 Third core: RNAlater 
 Fourth core: Sterile DMEM 
 Fifth core: 10% neutral buffered formalin 
 
If additional cores are obtained, they should be processed as follows: 
 Sixth core: RNAlater 
 Seventh core: 10% neutral buffered formalin 
 
If a skin punch is performed, the goal of tissue collection is 2, 5mm punches. The collection order 
should be as follows: 


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 First punch: 10% neutral buffered formalin 
 Second punch: RNAlater 
 
Guidelines for biopsy from various metastatic sites can be found in Appendix B. 
 
9.2.3 Handling and Shipping  
 
After being obtained, processing of the cores is as follows: 
 All samples should be de-identified and labeled with the Participant initials, Participant 
Study ID number and date of procedure. 
 One core in sterile DMEM should be brought as fresh tissue immediately to the lab of 
Mariano Severgnini at:  
Center for Immuno-Oncology 
Dana-Farber Cancer Institute 
1 Jimmy Fund Way, JF0406 
Boston, MA 02215 
Phone: (617) 632-2421 
Pager: 42093 
This core must arrive to the lab to be processed for TILs (as described below) within 90 
minutes of collection, though an additional 2-hour window is permitted..  In addition, a 
small piece of this core will be immediately frozen in liquid nitrogen upon arrival to 
Mariano Severgnini, for later use for RNA sequencing. 
 
 Two cores in formalin should be brought to the Brigham and Women’s SHL lab (with 
appropriate work order submitted and printed) on the 6th floor of the Thorn building, where 
a block will be made.  5 positively charged, 75mm x 25mm x 1mm slides should be cut 
from the block to be sent to QualTek for PD-L1 IHC assay. Shipping details will be 
provided in a lab manual.  The block should be brought to Dr. Scott Rodig on the 6th floor 
of the Thorn building at Brigham and Women’s. 
 
Specialized Histopathology Services – Longwood Core 
Brigham and Women’s Hospital, Thorn 604/603B 
75 Francis St.  
Boston MA 02115 
 
 
 
 One core in RNAlater should be brought to the DF/HCC Clinical Trial Core Laboratory 
(Deborah Dillon, MD) on Smith 9.  Please email the DF/HCC  Clinical Trials Core 
Laboratory (dfcibreastbank@partners.org) with patient name, study ID, date of collection, 
approximate time of collection, and study time point the day prior to collection.  
 
 
Tissue remaining after specific protocol testing described below will be banked in the Clinical 
Trial Core Laboratory (Deborah Dillon, MD) and may be used for additional or future analyses as 


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needed. 
 
 
9.2.4 Potential Testing 
 Assay 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.  In 
the research setting, all cases are reviewed by two pathologists and any discordant 
results resolved by consensus review.  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.[Salgado et al., 2015] 
 
After assessment of the TIL percentage, the pathologists will categorize the 
specimen as lymphocyte predominant breast cancer (LPBC), defined as a tumor 
that contains >60% stromal lymphocytes, or non-LPBC. 
 
 Assay 2: Immunohistochemistry 
 
Tissue will be collected and fixed by 10% neutral buffered formalin overnight, 
dehydrated, and paraffin embedded.  Four micrometer-thick sections will be cut.  
The paraffin blocks and unstained slides will be stored at room temperature.  All 
immunohistochemical staining will be performed in the Center for Immuno-
Oncology Pathology Core at Dana-Farber/Harvard Cancer Center (DF/HCC) 
Specialized Histopathology Core. 
 
Formalin fixed-paraffin embedded (FFPE) tumor slides will be prepared and H&E 
stained for assessment of TIL in pre- and post-treatment tumor samples.  To identify 
subsets of different immune populations (effector/memory CD8 cells, T regulatory 
cells, dendritic cells, tumor associated macrophages, and Tie-2 expressing 
monocytes (TEM)), immunohistochemical (IHC) staining will be performed on 
FFPE tumor slices using some or all of the following antibodies: 
Core set: CD8, PD-1, PD-L1, PD-L2 
Others: CD3, CD4, CD25, FoxP3, Indoleamine 2,3 deoxygenase-1 (IDO1), CD11c, 
CD83, CD86, CD56, CD14, CD16, Tie2. 
 
Investigators at our institution have developed IHC staining on paraffin embedded 
tissues for PD-L1, PD-L2, TIM-3, and LAG-3 through our center for Immuno-
Oncology Pathology Core (Scott Rodig MD, PhD Core Director, is a co-
investigator on this protocol).  PD-L1 IHC has recently been established in a CLIA 
approved laboratory and the remaining assays for CLIA laboratory conduct are 
being finalized. 
 


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These investigators have published the methods, protocols, and data establishing 
the sensitivity and specificity of IHC staining assays using the monoclonal 
antibodies recognizing PD-L1 (CD274, B7-H1, antibody clone 7G11, generated in 
the lab of Gordon Freeman, DFCI) and PD-L2 (CD273, B7-DC, clone 9E5, 
generated in the laboratory of Gordon Freeman, DFCI in two recent 
manuscripts.[Chen et al., 2013, Shi et al., 2014] 
 
 
Below is a schematic of the workflow for the tissue-based biomarker analysis. 
 
 
 
Tumor will be considered positive if >5% (PD-L1)[Topalian et al., 2012] or >10% 
(PD-L2) of the tumor cell population demonstrates unequivocal staining.  PD-1 
positivity will be defined as >3% positive cells/high power field.[Bachireddy et al., 
2014]  All IHC stained slides will be evaluated and scored by a pathologist.  A 
subset of slides will be reviewed by a second pathologist to ensure concordance of 
interpretation. 
 
The semi-quantitative scoring for this study is in accordance with those published 
previously and, as described above, will include scores for both the neoplastic and 
non-neoplastic cells within the tumor microenvironment.  Data derived from 
pathologist visual scoring (semi-quantitative, but with increased specificity for 


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42 
delineating neoplastic and non-neoplastic cells) and pathologist-assisted, 
automated scoring (quantitative, but without accurately delineating neoplastic and 
non-neoplastic cells) for each marker of interest will be assessed for its clinical 
value.  As necessary, the data from combinations of markers and multiplex 
immunofluorescent assays will also be considered (i.e. combined scores from PD-
L1 and PD-L2 expression).  All data will be analyzed in conjunction with the 
biostatistics group. 
 
Further details of the immunohistochemical assay and assessment are described in 
the lab manual. 
 
 Assay 3: Flow cytometry 
 
TILs will be isolated from the biopsy specimen as described in the lab manual 
 
Surface staining followed by flow cytometry on the resultant TILs will then be 
performed as described in the lab manual.   The following antibodies may be used 
on all specimens: (core set) 
CD8 
PD-1 
PD-L1 
PD-L2 
 
A selection of the following antibodies may also be used, and additional antibodies 
may be used as well, as deemed appropriate and informative based on the state of 
the immune profiling literature at the time of correlative science performance: 
CD4 
FOXP3 
CD127 
 
 Assay 4: RNA analysis 
 
RNA analysis may be performed, and tissue for RNA analysis will be stored, in the 
Clinical Trials Core Laboratory (Deborah Dillon, MD). 
 
Messenger RNA (mRNA) expression within tumor biopsy specimens may be 
assessed using the most current and informative methodologies at the time that 
correlative science is performed on all specimens.  NanoString signatures and 
comprehensive RNA sequencing may be used.  Potential genes of interest, based 
on prior immune profiling of breast tumors,[Denkert et al., 2015] include CXCL9, 
CCL5, CD8ACD80, CXCL13, IGKC, CD21, IDO1, PD-1, PD-L1, PD-L2, 
CTLA4, and FOXP3. 
 
