Preoperative Combination of Pembrolizumab and Radiation Therapy in Patients With Operable Breast Cancer
Phase: PHASE1
Conditions: Breast Cancer
Sponsor: Stephen Shiao

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IIT2017-07-HO-PEMBRORT 
 
Preoperative Combination of Pembrolizumab and Radiation 
Therapy in Patients with Operable Breast Cancer 
 
Principal Investigator:  
Stephen Shiao, MD, PhD 
*for overall study 
 
Samuel Oschin Comprehensive Cancer Institute 
 
 
 
 
Cedars-Sinai Medical Center 
 
 
 
 
8700 Beverly Blvd, Los Angeles CA 90048 
 
Principal Investigator:  
Reva Basho, MD 
*for ER+ cohort 
 
Samuel Oschin Comprehensive Cancer Institute 
 
 
 
 
Cedars-Sinai Medical Center 
 
 
 
 
8700 Beverly Blvd, Los Angeles, CA 90048 
 
 
Co-Investigators: 
 
Reva Basho, MD 
 
 
 
 
Samuel Oschin Comprehensive Cancer Institute 
 
 
 
 
Cedars-Sinai Medical Center 
 
 
 
 
8700 Beverly Blvd, Los Angeles, CA 90048 
 
 
 
 
 
 
Heather McArthur, MD, MPH 
 
 
 
 
Samuel Oschin Comprehensive Cancer Institute 
 
 
 
 
Cedars-Sinai Medical Center 
 
 
 
 
8700 Beverly Blvd, Los Angeles, CA 90048 
 
 
 
 
 
Investigators:  
 
 
 
 
 
C. Michele Burnison, MD 
 
Radiation Oncology 
 
 
 
 
Amin Mirhadi, MD 
 
 
Radiation Oncology 
 
 
 
 
Mitchell Kamrava, MD  
 
Radiation Oncology 
 
 
 
 
Monica Mita, MD 
 
 
Medical Oncology 
 
 
 
 
Dorothy Park, MD 
 
 
Medical Oncology 
 
 
 
 
 
Philomena McAndrew, MD 
 
Medical Oncology 
 
 
 
 
Maryliza El-Masry, MD  
 
Medical Oncology 
 
 
 
 
Michael Van Scoy-Mosher, MD 
Medical Oncology 
 
 
 
 
Armando Giuliano, MD 
 
Breast Surgery 
 
 
 
 
Farin Amersi, MD 
 
 
Breast Surgery 
 
 
 
 
Alice Chung, MD 
 
 
Breast Surgery 
 
 
 
 
Catherine Dang, MD 
 
 
Breast Surgery 
 
 
 
 
Scott Karlan, MD 
 
 
Breast Surgery 
 
 
 
 
Nimmi Kapoor, MD 
 
 
Breast Surgery 
 
 
 
 
Farnaz Dadmanesh, MD 
 
Pathology 
 
 
 
 
Xuemo Fan, MD 
 
 
Pathology 
 
 
Biostatistician: 
Mourad Tighiouart 
 
 
Biostatistics 
 
 Cedars-Sinai Medical Center 
 
116 N. Robertson Blvd, Suite 900C, Office 7H 
 
Los Angeles, CA 90048 
 
Funding Source:  
Cedars-Sinai; Department of Defense (TNBC cohort only) 
 
 

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IND Number:  
135725 
 
NCT Number:  
 
NCT03366844  
 
Current version: 
 
Protocol Version 11 dated: 15DEC2020 
 
Initial version:  
 
Protocol Version 1 dated: 09OCT2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Version 11: 15DEC2020 
 
Signature Page 
 
 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable U.S. federal regulations and ICH guidelines. 
 
 
 
 
______________________________________________  
 
_________________ 
Principal Investigator (PI) Signature 
   
 
 
 
 
Date 
 
 
 
 
 

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TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ............................................................................................1 
STUDY SCHEMA..............................................................................................................3 
1.0 
BACKGROUND AND RATIONALE ...................................................................6 
2.0 
STUDY OBJECTIVES.........................................................................................11 
3.0 
STUDY DESIGN...................................................................................................13 
5.0 
TREATMENT PLAN ............................................................................................18 
6.0 
STUDY PROCEDURES ......................................................................................27 
7.0 
ADVERSE EVENTS (AE) ...................................................................................33 
8.0 
CORRELATIVES/SPECIAL STUDIES ............................................................43 
9.0 
STATISTICAL CONSIDERATIONS .................................................................45 
10.0 
STUDY MANAGEMENT .................................................................................48 
12.0 
REFERENCES .................................................................................................54 

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LIST OF ABBREVIATIONS 
 
 
 
3D 
3-dimensional 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
ANC 
Absolute Neutrophil Count 
ASCO-CAP 
American Society of Clinical Oncology-College of American Pathologists 
AST 
Aspartate Aminotransferase 
BUN 
Blood Urea Nitrogen 
C 
Cycle 
CBC 
Complete Blood Count 
CD 
Cluster of differentiation 
Cm 
Centimeter 
CMP 
Comprehensive Metabolic Panel 
CR 
Complete Response 
CT 
Computed Tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTL 
Cytotoxic T-lymphocyte 
CTLA 
Cytotoxic T-lymphocyte-associated protein 4 
CTV 
Clinical target volume 
D 
Day 
DNA 
Deoxyribonucleic acid 
DSMB 
Data and Safety Monitoring Board 
ECOG 
Eastern Cooperative Oncology Group 
ER 
Estrogen Receptor 
FDA 
Food and Drug Administration 
FISH 
Fluorescence in-situ hybridization 
FOXP3 
Forkhead box P3 
FSH 
Follicle stimulating hormone 
Gy 
Gray 
H&P 
History & Physical Exam 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HER2 
Human Epidermal Growth Receptor 2 
HIV 
Human immunodeficiency virus 
HRT 
Hormone replacement therapy 
IB 
IDFS 
Investigator’s brochure 
Invasive Disease-Free Survival 
IFN 
Interferon 
IHC 
Immunohistochemistry 
IL-2 
Interleukin-2 
IMRT 
Intensity-modulated radiation therapy 
irAE 
Immune-related adverse event 
IV 
Intravenously 

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Kg 
Kilogram 
MerFISH 
Multiplexed error-robust FISH 
Mg 
Milligram 
mIHC 
Multispectral immunohistochemistry 
MK 
Merck 
MMG 
Mammogram 
Mo. 
Month(s) 
MRI 
Magnetic resonance imaging 
MTD 
Maximum Tolerated Dose 
N 
Nodal status 
NCI 
National Cancer Institute 
ORR 
Objective Response Rate 
PBMCs 
Peripheral Blood Mononuclear Cells 
PD 
Progressive Disease 
PD-L1 
Programmed Death-Ligand 1 
PFS 
Progression Free Survival 
PK 
Pharmacokinetic 
PR 
Progesterone Receptor 
PTV 
Planning target volume 
Q 
Every 
RNA 
Ribonucleic acid 
RT 
Radiation Therapy 
SAE 
Serious Adverse Event 
SBRT 
Stereotactic body radiation therapy 
SCOEPS 
Safety Committee on Early Phase Studies 
SD 
Stable Disease 
SEER 
Surveillance, epidemiology, and end results program 
SGOT 
Serum Glutamic Oxaloacetic Transaminase 
SOC 
Standard of care 
SPGT 
Serum Glutamic Pyruvic Transaminase 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
T 
Tumor size 
TCR 
T-Cell Receptor 
TIL 
Tumor infiltrating lymphocyte 
TNBC 
Triple-Negative Breast Cancer 
TNF- 
Tumor necrosis factor-alpha 
ULN 
Upper limit of normal 
US 
Ultrasound 
W 
Week 
WBC 
White Blood Cells 
WBRT 
Whole breast radiation therapy 
WOCBP 
Women of child-bearing potential 
YR 
Year 
 
 
 
 

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STUDY SCHEMA  
This is a prospective, phase Ib/II study of the preoperative combination of pembrolizumab (pembro) and 
radiation therapy (RT) in patients with operable breast cancer. Patients with newly diagnosed, Stage I-III 
breast cancer who are planned for breast-conserving surgery and adjuvant whole breast radiation therapy 
will be recruited into one of 2 cohorts as per their biomarker profile as depicted below. The TNBC cohort 
will have 50 subjects (10 TNBC subjects for phase Ib and 40 TNBC subjects for phase II) and the HR+ 
cohort will have 10 subjects. Enrollment will start with the phase Ib TNBC cohort. The trial will be 
conducted in a Bayesian design, wherein safety as assessed by lack of delay in standard of care 
treatment will be assessed after 3, 6, and 9 patients have initiated standard of care treatment in each 
cohort. Standard of care treatment is defined as breast-conserving surgery or initiation of neoadjuvant 
systemic chemotherapy. If the phase Ib TNBC trial meets a pre-specified safety endpoint,enrollment in 
the phase II TNBC trial and HR+ cohort will take place. 
Study treatment will consist of 2 doses of neoadjuvant pembro. The second dose will be given in 
combination with a RT boost. The primary objectives are: 1) to assess the feasibility of the preoperative 
combination of pembro and RT as defined by the number of patients who do not necessitate a delay in 
standard of care treatment, 2) to assess the change in tumor infiltrating lymphocyte (TIL) score with 
therapy. The secondary objectives are to describe the toxicity with preoperative pembro and RT up to 15 
weeks after administration of pembrolizumab, to assess the invasive disease-free (iDFS) survival, and to 
assess the pathological complete response (pCR) rates. 
 
• 
The first dose of pembro will be given on cycle 1, day 1 (C1D1). Each cycle will be 21 days. The 
second of pembro dose will be given on C2D1 (+/- 3 days). 
• 
RT will be administered concurrently within one week of the second dose of pembro (C2D1 + 5 
calendar days). 8Gy will be delivered in3 fractions over 3 to 5 business days (for a total dose of 
24Gy).   
• 
Standard of care treatment, including breast-conserving surgery or additional neoadjuvant 
systemic therapy, will be pre-set to start 8 weeks after study enrollment (+/- 5 days). 
• 
Research blood and stool will be obtained after consent up to C1D1. It will also be obtained on 
C2D1 (+/-3 days) and at the time of initiation of standard of care treatment (+/-5 days). 
• 
Research tumor biopsy will be performed after consent up to C1D1 (in the HR+ cohort, a 
diagnostic biopsy accepted if sufficient archived tissue is available) and on C2D1 (+/-3 days) prior 
to the initiation of radiation therapy. Biopsies will be processed for scSeq and tumor infiltrating 
High-Risk HR+ (N=10) 
 
• 
ER > 10% 
• 
HER2- by ASCO-CAP 
• 
T at least 2cm, any N 
 
High-risk defined by 2 
out of 3: 
• 
Grade II-III 
• 
Ki-67 > 20% 
• 
ER < 75% 
TNBC Phase Ib Trial (N=10) 
 
• 
ER/PR < 10% 
• 
HER2-by ASCO-CAP 
• 
T ≥ 2cm, any N 
TNBC Phase II Trial (N=40) 
 
• 
ER/PR < 10% 
• 
HER2-by ASCO-CAP 
• 
T ≥ 2cm, any N 
 

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lymphocytes (TIL) analysis. 
• 
Research tissue will also be obtained prior to the initiation of standard of care treatment at week 7 
(+/-5 days). If standard of care treatment consists of surgery, surgical tissue will be used. If it 
consists of further neoadjuvant systemic therapy, another research biopsy will be conducted. 
Fresh tissue may also be procured at the time of surgery if the surgery occurs after completion of 
neoadjuvant systemic therapy. This will be determined at the discretion of the treating physician. 

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STUDY SUMMARY 
Title 
 
Preoperative Combination of Pembrolizumab and Radiation Therapy in 
Patients with Operable Breast Cancer 
 
Protocol Number 
IIT2017-07-HO-PEMBRORT 
Phase 
Pilot Study; Phase Ib/II  
Methodology 
Prospective therapeutic trial 
Study Duration 
Approximately 5 years 
Study Center(s) 
Cedars-Sinai Medical Center 
Objectives 
Primary Objectives: 
• 
To assess the feasibility of the preoperative combination of 
pembro and RT as defined by the number of patients who do not 
necessitate a delay in standard of care treatment 
• 
To assess the change in tumor infiltrating lymphocyte score with 
therapy 
 
Secondary Objective: 
• 
To describe the toxicity with preoperative pembrolizumab and RT 
up to 15 weeks after the last dose. 
• 
To assess invasive disease-free survival (iDFS) rates. 
• 
To assess the pathological complete response (pCR) rates 
 
Number of Subjects 
60 
Diagnosis and Main 
Inclusion Criteria 
Operable hormone receptor positive (HR+) or triple negative breast  
cancer (TNBC) with planned breast-conserving surgery and adjuvant 
whole breast radiation therapy 
Study Product(s), Dose, 
Route, Regimen 
Pembrolizumab 200mg IV every 3 weeks x 2 doses 
RT 8Gy x 3 fractions 
Duration of administration 
6 weeks 
Reference therapy 
None 
Statistical Methodology 
Bayesian design 
 
 
 

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1.0 
BACKGROUND AND RATIONALE 
1.1 
Disease Background 
With more than 1 million new cases diagnosed yearly worldwide, breast cancer is a global public health 
burden.2 For most patients in the developed world, breast cancer is diagnosed at an early stage.3 
However, despite improvements with systemic therapies, approximately one-third of women with localized 
disease still develop distant metastases.4  Over the past decade, molecular subtyping of breast cancer 
has identified intrinsic subtypes that may be at enhanced risk for both local and distant recurrence. In 
2000, Perou et al first described the more modern concept of breast cancer subtypes5. They identified 
multiple breast tumor subgroups that could be defined by distinct expression patterns in genes related to 
proliferation, hormone receptor signaling, HER2 signaling and expression of basal epithelial markers. 
Today, there are 4 major subtypes (luminal A, luminal B, HER2-enriched and basal like/TNBC). Although 
these subtypes are most accurately defined by patterns of gene expression, characteristic 
immunohistochemistry patterns are commonly used as a surrogate in the literature. 
The prognostic value of these breast tumor subtypes has been demonstrated in multiple datasets. In 
addition, the 8th edition of the American Joint Committee on Cancer (AJCC) Manual has incorporated 
biologic factors including tumor grade, ER, PR and HER2 status. In this new staging system, an 
Oncotype Dx Recurrence Score (RS)<11 categorizes patients with hormone receptor (HR) positive, 
HER2 negative tumors into the prognosis of Stage 1 breast cancer, regardless of the size of the primary 
tumor. Of the 4 major subtypes, basal like/TNBC has the worst prognosis with disproportionately poorer 
clinical outcomes compared to other subtypes5,6. Compared to Luminal A tumors, Luminal B tumors are 
characterized by higher tumor grade, larger size, and increased incidence of lymph node positivity. In 
such patients who remain at high risk for relapse with standard therapy, harnessing the immune system 
to recognize and fight early stage breast cancer prior to definitive surgical intervention has the potential to 
confer long-term immunity, and ultimately, cure. 
Further, there is a growing body of evidence indicating that women with breast cancers of certain 
subtypes are ideally suited for immunotherapy trials. Specifically, dense lymphocytic infiltration has been 
observed in a significant proportion of TNBCs and HER2-enriched tumors, indicating an inherent interplay 
between the tumor and the immune system.7,8 Although HR+/HER2- tumors are less likely to be infiltrated 
by CD8+ cytotoxic T-cells compared to TNBCs and HER2+ tumors, approximately 40% of these tumors 
do display a CD8+ infiltrate. It has been suggested that expression of the estrogen receptor results in 
decreased immunogenicity 9,10. Thus, it is likely that the subset of HR+/HER2- tumors that display 
increased lymphocytic infiltrate are not as strongly driven by estrogen and belong to the Luminal B 
subtype. Thus, this study focuses on the immunogenicity of high-risk breast cancer subtypes that are 
likely to display a dense lymphocytic infiltration including TNBC and HR+/HER2- tumors that are more 
likely to be consistent with Luminal B subtypes, defined by 2 out of 3 of the following characteristics: 
grade II-III, Ki-67 > 20%, ER < 75%11. 
Locoregional therapies for patients with breast cancer consist of either mastectomy or lumpectomy 
followed by whole breast radiation therapy (WBRT). Both have been shown to be equivalent to 
mastectomy in terms of local control and overall survival.12 Recently, radiation therapy (RT) has been 
described to have numerous immune-modulatory effects. RT-induced tumor cell death increases the 
supply of tumor-specific antigens, leading to the release of signaling molecules that attract immune cells 
to the tumor microenvironment and to the upregulation of programmed death-ligand 1 (PD-L1) on tumor 
cells.13-15 Whether or not RT combined with PD-L1 blockade synergistically increases the tumor 
immunogenicity, compared to PD-L1 blockade alone, is the subject of this trial. Our study population will 
focus on women with immunogenic subtypes of breast cancer and/or those who are at high risk of 
recurrence. Our study design, which describes lead-in with administration of pembrolizumab alone prior to 
the addition of RT, will allow for examination of immune correlatives in a staged fashion and subsequent 
determination the efficacy of our strategy. 
1.2 
Rationale for Immune Augmentation in Breast Cancer 
Thus far, results from early clinical trials of checkpoint inhibition in breast cancer have been relatively 
modest but encouraging. In ER+/HER2- patients with locally advanced or metastatic disease treated with 
pembrolizumab, an objective response rate (ORR) of 12% was achieved.16 The Phase Ib JAVELIN study 