 
9.2.5 Sites Performing Correlatives 
 


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43 
BWH 
 
DFCI CIO  
 
9.3 
Blood Collection 
 
Research blood collection is mandatory for all patients for flow cytometry and potential DNA 
isolation.  The samples will be banked in the DFCI breast tissue repository for these and future 
research purposes. These specimens will become the property of the DF/HCC.  
 
Blood draws should not be performed on Friday afternoons, as there may not be time for processing 
of the blood.  If a blood draw must be performed on Friday morning, the lab of Mariano Severgnini 
must be notified ahead of time to ensure that there will be adequate time for processing the blood, 
since it cannot be stored over the weekend.   
 
The following research blood samples are required:  
Cycle 1 Day 1:  
 1-9mL Streck Tube for whole blood (cfDNA) 
 5- 10mL green top tubes for whole blood (Immune Markers) 
 
Every Cycle Day 1:  
 5- 10mL green top tubes for whole blood (Immune Markers) 
 
Restaging Visits: 
 1-9 mL Streck Tube for whole blood (cfDNA) 
 
 
Off Treatment (if for progressive disease): 
 1-9 mL Streck Tube for whole blood (cfDNA) 
 5- 10mL green top tubes for whole blood (Immune Markers) 
 
The following Time of Progression research blood samples are optional for patients who came off 
treatment for a reason other than progressive disease: 
 1-9 mL Streck Tube for whole blood (cfDNA) 
 5- 10mL green top tubes for whole blood (Immune Markers) 
 
If green top tubes for whole blood (Immune Markers) are unavailable, purple or CPT tubes are 
can be substituted. 
 
9.3.1 Immune Markers 
9.3.1.1 
Handling and Shipping  
 
All samples should be de-identified and labeled with the Participant initials, Participant Study ID 


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44 
number and date of collection and time point (e.g., “Baseline” or “Cycle 1” or “Progressive 
Disease”).  
 
 Green Top tubes: 
Must be processed within 3-4hrs of its being drawn at ambient temperature 
immediately after being drawn to Mariano Severgnini at: 
 
Center for Immuno-Oncology 
Dana-Farber Cancer Institute 
1 Jimmy Fund Way, JF0406 
Boston, MA 02215 
Phone: (617) 632-2421 
Pager: 42093 
 
9.3.1.2 
Potential Testing 
 
Assay 1: Flow cytometry 
 
PBMCs will be generated as described in the lab manual, and used to assess immune cell 
populations. 
 
Surface staining with a panel of antibodies and flow cytometry on PBMCs will then be 
performed as described in Appendices.  The following antibodies will be used on all 
specimens: (core set) CD8, PD-1, PD-L1, PD-L2,  
A selection of the following antibodies may also be used, and additional antibodies may be 
used as well, as deemed appropriate and informative based on the state of the immune 
profiling literature at the time of correlative science performance: CD4, FOXP3, CD127 
 
9.3.1.3 
Sites Performing Correlatives 
 
 
DFCI CIO  
 
 
 
9.3.2 Cell-free DNA (cfDNA) analysis 
 
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.   
 
9.3.2.1 Collection of cfDNA specimen(s) 
 
One 10 ml of whole blood will be collected in Streck Tubes.  The blood sample will be collected 
and processed at baseline, restaging visits and time of progression for evaluation of cfDNA.  Fill 
the Streck tube completely and immediately mix by gentle inversion 8 to 10 times.  Inadequate 
or delayed mixing may result in accurate results.  The banked samples will be used to analyze 
DNA, RNA and protein in future studies. 


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9.3.2.2 Handling and shipping of cfDNA specimens 
 
One 10 ml Streck tube will be collected and processed at baseline, restaging visits and at time of 
progression for evaluation of cfDNA.  Fill the Streck tube completely and immediately mix by 
gentle inversion 8 to 10 times.  Inadequate or delayed mixing may result in accurate results.  
Deliver to Clinical Trials Core Lab.  
 
Dana-Farber Cancer Institute 
Attn: Lynda Chichester 
Smith 9th Floor, Rm 948 
450 Brookline Avenue 
Boston, MA 02215 
dfcibreastbank@partners.org 
 
Email the blood bank (dfcibreastbank@partners.org) and the current Dana-Farber CRC with the 
sample information and tracking information the day before shipping specimens. 
 
Tube precautions: 
 If samples cannot be shipped within 24 hours of collection, contact DFCI.  DO NOT 
FREEZE OR REFRIDGERATE 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. 
 
Shipping Note:  Streck tube samples are sent ambient.  Frozen and ambient specimens 
obtained and shipped on the same day to the DFCI blood bank (e.g., Progression or Off 
Study Biopsy Specimens, Streck Tubes, and Circulating Tumor Cells) may be placed 
in a combination shipping box which contains separate compartments for frozen and 
ambient samples.  If a combination shipping box is not available, two shipping boxes 
should be used. 
 
 
9.3.2.3 
Sites Performing Correlatives 
 
DFCI Clinical Trials Core Lab 
 
9.4 
Stool Collection 
 
9.4.1 Handling and shipping 
 
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: 
 


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 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 in batches 
to an external lab vendor, Microbiome Dx, who will perform the analysis of the samples.  
 
 
9.4.2 Analysis of DNA extraction from stool samples 
 
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 
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.  
 
 
 


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9.4.3 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. 
 
 
9.4.4 Sites performing correlative analysis 
 
 BWH/Harvard Cohorts Biorepository  
 Microbiome Dx 
 
 
 
9.5 
Additional analysis 
 
The above-mentioned analyses may be altered based on novel developments in the field of cancer 
immune profiling at the time of correlative science.  Additional markers or alternative technologies 
(based on scientific developments and/or novel technologies) may also be used, to explore 
potential prognostic or predictive candidate markers/panels or markers related to treatment benefit 
and/or safety, to improve diagnostic tests, or to understand breast cancer biology. 
 
 
 
 
 
 
 
 
 
10. STUDY CALENDAR 
 
Screening evaluations are to be conducted within 28 days prior to start of protocol therapy unless 
otherwise specified.  Screening assessments occurring within 3 days prior to initiating study 
treatment do not need to be repeated on Cycle 1 Day 1.   
 
As detailed in the Study Calendar, laboratory assessments must be documented within 7 days 
before the first dose of study medication.  For women of childbearing potential, as defined in 
section 5.4.1, a pregnancy test must be completed within 7 days of receiving the first dose of study 
medication.  If a urine pregnancy test is positive or cannot be confirmed as negative, a serum 
pregnancy test will be required.   


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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. 
 
 


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Screeninga 
C1 
D1 
C2 
D1 
Cycle 3 + 
D1 
Off-
Treatmentl 
Follow-
Upp 
Informed consent 
X 
 
 
 
 
 
Radiotherapy Planning 
Assessments 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
Concurrent medications 
X 
X 
X 
X 
X 
X 
Physical exam 
X 
X 
X 
X 
X 
 
Radiation Oncologyb 
 
X 
 
 
 
 
Performance status 
X 
X 
X 
X 
X 
 
Adverse event evaluation 
 
X 
X 
X 
X 
 
Vital signsc 
X 
X 
X 
X 
X 
 
Hematology paneld  
X 
X 
X 
X 
X 
 
Chemistry paneld 
X 
X 
X 
X 
X 
 
TSHe 
X 
X 
X 
X 
 
 
Coagulation panel 
(PT/PTT) 
X 
 
 
 
 
 
Cortisolm 
X 
 
X 
X 
 
 
Pregnancy testf 
X 
 
 
 
 
 
Single 12- lead EKG 
X 
 
 
 
 
 
Tumor Measurementsg 
X 
 
 
X 
 
Xj 
Archival Tumorh 
X 
 
 
 
 
 
Research Biopsyi 
X 
 
X 
 
 
 
Research Bloodk 
 
X 
X 
X 
X 
X 
Research Stool 
Collectionn 
X 
 
 
X 
X 
 
Stool Questionnaireo 
X 
 
 
X 
X 
 
Survival 
 
 
 
 
 
Xp 
Pembrolizumab: 200mg given IV over 30 minutes (-5minutes/+10minutes q3weeks on Day 1 of each 
cycle 
Radiotherapy: Patients will start radiotherapy 2-7 days after their first infusion of pembrolizumab and 
be delivered over 5 treatments of 4Gy over a period of 1-2 weeks.  It should be delivered on 5 
consecutive business days but unavoidable delays due to machine maintanence, holidays, etc it is 
permitted to extend to 10 days. 
 
a. 
Screening assessments are to be conducted within 28 days prior to start of protocol therapy 
unless otherwise specified.  If these screening assessments occur within 3 days before start of 
study treatment, then they may serve as the baseline Cycle 1 Day 1 values.  
b. 
Patients will be seen at their final treatment (+/- 4 days) and 10 days after completion of RT 
(+/- 4 days) by a radiation oncologist in addition to their regularly scheduled medical 
oncology visits.  
c. 
Vitals to include: diastolic and systolic blood pressure, heart rate, temperature, and weight 
d. 
Labs to include: CBCA + Differential, 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) 
e. 
TSH will be performed on C1D1, C2D1, C3D1, C4D1 and then every other cycle on Day 1. 
f. 
In female subjects of child-bearing potential as defined in section 5.4, a pregnancy test must 
be performed within 7 days of the first dose of study medication.  If a urine test is positive or 
cannot be confirmed as negative, then a serum test is required. 
g. 
Tumor measurements are repeated every 6 weeks for the first 24 weeks and then every 9 
weeks thereafter.  Documentation (radiologic) must be provided for participants removed 
from study for progressive disease. Confirmatory scans 4 weeks after documented response 
should be obtained. 
h. 
Archival tumor sample should be collected (block or if not possible, 10-20 5micron, 
unstained, positively charged slides). 
i. 
A baseline tumor biopsy obtained within 28 days before starting protocol therapy is required 
for those with accessible tumor tissue outside the field of radiotherapy.  The C tumor biopsy 
should be performed as close to C2D1 as possible. It would be most preferable to perform the 
biopsy after the second treatment of pembrolizumab, but it may be performed up to 3 days 
before and 7 days after C2D1.    
j. 
For those taken off treatment for reasons other than progressive disease, tumor measurements 
should continue to be repeated every 6-9 weeks and after 1 year of assessments every 12 
weeks. 
k. 
cfDNA will be collected at screening, restaging visits and at time of progression 


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l. 
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.  SAEs and ConMeds need to be recorded for 30 days after the 
final treatment.  
m. 
Cortisol may be drawn at any time of day, though is most informative in the morning.  
n. 
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 collection 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. 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. 
o. 
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 at the required or optional 
timepoints will not constitute a protocol violation. 
p. 
Survival status should be collected annually, either by phone or medical record.  
 
 


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11. MEASUREMENT OF EFFECT 
 
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) 
[Eisenhauer et al., 2009].  Changes in the largest diameter (unidimensional measurement) of the 
tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the 
RECIST criteria. 
 
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 (every 9 weeks for the first 12 
months and then every 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 outside the field of radiation present at baseline, have received at least one cycle of 
therapy, and have had their disease re-evaluated will be considered evaluable for target 
disease response.  These participants will have their response classified according to the 
definitions stated below.  (Note:  Participants who exhibit objective disease progression 
prior to the end of cycle 1 will also be considered evaluable.) 
 
Evaluable Non-Target Disease Response.  Participants who have lesions present at baseline 
that are evaluable but do not meet the definitions of measurable disease, have received at 
least one cycle of therapy, and have had their disease re-evaluated will be considered 
evaluable for non-target disease.  The response assessment is based on the presence, 
absence, or unequivocal progression of the lesions.  
 


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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 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 


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also be recorded at baseline. Measurements of these lesions are not required, but the 
presence, absence, or in rare cases unequivocal progression of each should be noted 
throughout follow up.  
 
11.1.3 Methods for Evaluation of Disease 
 
All measurements should be taken and recorded in metric notation using a ruler, calipers, 
or a digital measurement tool.  All baseline evaluations should be performed as closely as 
possible to the beginning of treatment and never more than 4 weeks before the beginning 
of the treatment. 
 
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging-based 
evaluation is preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam.  
 
Clinical lesions. Clinical lesions will only be considered measurable when they are 
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:  


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(a) Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of 
PD based on a new lesion. 
(b)  No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive 
FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is 
PD.  If the positive FDG-PET at follow-up is not confirmed as a new site of disease on 
CT, additional follow-up CT  scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not 
PD. 
(c)  FDG-PET may be used to upgrade a response to a CR in a manner similar to a 
biopsy in cases where a residual radiographic abnormality is thought to represent 
fibrosis or scarring.  The use of FDG-PET in this circumstance should be prospectively 
described in the protocol and supported by disease-specific medical literature for the 
indication.  However, it must be acknowledged that both approaches may lead to false 
positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity. 
 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake 
greater than twice that of the surrounding tissue on the attenuation corrected image. 
 
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.  


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Planar: Anterior and posterior views from the top of the head to the proximal lower 
extremities are obtained for 10 minutes at 24 hours and occasionally at 48 hours 
following injection of 10 mCi/1.7 square meters of body surface area (~150 μCi/kg, 
maximum 10 mCi). Anterior views of the distal lower extremities are adequate. A large 
field of view dual head gamma camera with low energy collimators is preferred.  
 
SPECT: Most participants receiving I-123 MIBG also undergo SPECT at 24 hours, 
using a single or multi-headed camera with a low energy collimator. The camera is 
rotated through 360 degrees, 120 projections at 25 seconds per stop. Data are 
reconstructed using filtered back projections with a Butterworth filter and a cut off 
frequency of 0.2-0.5. SPECT/CT may be performed at institutions with this capacity. 
 
Ultrasound. Ultrasound is not useful in assessment of lesion size and should not be used as 
a method of measurement. Ultrasound examinations cannot be reproduced in their entirety 
for independent review at a later data and, because they are operator dependent, it cannot 
be guaranteed that the same technique and measurements will be taken from one 
assessment to the next. If new lesions are identified by ultrasound in the course of the study, 
confirmation by CT or MRI is advised. If there is concern about radiation exposure from 
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 


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nodes (whether target or non-target) must have reduction in short axis to <10 mm. 
 
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters. 
 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of target 
lesions, taking as reference the smallest sum on study (this includes the baseline sum if 
that is the smallest on study).  In addition to the relative increase of 20%, the sum must 
also demonstrate an absolute increase of at least 5 mm.  (Note:  the appearance of one 
or more new lesions is also considered progressions). 
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 
study. 
 
11.1.3.2 
Evaluation of Non-Target Lesions 
 
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
short axis). 
 
Note:  If tumor markers are initially above the upper normal limit, they must normalize 
for a patient to be considered in complete clinical response. 
 
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or maintenance 
of tumor marker level above the normal limits. 
 
Progressive Disease (PD):  Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions.  Unequivocal progression should not 
normally trump target lesion status.  It must be representative of overall disease status 
change, not a single lesion increase.     
 
Although a clear progression of “non-target” lesions only is exceptional, the opinion of 
the treating physician should prevail in such circumstances, and the progression status 
should be confirmed at a later time by the review panel (or Principal Investigator). 
 