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reported a response rate of 4.8% among breast cancer patients treated with the anti-PD-L1 antibody 
avelumab overall and an ORR of 8.6% and 2.8% in TNBC and ER+ or PR+/HER2- subtypes, 
respectively.17 
In PD-L1 positive TNBC patients, the Phase Ib trial KEYNOTE-012 showed a 18.5% ORR in 27 evaluable 
patients treated with pembrolizumab.18 The median duration of response was not yet reached (15 to ≥ 47 
weeks). Another Phase I trial using the PD-L1 antibody atezolizumab reported a response rate of 19% in 
21 evaluable patients with PD-L1 positive metastatic TNBC.19 The median duration of response was not 
yet reached (18 to ≥56 weeks). Of note, in both trials, only data from subjects that were PD-L1 positive by 
immunohistochemistry (IHC) were presented. The KEYNOTE-012 trial defined PD-L1 positivity as PD-L1 
expression in the stroma or in ≥ 1% of tumor cells. The atezolizumab trial defined PD-L1 positivity as PD-
L1 expression in ≥ 5% of infiltrating immune cells.  
Recently, a Phase I expansion cohort of patients with metastatic TNBC demonstrated a 10% ORR in 112 
evaluable patients treated with atezolizumab.20 ORR was 26% among patients that received 
atezolizumab as a first line treatment, 4% for patients that received atezolizumab as second line 
treatment, and 8% among those that received atezolizumab as third line or greater treatment. Notably, all 
11 patients who responded were alive after one and two years, while OS rates were 33% and 11% for 
non-responders after one year and two years, respectively. 
1.3 
Rationale for Combining Radiation Therapy and Immune Therapy 
In-situ tumor destruction releases a large amount of tumor antigens. Antigen-presenting cells, such as 
dendritic cells, then take up these antigens in the periphery and migrate to lymph nodes where they 
activate CD4+ and CD8+ T-lymphocytes that recognize these tumor antigens. Immune augmentation via 
immune co-stimulatory molecules then permits the ensuing immune response to strengthen and destroy 
cancer systemically.21,22 Thus, the anti-tumor immune response initiated by RT delivered to an intact 
tumor can be potentiated by combination with checkpoint blockade. 
1.3.1 
Preclinical Data 
Multiple pre-clinical studies support the combination of RT with checkpoint inhibition. The 
combination of radiotherapy and anti-CTLA-4 antibody delayed growth of irradiated tumor, 
inhibited lung metastases, and improved survival in a 4T1 murine carcinoma model.23 These 
findings were confirmed in a 9H10 model, in which combination therapy enhanced primary tumor 
regression and produced abscopal regression of non-irradiated lesions.24 Another murine model 
demonstrated that the combination of radiotherapy and antibodies against CD137 and PD-1 was 
curative and associated with tumor antigen specific CD8+ T-cell infiltration.25 Finally, Deng and 
colleagues showed that RT enhanced the effect of anti-PDL-1 in the TUBO breast cancer cell 
line, with reduced TUBO tumor growth after mice were re-challenged in the opposite flank, 
implying systemic immunity.13  
1.3.2 Clinical Data 
A systemic response to localized RT in combination with anti-CTLA-4 therapy, the so-called 
“abscopal effect”, was reported in a patient after disease progression with CTLA-4 blockade with 
ipilimumab alone.14 Specifically, a right hilar lymph node and spleen metastases, which was not 
the target of RT, showed regression only after the patient received palliative RT after several 
months of anti-CTLA-4.   
Based on the above phenomenon as well as pre-clinical data suggesting the synergism of RT 
with immune therapies, studies evaluating the efficacy of RT with checkpoint blockade in 
metastatic breast cancer are actively ongoing. In a phase II study evaluating pembrolizumab and 
RT in metastatic TNBC patients, 2 out of 5 evaluable patients had a response outside of the 
irradiated field.26 Although assessing distant response is not one the endpoints of this protocol, 
which includes women with non-metastatic breast cancer, the study nevertheless provides proof-
of-principle for the concept of systemic immune modulation by RT when added to checkpoint 
inhibition. 
Once safety of the pembro-RT regimen has been established, the efficacy of the regimen remains 
to be tested.  Current chemotherapy regimens (GeparSixto) report pathologic complete response 

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rates of approximately 50%27 and in the metastatic setting addition of atezolizumab (PD-L1) to 
nab-paclitaxel (IMpasion130) showed increased PFS in triple negative breast cancer28.  Based on 
these two recent observations, the phase II portion of this trial will test the efficacy of adding 
pembro-RT to neoadjuvant chemotherapy in early-stage breast cancer and determine the 
pathologic complete response rate in comparison to best available chemotherapy which is the 
current standard of care.  
1.4 
Whole Breast Radiation Plus Boost for Localized Breast Cancer 
1.4.1 Rationale 
A radiation boost targeted at the tumor bed is routinely administered to breast cancer patients as 
part of breast-conserving therapy. The rationale for a boost is based on the patterns of recurrence 
data that has demonstrated that the vicinity of the original index tumor is the most common site of 
local recurrence and therefore requires a higher tumoricidal dose from RT compared to the rest of 
the breast29. Several randomized controlled trials have shown that the addition of a boost to 
standardly fractionated whole breast RT (WBRT) in women with localized breast cancer led to an 
improvement in local control 30,31. The largest of these trials, the EORTC 22822, included 5318 
patients with stage I-II breast cancer who underwent lumpectomy + axillary lymph node 
dissection30. Following WBRT (50 Gy/25 fractions), patients were randomized to +/- 16 Gy boost. 
With a median of 17.8 years of follow-up, the cumulative incidence of local failure was 6.4%, 
8.8%, and 12.0%, respectively, in boosted patients compared with 10.2%, 13.1%, and 16.4% in 
the non-boost group. The benefit of a boost was observed in all age groups.  
A boost to the lumpectomy bed is most commonly delivered sequentially following the completion 
of whole breast RT. However, this is generally out of convention rather than due to scientific 
rationale. A boost may be equally efficacious, if prior to whole breast RT. In this protocol, we 
propose the delivery of a boost in the preoperative setting prior to lumpectomy, so that it may 
engage the immune system while the primary tumor remains intact, providing a rich supply of 
tumor antigens. 
1.4.2 Safety of Preoperative RT 
Radiation therapy is routinely administered preoperatively in other tumor types such as sarcoma 
and rectal cancer.32,33 Studies have shown that RT can be safely administered to early stage 
breast cancer patients in the preoperative setting.34,35 In a phase I dose-escalation trial of single-
dose preoperative partial breast irradiation in early stage breast cancer patients, no acute dose-
limiting toxicity was observed, no post-operative wound dehiscence was seen, and cosmetic 
results were favorable.34 A dose escalation study of stereotactic body radiation therapy (SBRT) 
combined with neoadjuvant chemotherapy in breast cancer patients demonstrated that the 
combination was well tolerated with the MTD not reached in the study.35 These data support 
administration of RT preoperatively in localized breast cancer patients.  
1.4.3 
Dose and Fractionation of RT with Immune Therapy  
Multiple studies have examined the role of radiation dose and fractionation in generating systemic 
anti-tumor immune responses.36 In murine models of RT, several groups have demonstrated 
enhanced production of circulating IFN-gamma expressing, tumor-specific CD8+ T cells with a 
single fraction of RT (15 Gy) delivered focally to tumors.37,38 Dewan et al reported that a 
hypofractionated dose of 8 Gy x 3 fractions was more effective than single fraction therapy when 
combined with anti-CTLA-4 therapy and resulted in increased TILs.24 Furthermore, in a 4T1 
mammary carcinoma model, when CTLA-4 was combined with RT, two fractions of 12Gy each 
improved survival compared to single fraction.23 In a phase I dose-escalation trial of single-dose 
preoperative partial breast irradiation in early stage breast cancer patients, expression of genes 
immune-modulatory genes was increased in a dose-dependent fashion with single fraction RT.34  
Although there is no consensus regarding the optimal dose and fractionation of RT to utilize with 
immune therapies, it appears reasonable that higher total doses and that relative to single 
fraction, short, hypofractionated courses of fractionation generate greater antigen release, leading 

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to improved anti-tumor immune responses. In this study, we plan to use 8 Gy x 3 fractions on the 
basis of preclinical data from Dewan et al..24 
1.5 
Rationale for Pembrolizumab Dose Selection/Regimen  
Pembrolizumab is an optimal immunotherapy agent to study, as this agent has recently been FDA-
approved for use in multiple tumor types. It is therefore ready to be tested for efficacy in other disease 
sites and in combination with other treatments. It is currently in being tested in combination with RT for 
metastatic TNBC (NCT02730130). 
An open-label Phase I trial was conducted to evaluate the safety and clinical activity of single agent MK-
3475 (pembrolizumab).39 The dose escalation portion of this trial evaluated three dose levels, 1 mg/kg, 3 
mg/kg, and 10 mg/kg, administered every 2 weeks (Q2W) in subjects with advanced solid tumors. All 
three dose levels were well tolerated and no dose-limiting toxicities were observed. This first in-human 
study of MK-3475 showed evidence of target engagement and objective evidence of tumor size reduction 
at all dose levels (1 mg/kg, 3 mg/kg and 10 mg/kg Q2W). No MTD has been identified to date. Recent 
data from other clinical studies within the MK-3475 program has shown that a lower dose of MK-3475 and 
a less frequent schedule may be sufficient for target engagement and clinical activity.  
PK data analysis of MK-3475 administered Q2W and Q3W showed slow systemic clearance, limited 
volume of distribution, and a long half-life (refer to IB). Pharmacodynamic data (IL-2 release assay) 
suggested that peripheral target engagement is durable (>21 days). This early PK and pharmacodynamic 
data provides scientific rationale for a Q2W and Q3W dosing schedule.  
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.  
The choice of the 200 mg Q3W as an appropriate dose for the switch to fixed dosing is based on 
simulations performed using the population PK model of pembrolizumab showing that the fixed dose of 
200 mg every 3 weeks will provide exposures that 1) are optimally consistent with those obtained with the 
2 mg/kg dose every 3 weeks, 2) will maintain individual patient exposures in the exposure range 
established in melanoma as associated with maximal efficacy response and 3) will maintain individual 
patients exposure in the exposure range established in melanoma that are well tolerated and safe.  

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1.6 
Correlative Studies 
Tumor Tissue: 
Infiltration of immune cells has predicted improved prognosis and 
response to therapy in many different tumor types including 
breast cancer.40,41 The greatest clinical benefit is seen when TILs 
comprise at least 50-60% of the tumor.40 While a baseline TIL 
score indicates the immunogenicity of a tumor, the influx of TILs 
with therapy indicates the engagement of the immune system as 
a result of therapy (Figure 1).1 
In addition to the amount of lymphocytic infiltrate, the phenotype 
of that infiltrate has also been shown to be prognostic. Type 1 T-
cells are associated with favorable prognosis. Type 1 CD4+ T-
helper cells facilitate antigen presentation and activation of CD8+ 
cytotoxic T-cells (CTL), which are essential for tumor 
destruction.42 On the other hand, Type 2 CD4+ T-helper cells, 
including Forkhead box P3 (FOXP3) CD4+ regulatory T-cells, 
contribute to a tumor environment that inhibits CTL function and 
promotes tumor growth.43 
Multiplex IHC (mIHC) allows for multiple fluorescently tagged 
antibodies to be applied to paraffin-embedded tissue 
quantification of cell types including macrophages (CD163+), 
effector T-cells (CD3+CD8+), and regulatory T-cells 
(CD3+CD4+FOXP3+) (Figure 2). Assays can also be used to assess 
the activation and proliferation of TILs using markers such as Ki-67, PD-
1, CTLA-4.  
 
Figure 2 
 
 
Flow cytometry can similarly be used on fresh or frozen tissue to quantify cell types including effector T-
cells (CD3+CD8+), and regulatory T-cells (CD3+CD4+FOXP3+) cells. 
Activation of the immune system can be further assessed using sequencing technology. Using TCR 
sequencing analysis on extracted DNA from paraffin-embedded tissue, increase in shared clones 
suggests that therapy is enhancing proliferation of antigen experienced cells within the tumor. 
Single cell gene expression profiling can be used to assess the transcriptome of individual cells. 
Innovative technology allows for the evaluation of the transcriptome in single cells within the tumor and 
 
Figure 1Figure 1 
Increase in TILs in a 
breast cancer patient 
after treatment with 
neoadjuvant paclitaxel.1 
 
Epithelium/Pan-CK = green 
T-cell/CD4 = yellow 
T
-
-cell/CD8 = purple 
Macrophage = red 
Nuclear hematoxylin = blue 
Multispectral IHC to quantify immune cell 
subsets on a paraffin-embedded tumor slide. 
Slide courtesy of Dr. Stephen Shiao, study 
collaborator. 

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the tumor microenvironment.44 Serial assessment of the transcriptome through the course of therapy can 
identify genes that are indicators and/or effectors of safety and response. 
Whole Genome/Exome Sequencing: Frozen or FFPE tumor samples will be submitted for DNA extraction 
and whole genome/exome sequencing to assess the expression level of different genes within the tumor.   
Whole Blood: 
An increase in circulating cytokines is an indicator of systemic immune activation. Further, flow cytometry 
can also be performed on fresh or frozen blood to assess the composition of systemic immune cells. 
Similarly, TCR sequencing can be conducted on blood to assess the clonality of systemic immune cells. 
Optional Research Imaging: 
One of the most critical questions facing immunotherapy is how to determine which patients will respond.  
There is currently no biomarker for early response, however recent data indicates that DCE-MRI can 
assess inflammation and that this may be an early marker of the response to immunotherapy.  MR 
imaging is incorporated to assess possible responses to treatment and may aide in the decision for 
additional therapy after completion of the experimental portion of the trial.  The unique tissue 
characterization of MRI will be utilized and compared to tissue analysis from biopsy samples at the same 
timepoints; before, during and after neoadjuvant immunotherapy and radiation treatment.   
 
Optional Fecal Samples for Analysis 
Many factors have been shown to regulate the anti-tumor immune response and, in particular, the 
immune set point as determined by the basal immune responses.  Recent evidence has found that most, 
if not all, immune responses are shaped by the interaction of the immune system with the microbiome.45 
Several experimental models revealed that the efficacy of immune therapies including CpG and 
checkpoint inhibitors as well as immune-related toxicity can be influenced by the microbiome.46 Thus, for 
this trial we plan to collect optional fecal samples for microbiome analysis as a potential biological 
correlate for efficacy and/or toxicity. 
 
 
2.0 
STUDY OBJECTIVES 
2.1 
Primary Objectives   
 
2.1.1 
To assess the feasibility of the preoperative combination of pembrolizumab and 
RT as defined by the number of patients who do not necessitate a delay in 
standard of care treatment 
 
2.1.2 
To assess the change in TIL score with preoperative pembrolizumab and RT.  
 
2.2 
Secondary Objectives  
 
2.2.1 
To describe the toxicity with preoperative pembrolizumab and RT up to 15 weeks 
after administration of pembrolizumab. 
 
2.2.2 
To assess invasive disease-free survival (iDFS) rates. 
 
2.2.3  
To assess the pathological complete response (pCR) rates. 

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2.3 
Exploratory Objectives 
2.3.1 To assess changes in intratumoral and serum lymphocyte composition and 
serum cytokines with therapy.  
2.3.2 To explore T-cell diversity and clonality by deep sequencing. 
2.3.3 To analyze the RNA transcriptome in tumor, intratumoral lymphocytes, and 
adjacent normal tissue. 
 