11.1.3.3 
Evaluation of New Lesions 
 
The finding of a new lesion should be unequivocal (i.e. not due to difference in 
scanning technique, imaging modality, or findings thought to represent something other 
than tumor (for example, some ‘new’ bone lesions may be simply healing or flare of 
pre-existing lesions). However, a lesion identified on a follow-up scan in an anatomical 
location that was not scanned at baseline is considered new and will indicate PD. If a 
new lesion is equivocal (because of small size etc.), follow-up evaluation will clarify if 
it truly represents new disease and if PD is confirmed, progression should be declared 
using the date of the initial scan on which the lesion was discovered. 


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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. 
 
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). 
 


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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 (CBR): defined as CR, PR and stable disease (SD) ≥  24 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 (irRC) 
 
The sum of the longest diameter of lesions (SPD) at tumor assessment using the immune-
related response criteria (irRC) for progressive disease incorporate the contribution of new 
measurable lesions.  Each net Percentage Change in Tumor Burden per assessment using 
irRC criteria accounts for the size and growth kinetics of both old and new lesions as they 
appear. 
 
11.3.1.1 
Impact of New Lesions on irRC 
 
New lesions in and of themselves do not qualify as progressive disease.  However, their 
contribution to total tumor burden is included in the SPD which in turn feeds into the irRC 
criteria for tumor response.  Therefore, new non-measurable lesions will not discontinue 
any subject from the study. 
 
11.3.1.2 
Definition of Target Lesions Response Using irRC 
 
 irComplete Response (irCR): Complete disappearance of all target lesions.  This category 
encompasses exactly the same subjects as “CR” by the mWHO criteria. 
 irPartial Response (irPR): Decrease, relative to baseline, or 50% or greater in the sum of 
the products of the two largest perpendicular diameters of all target and all new measurable 
target lesions (i.e., Percentage Change in Tumor Burden).  Note: the appearance of new 
measurable lesions is factored into the overall tumor burden, but does not automatically 
qualify as progressive disease until the SBD increases by >25% when compared to SPD at 
nadir. 


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 irStable Disease (irSD): Does not meet criteria for irCR or irPR, in the absence of 
progressive disease. 
 irProgressive Disease (irPD): At least 25% increase Percentage Change in Tumor Burden 
(i.e. taking SPD of all target lesions and any new lesions) when compared to SPD at nadir. 
 
11.3.1.3 
Definition of Non-Target Lesions Response Using irRC 
 
 irComplete Response (irCR): Complete disappearance of all non-target lesions.  This 
category encompasses exactly the same subjects as “CR” by the mWHO criteria. 
 irPartial Response (irPR) or irStable Disease (irSD): Non-target lesion(s) are not 
considered in the definition of PR; these terms do not apply. 
 irProgressive Disease (irPD): Increases in number or size of non-target lesion(s) does not 
constitute progressive disease unless/until the Percentage Change in Tumor Burden 
increases by 25% (i.e. the SPD at nadir of the target lesions increases by the required 
amount). 
 
11.3.1.4 
Definition of Overall Response Using irRC 
 
Overall response using irRC will be based on these criteria: 
 
 Immune-Related Complete Response (irCR): Complete disappearance of all tumor 
lesions (target an non-target) together with no new measurable/unmeasurable lesions for at 
least 4 weeks from the date of documentation of complete response. 
 Immune-Related Partial Response (irPR): The sum of the products of the two largest 
perpendicular diameters of all target lesions is measured and captured as the SPD baseline.  
At each subsequent tumor assessment, the SPD of the two largest perpendicular diameters 
of all target lesions and of new measurable lesions are added together to provide the 
Immune Response Sum of Product Diameters (irSPD).  A decrease, relative to baseline, of 
the irSPD compared to the previously SPD baseline  of 50% or greater is considered an 
irPR. 
 Immune-Related Stable Disease (irSD): irSD is defined as the failure to meet criteria for 
immune complete response or immune partial response, in the absence of progressive 
disease 
 Immune-Related Progressive Disease (irPD): It is recommended in difficult cases to 
confirm PD by serial imaging.  Any of the following will constitute PD: 
 At least 25% increase in the SPD of all target lesions over baseline SPD calculated for 
the target lesions. 
 At least 25% increase in the SPD of all target lesions and new measurable lesions 
(irSPD) over the baseline SPD calculated for the target lesions. 
 
Criteria for determining overall response by irRC are summarized as follows: 


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11.3.1.5 
Immune-Related Best Overall Response Using irRC (irBOR) 
 
irBOR is the best confirmed overall response over the study as a whole, recorded between 
the date of first dose until the last tumor assessment before subsequent therapy (except for 
local palliative radiotherapy for painful bone lesions) for the individual subject in the study.  
For the assessment of irBOR, all available assessments per subject are considered. 
 
irCR or irPR determinations included in the irBOR assessment must be confirmed by a 
second (confirmatory) evaluation meeting the criteria for response and performed no less 
than 4 weeks after the criteria for response are first met. 
 
 
11.3.2 Definition of Abscopal response 
As described previously, abscopal response is defined as a decrease in the longest 
diameter of at least 30% in any measurable (≥1 cm) non-irradiated lesion from 
baseline[Golden et al., 2015]. In patients with more than three lesions, the non-irradiated 
lesions will be measured individually for response to treatment. The best abscopal 
responding lesion will be reported. 
 
11.3.2.1 
Definition of overall response according abscopal response definition 
 
 Complete abscopal response is defined as the complete disappearance of a measurable non-
irradiated lesion. 
 Partial abscopal response is defined as at least a 30% decrease in the longest diameter. 


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 Progressive disease is defined as at least a 20% increase in the longest diameter of the 
best measurable non-irradiated lesion.  
 Stable disease is defined as insufficient shrinkage or growth to qualify for a partial 
abscopal or complete abscopal response or progressive disease. 
 
12. DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
 
12.1 
Data Reporting 
 
12.1.1 Method 
 
The ODQ will collect, manage, and perform quality checks on the data for this study. 
 
12.1.2 Responsibility for Data Submission 
 
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the ODQ according to the schedule set by the ODQ. 
 
12.2 
Data Safety Monitoring 
 
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity 
and accrual data from this study. The committee is composed of clinical specialists with experience 
in oncology and who have no direct relationship with the study. Information that raises any 
questions about participant safety will be addressed with the Overall PI and study team. 
 
The DSMC will review each protocol up to four times a year or more often if required to review 
toxicity and accrual data. Information to be provided to the committee may include: up-to-date 
participant accrual; current dose level information; DLT information; all grade 2 or higher 
unexpected adverse events that have been reported; summary of all deaths occurring with 30 days 
of intervention for Phase I or II protocols; for gene therapy protocols, summary of all deaths while 
being treated and during active follow-up; any response information; audit results, and a summary 
provided by the study team. Other information (e.g. scans, laboratory values) will be provided 
upon request.  
12.3 
Multicenter Guidelines 
 
N/A 
 
12.4 
Collaborative Agreements Language 
 
N/A 
 


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13. STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Endpoints 
 
This is a single-arm phase 2 study to evaluate efficacy of pembrolizumab in combination with 
radiation therapy for patients with metastatic HR positive breast cancer.  
 
This study uses a Simon “optimal” two-stage design. 8 patients will be enrolled in the first stage, 
and if the study continues to the second stage, another 19 patients would be enrolled. A total of 27 
patients will be enrolled in this study.  
 
Primary Endpoint 
The primary endpoint of this trial is ORR outside the field of radiation, according to RECIST 1.1 
as defined in Section 11.1.  
 
Secondary endpoints include: 
Secondary endpoints include ORR according to immune-related response criteria (irRC), ARR 
according abscopal response definition (as defined in Section 11.3.2), PFS according to RECIST 
1.1 and irRC (as defined in Section 11.3), CBR, irCBR, safety and tolerability.  
 
Blood and Tissue correlative science objectives include:  
 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). 
 To explore how different immunosuppressive and/or immune-stimulating immune marker 
profiles at baseline correlate with disease response to therapy (response assessed by RECIST 
1.1 and immune-related response criteria). 
 To characterize changes in tumor-infiltrating lymphocytes and PD-L1 expression immune 
marker profile in tissue microenvironment (TME) from baseline to after 2 cycles of 
Pembrolizumab. 
 To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in TME correlates with disease response to therapy 
(response assessed by RECIST 1.1, irRC and ARR). 
 To characterize serial changes in immune marker profile in peripheral blood mononuclear 
cells (PBMCs) over the course of the trial treatment. 
 To explore whether induction of changes in the immunosuppressive and/or immune-
stimulating immune marker profile in PBMCs correlates with disease response to therapy 
(response assessed by RECIST 1.1, irRC and ARR). 
 To investigate whether there is an immune marker in circulating PBMCs that corresponds to 
tumor infiltrating lymphocyte (TIL) percentage in baseline tumor  
 To collect blood to study cell-free DNA for comparison to tumor specimens before and after 
immunotherapy 
 
Stool and microbiome correlative science endpoints: 
 


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Overall, we plan to describe the landscape of gut microbiota in patients with BC who will 
receive the combination of radiation therapy plus 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. 
 
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.  
 
 
13.2 
Sample Size, Accrual Rate and Study Duration 
 
Based on data of recently presented Javelin (cohort of metastatic breast cancer) and Keynote-028 
trials[Dirix et al., 2015, Hugo et al., 2015], and considering that our population will not be 
previously selected by PD-L1 expression status, a true ORR outside the field of radiation of 3% or 
less would not be of clinical interest, and is the null hypothesis to the Simon optimal two-stage 
design.  A true rate of 20% would be considered a clinically meaningful level of response, so the 
sample size was chosen to have higher power (80%) to declare the combination effective at this 
rate, while controlling the one-sided Type I error at no more than 5% under the null.  
 
Using the Simons “optimal” method, in the first stage, 8 patients will be enrolled. If there is at 
least 1 response, accrual will continue to the second stage where up to 19 additional patients will 
be enrolled. If at least 3 of these 27 patients have an objective response (≥10%), the regimen will 
be considered worthy of further study.  With this design, the probability of stopping the trial early 


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is 78% if the true response rate is 3%. If the true response rate is 20% the chance that the regimen 
is declared worthy of further study is 80%. 
 
The expected accrual rate is 0-1 patients per month, and the accrual is expected to complete within 
approximately 54 months. 
 
13.3 
Stratification Factors 
 
NA   
 
13.4 
Interim Monitoring Plan 
 
An interim analysis will happen after 8 patients enrolled in the first stage.  If there is at least 1 
response, accrual will continue to the second stage where up to 19 additional patients will be 
enrolled. 
 
13.5 
Analysis of Primary Endpoint 
 
The primary endpoint is objective response outside the field of radiation, which will be assessed 
among all patients who initiated protocol therapy. Radiographic response will be assessed using 
RECIST 1.1 criteria as defined in section 11.1. Objective response will require confirmatory scans 
as indicated.  The ORR (CR + PR) will be reported with 90% exact confidence intervals.   
 
13.6 
Analysis of Secondary Endpoints 
 
Efficacy Endpoints  
All patients who initiated protocol therapy will also be evaluated for ORR outside the field of 
radiation according irRC, and for CBR and PFS, according RECIST and irRC. In addition, these 
patients will be evaluated for ARR according abscopal response definition (Section 11.3.2).  
Clinical benefit is defined as CR, PR or SD ≥ 24 weeks according RECIST 1.1. Immune-related 
clinical benefit is defined as CR, PR or SD ≥ 24 weeks according irRC. ORR according to irRC 
and ARR will be reported with 90% exact confidence intervals. CBR will be reported respectively 
with 95% exact confidence intervals. The radiation plan will be reviewed and the treated volume 
(clinical target volume, planning target volume, area receiving 100% and 95% of the planned 
dose), and the site of treatment will be also evaluated; we will explore if there is any correlation 
between these parameters and the likelihood of clinical benefit. PFS,  irPFS, and OS will be 
analyzed using Kaplan–Meier product-limit estimates and will be plotted using Kaplan-Meier 
plots. PFS is defined as the time from study randomization to disease progression according 
RECIST 1.1, medical judgment or death due to any cause, whichever occurred first.  Immune-
related PFS is defined as the time from study randomization to disease progression according irRC, 
medical judgment or death due to any cause, whichever occurred first. Patients alive without 
disease progression are censored at the date of last disease evaluation.  The hazard ratio for each 
time-to-event endpoint will be estimated with 95% confidence intervals derived from the Cox 
proportional hazard model, but no hypothesis testing will be conducted.   
 
Safety and tolerability  


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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 Fisher’s exact test. 
 
Correlative 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 expression, and 
mRNA expression) 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 
immune-related response criteria) using odds ratios and 95% confidence intervals. 
 PD-L1 positivity seen at baseline and C3D1 samples will be summarized using contingency 
tables. An exploratory analysis is planned to evaluate PD-L1 change in continuous scale. 
 We will explore the correlation of immunosuppressive and/or immune-stimulating immune 
marker profiles in TME to disease response to therapy (response assessed by RECIST 1.1, irRC 
and ARR) using odds ratios and 95% confidence intervals. Descriptive statistics will be used to 
characterize serial changes in immune marker profile in peripheral blood mononuclear cells 
(PBMCs) over the course of the trial treatment 
 We will explore the correlation of immunosuppressive and/or immune-stimulating immune 
marker profiles in PBMCs to disease response to therapy (response assessed by RECIST 1.1, 
irRC and ARR) using odds ratios and 95% confidence intervals. 
 We will explore whether there is an immune marker in circulating PBMCs that corresponds to 
tumor infiltrating lymphocyte (TIL) percentage in baseline tumor. T-test, ANOVA, or regression 
will be used, depending on whether immune markers are characterized as categorical or 
continuous variables.      
 Descriptive statistics will be used to characterize cell-free DNA before and after immunotherapy. 
 Descriptive statistics will be used to characterize tumor genomic profile by disease response to 
therapy (response assessed by RECIST 1.1, irRC, and ARR). PFS and OS will be estimated 
using Kaplan-Meier methods among patients with and without certain gene mutations, 
respectively. 
 
 
Previous studies demonstrated that, in addition to its direct cytoreductive effect, RT-induced cell 
death can be immunogenic, facilitating the recruitment and activation of antigen presenting cells 
(APCs) and priming of tumor antigen-specific T-cells[Shahabi et al., 2015]. Recently, different 
groups demonstrated that RT to the tumor bed led to upregulation of PD-L1 on tumor cells, 
dendritic cells, and on myeloid-derived suppressive cells (MDSCs), which may contribute to 
impairment of T-cell function in the tumor[Liang et al., 2013, Deng et al., 2014, Sharabi et al., 
2014]. Furthermore, these groups also demonstrated that the combination of RT plus blockade of 
the PD-1/PD-L1 axis improved outcomes in different preclinical models compared with RT or 
anti-PD1/PD-L1 alone, including breast cancer models.  


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Recently, Herbst et al have demonstrated that patients who presented an increase of at least 5% in 
expression of PD-L1 in tumor microenvironment experienced a bigger likelihood to respond to 
treatment with the anti-PD-L1 Atezolizumab[Herbst et al., 2014]. Also, modifications in molecular 
signature of tumor microenvironment also correlated with response rate to this drug. Because of 
this rationale, we plan to perform two research biopsies in tumor lesion outside the field of 
radiation: one at baseline and the other one just before the begging of cycle 3 of pembrolizumab.  
We anticipate that 25 (93%) pairs of tumor specimens will be evaluable for PD-L1 expression.  
PD-L1 positivity is defined as ≥5%. Assuming 2% of patients unexpectedly show PD-L1 
positivity only in the baseline assessment, we have 80% power to detect 30% increases of PD-L1 
positivity rate (e.g. 20% at baseline vs. 50% at C3D1). The power calculation is based on 
McNemar’s test with 1-sided alpha of 0.05. 
 