2.3.4 To assess changes in the microbiome.  
 
2.3.5 To assess changes in breast cosmesis. 
 
2.3.6   To assess disease status by breast MR imaging 
 
2.3.7  To assess the pathological complete response (pCR) rates 
 
2.4 
Endpoints 
• 
Primary Efficacy Endpoint: Feasibility 
Feasibility will be defined by the number of patients who do not necessitate a 
delay in standard of care treatment. Since the combination of pembrolizumab and 
RT has not been tested in the preoperative setting in patients with potentially 
curative disease, it is necessary to establish whether such preoperative treatment 
can be safely given without delaying curative-intent treatment. 
• 
Co-Primary Efficacy Endpoint: Change in TILs 
An increase in TILs is an indicator of immune system engagement. A lead in with 
pembrolizumab alone followed by the combination of pembrolizumab with RT will 
allow for serial assessment of TILs. This will establish the contribution of RT to the 
immune response generated by pembrolizumab. 
• 
Safety Evaluations 
Immune related adverse events that result from checkpoint blockade can occur 
weeks after the drugs are administered. Therefore, adverse events will be 
recorded up to 15 weeks after administration of pembrolizumab and radiation 
therapy and graded according to CTCAE, Version 4.03. Immune related adverse 
events will also be documented for up to 1-year post treatment. When considering 
if an adverse event is immune related, a general principle is that differential 
diagnoses should be diligently evaluated according to standard medical practice. 
Non-inflammatory etiologies should be considered.  
• iDFS 
iDFS will be defined as the time from completion of surgery to the first occurrence 
of the following events: invasive ipsilateral, local, regional, or distant recurrence, 
or death due to breast cancer.60 
• pCR 
 
pCR will be defined as the absence of invasive disease in the breast and lymph 
nodes at the time of curative-intent surgery. 
 
 
 

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3.0 
STUDY DESIGN 
Study Design 
This is a prospective, single-institution, Phase Ib/II study of the preoperative combination of 
pembrolizumab (pembro) and radiation therapy (RT) in patients with operable breast cancer 
(TNBC). The two cohorts will consist of 50 TNBC and 10 HR+ women each. Enrollment will start 
with the Phase Ib TNBC safety cohort. The trial will be conducted in a Bayesian design, wherein 
safety as assessed by lack of delay in standard of care treatment will be assessed after 3, 6, and 
10 patients have initiated standard of care treatment. If at least 80% of the first 10 subjects in the 
TNBC cohort proceed with standard of care therapy without delay, the combination will be 
deemed safe in the cohort, and enrollment in the HR+ cohort, as well as continued enrollment in 
the TNBC cohort (40 subjects), will occur at that time.  
Each cohort will be evaluated for delay in curative-intent standard of care treatment every time 3 
patients complete study therapy and undergo standard of care treatment. At each of those 
timepoints, if the posterior probability that a patient experiences a delay in standard of care 
treatment exceeds 20%, the cohort will stop enrollment. In Bayesian statistics, the posterior 
probability of a random event or an uncertain proposition is the conditional probability that is 
assigned after the relevant evidence or background is taken into account. This design will assure 
that a cohort does not continue enrollment if early delays in standard of care treatment are seen. 
Enrollment will be carried out continuously and will not be stopped in order to evaluate the 
posterior probability of a delay. However, the number of patients undergoing assessment for a 
possible delay simultaneously will not exceed four in each of the 2 cohorts. 
Study Population 
Subjects with pathologically confirmed, localized (Stage I-III) breast cancer with a primary tumor 
size of at least 2cm for whom breast-conserving surgery and adjuvant whole breast RT is planned 
will be recruited into one of 2 cohorts, delineated by breast cancer subtype (TNBC or HR+). We 
anticipate that a low proportion of patients who are planning breast-conserving surgery may 
ultimately choose mastectomy. The receipt of a pre-operative boost should not preclude the 
ability to receive mastectomy with immediate reconstruction. The outcomes of these patients will 
be captured and followed on protocol. 
The magnitude of TILs is reported to be higher in TNBC tumors than HR+/HER2- tumors.47 It is 
postulated that TNBCs have a higher lymphocytic infiltrate due to higher mutational load, which 
enhances immunogenicity and results in increased TIL recruiting.48 Clinical data with checkpoint 
blockade therapy supports the increased immunogenicity of TNBC and HER2+ tumors compared 
to HR+/HER2- tumors. For example, the Phase Ib JAVELIN study reported an ORR of 8.6%, 
3.8%, and 2.8% in TNBC, HER2+, and ER+ or PR+/HER2- subtypes, respectively.17 Due to the 
differences in intrinsic risk and baseline immunogenicity, the cohorts in this study are delineated 
by biomarker subtype. 
As such, the cohorts include: 
1) Cohort 1: TNBC patients with primary tumors measuring at least 2 cm 
 
2) Cohort 2: High-risk, ER-positive and HER2-negative breast cancer patients with primary 
tumors measuring at least 2 cm 
ER-positive and HER2-negative breast cancer will be defined by ER expression  10% by 
immunohistochemistry (IHC) and HER2-negativity per ASCO-CAP guidelines.49 High-risk disease 
will be defined by the presence of at least 2 of the following 3 criteria: histologic grade II-III, Ki-67 
> 20%, ER expression < 75% by IHC.  TNBC will be defined by ER and PR expression < 10% by 
IHC and HER2-negativity by ASCO-CAP guidelines49.  
It is well known that systemic therapy activates the immune system.50 Therefore, clear, 
discriminate assessment of the contribution of RT to tumor immunogenicity in each of the 2 
subsets requires that this trial be conducted in the absence of prior therapy. We recognize the 

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importance of other standard of care therapy, including neoadjuvant systemic therapy in TNBC 
tumors.51,52 Therefore, the standard of care curative-intent treatment will be set to start 8 weeks 
(+/-5 days) after trial initiation, and the primary endpoint is to assess delays in this pre-planned 
date. 
Bleicher, et al reported increased breast cancer-specific mortality with delays from time of 
diagnosis to surgery of > 60 days in a retrospective cohort analysis from the SEER Medicare 
database.53 This was particularly true in Stage I patients. In a similar retrospective analysis from 
the California Cancer Registry, Chavez-Macgregor, et al reported worse overall survival and 
breast cancer-specific survival in patients whose adjuvant chemotherapy was delayed beyond 90 
days after surgery.54 The planned standard of care treatments in this study are within the 
timeframes of these analysis. The Bayesian design of the trial will prevent ongoing recruitment 
into each of the cohorts if the probability of surgical delays exceeds pre-specified limits after 3, 6, 
and 9 patients undergo standard of care treatment in each cohort.  
This study requires tumor samples collected at 3 different time points for each patient to complete 
the immune correlative analysis. Therefore, a minimum tumor size of 2 cm was chosen to 
maximize the probability of having sufficient tumor tissue available for analysis.  
Study Intervention 
Patients will receive pembrolizumab Q3W for 2 doses. The second dose will be given in 
conjunction with an RT boost, consisting of 8 Gy for 3 fractions. We estimate that approximately 
10% of patients who are planned to undergo breast-conserving therapy may undergo 
mastectomy for reasons including positive margins and patient choice.55 It is anticipated that 
receipt of a boost dose, which is directed to a small portion (<30%) of the total breast volume, will 
not preclude the ability to undergo mastectomy.  
Approximately 3 weeks after the second dose of pembrolizumab, patients will initiate standard of 
care curative-intent treatment, consisting of breast surgery or neoadjuvant systemic treatment. 
Patients will have a pre-planned date for initiation of standard of care treatment scheduled 
approximately 8 weeks (+/- 5 days) after study initiation. Research study 
appointments/interventions will not interfere with the pre-planned, standard of care 
treatment arrangements. 
Recruitment Plan 
At the time of the initial surgical consultation at the Saul and Joyce Brandman Breast Center at 
Cedars-Sinai, medical oncology or radiation oncology consultation at the Samuel Oschin 
Comprehensive Cancer Institute at Cedars-Sinai, potentially eligible patients will be identified, 
informed consent forms provided, and initiation of standard of care treatment (breast conserving 
surgery or neoadjuvant systemic therapy) dates established. Potentially eligible patients will be 
evaluated by 1) A designated consenting medical oncologist and 2) consenting radiation 
oncologist for assessment of amenability to preoperative radiation. All available (per the standard 
of care) imaging (mammogram +/- breast ultrasound +/- breast MRI) as well as clinical breast 
exam will be reviewed to assess tumor size and determine feasibility of preoperative radiation 
boost delivery and thus, study participation. Whenever feasible, the RT evaluation will be 
arranged prior to the testing date for standard of care treatment so that baseline research bloods 
may be collected after the patient is confirmed eligible and together with the non-study 
pretreatment bloods. Prior to protocol intervention, consent will take place at the first available 
appointment with a consenting professional.   
 
 
4.0 
PATIENT ELIGIBILITY  
4.1 
Inclusion Criteria  
Subjects must meet the following criteria at screening to be eligible to participate in the study.  
4.1.2 
Women age 18 years or older 

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4.1.3 
Confirmed histologic diagnosis of invasive adenocarcinoma of the breast, and 
4.1.4 
ER, PR and HER2 testing (on outside or Cedars-Sinai biopsy report), and 
4.1.5 
High-risk, ER-positive and HER2-negative breast cancer patients. ER-positive 
disease is defined as ER>10%, any PR and HER2-negative by ASCO CAP guidelines. 
High-risk disease will be defined by the presence of at least 2 of the following 3 criteria: 
histologic grade II-III, Ki-67 > 20%, ER expression < 75% by IHC) 
4.1.6  
TNBC patients (defined as ER<10%, PR<10%, HER2-neu 0-1+ by IHC or FISH-
negative; or as per MD discretion) 
 
4.1.7 
Operable tumor measuring ≥2 cm in maximal diameter as measured by any 
available standard of care imaging (examples include mammogram, breast ultrasound, 
breast MRI, CT) and/or by clinical exam 
 
4.1.8 Any nodal status 
 
4.1.9 
Multifocal disease is permitted; largest focus must measure ≥2 cm 
 
4.1.10 Synchronous bilateral invasive breast cancer is permitted  
 
4.1.11 No indication of distant metastases 
 
4.1.12 Candidate for breast-conserving therapy 
 
4.1.13 Tumor amenable to preoperative radiation therapy boost as determined by 
radiation oncologist 
 
4.1.14 ECOG performance status score of 0 or 1 
 
4.1.15 Screening laboratory values must meet the following criteria:  
i. White blood cells (WBCs) ≥ 2000/μL  
ii. Absolute neutrophil count (ANC) ≥ 1500/μL 
iii. Platelets ≥ 100 x 103/μL  
iv. Hemoglobin ≥ 9.0 g/dL  
v. Serum creatinine ≤ 2 mg/dL (or glomerular filtration rate ≥ 40 ml/min)  
vi. AST ≤ 2.5 x upper limit of normal (ULN) 
vii. ALT ≤ 2.5 x ULN  
viii. Total bilirubin within normal limits (except subjects with Gilbert’s syndrome, who 
must have total bilirubin < 3.0 mg/dL)  
ix. INR ≤ 1.5 x ULN unless participant is receiving anticoagulant therapy as long as 
PT or aPTT is within therapeutic range of intended use of anticoagulant(s) 
x. Negative HIV screening test 
xi. Negative screening tests for Hepatitis B and Hepatitis C.  
Patients with positive results that do not indicate true active or chronic 
infection may enroll after discussion and consensus agreement by the 
treating physician and principal investigator. 
 
4.1.16 Women of childbearing potential (WOCBP) must be using an acceptable method 
of contraception to avoid pregnancy throughout the study and for at least 4 months after 
the last dose of pembrolizumab in such a manner that the risk of pregnancy is minimized. 
See below for the definition of WOCBP. 

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4.1.17 WOCBP must have a negative urine or serum (preferred) pregnancy test within 
14 days prior to the first dose of pembrolizumab. Note: Patients undergoing egg 
harvesting are exempt from being required to take a pregnancy test within 72 hours of 
C1D1 due to the likelihood of false positive results. These patients may have false 
positive results for at least two weeks and may be exempt from pregnancy testing 
requirements within 14 days of C1D1 and may proceed to treatment per investigator 
discretion. 
Definition of WOCBP  
Women of childbearing potential include any female who has experienced 
menarche and who has not undergone successful surgical sterilization 
(hysterectomy, bilateral tubal ligation, or bilateral oophorectomy) or is not 
postmenopausal. Post menopause is defined as:  
• Amenorrhea ≥ 12 consecutive months without another cause and a 
documented serum follicle stimulating hormone (FSH) level >35 mIU/mL  
• Women with irregular menstrual periods and a documented FSH level > 
35 mIU/mL  
• Women on hormone replacement therapy (HRT)  
 
Women who are using oral contraceptives, other hormonal contraceptives 
(vaginal products, skin patches, or implanted or injectable products), or 
mechanical products such as an intrauterine device or barrier methods 
(diaphragm, condoms, spermicides) to prevent pregnancy, or are practicing 
abstinence or where their partner is sterile (e.g., vasectomy) should be 
considered to be of childbearing potential. 
 
4.1.18 Women must not be breastfeeding. 
 
4.1.19 Willingness to adhere to the study visit schedule and the prohibitions and 
restrictions specified in this protocol. 
 
4.1.20 Willingness to undergo mandatory Week 4 research biopsy 
 
4.1.21 Written informed consent obtained from subject and ability for subject to comply 
with the requirements of the study. 
 
4.2 
Exclusion Criteria 
 
4.2.1 
HER2-positive breast cancer defined as IHC3+ or IHC2+ with FISH>2 AND copy 
number >4 OR FISH <2 AND copy number >6 
 
4.2.2 
Inflammatory breast cancer 
 
4.2.3 
Contraindication(s) to breast-conserving therapy 
 
4.2.4 
Contraindication to radiation therapy or planned partial breast irradiation 
 
4.2.5 
Patients with cosmetic breast augmentations, specifically sub glandular implants 
with altered breast tissue, at the time of diagnosis 
 

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4.2.6 
Evidence of metastatic disease. 
 
4.2.7 
Diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy (in 
dosing exceeding 10 mg daily of prednisone equivalent) or any other form of 
immunosuppressive therapy within 7 days prior to the first dose of study drug. 
 
4.2.8 
Known additional malignancy that is progressing or has required active treatment 
within the past 3 years.  Note:  Participants with basal cell carcinoma of the skin, 
squamous cell carcinoma of the skin, or carcinoma in situ (e.g. breast carcinoma, 
cervical cancer in situ) that have undergone potentially curative therapy are not 
excluded. 
 
4.2.9 
Medical history and concurrent diseases 
 
o 
Active autoimmune disease that has required systemic treatment in the past 2 
years (i.e. with use of disease modifying agents, corticosteroids or 
immunosuppressive drugs). Replacement therapy (eg., thyroxine, insulin, or 
physiologic corticosteroid replacement therapy for adrenal or pituitary 
insufficiency, etc.) is not considered a form of systemic treatment. 
 
o 
History of (non-infectious) pneumonitis that required steroids or has current 
pneumonitis. 
o 
Active infection requiring systemic therapy. 
 
o 
Any serious or uncontrolled medical disorder that, in the opinion of the investigator, 
may increase the risk associated with study participation or study drug 
administration, impair the ability of the subject to receive protocol therapy, or 
interfere with the interpretation of study results.  
 
o 
Known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies). 
 
o 
Known history of Hepatitis B (e.g., HBsAg reactive) or known active Hepatitis C 
(e.g., HCV RNA [qualitative] is detected).  
 
4.2.10 Prohibited Treatments and/or Therapies 
o 
Chronic use of immunosuppressants and/or systemic corticosteroids (used in the 
management of cancer or non-cancer-related illnesses). However, use of 
corticosteroids is allowed for the treatment of immune related Adverse Events 
(irAEs), or adrenal insufficiency. 
o 
Live vaccines within 30 days prior to the first dose of study treatment and while 
participating in the study.  Examples of live vaccines include, but are not limited to, 
the following: measles, mumps, rubella, varicella/zoster, yellow fever, rabies, BCG, 
and typhoid vaccine. Seasonal influenza vaccines for injection are generally killed 
virus vaccines and are allowed; however, intranasal influenza vaccines (eg, 
FluMist®) are live attenuated vaccines and are not allowed. Prior treatment with 
an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or with an agent directed to another 
stimulatory or co-inhibitory T-cell receptor (eg, CTLA-4, OX-40, CD137) 
 
o 
Prior systemic anti-cancer therapy including investigational agents within 4 weeks 
prior to study start. 
Note: Participants must have recovered from all AEs due to previous therapies to 
≤Grade 1 or baseline. Participants with ≤Grade 2 neuropathy may be eligible. 

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Note: If participant received major surgery, they must have recovered adequately 
from the toxicity and/or complications from the intervention prior to starting study 
treatment. 
 
o 
Prior radiotherapy within 2 weeks of start of study treatment to sites outside the 
breast.  Participants must have recovered from all radiation-related toxicities, not 
require corticosteroids, and not have had radiation pneumonitis.   
 
o 
Currently participating in or has participated in a study of an investigational agent 
or has used an investigational device within 4 weeks prior to the first dose of study 
treatment. 
Note:  Participants who have entered the follow-up phase of an 
investigational study may participate as long as it has been 4 weeks after 
the last dose of the previous investigational agent. 
 
4.2.11 For subjects who agree to the research breast MRI sub-study: Four or more 
previous gadolinium contrast scans due to the risk of brain deposits following 
repeated use of gadolinium-based contrast agents. 
 
 
5.0 
TREATMENT PLAN 
5.1 
Treatment Dosage and Administration 
5.1.1 Pembrolizumab 
Subjects will receive pembrolizumab 200 mg as an IV infusion. Pembrolizumab will be 
administered as a 30-minute IV infusion (every effort should be made to target infusion timing to 
be as close to 30 minutes as possible. However, given the variability of infusion pumps from site 
to site, a window of -5 minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 
min/+10 min).  
5.1.1.1 Pembrolizumab Agent 
Pembrolizumab is manufactured by Merck. Please refer to investigator brochure and MK-3475 
Drug Preparation Instructions’ manual. 
Table 1: Product Descriptions 
Product Name & Potency 
Dosage Form 
Pembrolizumab 50 mg 
Lyophilized Powder for Injection 
Pembrolizumab 100 mg/ 4mL 
Solution for Injection 
The investigator shall take responsibility for and shall take all steps to maintain appropriate 
records and ensure appropriate supply, storage, handling, distribution and usage of 
investigational produce in accordance with the protocol and any applicable laws and regulations. 
Clinical supplies will be affixed with a clinical label in accordance with regulatory requirements. 
 
5.1.2 RT Boost 
The Gross Target Volume (GTV) for the boost will be defined by preoperative imaging +/- clips 
placed into the tumor at the time of the biopsy. A planning target volume (PTV) will be created 
from the GTV as per departmental partial breast irradiation treatment planning guidelines. The 
PTV will be treated with 8 Gy x 3 fractions (24 Gy total) with external beam techniques. RT will 

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begin within 5 calendar days of the second dose of pembrolizumab.  
 
A total dose of 24 Gy will be delivered over 3 fractions within a 3 to 5-day period.  Doses of 8 Gy 
will be given once daily for 3 treatment days.  
 
Figure 3 
A: Beams-eye view of the boost directed to the PTV. B: Axial and coronal views of the PTV with 
isodose lines denoting the radiation doses delivered with partial breast irradiation. 
 
 
5.1.2.1 Modality and Energy 
Any combinations of photon beams of energy 6 MV or higher, with or without the addition of 
electrons of any energy, may be used for treatment, provided the dosimetric requirements of 
adequately treating the planning target volume (PTV) and homogeneity are met, although mixed 
energy (photons and electrons) is encouraged to minimize the exposure of the lung to radiation. 
5.1.2.2 Patient Positioning and Simulation 
The patient will be simulated in the supine positioning in a breast board or alpha cradle.  
5.1.2.3 Target and Normal Structure Delineation 
The radiation oncologist will identify the tumor by utilizing diagnostic MRI and mammographic 
imaging as the presence of clips in the breast. A radiopaque wire will be placed around the extent 
of the palpable breast tissue to delineate the whole breast volume. Axial non-contrast CT images 
will be obtained in 2.5 mm thick slices, superiorly from the angle of the mandible through the lung 
bases, except for the region of the tumor, where slice thickness will be narrowed to 1.25 mm to 
ensure precision in identifying the tumor. Following the initial scan, a review of the CT images will 
be performed to confirm the location of the primary tumor to be targeted. Tattoos will be placed in 
the same number and configuration as those for traditional whole breast treatments.  
The radiation oncologist will outline the tumor, utilizing MRI or mammographic imaging to 
delineate the volume. A margin of 2 - 5 mm will be added superiorly, inferiorly, medially, and 
laterally to the tumor gross treatment volume (GTV) in order to create the PTV; the border of the 
PTV will be limited anteriorly to within 5 mm below the skin surface and posteriorly to the anterior 
chest wall, i.e. the anterior surface of the ribs. No attempt will be made to include the entire length 
of the incision scar. 
The margins to be given around the excision volume will be determined in relation to the 
maximum diameter of the tumor GTV. If the maximum diameter of the tumor GTV is 4 cm or less, 
then up to 5 mm margins around it should be used. If the maximum diameter is larger than 4 cm, 
then 2 – 4 mm margins should be used. It is preferred the PTV does not exceed 30-35% of the 

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breast volume. Estimation of the PTV will be performed immediately after the simulation. 
It is also required that 50% of the non-target breast tissue (ipsilateral breast volume minus the 
PTV) does not receive more than 50% of the prescribed dose.  Additional margin beyond the PTV 
to the field edges will be added as required to achieve full dose (100%) to the PTV. Blocks or 
multileaf collimators should be used to define the treatment area. 
 
5.1.2.4 Dose-Volume Histogram and Normal Tissue Constraints 
The following structures should be contoured for all patients by the radiation oncologist: 
1. Whole breast 
2. Tumor CTV 
3. Tumor PTV  
4. Ipsilateral lung 
5. Contralateral lung 
6. Heart 
7. Nipple 
 
A dose-volume histogram (DVH) with volumes expressed as % total will be submitted and the 
dosimetry forms will be completed using the DVH data of the following: 
1. Ipsilateral lung 
2. Contralateral lung 
3. Heart 
4. Skin 
5. PTV 
6. Breast minus PTV 
 
5.1.2.5 Treatment Dose 
The dose delivered will be 8 Gy in 3 fractions (for a total of 24 Gy), given once daily over 3-5 days. 
Treatment may be started on any day of the week, but preferably on a Monday or Tuesday in order to 
allow for completion of RT prior to the weekend.  
 
5.1.2.6 Prescription Point 
The minimum isodose line that completely encompasses the PTV on the transverse, sagittal and coronal 
planes through the center of the PTV. Inhomogeneity corrections are to be used. 
 
5.1.2.7 Time-Dose Considerations 
• 
Dose per fraction: 24 Gy to the isodose line that completely encompasses the PTV 
• 
Deliver one fraction per day to the isodose line that completely encompasses the PTV 
• 
Total elapsed time: 3 to 5 days, unless there is a treatment break or interruption 
5.1.2.8 Homogeneity and Reference Points 
• The dose within the PTV must be within 100-125% of the prescribed dose 
• The minimum isodose line that completely encloses the PTV on the transverse, sagittal and 
coronal planes through the center of the PTV. 
5.1.2.9 Dose Restraints for Uninvolved Ipsilateral Breast, Lungs, Heart and Liver Volumes57 
5.1.2.9.1 Ipsilateral Lung 
• 
Volume receiving 20 Gy should not exceed 3% 
• 
Volume receiving 20 Gy should not exceed 3% 
• 
Volume receiving 20 Gy should not exceed 3% 
5.1.2.9.2 Contralateral Lung 
• 
Volume receiving 20 Gy should not exceed 3% 
• 
Volume receiving 20 Gy should not exceed 3% 

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• 
Volume receiving 20 Gy should not exceed 3% 
5.1.2.9.3 Heart 
• 
Maximum dose constraint: 90% of Rx dose 
5.1.2.9.4 Liver 
• 
Maximum dose constraint: 90% of Rx dose 
• 
20% of the liver should not receive more than 5 Gy 
5.1.2.9.6 Skin  
• 
Maximum dose constraint: 12 Gy to a point dose 
• 
No more than 1 cc of the skin should receive greater than 10 Gy 
5.1.2.9.5 Treatment Technique  
Treatment must be performed using CT-guided planning with the capability of performing 
true 3D reconstruction 
5.1.2.10 Daily Imaging Guidance and Respiratory Motion Management 
Radiation treatment will be delivered with multiple beams utilizing either intensity-modulated or 3D-
conformal techniques. One of the following three techniques will be employed for accurate positioning of 
the patient on the treatment machine, for each treatment received. Each patient will only use one 
technique per treatment during the course of radiation. 
 
1. Two orthogonal pair kilovoltage images can be acquired with the patient positioned for treatment, 
and registered to digitally reconstructed radiographs (DRRs) from the planning CT. Shifts will be 
determined based on registration to biopsy clips. 
 
2. Cone beam (CBCT) imaging is a new technique that utilized X-ray imaging equipment that has 
recently been installed on the gantry of the radiation-treatment machines, manufactured by 
Varian Medical Systems, Palo Alto, CA. During CBCT scan, the treatment-machine gantry makes 
a full rotation while taking ~600 projection images. The projection images are then converted into 
axial slices, similar in nature to a CT scan. The time taken from the beginning of the CBCT scan 
to the generation of the axial slices usually takes several minutes. The use of the imaging 
equipment for the purpose of setting up patients for external-beam radiotherapy has received 
FDA approval. Further information regarding this approval can be accessed via 
https://www.accessdata.fda.gov/cdrh_docs/pdf4/K040192.pdf. The software used to control the 
imaging equipment for the CBCT scan is also FDA approved. 
 
3. Align RT® employs two ceiling-mounted 3D camera units to produce high-resolution and 
accurate 3D surface data referenced to the treatment isocenter. At each treatment fraction, the 
system images the current patient position instantaneously. Align RT® is able to gate its image 
capture so that 3D data are acquired at a reproducible point in the breathing cycle. State-of-the-
art surface-matching software registers these data to the reference surface within seconds. 
Couch shifts are displayed to show discrepancies between existing and ideal treatment positions. 
New coordinates for the optimal couch position are then displayed and may be applied. This 
system has been FDA approved. The use of AlignRT for positioning of patient for partial breast 
irradiation has been published by Bert58 et al and Gierga59 et al. Further information regarding this 
approval is available on the website: http://www.visionrt.com/wp-
content/uploads/2016/04/AlignRTPlus-510kClearance-Jan-2013.pdf 
 
 
Standard of Care Therapy 
The decision to proceed to breast-conserving surgery versus neoadjuvant systemic therapy will 
be at the discretion of the treating physician. Patients who are planned for neoadjuvant systemic 

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therapy will initiate treatment after study treatment completion. Either after neoadjuvant systemic 
therapy if planned or after study treatment, all patients will proceed to breast-conserving surgery. 
After surgery, patients will undergo adjuvant systemic treatment consisting of chemotherapy 
and/or endocrine therapy, as per physician’s choice. 
Following breast-conserving surgery, whole breast RT (WBRT) will be delivered as per standard 
of care. Given the hypofractionated boost approach, a standard fractionation of 200 cGy x 25 
fractions (5 days a week for 5 weeks) will be utilized. Patients will begin WBRT approximately 4-6 
weeks postoperatively or following chemotherapy. 
5.3 
Method of Assigning Subjects to Treatment Groups  
There is no randomization. Subjects will be assigned to one of two clinically relevant cohorts per 
their biomarker profile.  
5.4 
Toxicities and Dosing Delays/Dose Modifications  
Adverse events (both non-serious and serious) associated with pembrolizumab exposure may 
represent an immunologic etiology. These adverse events may occur shortly after the first dose or 
several months after the last dose of treatment. Therefore, early recognition and initiation of 
treatment is critical to reduce complications.  Based on existing clinical study data, most irAEs 
were reversible and could be managed with interruptions of pembrolizumab, administration of 
corticosteroids and/or other supportive care. For suspected irAEs, ensure adequate evaluation to 
confirm etiology or exclude other causes. Additional procedures or tests such as bronchoscopy, 
endoscopy, skin biopsy may be included as part of the evaluation. Based on the severity of irAEs, 
withhold or permanently discontinue pembrolizumab and administer corticosteroids. Dose 
modification and toxicity management guidelines for irAEs associated with pembrolizumab are 
provided in Tables 2 and 3. 
Pembrolizumab dose modifications will not be allowed. Pembrolizumab must be withheld for 
drug-related toxicities and severe or life-threatening AEs as per Table 2 and 3 below. To avoid 
delay in standard of care treatment, study treatment should be discontinued if toxicities preclude 
administration of the second dose pembrolizumab as planned. See Pembrolizumab Program 
Events of Clinical Interest Guidance Document for supportive care guidelines, including use of 
corticosteroids to treat immune related AEs.  
Any patient who receives treatment on this protocol will be evaluable for toxicity. 
Radiotherapy is given in a continuous course. It is not anticipated that a treatment break should 
be needed, as the development of acute skin-related or other side effects is extremely rare. If a 
break seems necessary due to the development of CTCAE version 4.0 Grade 4 skin or 
subcutaneous side effects during treatment, or must be given due to unavoidable medical 
necessity (i.e., intercurrent illness), the situation should be discussed with the treating radiation 
oncologist. In the event that a patient must miss a scheduled treatment for non-toxicity related 
reasons (i.e.: weather, sickness, family emergencies, etc) the treatment visit will be made up at 
the end.  
 
 
  
 
 
 

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Table 2: Dose Modification and Toxicity Management Guidelines for Immune-related AEs Associated with 
Pembrolizumab General instructions: 
1. Corticosteroid taper should be initiated upon AE improving to Grade 1 or less 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 Grade 1 or 0 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 

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AST / ALT 
elevation or 
Increased 
bilirubin 
Grade 2 
Withhold 
• Administer corticosteroids (initial 
dose of 0.5- 1 mg/kg prednisone or 
equivalent) followed by taper 
• 
Monitor with liver function tests (consider 
weekly or more frequently until liver enzyme 
value returned to baseline or is stable 
Grade 3 or 4  
Permanently 
discontinue 
• 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 causes 
 
Grade 3 or 4 
Permanently 
discontinue 

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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, 
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.   
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).   
 
 
 
 
 

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Table 3  Pembrolizumab Infusion Reaction Dose modification and Treatment Guidelines 
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 participant is 
deemed medically stable in the opinion of the investigator. 
None 
Grade 2 
Requires therapy or infusion 
interruption but responds promptly 
to symptomatic treatment (e.g., 
antihistamines, NSAIDs, narcotics, 
IV fluids); prophylactic medications 
indicated for ≤24 hrs 
Stop Infusion. 
Additional appropriate medical therapy may include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDs 
Acetaminophen 
Narcotics 
Increase monitoring of vital signs as medically indicated until the participant is 
deemed medically stable in the opinion of the investigator. 
If symptoms resolve within 1 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 participant should be 
premedicated for the next scheduled dose. 
Participants who develop Grade 2 toxicity despite adequate premedication 
should be permanently discontinued from further study drug treatment 
Participant may be premedicated 1.5h (± 30 minutes) 
prior to infusion with: 
Diphenhydramine 50 mg po (or equivalent dose of 
antihistamine). 
Acetaminophen 500-1000 mg po (or equivalent dose 
of analgesic). 
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: 
Epinephrine** 
IV fluids 
Antihistamines 
NSAIDs 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Increase monitoring of vital signs as medically indicated until the participant is 
deemed medically stable in the opinion of the investigator. 
Hospitalization may be indicated. 
**In cases of anaphylaxis, epinephrine should be used immediately. 
Participant is permanently discontinued from further study drug treatment. 
No subsequent dosing 
Appropriate resuscitation equipment should be available at the bedside and a physician readily available during the period of drug administration. 
For further information, please refer to the Common Terminology Criteria for Adverse Events v4.0 (CTCAE) at http://ctep.cancer.gov 
 
 
 
 
 

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5.4 
Concomitant Medications/Treatments 
Permitted Concomitant Medications  
Investigators may prescribe concomitant medications or treatments (e.g., 
acetaminophen, diphenhydramine) deemed necessary to provide adequate prophylactic 
or supportive care except for those medications identified as “excluded” as listed below.  
Excluded Concomitant Medications  
Subjects must be instructed not to take any medications, including over-the-counter 
products, without first consulting with the investigator.  
The following medications are considered exclusionary during the study. The Principal 
Investigator must be notified if a subject receives any of these during the study.  
• 
Any investigational anticancer therapy.   
• 
Any concurrent chemotherapy (except as per study), immunotherapy, or biologic 
therapy. 
• 
Concurrent use of hormones for non-cancer-related conditions (e.g., insulin for 
diabetes and hormone replacement therapy) is acceptable.   
• 
Immunosuppressive medications including, but not limited to systemic corticosteroids 
(>10 mg/day prednisone or equivalent), methotrexate, azathioprine, and tumor 
necrosis factor alpha (TNF-α) blockers. Use of immunosuppressive medications for 
the management of investigational product- related AEs, in subjects with contrast 
allergies is acceptable. In addition, use of inhaled and intranasal corticosteroids is 
permitted.   
5.5 
Other Modalities or Procedures/ Research vs. Standard of Care Procedures 
A RT boost is a standard component of RT planning and usually follows WBRT after 
breast-conserving surgery. In this study, the boost will be administered preoperatively at 
a dose of 8 Gy for 3 fractions, to begin within 5 days of the second dose of 
pembrolizumab. RT will be performed using external beam ionizing radiation in 
accordance with institutional standard practice. 
5.6 
Duration of Study Participation  
The study duration per subject will be up to 150 days, with up to 14 days of screening, up 
to 47 days on treatment, and 15 weeks of follow-up after the second pembrolizumab 
dose. 
5.7 
Removal of Patients from Protocol  
Patients will be removed from the study when any of the criteria listed in Section 6.5 
apply. Notify the Principal Investigator, and document the reason for study removal and 
the date the patient was removed on the Case Report Form. The patient should be 
followed-up per protocol.  
5.8 
Subject Replacement 
Subjects who withdraw from the study treatment prior to starting or during the study 
intervention and fail to complete the intervention may need to be replaced at the 
discretion of the study PI.  Subjects who complete the study intervention, but are not 
evaluable for the primary endpoint of tumor infiltrating lymphocytes will also be replaced 
at the discretion of the study PI.   
 