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 patients who initiate protocol 
therapy, even if there are major protocol therapy deviations.   
Subanalyses may then be performed on the basis of a subset of participants, excluding those 
for whom major protocol deviations have been identified (e.g., early death due to other 
reasons, early discontinuation of treatment, major protocol violations, etc.).  However, 
these subanalyses may not serve as the basis for drawing conclusions concerning treatment 
efficacy, and the reasons for excluding participants from the analysis should be clearly 
reported.  If applicable to the endpoint, the 95% confidence intervals should also be 
provided.  
 
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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REFERENCES 
 
 
Adamow, M., Ritter, E., Sedrak, C., Roman, R.-A., Rosner, S., Benson, B. et al. (2012) Effect in 
a Patient with Melanoma.  
 
Adams, S., Gray, R.J., Demaria, S., Goldstein, L., Perez, E.A., Shulman, L.N. et al. (2014) 
Prognostic Value of Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancers from 
Two Phase Iii Randomized Adjuvant Breast Cancer Trials: Ecog 2197 and Ecog 1199. Journal 
of Clinical Oncology 32: JCO.2013.2055.0491--JCO.2013.2055.0491-. 
 
Ali, H.R., Provenzano, E., Dawson, S., Blows, F.M., Liu, B., Shah, M. et al. (2014) Association 
between Cd8 + T-Cell in Fi Ltration and Breast Cancer Survival in 12 439 Patients. 1536-1543. 
 
Bachireddy, P., Hainz, U., Rooney, M., Pozdnyakova, O., Aldridge, J., Zhang, W. et al. (2014) 
Reversal of in Situ T-Cell Exhaustion During Effective Human Antileukemia Responses to 
Donor Lymphocyte Infusion. Blood 123: 1412-1421. 
 
Burstein, H.J., Prestrud, A.A., Seidenfeld, J., Anderson, H., Buchholz, T.A., Davidson, N.E. et 
al. (2010) American Society of Clinical Oncology Clinical Practice Guideline: Update on 
Adjuvant Endocrine Therapy for Women with Hormone Receptor-Positive Breast Cancer. 
Journal of Clinical Oncology 28: 3784-3796. 
 
Casey, S.C., Tong, L., Li, Y., Do, R., Walz, S., Fitzgerald, K.N. et al. (2016) Myc Regulates the 
Antitumor Immune Response through Cd47 and Pd-L1. Science 352: 227-231. 
 
Chen, B.J., Chapuy, B., Ouyang, J., Sun, H.H., Roemer, M.G., Xu, M.L. et al. (2013) Pd-L1 
Expression Is Characteristic of a Subset of Aggressive B-Cell Lymphomas and Virus-Associated 
Malignancies. Clin Cancer Res 19: 3462-3473. 
 
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:  
 
Deng, L., Liang, H., Burnette, B., Beckett, M., Darga, T., Weichselbaum, R.R. et al. (2014) 
Irradiation and Anti – Pd-L1 Treatment Synergistically Promote Antitumor Immunity in Mice. 
The journal of clinical investigation 124: 687-695. 
 
Denkert, C., Loibl, S., Noske, A., Roller, M., Mu, B.M., Komor, M. et al. (2015) J Ournal of C 
Linical O Ncology Tumor-Associated Lymphocytes as an Independent Predictor of Response to 
Neoadjuvant Chemotherapy in Breast Cancer. 28: 105-114. 
 
Denkert, C., Loibl, S., Noske, A., Roller, M., Muller, B.M., Komor, M. et al. (2010) Tumor-
Associated Lymphocytes as an Independent Predictor of Response to Neoadjuvant 
Chemotherapy in Breast Cancer. J Clin Oncol 28: 105-113. 
 
Denkert, C., Von Minckwitz, G., Brase, J.C., Sinn, B.V., Gade, S., Kronenwett, R. et al. (2015) 


NCI Protocol #: N/A 
DF/HCC Protocol #: 16-588 
Protocol Version Date: 4/9/2019 
 
69 
Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy with or without 
Carboplatin in Human Epidermal Growth Factor Receptor 2-Positive and Triple-Negative 
Primary Breast Cancers. Journal of Clinical Oncology 33: 983-991. 
 
Denkert, C., Von Minckwitz, G., Brase, J.C., Sinn, B.V., Gade, S., Kronenwett, R. et al. (2015) 
Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy with or without 
Carboplatin in Human Epidermal Growth Factor Receptor 2-Positive and Triple-Negative 
Primary Breast Cancers. J Clin Oncol 33: 983-991. 
 
DeSantis CE, Fedewa SA, Goding Sauer A, Kramer JL, Smith RA, Jemal A. Breast cancer 
statistics, 2015: Convergence of incidence rates between black and white women. CA: a cancer 
journal for clinicians. Jan-Feb 2016;66(1):31-42. 
 
Dirix, L.Y., Takacs, I., Nikolinakos, P., Jerusalem, G., Arkenau, H.-T., Hamilton, E.P. et al. 
(2015) Avelumab (Msb0010718c), an Anti-Pd-L1 Antibody, in Patients with Locally Advanced 
or Metastatic Breast Cancer: A Phase Ib Javelin Solid Tumor Trial, SABCS. 
 
Dovedi, S.J. and Illidge, T.M. (2015) The Antitumor Immune Response Generated by 
Fractionated Radiation Therapy May Be Limited by Tumor Cell Adaptive Resistance and Can 
Be Circumvented by Pd-L1 Blockade. OncoImmunology 4: e1016709-e1016709. 
 
Dushyanthen, S., Beavis, P.A., Savas, P., Teo, Z.L., Zhou, C., Mansour, M. et al. (2015) 
Relevance of Tumor-Infiltrating Lymphocytes in Breast Cancer. BMC Medicine 1: 1-13. 
 
Eisenhauer, E.A., Therasse, P., Bogaerts, J., Schwartz, L.H., Sargent, D., Ford, R. et al. (2009) 
New Response Evaluation Criteria in Solid Tumours: Revised Recist Guideline (Version 1.1). 
Eur J Cancer 45: 228-247. 
 
Gajewski, T.F. (2015) The Next Hurdle in Cancer Immunotherapy_ Overcoming the Non–T-
Cell–Inflamed Tumor Microenvironment. Seminars in Oncology 42: 663-671. 
 
Gao, J., Shi, L.Z., Zhao, H., Chen, J., Xiong, L., He, Q. et al. (2016) Loss of Ifn-Gamma 
Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-Ctla-4 Therapy. Cell 167: 
397-404.e399. 
 
Gatalica, Z., Snyder, C., Maney, T., Ghazalpour, A., Holterman, D.A., Xiao, N. et al. (2014) 
Programmed Cell Death 1 (Pd-1) and Its Ligand (Pd-L1) in Common Cancers and Their 
Correlation with Molecular Cancer Type. Cancer Epidemiology Biomarkers & Prevention 23: 
2965-2970. 
 
George, S., Miao, D., Demetri, G.D., Adeegbe, D., Rodig, S.J., Shukla, S. et al. (2017) Loss of 
Pten Is Associated with Resistance to Anti-Pd-1 Checkpoint Blockade Therapy in Metastatic 
Uterine Leiomyosarcoma. Immunity 46: 197-204. 
 