 

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6.0 
STUDY PROCEDURES 
6.1 
Screening/Baseline Procedures 
Assessments performed exclusively to determine eligibility for this study will be done only 
after obtaining informed consent. Assessments performed for clinical indications (not 
exclusively to determine study eligibility) may be used for baseline values even if the 
studies were done before informed consent was obtained. 
 
Following consent, a research biopsy of the primary tumor will be performed on all 
patients in the TNBC cohort.  
 
All screening procedures must be performed within 28 calendar days prior to the first 
dose of pembro (C1D1) unless otherwise stated. The screening procedures include: 
6.1.1 Informed Consent 
6.1.2 Medical history and record review 
Relevant medical history, including history of current disease, other 
pertinent history, current medication use, and information regarding 
underlying diseases will be recorded at Screening. 
6.1.3 Demographics 
6.1.4 Review subject eligibility criteria 
6.1.5 Complete physical exam including ECOG PS 
6.1.6 Adverse event assessment 
Baseline adverse events will be assessed. See Sections 6.2 and 7.0 for 
Adverse Event monitoring and reporting.  
 
6.1.7 Standard Laboratory Evaluations Including: 
Comprehensive metabolic panel (CMP): aspartate aminotransferase 
(AST), alanine aminotransferase (ALT), albumin, alkaline phosphatase 
(ALP), total bilirubin, calcium, creatinine, glucose, total protein, BUN, 
sodium, potassium, chloride, bicarbonate  
Complete blood cell count with differential and platelet count (CBC) 
Amylase, lipase, lactate dehydrogenase (LDH) 
Prothombin time (PT), international normalized ratio (INR), partial 
thromboplastin time (aPTT) 
HIV testing 
Hepatitis serologies including hepatitis B surface antigen (HBsAg), 
hepatitis B core antibody (HBcAb) and hepatitis C antibody 
 
6.1.8 
Research-Related Laboratory Evaluation including: 
Thyroid stimulating hormone (TSH), free T4, T3, ACTH, FSH/LH, 
prolactin, testosterone levels. Labs must be drawn, but do not require 
results to determine eligibility or treatment continuation. 
6.1.9 
EKG 

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6.1.10  Pregnancy Testing for WOCBP 
Urine or serum (preferred) pregnancy testing within 14 days of first dose 
of pembrolizumamb for screening (C1D1) and also 72 hours prior to 
C1D1. If screening pregnancy test was done 72 hours of C1D1, it does 
not have to be repeated.  
Note: In the setting of egg harvesting, the test for pregnancy looks for 
beta-HCG which is the agent used for triggering the egg harvest. Given 
the sensitivity of the test, it will remain positive for about two weeks. This 
will be true for all egg harvesting patients. Therefore, the 72-hour test is 
not required for patients undergoing egg harvesting because of the high 
likelihood of false positive results. These patients may have false positive 
results for at least two weeks and may be exempt from pregnancy testing 
requirements within 14 days of C1D1, and may proceed per investigator 
discretion. 
6.1.11 Collection for correlative research tests including: 
 
Blood (3 CPT tubes,1 red-top tube, and 1 green-top tube) for immune 
response studies 
 Core needle biopsy of primary tumor with 2 cores obtained for immune       
response studies 
 
 
 
Optional fecal sample for microbiome analysis 
 
6.1.12 Cosmesis Evaluation 
Cosmesis will be scored by the treating physician using the four-tier 
Harvard Scale (Excellent, Good, Fair, Poor) to compare the treated 
breast to the control breast (Appendix C) 
Digital photographs (frontal, with arms at side) will be recorded. 
6.1.13 Optional breast MRI 
 
6.2 
Procedures During Treatment  
Prior to Each Treatment Cycle 
History and physical exam including vital signs and ECOG PS 
Adverse event assessment 
Duration (start and stop dates and times), severity/grade, outcome, treatment 
and relation to study drug and radiation therapy of adverse events will be 
recorded on the case report form (CRF). 
 
 
Medication reconciliation 
 
 
 
Labs: CBC & CMP 
6.2.1 
Prior to Cycle 2 
Research tests including: 
• 
Blood (3 CPT tubes, 1 red-top tube, and 1 green-top tube) for immune 
response studies. 
• 
Core needle biopsy of primary tumor with 2 cores obtained for immune 
response studies. 
• 
Optional breast MRI 
 

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• 
Optional fecal sample for microbiome analysis 
 
6.2.2 
Prior to standard of care treatment or at the time of surgery if that is the 
planned standard of care treatment 
Research tests including: 
• 
Blood (3 CPT tubes,  1 red-top tube, and 1 green-top tube) for immune 
response studies. 
• 
Core needle biopsy of primary tumor with 2 cores obtained for immune 
response studies or surgical sample with wedge of tumor tissue and 
benign tissue obtained. 
o 
If subject receives neoadjuvant systemic therapy before 
proceeding to surgery as part of SOC treatment, fresh specimen 
may also be collected at the time of surgery. This will be 
determined at the discretion of the treating investigator. 
 
• 
Optional breast MRI 
• 
Optional fecal sample for microbiome analysis 
• 
Cosmesis Evaluation (#2 if surgery is planned prior to SOC 
treatment) 
Cosmesis will be scored by the treating physician using the four-tier 
Harvard Scale (Excellent, Good, Fair, Poor) to compare the treated 
breast to the control breast.  
• 
Digital photographs (frontal, with arms at side) will be recorded. 
 
6.3 
Follow-up Procedures 
Patients will be followed at minimum every 3 weeks after completion of (or early 
withdrawal from) study treatment until week 19 after study initiation after which they will 
be followed every 6 months for 1 year post treatment.  The assessments are listed below: 
6.3.1 
History and physical exam including vital signs and ECOG PS 
 
6.3.2 
Adverse event assessment: 
AE collection will continue until week 19, after which time only immune related 
AE’s listed in table 2 will be collected post treatment for 1-year post treatment as 
well as through chart reviews at these time points. For study patients who are 
receiving standard chemotherapy (usually at Weeks 7 -25) at their treating 
physician’s office, AE collection may also be done via phone call and 
documented in the subject’s study record.  
6.3.3 
Medication reconciliation 
6.3.4  
Cosmesis Evaluations  
(#2 prior to surgery if after SOC treatment, #3 prior to whole breast 
radiation, approx. 4 weeks post-operative, #4 at 6 months post-op) 
Cosmesis will be scored by the treating physician using the four-tier Harvard 
Scale (Excellent, Good, Fair, Poor) to compare the treated breast to the control 
breast.  
Digital photographs (frontal, with arms at side) will be recorded. 
6.3.5     Endocrine Toxicity Assessment  

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At one year +/- 2 months post-treatment follow-up, a full thyroid function panel 
(including TSH, free and total T3, and free T4) will be completed. Cortisol results 
from adrenal glands will also be obtained as part of this assessment. Results will 
be obtained if feasible or if already available in the medical chart. 
6.3.6 
Survival Status 
 
 
Survival status assessment and any documentation of recurrence (if possible) 
will be conducted every 6 months for up to 5 years following the end of treatment. 
Study staff will record survival status and recurrence via medical record review, 
phone call, or at standard clinical visits.  
 
 
 

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6.4 Time and Events Table  
 
 
Screening 
 28 days4 
Week 1 
(C1D1) 
Week 4 
(C2D1) 
Week 7 
(SOC Tx) 
Post 
Treatment 
Follow Up12 
Q3W until 
week 19 & 
Q6 Mo.post 
Tx for 1 YR  
Long-Term 
Follow Up 
 
Q6mos for up 
to 5 years 
post Tx 
Pembrolizumab1 
 
X 
X 
 
 
 
Radiation Treatment2 
 
 
X 
 
 
 
Initiation of Standard of Care 
Treatment3 
 
 
 
X 
 
 
Eligibility Criteria 
X 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
Pathology Reviewed by 
Investigator 
X 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
Height and weight 
X 
 
 
 
 
 
History of present illness5 
X 
X 
X 
X 
X 
 
Safety bloodwork6 
X 
X 
X 
X 
 
 
Full thyroid function panel 
(TSH, free and total T3 and 
free T4), 
X13 
 
X 
X 
X16 
 
Cortisol 
 
 
 
 
X16 
 
ACTH, FSH/LH, prolactin, 
testosterone 
X 
 
 
 
 
 
Amylase, lipase, LDH, 
X 
 
 
 
 
 
PT/INR and aPTT 
X 
 
 
 
 
 
HIV, Hepatitis B and C7 
X 
 
 
 
 
 
Pregnancy test8 
X 
X 
 
 
 
 
Research bloods9 
X 
 
X 
X 
 
 
Research tumor biopsy10 
X 
 
X 
X 
X 
 
Optional research breast 
MRI 
X 
 
X 
X 
 
 
Optional fecal samples for 
microbiome analysis11 
X 
 
X 
X 
 
 
Cosmesis Evaluation and 
Digital Photos14 
X 
 
 
X 
X14 
 
Survival Status 
 
 
 
 
 
X 
 
1. Each cycle, 3 weeks (21 days) in duration, corresponds to 1 completed pembrolizumab 
treatment. Pembrolizumab will begin on C1D1. The second dose will be given on C2D1 (+/- 3 
days. 
2. RT will consist of 8Gy for 3 fractions. RT will be delivered concurrently with the second dose of 
pembrolizumab within 5 business days of C2D1  
3. Initiation of standard of care treatment consisting of either breast-conserving surgery or 
neoadjuvant systemic therapy will begin 3 weeks after the second dose of pembrolizumab (+/- 
5 days).  

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4. Procedures must be performed within 28 calendar days prior to C1D1. 
5. A history of present illness (HPI) consisting of physical examination, vital signs (including 
temperature, pulse rate, respiratory rate, blood pressure), performance status assessment, 
medication reconciliation, and toxicity/adverse event assessment will occur at each visit, prior 
to study treatment administration if due.AE collection will continue until week 19, immune 
related AE collection (listed in table 2) will continue until 1 year post treatment 
6. Safety bloodwork consisting of CBC, CMP, AST, ALT, albumin, alkaline phosphatase (AP), 
and total bilirubin will be obtained at the time of screening, on C2D1 (+/- 3 days), at the time of 
initiation of standard of care treatment (+/- 5 days). Bloodwork from screening to be used for 
C1D1 dosing if drawn within 14 days of C1D1. 
7. Serology for HIV antibodies, HBsAg, HBcAb and hepatitis C antibody (unless previously tested 
negative).  
8. Urine or serum pregnancy test for WOCBP. Serum is preferred to urine test; within 14 days of 
first dose of pembrolizumab for screening (C1D1) and also 72 hours prior to C1D1. If 
screening pregnancy test was done within 72 hours of C1D1, it does not have to be repeated. 
Patients undergoing egg harvesting are exempt from the 72-hour test. 
9. Blood draws for research purposes performed after obtaining consent but prior to C1D1, then 
C2D1 (+/- 3 days), and at the time of initiation of standard of care treatment (+/- 5 days). A 
minimum of 3 CPT tubes,  1 red top tube, and 1 green top tube of blood will be required at all 
timepoints. All research blood draws will be performed in conjunction with safety labs in order 
to minimize the number of venipunctures.  
10. Research tumor biopsy will be performed after consent at baseline, within 14 days prior to 
C1D1 (for the ER+ cohort, archived diagnostic biopsy is allowed if sufficient tissue available, 
including 20-5 μm unstained slides; for the TNBC cohort, a baseline research biopsy of fresh 
tumor is required), prior to C2D1 (+/- 5 days), and prior to Week 7(+/- 5 days) at the time of 
initiation of standard of care treatment. If standard of care treatment is surgery, surgical tissue 
will be used. If it consists of further neoadjuvant systemic therapy, a research biopsy will be 
obtained. If FFPE diagnostic tissue alone is accepted, flow cytometry and MerFISH at baseline 
will not be performed. If subject receives neoadjuvant systemic therapy before proceeding to 
surgery as part of SOC treatment, then fresh specimen collection may also be done at the 
time of surgery. 
11. All fecal samples for microbiome analysis are optional. Fecal samples will be collected at 
baseline, within 14 days prior to C1D1, C2D1 (+/- 5 days), and at the time of initiation of 
standard of care treatment (+/- 5 days). 
12. Follow up appointments will occur through week 19 after study initiation, at minimum every 3 
weeks (+/- 5 business days) and continue every 6 months for 1-year post treatment. Patients 
receiving NAC may be seen more frequently (every 2 weeks) depending upon SOC treatment 
schedule. 
13. Thyroid panel results from screening will be used for C1D1 dosing, if drawn within 14 days of 
C1D1.  
14. Cosmesis Evaluation performed at baseline, prior to surgery, approximately 4 weeks 
postoperatively (prior to whole breast radiation), and 6-months postoperatively 
15. Imaging will be performed as an optional sub-study after consent at baseline, within 14 days 
prior to C1D1, prior to C2D1 (+/- 5 days), and prior to Week 7(+/- 5 days) at the time of 
initiation of standard of care treatment.  Timing to coincide with research biopsies. 
16. Cortisol lab results along with full thyroid function panel will be obtained at one year (+/- 2 
months) post-treatment follow-up, if feasible or available in medical chart.  
 
6.5 
 Removal of Subjects from Study 
Patients can be taken off the study treatment and/or study at any time at their own 
request, or they may be withdrawn at the discretion of the investigator for safety, 
behavioral or administrative reasons. The reason(s) for discontinuation will be 
documented and may include: 

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6.5.1 
Patient voluntarily withdraws (follow-up permitted);  
6.5.2 
Patient withdraws consent (termination of treatment and follow-up); 
6.5.3 
Patient is unable to comply with protocol requirements; 
6.5.4 
Patient demonstrates disease progression (unless continued treatment with 
study drug is deemed appropriate at the discretion of the investigator); 
6.5.5 
Patient experiences toxicity that makes continuation in the protocol unsafe; 
6.5.6 
Treating physician determines continuation on the study would not be in the 
patient’s best interest; 
 
6.5.7 
Patient becomes pregnant (pregnancy to be reported along same timelines as a 
serious adverse event); 
6.5.8 
Development of second malignancy (except for basal cell carcinoma or 
squamous cell carcinoma of the skin) that requires treatment, which would 
interfere with this study; 
6.5.9 
Lost to follow-up. If a research subject cannot be located to document toxicities 
after a period of up to 20 weeks following study enrollment or cannot be located 
to document survival after a period of 2 years, the subject may be considered 
“lost to follow-up.” All attempts to contact the subject during the two years must 
be documented. This will be reviewed during an interim data monitoring visit. 
 
6.6       Subject Withdrawal 
Participants may discontinue study treatment at any time for any reason or be dropped 
from the study treatment at the discretion of the investigator should any untoward effect 
occur.  In addition, a participant may be discontinued from study treatment by the 
investigator or the Sponsor if study treatment is inappropriate, the trial plan is violated, or 
for administrative and/or other safety reasons.  
 