Goedert JJ, Jones G, Hua X, et al. Investigation of the association between the fecal microbiota 
and breast cancer in postmenopausal women: a population-based case-control pilot study. J Natl 


NCI Protocol #: N/A 
DF/HCC Protocol #: 16-588 
Protocol Version Date: 4/9/2019 
 
70 
Cancer Inst. Aug 2015;107(8). 
 
Golden, E.B., Chhabra, A., Chachoua, A., Adams, S., Donach, M., Fenton-Kerimian, M. et al. 
(2015) Local Radiotherapy and Granulocyte-Macrophage Colony-Stimulating Factor to Generate 
Abscopal Responses in Patients with Metastatic Solid Tumours: A Proof-of-Principle Trial. 
Lancet Oncol 16: 795-803. 
 
Greenberg, B.P.a.C., Hortobagyi, G.N., Smith, T.L., Ziegler, L.D., Frye, D.K., and Buzdar, A.U. 
(2015) Long-Term Follow-up of Patients with Complete Remission Following Combination 
Chemotherapy for Metastatic Breast Cancer. 14: 2197-2205. 
 
Hartsell, W.F., Scott, C.B., Bruner, D.W., Scarantino, C.W., Ivker, R.A., Roach, M. et al. (2005) 
Randomized Trial of Short- Versus Long-Course Radiotherapy for Palliation of Painful Bone 
Metastases. JNCI Journal of the National Cancer Institute 97: 798-804. 
 
Herbst, R.S., Soria, J.-C., Kowanetz, M., Fine, G.D., Hamid, O., Gordon, M.S. et al. (2014) 
Antibody Mpdl3280a in Cancer Patients. Nature 515: 563-567. 
 
Hugo, H.S., Delord, J., Im, S., Ott, P.A., P, S.A., Bedard, P.L. et al. (2015) Preliminary Efficacy 
and Safety of Pembrolizumab in Patients with Pd-L1-Positive, Estrogen Receptor-Positive/Her2-
Negative Advanced Breast Cancer Enrolled in Keynote-028, SABCS. 
 
Human Microbiome Project C. Structure, function and diversity of the healthy human 
microbiome. Nature. Jun 13 2012;486(7402):207-214. 
 
Hwu, W.-J., Ph, D., Topalian, S.L., Hwu, P., Chen, S., Ph, D. et al. (2012) Safety and Activity of 
Anti–Pd-L1 Antibody in Patients with Advanced Cancer. 2455-2465. 
 
Intlekofer, A.M. and Thompson, C.B. (2013) F Basic-Translational Review at the Bench : 
Preclinical Rationale for Ctla-4 and Pd-1 Blockade as Cancer Immunotherapy. 94: 25-39. 
 
Jemal, A., Bray, F., and Ferlay, J. (2011) Global Cancer Statistics. 61: 69-90. 
 
Kroemer, G., Senovilla, L., Galluzzi, L., André, F., and Zitvogel, L. (2015) Review Natural and 
Therapy-Induced Immunosurveillance in Breast Cancer. Nature Publishing Group 21: 1128-
1138. 
 
Leighl, N., Balmanoukian, A.S., Eder, J.P., Patnaik, A., Aggarwal, C., Gubens, M. et al. (2015) 
Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. 2018-2028. 
 
Li, S., Zhu, M., Pan, R., Fang, T., Cao, Y.Y., Chen, S. et al. (2016) The Tumor Suppressor Pten 
Has a Critical Role in Antiviral Innate Immunity. Nat Immunol 17: 241-249. 
 
Liang, H., Deng, L., Chmura, S., Burnette, B., Liadis, N., Darga, T. et al. (2013) Radiation-
Induced Equilibrium Is a Balance between Tumor Cell Proliferation and T Cell-Mediated 
Killing. The Journal of Immunology 190: 5874-5881. 


NCI Protocol #: N/A 
DF/HCC Protocol #: 16-588 
Protocol Version Date: 4/9/2019 
 
71 
 
Loi, S., Sirtaine, N., Piette, F., Salgado, R., Viale, G., Van Eenoo, F. et al. (2013) Prognostic and 
Predictive Value of Tumor-Infiltrating Lymphocytes in a Phase Iii Randomized Adjuvant Breast 
Cancer Trial in Node-Positive Breast Cancer Comparing the Addition of Docetaxel to 
Doxorubicin with Doxorubicin-Based Chemotherapy: Big 02-98. Journal of Clinical Oncology 
31: 860-867. 
 
Marks LB, Int J Radiat Oncol Biol Phys. 2010 Mar 1;76(3 Suppl):S10-9.), PMID 20171502 
 
Mittal, D., Gubin, M.M., Schreiber, R.D., and Smyth, M.J. (2015) Hhs Public Access. 16-25. 
 
Mlecnik, B., Bindea, G., Angell, H.K., Berger, A., Lagorce, C., Lugli, A. et al. (2014) Towards 
the Introduction of the ‘ Immunoscore ’. 199-209. 
 
Nancy, I.V.-L., Nancy, U.L., Barry, W.T., Lii, H., Winer, E.P., Freedman, R.A. et al. (2015) 
Racial Differences in Outcomes for Patients with Metastatic Breast Cancer by Disease Subtype. 
Breast Cancer Research and Treatment 151: 697-707. 
 
Nanda R, Chow LQ, Dees EC, et al. Pembrolizumab in Patients With Advanced Triple-Negative 
Breast Cancer: Phase Ib KEYNOTE-012 Study. J Clin Oncol. Jul 20 2016;34(21):2460-2467. 
 
Perez, E.A., Thompson, E.A., Ballman, K.V., Anderson, S.K., and Asmann, Y.W. (2015) 
Genomic Analysis Reveals That Immune Function Genes Are Strongly Linked to Clinical 
Outcome in the North Central Cancer Treatment Group N9831 Adjuvant Trastuzumab Trial. 33:  
 
Peng, W., Chen, J.Q., Liu, C., Malu, S., Creasy, C., Tetzlaff, M.T. et al. (2016) Loss of Pten 
Promotes Resistance to T Cell-Mediated Immunotherapy. Cancer Discov 6: 202-216. 
 
Pritchard, K.I., Lebrun, F., Beck, J.T., Ito, Y., Yardley, D., Deleu, I. et al. (2012) Everolimus in 
Postmenopausal Hormone- Receptor–Positive Advanced Breast Cancer.  
 
Qin J, Li R, Raes J, et al. A human gut microbial gene catalogue established by metagenomic 
sequencing. Nature. Mar 04 2010;464(7285):59-65. 
 
Quine, M.A., Bell, G.D., Mccloy, R.F., Charlton, J.E., Devlin, H.B., and Hopkins, A. (1995) 
Prospective Audit of Upper Gastrointestinal Endoscopy in Two Regions of England: Safety, 
Staffing, and Sedation Methods. Gut 36: 462-467. 
 
Ribas, A. (2015) Adaptive Immune Resistance : How Cancer Protects from Immune Attack. 915-
920. 
 
Rizvi, N.A., Hellmann, M.D., Snyder, A., Kvistborg, P., Makarov, V., Havel, J.J. et al. (2015) T 
( 9 ). 348: 124-129. 
 
Roy S, Trinchieri G. Microbiota: a key orchestrator of cancer therapy. Nat Rev Cancer. Mar 17 
2017. 


NCI Protocol #: N/A 
DF/HCC Protocol #: 16-588 
Protocol Version Date: 4/9/2019 
 
72 
 
Rugo H, Delord J-P, Im S-A, et al. Abstract S5-07: Preliminary efficacy and safety of 
pembrolizumab (MK-3475) in patients with PD-L1–positive, estrogen receptor-positive 
(ER+)/HER2-negative advanced breast cancer enrolled in KEYNOTE-028. Cancer Research. 
2016;76(4 Supplement):S5-07-S05-07. 
 
Salgado, R., Denkert, C., Demaria, S., Sirtaine, N., Klauschen, F., Pruneri, G. et al. (2014) The 
Evaluation of Tumor-Infiltrating Lymphocytes (Tils) in Breast Cancer: Recommendations by an 
International Tils Working Group 2014. Annals of Oncology 26: 259-271. 
 