 
A participant must be discontinued from study treatment but continue to be monitored in 
the study for any of the following reasons: 
• 
The participant  or participant’s legally acceptable representative requests to 
discontinue study treatment 
• 
Confirmed radiographic disease progression  
• 
Any progression or recurrence of any malignancy, or any occurrence of another 
malignancy that requires active treatment 
• 
Unacceptable adverse experiences  
• 
The participant has a medical condition or personal circumstance which, in the 
opinion of the investigator and/or sponsor, placed the participant at unnecessary risk 
from continued administration of study treatment. 
• 
The participant  has a confirmed positive serum pregnancy test (except for patients 
undergoing egg harvesting. See notes in Section 6.1.10 for further details) 
• 
Noncompliance with study treatment or procedure requirements 
• 
Recurrent Grade 2 pneumonitis 
• 
The participant is lost to follow-up 
 
 
7.0 
ADVERSE EVENTS (AE) 

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7.1 
Definitions 
7.1.1 
Adverse Event 
An adverse event is any untoward medical occurrence in a patient receiving 
study treatment and which does not necessarily have a causal relationship with 
this treatment. An AE can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of an experimental intervention, whether or not related to 
the intervention.  
7.1.2 
Serious Adverse Events (SAE) 
A “serious” adverse event is defined in regulatory terminology as any untoward 
medical occurrence that: 
7.1.2.1 
Results in death. 
If death results from (progression of) the disease, the disease should 
be reported as event (SAE) itself. 
7.1.2.2 
Is life-threatening. 
(the patient was at risk of death at the time of the event; it does not 
refer to an event that hypothetically might have caused death if it were 
more severe). 
7.1.2.3 
Requires in-patient hospitalization for ≥ 24 hours or prolongation of 
existing hospitalization. 
7.1.2.4 
Results in persistent or significant disability or incapacity. 
7.1.2.5 
Is a congenital anomaly/birth defect 
7.1.2.6 
Is an important medical event 
 
Any event that does not meet the above criteria, but that in the judgment of the 
investigator jeopardizes the patient, may be considered for reporting as a serious 
adverse event. The event may require medical or surgical intervention to prevent 
one of the outcomes listed in the definition of “Serious Adverse Event “.  
For example: allergic bronchospasm requiring intensive treatment in an 
emergency room or at home; convulsions that may not result in hospitalization; 
development of drug abuse or drug dependency. 
 
7.1.3 
Unanticipated Problem (UP) 
Unanticipated problems include any incident, experience, or outcome that meets all of 
the following criteria: 
• 
Unexpected (in terms of nature, severity, frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 

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• 
Related or possibly related to participation in the research (possibly related 
means there is a reasonable possibility that the incident, experience, or outcome 
may have been caused by the procedures involved in the research); and 
• 
Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known to an individual or group of individuals (including research subjects, 
research staff, or others not directly involved in the research). 
 
7.1.3.1  
Evaluable for toxicity  
All patients will be evaluable for toxicity from the time of their first treatment with 
study drug. 
7.2 
Severity of Adverse Events 
All non-hematologic adverse events will be graded according to the NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 4.0. The CTCAE current version is available at 
http://ctep.cancer.gov/reporting/ctc.html. 
 
7.3 
Adverse Event Monitoring  
The investigator or designee is responsible for ensuring that all adverse events (both serious and 
non-serious) observed by the clinical team or reported by the subject which occur after the 
subject has signed the informed consent are fully recorded in the subject’s medical records. 
Source documentation must be available to support all adverse events.  
 
Note: For study patients who are receiving standard chemotherapy (usually at Weeks 7 – 25) at 
their treating physician’s office, AE collection may also be done via phone call and documented in 
the subject’s study record. 
 
A laboratory test abnormality considered clinically relevant (e.g., causing the subject to withdraw 
from the study, requiring treatment or causing apparent clinical manifestations result in a delay or 
dose modification of study treatment, or judged relevant by the investigator), should be reported 
as an adverse event.   
 
The investigator or sub-investigator (treating physician if applicable) will provide the following for 
all adverse events (both serious and non-serious): 
 
• Event term (as per CTCAE) - Descriptions and grading scales found in the NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be 
utilized for AE reporting.  
• Description of the event 
• Date of onset and resolution 
• Expectedness of the toxicity 
• Grade of toxicity  
• Attribution of relatedness to the investigational agent- (this must be assigned by 
an investigator, sub-investigator, or treating physician) 
• Action taken as a result of the event, including but not limited to; no changes, 
dose interrupted, reduced, discontinued, etc. or action taken with regard to the 
event, i.e. no action, received conmed or other intervention, etc. 
 
Outcome of event 
All patients experiencing an adverse event, regardless of its relationship to study drug, will 
be monitored until:  

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• the adverse event resolves or the symptoms or signs that constitute the adverse  
 
event return to baseline 
• any abnormal laboratory values have returned to baseline 
• Week 19 for all AEs 
• 1-year post treatment for all immune related AE’s listed in table 2 
• there is a satisfactory explanation other than the study drug for the changes  
 
observed, or death. 
 
7.4 
Potential AEs 
Radiotherapy: 
Toxicities may occur from radiation treatment. The type and risk of toxicity will depend on the 
presence of normal tissue structures in close proximity to the target. These normal tissue doses 
will be constrained by the treatment plan to deliver doses no more than those recommended in 
the Cedars-Sinai Department of Radiation Oncology guidelines. 
The estimated risks of each type of toxicity are noted below, and include:  
1. Grade 2-3 skin (erythema, dry or moist desquamation, patchy ulceration) - 10% 
2. Grade 1-2 non-debilitating fatigue - 10% 
3. Any grade brachial plexopathy - 5% 
4. Grade 2-4 pneumonitis- 2-5% 
  
 
Pembrolizumab: 
Pembrolizumab is an FDA-approved agent on formulary for the treatment of metastatic melanoma, 
non-small cell lung cancer, head and neck squamous cell cancer, classical Hodgkin’s lymphoma, 
primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high 
cancer, gastric cancer, cervical cancer, hepatocellular cancer, and Merkel cell carcinoma. It is 
investigational for use in breast cancer. A comprehensive list of toxicities may be found in Lexicomp 
and in the investigator’s brochure. Principal side effects of pembrolizumab are listed in Table 4.  
 
Table 4: Principal side effects of pembrolizumab  
Cardiovascular 
Peripheral edema 
Central Nervous System 
Fatigue, headache, chills, insomnia, dizziness, 
optic neuritis (rare), uveitis (rare) 
Endocrine 
Hyperglycemia, hyponatremia, hypoalbuminemia, 
hypertriglyceridemia, hypocalcemia, adrenal 
insufficiency (rare), hypophysitis (rare) 
GI 
Nausea, decreased appetite, constipation, 
diarrhea, vomiting, abdominal pain, pancreatitis 
(rare), increased serum AST, hepatitis (rare) 
Hematologic 
Anemia, hemolytic anemia (rare) 
Neurological/Muscular 
Arthralgias, limb pain, myalgia, back pain, arthritis 
(rare), Lambert-Eaton syndrome (rare), myositis 
(rare), partial epilepsy (rare), rhabdomyolysis 

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(rare) 
Respiratory 
Cough, dyspnea, upper respiratory tract infection 
Miscellaneous 
Fever 
Dermatologic 
Exfoliative dermatitis (rare) 
Renal 
Interstitial nephritis (rare), nephritis (rare) 
 
7.5 
Supportive Care Guidelines 
Subjects should receive appropriate supportive care measures as deemed necessary by the 
treating investigator. Suggested supportive care measures for the management of adverse 
events with potential immunologic etiology are outlined below. Where appropriate, these 
guidelines include the use of oral or intravenous treatment with corticosteroids as well as 
additional anti- inflammatory agents if symptoms do not improve with administration of 
corticosteroids. 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.  
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or 
skin photography as part of evaluation of the event.  
Management of Pneumonitis  
• 
For Grade 2 events, treat with systemic corticosteroids. When symptoms improve to Grade 1 
or less, steroid taper should be started and continued over no less than 4 weeks.   
• 
For Grade 3-4 events, immediately treat with intravenous steroids. Administer additional anti-
inflammatory measures, as needed.   
• 
Add prophylactic antibiotics for opportunistic infections in the case of prolonged steroid 
administration.   
Management of Diarrhea/Colitis   
Subjects should be carefully monitored for signs and symptoms of enterocolitis (such as diarrhea, 
abdominal pain, blood or mucus in stool, with or without fever) and of bowel perforation (such as 
peritoneal signs and ileus).   
• 
All subjects who experience 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. For Grade 2 or higher diarrhea, consider GI consultation and 
endoscopy to confirm or rule out colitis.  
• 
For Grade 2 diarrhea/colitis that persists greater than 3 days, administer oral corticosteroids.  
• 
For Grade 3 or 4 diarrhea/colitis that persists > 1 week, treat with intravenous steroids 
followed by high dose oral steroids.   
• 
When symptoms improve to Grade 1 or less, steroid taper should be started and continued 
over no less than 4 weeks. 
Management of Type 1 diabetes mellitus (if new onset, including diabetic ketoacidosis 
[DKA]) or ≥ Grade 3 Hyperglycemia, if associated with ketosis (ketonuria) or metabolic 

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acidosis (DKA)   
• 
For T1DM or Grade 3-4 Hyperglycemia  
• 
Insulin replacement therapy is recommended for Type I diabetes mellitus and for Grade 3-4 
hyperglycemia associated with metabolic acidosis or ketonuria.  
• 
Evaluate patients with serum glucose and a metabolic panel, urine ketones, glycosylated 
hemoglobin, and C-peptide.  
 
Management of Hypophysitis  
• 
For Grade 2 events, treat with corticosteroids. When symptoms improve to Grade 1 or less, 
steroid taper should be started and continued over no less than 4 weeks. Replacement of 
appropriate hormones may be required as the steroid dose is tapered.   
• 
For Grade 3-4 events, treat with an initial dose of IV corticosteroids followed by oral 
corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started 
and continued over no less than 4 weeks. Replacement of appropriate hormones may be 
required as the steroid dose is tapered. 
Management of Hyperthyroidism or Hypothyroidism 
Thyroid disorders can occur at any time during treatment. Monitor patients for changes in thyroid 
function (at the start of treatment, periodically during treatment, and as indicated based on clinical 
evaluation) and for clinical signs and symptoms of thyroid disorders.   
• 
Grade 2 hyperthyroidism events (and Grade 3-4 hypothyroidism): 
o 
In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are suggested 
as initial therapy.  
o 
In hypothyroidism, thyroid hormone replacement therapy, with levothyroxine or 
liothyronine, is indicated per standard of care.  
• 
Grade 3-4 hyperthyroidism. Treat with an initial dose of IV corticosteroid followed by oral 
corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started 
and continued over no less than 4 weeks. Replacement of appropriate hormones may be 
required as the steroid dose is tapered.  
Management of Hepatic toxicities  
• 
For Grade 2 events, monitor liver function tests more frequently until returned to 
baseline values (consider weekly).  
o 
Treat with IV or oral corticosteroids  
• 
For Grade 3-4 events, treat with intravenous corticosteroids for 24 to 48  hours.   
• 
When symptoms improve to Grade 1 or less, a steroid taper should be started and 
continued over no less than 4 weeks.   
Management of Renal Failure or Nephritis   
• 
For Grade 2 events, treat with corticosteroids.  
• 
For Grade 3-4 events, treat with systemic corticosteroids.  
• 
When symptoms improve to Grade 1 or less, steroid taper should be started and 
continued over no less than 4 weeks. 
Management of Infusion Reactions 
Signs and symptoms usually develop during or shortly after drug infusion and generally resolve 
completely within 24 hours of completion of infusion.  
Table 5 shows treatment guidelines for subjects who experience an infusion reaction associated 
with administration of pembrolizumab. 
Table 5: Pembrolizumab Infusion Reaction Treatment Guidelines 

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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  
Increase monitoring of vital 
signs as medically 
indicated until the subject 
is deemed medically stable 
in the opinion of the 
investigator.  
If symptoms resolve within 
one hour of stopping drug 
infusion, the infusion may 
be restarted at 50% of the 
original infusion rate (e.g., 
from 100 mL/hr to 50 
mL/hr). Otherwise dosing 
will be held until symptoms 
resolve and the subject 
should be premedicated 
for the next scheduled 
dose.  
Subjects who develop 
Grade 2 toxicity despite 
adequate premedication 
should be permanently 
discontinued from further 
trial treatment 
administration.  
Subject may be 
premedicated 1.5h (± 30 
minutes) prior to infusion of 
pembrolizumab (MK- 3475) 
with:  
Diphenhydramine 50 mg po 
(or equivalent dose of 
antihistamine).  
Acetaminophen 500-1000 
mg po (or equivalent dose 
of antipyretic).  
Grades 3 or 4  
Grade 3: Prolonged (i.e., not 
rapidly responsive to symptomatic 
Stop Infusion.  
Additional appropriate 
medical therapy may 
No subsequent dosing 

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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  
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.  
Appropriate resuscitation equipment should be available in the room and a physician readily 
available during the period of drug administration.  
 
7.6 
Reporting Requirements for Adverse Events 
7.6.1 
Steps to Determine If an Adverse Event Requires Expedited Reporting 
 
Step 1: Identify the type of adverse event using the NCI Common Terminology Criteria for 
Adverse Events (CTCAE VERSION 4.0).  
 
Step 2: Grade the adverse event using the NCI CTCAE VERSION 4.0 
 
Step 3: Determine whether the adverse event is related to the protocol therapy  
 
Attribution categories are as follows: 
• 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. 
Note: This includes all events that occur within 90 days of the last dose of protocol treatment 
(pembrolizumab and RT). Any event that occurs more than 90 days after the last dose of 
treatment (pembrolizumab and/or RT) and is attributed (possibly, probably, or definitely) 
to the agent(s) must also be reported accordingly. 
 
Step 4: Determine the prior experience of the adverse event.  
 
Expected events are those that have been previously identified as resulting from 
administration of the agent. An adverse event is considered unexpected, for expedited 
reporting purposes only, when either the type of event or the severity of the event is not 
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• the current known adverse events listed in the Agent Information Section of this 
protocol; 
• the drug package insert; 
• the current Investigator’s Brochure 
7.6.2 
Expedited Reporting 
7.6.2.1 
The Principal Investigator must be notified by study staff or co-investigators 
within 24 hours of learning of any serious adverse events, regardless of attribution, 
occurring during the study or within 30 days of the last administration of the study drug.  
 
Phone Number for Expedited Reporting: 
Stephen Shiao, MD, PhD 
310-423-2836 
 
Alternate Phone Number for Expedited Reporting: 
Reva Basho, MD 
832-423-8255 
 
 
Serious Adverse Event deemed to be related to the protocol and on-study deaths, 
including death of a research subject unless the death is expected (e.g. due to disease 
progression) to be reported to the DSMC within 24 hours for medical monitor ad hoc 
review between meetings to determine if immediate action is required. Reports may be 
emailed to the DSMC Admin at GroupSOCCICCTODSMCAdmin@cshs.org.   
 
7.6.2.2 
Reporting to the Institutional Review Board (IRB) 
The IRB must be notified within 10 business days of “any unanticipated problems 
involving risk to subjects or others.” 
1. Any serious event (injuries, side effects, deaths or other problems), which in the opinion 
of the Principal Investigator was unanticipated, involved risk to subjects or others, and 
was possibly related to the research procedures. 
2. Any serious accidental or unintentional change to the IRB-approved protocol that alters 
the level of risk. 
3. Any deviation from the protocol taken without prior IRB review to eliminate apparent 
immediate hazard to a research subject. 
4. Any new information (e.g., publication, safety monitoring report, updated sponsor safety 
report), interim result or other finding that indicates an unexpected change to the 
risk/benefit ratio for the research. 
5. Any breach in confidentiality that may involve risk to the subject or others. 
6. Any complaint of a subject that indicates an unanticipated risk or that cannot be resolved 
by the Principal Investigator. 
7.6.3 
Routine Reporting 
All other adverse events- such as those that are expected, or are unlikely or 
definitely not related to the study participation are to be reported annually as part 
of regular continuing reviews or data submissions. 
7.6.4 
 Reporting to the Food and Drug Administration 

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The investigator or his designee must submit SAEs on FDA Form 3500A 
(MedWatch) according to the following reporting criteria: 
• Reporting any unexpected fatal or life-threatening suspected adverse 
reactions no later than 7 calendar days after initial receipt of the 
information 
• Reporting any (1) serious, unexpected suspected adverse reactions 
(SUSAR), (2) findings from other clinical, animal, or in-vitro studies that 
suggest significant human risk, and (3) a clinically important increase in 
the rate of a serious suspected adverse reaction no later than 15 calendar 
days after determining that the information qualifies for reporting 
 
7.7 
Reporting to US Army Medical Research and Material Command Office 
(USARMC) of Research Protections (ORP) Human Research Protection Office 
(HRPO) 
The protocol will not be initiated until written notification of approval of the research 
project is issued by the USARMC ORP HRPO.  
 
(1) Substantive modifications to the research protocol and any modifications that could 
potentially increase risk to subjects must be submitted to the HRPO for approval prior 
to implementation. The USAMRMC ORP HRPO defines a substantive modification as 
a change in Principal Investigator, change or addition of an institution, elimination or 
alteration of the consent process, change to the study population that has regulatory 
implications (e.g. adding children, adding active duty population, etc), significant change 
in study design (i.e. would prompt additional scientific review) or a change that could 
potentially increase risks to subjects.  
 
(2) Any changes of the IRB used to review and approve the research will be promptly 
reported to the USAMRMC ORP HRPO.  
 
(3) All unanticipated problems involving risk to subjects or others must be promptly 
reported: 
 
by telephone (301-619-2165),  
 
by email (usarmy.detrick.medcom-usamrmc.other.hrpo@mail.mil), or  
 
by facsimile (301-619-7803) to the HRPO.  
A complete written report will follow the initial notification. In addition to the methods 
above, the complete report can be sent to the US Army Medical Research and Materiel 
Command,  
 
ATTN: MCMR-RP,  
 
810 Schreider Street,  
 
Fort Detrick, Maryland 21702-5000. 
 
(4) Suspensions, clinical holds (voluntary or involuntary), or terminations of this research 
by the Institutional Review Board (IRB), the institution, the Sponsor, or regulatory 
agencies will be promptly reported to the USAMRMC ORP HRPO.  
 
(5) A copy of the continuing review approval notification by the IRB of Record must be 
submitted to the HRPO as soon as possible after receipt. For greater than minimal risk 
research, a copy of the continuing review report approved by the IRB must also be 
provided. Please note that the HRPO also conducts random audits at the time of 
continuing review. Additional information and documentation may be requested at that 
time.  
 

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(6) The final study report, including any acknowledgement documentation and supporting 
documents, must be submitted to the HRPO when available. 
(7) The knowledge of any pending compliance inspection/visit by the FDA, DHHS Office 
of Human Research Protections (OHRP), or other government agency concerning this 
research, the issuance of Inspection Reports, FDA Form 483, warning letters or actions 
taken by any regulatory agencies including legal or medical actions and any instances of 
serious or continuing noncompliance with the regulations or requirements, must be 
promptly reported to the HRPO  
 
8.0 
CORRELATIVES/SPECIAL STUDIES 
8.1 
Assay Methodology 
H&E TIL Score: A dedicated breast pathologist will score TILs on fresh frozen paraffin embedded 
(FFPE) tumor specimens.56 Guidelines for TIL score measurement will be according to the 
Salgado criteria, and can be found in Appendix B.56 TILs score will be rounded to the nearest 
tenth percentile. TIL scores will then be averaged, and mean/SD fold-changes will be reported for 
each cohort. Residual cancer burden (RCB) values will also be obtained for post-neoadjuvant 
pathologic tumor staging to assess pCR at the time of curative-intent surgery. 
 
Multispectral IHC (mIHC): In FFPE tumor samples, we will use mIHC to quantify TIL subsets 
including regulatory T-cells, effector T-cells, macrophages, and PD-L1+ subsets. Using the 
immediately adjacent H&E as reference, the study pathologist will identify and circle areas of 
viable tumor tissue, and within this area, intratumoral versus stromal areas will be gated on the 
basis of cytokeratin-positivity. Densities of various cell types will be quantified in both intratumoral 
and stromal compartments, and spatial relationships between effectors and suppressors will be 
analyzed. Staining will include CD3 (T-cell), CD8 (effector T-cell), FOXP3 (regulatory T-cell), 
CD163 (macrophage), PD-L1, and Ki-67 (activated T-cell). 
 
Flow Cytometry: Frozen blood and tumor samples will undergo fluorescent immunophenotyping 
to assess baseline and changes in composition/activation status of lymphocyte subsets including 
CD8+ regulatory T-cells and FOXP3+ effector T-cells through the course of treatment. Expression 
of markers of activation and proliferation will also be evaluated.  
 
Circulating Cytokines: Frozen blood will also be evaluated for the prevalence of circulating 
cytokines.  
 
T-Cell Receptor Sequencing: Frozen blood and FFPE tumor samples will be submitted for DNA 
extraction and T-cell receptor deep sequencing to assess clonality/diversity of the T-cell 
population. 
 
Whole Genome/Exome Sequencing: Frozen or FFPE tumor samples will be submitted for DNA 
extraction and whole genome/exome sequencing to assess the expression level of different 
genes within the tumor.   
 
MerFISH: In conjunction with Dr. Simon Knott (Biomedical Sciences, Cedars-Sinai), frozen tumor 
samples will be submitted for copy number and spatial assessment of thousands of different RNA 
species in single cells using fluorescence in situ hybridization (FISH).44 The eventual goal of this 
technology is to build a 3-D model of the tumor microenvironment with data on expression levels 
of various RNAs in tumor, stroma, and immune infiltrate. Changes in gene expression levels 
through the course of therapy will allow for the identification of drivers of safety and response. 
 
Single-cell RNA Sequencing: In collaboration with Dr. Simon Knott, correlative endpoints have 
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and define the molecular signatures that are associated with amplified tumor immunogenicity. 
Single cell gene expression profiling will be applied to research biopsies performed prior to 
pembrolizumab (in the TNBC cohort only) and in all cohorts, sequentially following administration 
of pembrolizumab and pembrolizumab + RT to identify tumors with an immunogenic response. 
For each biopsy, 10,000 live single cells will be isolated from the mixture using Fluorescence 
Activated Cell Sorting (FACS), disregarding cell type. These cells will be profiled individually 
using the 10X genomics single cell RNA-sequencing platform. The result of this processing will be 
a set of 10,000 gene expression signatures for each biopsy, where each profile informs on the 
type and molecular state of a single tumor cell. 
 
Optional MR Breast Imaging: 
Imaging will be performed as an optional sub-study after consent at baseline, within 14 days prior 
to C1D1, prior to C2D1 (+/- 5 days), and prior to Week 7(+/- 5 days) at the time of initiation of 
standard of care treatment.  Timing to coincide with research biopsies.   
 
If the patient agrees to participate in the study, he/she will undergo the following procedures: 
• The patient will be set up in the prone position.  A breast MR coil will be placed on the patient 
body. 
• The patient will be asked to breathe freely. Standard breast MR sequences, including T2 
weighted sequences (anatomy and analysis), T1 gradient-echo sequences will be performed 
and used as a comparison.  
• Research MR sequence including free-breathing dynamic contrast-enhanced (DCE) MR, 
diffusion MR, multi-tasking 4D-MR with T1/T2 map and Chemical exchange saturation 
transfer (CEST) will be performed on the patient. 
• The patient will be given contrast for the DCE sequence. 
• The MR scan will take 1-1.5hrs. A patient arrives at the research imaging core 45min before 
the scan for consent and IV placement. 
 
Data will be collected by the imaging core and analyzed by the Shiao Lab in conjunction with his 
collaborators in the Cedars-Sinai Biomedical Imaging Research Institute (BIRI).   
 
 
Optional Fecal Samples for Microbiome Analysis:  
In conjunction with Dr. Stephen Shiao (Radiation Oncology, Cedars-Sinai), optional fecal samples 
will undergo microbiome analysis. Kits for sample collection will be provided to all participants 
who opt to participate in this optional part of the trial, upon registration to the trial. Each kit will be 
provided with a pre-labeled box to be shipped back by the patient for analysis to the laboratory of 
the CSMC PI who will be conducting this analysis: 
 
Dr. Stephen Shiao 
 
Cedars-Sinai Medical Center 
 
110 N. George Burns Rd #D4094D 
 
Los Angeles, CA 90048 
All specimens will contain the subject’s name and date of collection. The shipping and receipt of 
specimens will be tracked in a database and by emails sent to Dr. Shiao and the PI. Alternatively, 
samples may be delivered to Dr. Shiao’s lab via the study coordinator. 
8.2 
Sample Collection / Specimen Banking 
Research bloods to be used for flow cytometry analysis, measurement of circulating 
cytokines, and TCR sequencing will be collected prior to C1D1, on C2D1, and at the time 

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of initiation of standard of care treatment. Samples will be frozen and stored for 
subsequent analysis in the laboratory of Dr. Stephen Shiao. 
Research biopsies will be conducted prior to C1D1 (standard of care diagnostic biopsy 
can be used if sufficient archived tissue available for the ER+ cohort), on C2D1, and at 
the time of initiation of standard of care treatment. If subsequent standard of care 
treatment is breast-conserving surgery, excision of tumor tissue+/- surrounding normal 
tissue can be collected in lieu of biopsy. At each time point, at least 2 cores (1cm each) 
will be collected. One will be frozen for flow cytometry analysis and MerFISH and the 
other will be paraffin embedded for TIL score, mIHC, and TCR and exome sequencing. 
For the ER+ cohort, if archived tissue is used in lieu of the first research biopsy, flow 
cytometry and MerFISH will not be conducted on this sample as fresh or frozen tissue is 
required for these studies. 
 
 
If subjects receive neoadjuvant systemic therapy prior to proceeding to surgery as part of  
 
SOC treatment, fresh specimens may be collected at the time of surgery. 
9.0 
STATISTICAL CONSIDERATIONS 
9.1 
Sample Size  
 
Phase Ib: 
It is imperative that any experimental pre-operative therapeutic innovation not undermine 
the timing of curative-intent treatment in localized breast cancer, the primary endpoint for 
the Phase Ib study is safety/tolerability, as defined by the number of patients who do not 
necessitate a delay in standard of care treatment (breast surgery or neoadjuvant 
systemic therapy followed by breast surgery). A small number of patients of TNBC will be 
included in this cohort to establish safety of the regimen as described below for 
consideration for further study. Along with safety, assessment of TILs through the course 
of treatment in this cohort will establish the immunogenicity of this combination. If at least 
80% of the first 10 subjects in the TNBC cohort proceed with standard of care therapy 
without delay, the combination will be deemed safe in the cohort, and enrollment in the 
HR+ cohort (10 subjects) will occur at that time.  
 
 
Phase II:  
Once the protocol has been determined to be safe and the protocol modified accordingly, 
we will employ Simon’s two stage trial design56.  For this cohort, the null hypothesis that 
the true response rate is 10% will be tested against a one-sided alternative. In the first 
stage, 22 patients will be accrued. If there are 2 or fewer responses in these 22 patients, 
the study will be stopped. Otherwise, 18 additional patients will be accrued for a total of 
40 patients. The null hypothesis will be rejected if 8 or more responses are observed in 
40 patients. This design yields a type I error rate of 0.04 and power of 0.80 when the true 
response rate is 25%. 

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9.2 
Data Sets Analyzed  
Data Analyses/Study Endpoints 
 
9.2.1 
Efficacy Analysis 
The primary analysis will be based on an intention-to-treat approach and will include all 
subjects who receive a dose of study drug.   
Phase Ib: 
The primary efficacy endpoint will be feasibility as defined by the number of patients who 
do not necessitate a delay in standard of care treatment. Let Pd be the true probability 
that a patient experiences a delay. The trial will stop if there is statistical evidence that Pd 
exceeds 20%. We will use a Bayesian sequential design by checking whether Pd 
exceeds this threshold value after 3, 6, 9, … patients are evaluable for feasibility. The 
decision rule is to stop the trial if the posterior probability that Pd exceeds 0.20 is 0.95 or 
more; P(Pd > 0.2 | data) > 0.95. A non-informative prior distribution for Pd will be used. 
Table 9.2.1 gives the stopping rules for the design at each look and column 2 gives the 
maximum number of patients with delay in order for the trial to proceed.  Stopping rules 
will also apply for any patients with clinical progression of disease during study treatment, 
although this is expected to be uncommon. For example, if 4 or more delays in treatment 
are observed after enrolling 6 patients, the trial stops. The third column gives the 
probability of stopping the trial when in fact, the true Pd = 0.20. This is the equivalent of 
the Bayesian type I error probability. The target type I error probability was set at 0.05.  
 
 
Table 9.2.1 Stopping rules based on four interim looks 
Number of  
Patients 
Number to 
Continue 
Probability to  
Stop 
Cumulative Probability 
to Stop 
3 
2 
0.008 
0.008 
6 
3 
0.013 
0.021 
9 
4 
0.010 
0.031 
10 
4 
0.011 
0.042 
Number to continue is the maximum number of delays in standard of care treatment for 
not stopping the trial. 
 
Table 9.2.2 gives the design operating characteristics under selected values of the true 
probability Pd for the HR+ cohort. It gives the probability of stopping the trial under the 
alternative hypothesis, the expected sample size, and the average sample size given that 
the trial stopped. For example, if the true value of Pd is 0.5, then there is an 64% chance 
that the trial is stopped early and the average sample size is about 8. On the other hand, 
there is a small chance of stopping the trial if Pd is small; 0.3% chance of stopping the trial 
when in fact Pd = 0.1. Tables 9.2.3 and 9.2.4 list the same operating characteristics for the 
TNBC cohort. 
 
Table 9.2.2 Design operating characteristics under different scenarios for the true 
probability of Pd 
True Value 
of Pd 
Probability to  
Stop 
Expected 
N 
Expected 
N given that we Stopped 
0.10 
0.003 
9.99 
6.32 
0.30 
0.17 
9.55 
7.38 
0.50 
0.64 
8.01 
6.90 
0.60 
0.84 
6.92 
6.35 

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Table 9.2.3 Stopping rules based on seven interim looks 
Number of 
Patients 
Number to 
Continue 
Probability to 
Stop 
Cumulative Probability 
to Stop 
3 
2 
0.008 
0.008 
6 
3 
0.013 
0.021 
9 
4 
0.010 
0.031 
12 
5 
0.007 
0.038 
15 
6 
0.005 
0.044 
18 
7 
0.004 
0.047 
20 
8 
0.009 
0.048 
 
Table 9.2.4 Design operating characteristics under different scenarios for the true 
probability of Pd for TNBC cohort 
True Value 
of Pd 
Probability to  
Stop 
Expected 
N 
Expected 
N given that we Stopped 
0.10 
0.003 
19.96 
6.48 
0.30 
0.233 
17.76 
10.38 
0.50 
0.834 
11.01 
9.28 
0.60 
0.967 
8.11 
7.70 
 
 
 
 
The co-primary efficacy endpoint with be change in TILs. TILs will be assessed in paraffin 
embedded samples after the study is completed and will have no bearing on impact on 
continued enrollment for the trial. For each cohort, the mean percent change in TIL score 
before treatment and after pembrolizumab alone μ1 will be compared to the mean change 
in TIL score before treatment and after pembrolizumab plus radiation μ2 using the paired t-
test or Wilcoxon sign test depending on the distribution of the data. A significance of 0.05 
will be used to declare statistical significance. Let SD(diff) be the standard deviation of the 
difference in percent change in TIL score before treatment and after pembrolizumab and 
before and after treatment with pembrolizumab and radiation. Data from 8 evaluable 
patients in the HR+ cohort achieve 80% power to detect an effect size of 1.16 using a two-
sided paired t-test at the 0.05 level of significance. The effect size is defined as the 
difference between the two means divided by SD(diff), (μ1 – μ2) / SD(diff). This is equivalent 
to an absolute difference of 20% between the mean change in TIL scores assuming an 
SD(diff) of 17%. For the TNBC cohort, assuming 16 patients are evaluable, the effect size 
that is detectable with the same power and type I error is 0.75. 
 
Comparisons of clinical characteristics among cohorts will be conducted using descriptive 
summary statistics using median and range. Changes in TIL score will be compared 
between cohorts using Fishers exact test. 
 