Salgado, R., Denkert, C., Demaria, S., Sirtaine, N., Klauschen, F., Pruneri, G. et al. (2015) The 
Evaluation of Tumor-Infiltrating Lymphocytes (Tils) in Breast Cancer: Recommendations by an 
International Tils Working Group 2014. Ann Oncol 26: 259-271. 
 
Schreiber, R.D. (2012) Cancer Immunoediting : Integrating Suppression and Promotion. 1565:  
 
Schreiber, R.D., Old, L.J., and Smyth, M.J. Cancer Immunoediting : Integrating Suppression and 
Promotion.  
 
Shahabi, V., Postow, M.A., Tuck, D., and Wolchok, J.D. (2015) Immune-Priming of the Tumor 
Microenvironment by Radiotherapy. American Journal of Clinical Oncology 38: 90-97. 
 
Sharabi, A.B., Nirschl, C.J., Kochel, C.M., Nirschl, T.R., Francica, B.J., Velarde, E. et al. (2014) 
Stereotactic Radiation Therapy Augments Antigen-Specific Pd-1-Mediated Antitumor Immune 
Responses Via Cross-Presentation of Tumor Antigen. Cancer Immunology Research: 345-356. 
 
Sharma, P. and Allison, J.P. (2015) The Future of Immune Checkpoint Therapy. 348:  
 
Shi, M., Roemer, M.G., Chapuy, B., Liao, X., Sun, H., Pinkus, G.S. et al. (2014) Expression of 
Programmed Cell Death 1 Ligand 2 (Pd-L2) Is a Distinguishing Feature of Primary Mediastinal 
(Thymic) Large B-Cell Lymphoma and Associated with Pdcd1lg2 Copy Gain. Am J Surg Pathol 
38: 1715-1723. 
 
Siegel, R., Desantis, C., Virgo, K., Stein, K., Mariotto, A., Smith, T. et al. (2013) Cancer 
Treatment and Survivorship Statistics , 2012.  
 
Sivan A, Corrales L, Hubert N, et al. Commensal Bifidobacterium promotes antitumor immunity 
and facilitates anti-PD-L1 efficacy. Science. Nov 27 2015;350(6264):1084-1089. 
 
Smith, D.C., Mcdermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B. et al. 
(2012) New England Journal. 2443-2454. 
 
Snyder, A., Makarov, V., Merghoub, T., Yuan, J., Zaretsky, J.M., Desrichard, A. et al. (2014) 
Genetic Basis for Clinical Response to Ctla-4 Blockade in Melanoma. The New England journal 
of medicine: 2189-2199. 
 


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Spranger, S., Bao, R., and Gajewski, T.F. (2015) Melanoma-Intrinsic Beta-Catenin Signalling 
Prevents Anti-Tumour Immunity. Nature 523: 231-235. 
 
Stanton SE, Disis ML. Clinical significance of tumor-infiltrating lymphocytes in breast cancer. J 
Immunother Cancer. 2016;4:59. 
 
Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., Mcdermott, D.F. et al. 
(2012) Safety, Activity, and Immune Correlates of Anti-Pd-1 Antibody in Cancer. N Engl J Med 
366: 2443-2454. 
 
Tosolini, M., Camus, M., Berger, A., Wind, P., and Lagorce-Page, C. (2006) References and 
Notes 1. 313: 1960-1965. 
 
Trinchieri G. Cancer Immunity: Lessons From Infectious Diseases. J Infect Dis. Jul 15 2015;212 
Suppl 1:S67-73. 
 
Vetizou M, Pitt JM, Daillere R, et al. Anticancer immunotherapy by CTLA-4 blockade relies on 
the gut microbiota. Science. Nov 27 2015;350(6264):1079-1084. 
 
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. 
 
Wolff, A.C., Hammond, M.E., Hicks, D.G., Dowsett, M., Mcshane, L.M., Allison, K.H. et al. 
(2013) Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast 
Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical 
Practice Guideline Update. J Clin Oncol 31: 3997-4013. 
 
Wu, J.S.Y., Wong, R., Johnston, M., Bezjak, A., and Whelan, T. (2003) Meta-Analysis of Dose-
Fractionation Radiotherapy Trials for the Palliation of Painful Bone Metastases. International 
Journal of Radiation Oncology Biology Physics 55: 594-605. 
 
Zaretsky, J.M., Garcia-Diaz, A., Shin, D.S., Escuin-Ordinas, H., Hugo, W., Hu-Lieskovan, S. et 
al. (2016) Mutations Associated with Acquired Resistance to Pd-1 Blockade in Melanoma. N 
Engl J Med:  
 
Zhang, K., Ph, D., Giorgetti, C., Ph, D., Randolph, S., Ph, D. et al. (2015) New England Journal. 
209-219. 
 
 
 
 


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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 
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 5-7 core biopsy specimens will be obtained using an 18-gauge needle. 
 
Lung: Because of the risk of pneumothorax associated with core needle biopsies of lung nodules, 
no core biopsies of lung nodules are mandated on this protocol, unless they are clinically 
indicated. 
 
Bone: Because the yield of malignant tissue from bone biopsies tends to be relatively low, if a 
patient has another accessible site of disease (i.e. skin, lymph node, liver), that site should be 
biopsied preferentially. If bone is the only biopsy-accessible site, then a goal of 5-7 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 


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the biopsy procedure. Patients will be queried if they have had previous allergic 
reactions to local anesthetics. 
 
Intravenous Conscious Sedation 
Certain biopsy procedures, such as lymph node, liver, or bone biopsies, may require 
intravenous conscious sedation (IVCS). IVCS is a minimally depressed level of consciousness 
that retains the patient’s ability to maintain a patent airway independently and continuously and 
respond appropriately to physical stimulation and verbal commands. 
The risks of intravenous conscious sedation include: inhibition of the gag reflex and concomitant 
risk of aspiration, cardiopulmonary complications (myocardial infarction, cardiac arrhythmias, 
hypoxemia), and allergic reactions to the sedative or analgesic medications. These risks are small 
but real; for example, in a prospective study of 14,149 patients undergoing IVCS during upper 
gastrointestinal endoscopies, the rate of immediate cardiopulmonary events was 2 in 
1000.[Quine et al., 1995] The 30-day mortality was 1 per 2,000 cases. In this study, there was a 
strong association between lack of monitoring and use of high-dose benzodiazepines with 
adverse outcomes. There was also an association between the use of local anesthetic sprays to the 
oropharynx and the development of pneumonia. In order to minimize the risk of intravenous 
conscious sedation, only qualified personnel will be responsible for conscious sedation. A 
minimum of two individuals will be involved in the care of patients undergoing conscious 
sedation—the physician performing the biopsy procedure, and the individual (M.D. or R.N.) who 
monitors the patients and his/her response to both the sedation and the procedure, and who is 
capable of assisting with any supportive or resuscitative measures. The room where the 
procedure utilizing IVCS takes place will have adequate equipment to provide supplemental 
oxygen, monitor vital signs, and maintain an airway should this be necessary. An emergency cart 
will also be immediately accessible to the room where the procedure is to take place, and 
emergency support services will be available on page. Patients will be screened and evaluated for 
their fitness to undergo conscious sedation by a trained physician. Patients with active cardiac 
disease are excluded from this study. No local anesthetic spray to the oropharynx will be 
necessary, given that endoscopy is not a planned procedure. Following the procedure, patients 
will be observed closely in the recovery room for a minimum of 2 hours. 
 
General Anesthesia 
Because of the higher risk of general anesthesia compared with local anesthesia or intravenous 
conscious sedation, biopsies that would require general anesthesia in order to be performed are 
not permitted on this protocol, 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.