Phase II: 
 
The efficacy endpoint with be pathologic complete response rate (pCR) as determined at 
the time of surgery assuming that the trial continues through both stages.  For this cohort, 
the pCR rate at the time of surgery which is after RT, pembrolizumab and, in most cases, 
chemotherapy will be determined.  A significance of 0.05 will be used to declare statistical 
significance. Data from 50 evaluable patients achieve 80% power to detect an effect size 

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of 1.20 using a two-sided paired t-test at the 0.05 level of significance compared to the 
historical pCR rate for triple negative breast cancer90. The effect size is defined as the 
difference between the mean pCR rate for this trial compared to historical data for triple-
negative breast cancer. This is equivalent to an absolute difference of 20% between the 
mean pCR rate between the two groups assuming a standard deviation (SD) of 15%. 
 
Comparisons of clinical characteristics for this cohort will be conducted using descriptive 
summary statistics using median and range. Changes in TIL score, immune populations, 
gene expression, microbiome populations and cytokine expression will be compared 
between different time points and responder/non-responder groups if sufficient numbers 
for analysis are available using the paired t-test or Wilcoxon sign test depending on the 
distribution of the data. 
 
9.2.2 
Safety Analysis 
All subjects entered into the study on C1D1 will be included in the safety analysis. The 
frequencies of AEs that occur through week 15 after the second dose of pembrolizumab 
by type, body system, severity and relationship to study drug will be summarized. SAEs 
(if any) will be described in detail. Potential association with study treatment will be 
assessed. 
9.2.3 
 
Invasive Disease-Free Survival (IDFS) 
Secondary objective of IDFS will be evaluated by the Kaplan-Meier method. IDFS is defined 
as the time from completion of surgery to the first occurrence of the following events: 
invasive ipsilateral, local, regional, or distant recurrence, or death due to breast cancer60. 
10.0 
STUDY MANAGEMENT 
10.1 Conflict of Interest 
Any reportable conflict of interest by study investigators will be disclosed to the local IRB 
and will be outlined in the Informed Consent Form. Conflict of interest will be defined by 
a situation in which a study investigator is involved in an interest, financial or otherwise, 
which could affect the decision-making of that individual as it relates to this study. 
10.2 Institutional Review Board (IRB) Approval and Consent  
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations. The IRB should approve the consent form and 
protocol. 
 
In obtaining and documenting informed consent, the investigator should comply with the 
applicable regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) 
and to ethical principles that have their origin in the Declaration of Helsinki. 
 
 
Before recruitment and enrollment onto this study, the patient will be given a full 
explanation of the study and will be given the opportunity to review the consent form. 
Each consent form must include all the relevant elements currently required by the FDA 
Regulations and local or state regulations. Once this essential information has been 
provided to the patient and the investigator is assured that the patient understands the 
implications of participating in the study, the patient will be asked to give consent to 
participate in the study by signing an IRB-approved consent form. 
 
Prior to a patient’s participation in the trial, the written informed consent form should be 
signed and personally dated by the patient and by the person who conducted the 
informed consent discussion. 

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10.3 Registration Procedures 
 
All subjects that sign informed consent will be assigned a subject number sequentially by 
their date of consent.  Those subjects that do not pass the screening phase will be listed 
as screen failures on the master list of consented subjects.  Eligible subjects, as 
determined by screening procedures and verified by a treating investigator, will be 
registered on study at Cedars Sinai Medical Center (CSMC) by the Study Coordinator.   
 
Issues that would cause treatment delays after registration should be discussed with the 
Principal Investigator (PI). If a patient does not receive protocol therapy following 
registration, the patient’s registration on the study may be canceled.  The Study 
Coordinator should be notified of cancellations as soon as possible. 
 
Assignment of Subject ID: The study team will track all subjects who sign consent on a 
subject screening/enrollment log using a unique screening ID (S001, S002, etc.). 
Subjects found to be ineligible will be recorded as screen failures. Subjects found to be 
eligible will be registered using only the three-digit numeric ID assigned at screening that 
follows the standard SOCCI format (001, 002, etc.). 
 
A) 
Eligibility Verification 
 
Prior to registration, all subjects must undergo eligibility verification by the SOCCI Cancer 
Clinical Trial Office (CCTO). The following documents will be completed and provided for 
review: 
 
• 
Registration form (or equivalent) 
• 
Copy of applicable source documents 
• 
Eligibility checklist (signed by investigator) 
• 
Signed patient consent form and Subject’s Bill of Rights 
• 
HIPAA authorization form 
 
B) 
Registration 
 
After eligibility is verified, registration is completed as follows: 
 
• 
Assign a patient study number 
• 
Entry of the patient in OnCore by the Study Coordinator 
• 
Notify the investigational pharmacy and treating physicians that a subject has 
gone on study and anticipated treatment start date 
 
Oversight by the principal investigator is required throughout the entire registration 
process. 
 
10.4 Data Management and Quality Control and Reporting 
The data will be entered into a HIPAA-compliant database. The Study Staff will be 
responsible for data processing, in accordance with procedural documentation. Database 
lock will occur once quality assurance procedures have been completed. 
 
All procedures for the handling and analysis of data will be conducted using good 
computing practices meeting FDA guidelines for the handling and analysis of data for 
clinical trials. 

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10.5 Data and Safety Monitoring 
 
10.5.1 
Data Monitoring and Quality Assurance 
 
High Risk Monitoring 
 
Adherence to the protocol, Good Clinical Practices (GCP) and institutional policy will be 
monitored by the PI during the course of the study through routine Disease Research 
Group (DRG) meetings (or equivalent). In addition, the SOCCI Cancer Clinical Trials 
Office (CCTO) Quality Management Core (QMC) will conduct internal monitoring visits 
and audits for data quality and protocol adherence. QMC reports will then be forwarded 
to the SOCCI Data and Safety Monitoring (DSMC).   
QMC will also conduct the following:  
1. Central eligibility verification for all subjects enrolled as described in protocol 
Section 4.   
2. Central review of all eligibility waivers by a SOCCI Medical reviewer to assess 
appropriateness and risk to ensure quality data and ensure subject safety 
protections for investigator-initiated research 
 
 
10.5.2 
Safety Monitoring 
High Risk Monitoring 
 
Oversight of the progress and safety of the study will be provided by the PI. The PI will 
maintain continuous safety monitoring for the duration of the study by reviewing 
subject/study data. Adverse events and unanticipated problems are not expected, but if 
they occur they will be documented and reported according to CSMC IRB policies and 
procedures. If the PI becomes aware of any new safety information that may place 
subjects at increased risk than what was previously known the IRB will be promptly 
notified and if warranted, enrollment may be held until the PI determines whether a 
modification to the study is necessary and/or the informed consent documents are 
updated accordingly. 
 
In addition, this protocol will utilize the SOCCI Data and Safety Monitoring Committee will 
provide another layer of data and safety oversight. Committee membership includes 
experts in the field of oncology, nursing, pharmacy, and biostatistics in reviewing the over 
data, safety, quality, and study integrity of SOCCI interventional IITs. DSMC membership 
and responsibilities are governed by the committee charter. The DSMC findings and 
recommendations will be reported in writing to the Principal Investigator. A summary 
letter will be forwarded by the Principal Investigator or his/her designee to the Cedars-
Sinai Medical Center IRB.  . 
 
DSMC Medical Monitor: Ronald L. Paquette, MD 
The DSMC Medical Monitor appointed to this study is independent of the research team 
listed in this protocol. The DSMC Medical Monitor also assumes the specific duties and 
responsibilities of the Department of Defense (DoD) Research monitor as listed below.  
 
DSMC Monitors responsibilities include: 
• Attend the DSMC meeting at which the submitted protocol is to be reviewed in order to 
present a brief synopsis of the reported study outcomes. 
• Assessment of DSM risk category and the proposed DSMP. Provide recommendation 
for PRMC’s final determination. The Data and Safety Monitoring (DSM) Risk Category 
Worksheet may be used. The DSMP Reviewer Worksheet may be used. 
• Submit a written review summary to the DSMC Coordinator prior to the meeting. The 
DSMC medical/biostatistician monitor reviewer worksheet may be used. In the rare event 

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the medical monitor or biostatistician are unable to attend and a substitute appointed 
DSMC Chair or Vice Chair, the monitor’s written review summary may be read during the 
room. Proxy voting is not allowed. 
• Review of data and safety monitoring reports for each study is complete and accurate.  
 
DoD Research Monitor Duties, Authorities, and Responsibilities:  
• 
Observe recruitment and enrollment procedures and the consent process for 
individuals, groups or units 
• 
Oversee study interventions and interactions 
• 
Review monitoring plans and UPIRSO reports 
• 
Oversee data matching , data collection, and analysis 
• 
At a minimum, the research monitor: may discuss the research protocol with the 
investigators, interview human subjects, and consult with others outside of the study 
about the research; shall have authority to stop a research protocol in progress, 
remove individual human subjects from a research protocol, and take whatever steps 
are necessary to protect the safety and well-being of human subjects until the IRB 
can assess the monitor’s report; shall have the responsibility to promptly report their 
observations and findings to the IRB or  other designated official and the HRPO.  
 
10.6 
Record Retention 
 
Study documentation includes all Case Report Forms, data correction forms or queries, 
source documents, monitoring/auditing logs/letters, and regulatory documents (e.g., 
protocol and amendments, IRB correspondence and approval, signed patient consent 
forms). 
 
 
Source documents include all recordings of observations or notations of clinical activities 
and all reports and records necessary for the evaluation and reconstruction of the clinical 
research study. Government agency regulations and directives require that the study 
investigator must retain all study documentation pertaining to the conduct of a clinical 
trial. Study documents should be kept on file per local guidelines. 
 
10.7 
Adherence to Protocol 
 
Except for an emergency situation in which proper care for the protection, safety, and 
well-being of the study patient requires alternative treatment, or a protocol exception 
request approved by the SOCCI Medical Director and CSMC IRB, the study shall be 
conducted exactly as described in the approved protocol. 
10.7.1 
Emergency Modifications 
Investigators may implement a deviation from, or a change of, the protocol to eliminate 
an immediate hazard(s) to trial subjects without prior IRB approval. For any such 
emergency modification implemented, the IRB must be notified as soon as possible, but 
no more than 10 days from the investigator’s awareness of the event. 
10.7.2 
Protocol Exceptions and Eligibility Waivers  
An exception is an anticipated or planned deviation from the IRB-approved research 
protocol, as described in the IRB Policy, Reporting Possible Unanticipated Problems 
Involving Risks to Subject or Others (UPIRSO) Policy: Institutional Review 
Board/Research Compliance and Quality Improvement.  

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A protocol exception most often involves a single subject and is not a permanent revision 
to the research protocol. Protocol exceptions that extend beyond a single subject should 
result in a protocol amendment to avoid serial violations.  
All exception requests must be reviewed by the SOCCI CCTO Medical Director and the 
Institutional Review Board prior to implementation. The PI or her/his designee is 
responsible for submitting a protocol exception and its supporting documents to the 
SOCCI Medical Director for review. Planned exceptions to the protocol that are more 
than logistical and/or have the potential to affect the subject’s safety and/or study integrity 
may not be implemented without prior approval from the SOCCI Medical Director and 
IRB. 
 
Special considerations for Eligibility Waivers (EW) 
In general, subjects who do not meet the eligibility requirements should not be enrolled. 
In the rare event that it is appropriate for subject inclusion, the rationale/justification and 
subject case history should be forwarded to the SOCCI CCTO Medical Director for 
assessment prior to submission to the IRB for approval.  
 
The CCTO Medical Director will review the case and contact the PI if additional 
information is needed or further discussion is warranted. The CCTO Medical Director will 
provide a written assessment/recommended course of action. The CCTO Medical 
Director’s assessment must be uploaded into CS-IRB with the waiver request for IRB 
review and consideration. The CCTO Medical Director may recommend future protocol 
changes.  
 
Eligibility Waiver Submission Process  
The PI and/or treating physician should provide written request for a waiver which 
includes case history and justification for prospective deviation from the study design to 
the SOCCI CCTO Medical Director.  
 
10.7.3 
Other Protocol Deviations 
Logistical deviations from the protocol (e.g., minor changes to the study schedule for an 
individual subject) do not require prior IRB approval unless the deviation has the potential 
to affect the subject’s safety. Such planned deviations that do meet this definition and do 
not affect the subject’s safety should be noted in the subject’s research record or 
deviation log as described in the SOCCI CCTO’s Standard Operating Procedure 12: 
Deviation and Noncompliance Reporting. 
 
Unintentional deviations from the protocol that might affect subject safety or study 
integrity should be reported to the IRB within 10 days from when the investigator 
becomes aware that such a deviation has occurred, as outlined in the SOCCI CCTO’s 
Standard Operating Procedure 12: Deviation and Noncompliance Reporting. In this case, 
a Protocol Deviation report must be submitted in CS-IRB, per IRB policy, Reporting 
Possible Unanticipated Problems Involving Risks to Subject or Others (UPIRSO) Policy: 
Institutional Review Board/Research Compliance and Quality Improvement. All 
submissions should include a description of the plan to avoid similar deviations or 
exceptions in the future.   
 
10.7.4 
Amendments to the Protocol  
Should amendments to the protocol be required, the amendments will be originated and 
documented by the PI. It should also be noted that when an amendment to the protocol 
substantially alters the study design or the potential risk to the patient, a revised consent 
form might be required.  

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The written amendment, and if required the amended consent form, must be sent to the 
IRB for approval prior to implementation. Repeat exceptions or deviations to the protocol 
may suggest a protocol amendment is needed. 
10.8 
Obligations of Investigators 
 
The PI is responsible for the conduct of the clinical trial at the site in accordance with Title 
21 of the Code of Federal Regulations and/or the Declaration of Helsinki. The PI is 
responsible for personally overseeing the treatment of all study patients. The PI must 
assure that all study site personnel, including co-investigators and other study staff 
members, adhere to the study protocol and all FDA/GCP/NCI regulations and guidelines 
regarding clinical trials both during and after study completion. 
 
 
The PI will be responsible for assuring that all the required data will be collected and 
entered onto the Case Report Forms and/or into a HIPAA-compliant study database. 
Periodically, monitoring visits will be conducted and the PI will provide access to his/her 
original records to permit verification of proper entry of data. At the completion of the 
study, all case report forms will be reviewed by the PI and will require her final signature 
to verify the accuracy of the data. 
 
 
 
 

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12.0 
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Anti-PD-1 Monoclonal Antibody) in Patients with Advanced Solid Tumors. Clin Cancer Res 
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Cortazar P, Zhang L, Untch M, et al: Pathological complete response and long-
term clinical benefit in breast cancer: the CTNeoBC pooled analysis. Lancet 384:164-72, 2014 
 
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chemotherapy (CREATE-X, JBCRG-04). Cancer Research 76:Abstract S1-07, 2016 
 
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Survival in the United States. JAMA Oncol 2:330-9, 2016 
 
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Adjuvant Chemotherapy Among Patients With Breast Cancer. JAMA Oncol 2:322-9, 2016 
 
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Salgado R, Denkert C, Demaria S, et al: The evaluation of tumor-infiltrating 
lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 
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APPENDIX A: PROTOCOL SUMMARY OF CHANGES 
 
Amendment 1(Changes made in Protocol Version 2 dated 13MAR2018) 
 
1. PI change from Alice Ho, MD to Stephen Shiao, MD, PhD 
2. Revisions to study schema for clarification  
3. Throughout document: research MRI has been removed   
4. Formatting throughout document  
5. Addition of Table 3: Dose Modification and Toxicity Management Guidelines for Immune-related 
AEs Associated with Pembrolizumab 
6. Section 6.1.8: Research-Related Laboratory Evaluation 
Clarification that “Labs must be drawn, but do not require results to determine eligibility or 
treatment continuation.”  
7. Section 6.4: Time and Events Table 
o 
Clarification of Pathology review 
o 
Clarification of safety bloodwork and thyroid function panel (to be obtained at screening; 
additional specimens not required C1D1)  
o 
Clarification of follow up schedule 
8. Section 7.6: Reporting Requirements for Adverse Events  
o 
Change AE collection from within 30 days of last dose of pembrolizumab to 90 days 
o 
Updated expedited reporting contact and phone number 
• 
10.  Section 11.0: Removal of reference 55 
 
 
 
Amendment 2 (Changes made in Protocol Version 3 dated 09APR2018) 
 
1. List of abbreviations updated to include YR and SUSAR 
2. Radiation treatment timing and dose clarified throughout document 
3. Section 2.3: added new exploratory objective 2.3.5 to assess changes in breast cosmesis 
4. Section 2.4: Safety evaluations clarified to include those related to both pembrolizumab and 
radiation therapy 
5. Section 3.0: updated to clarify cohort number as 2 cohorts only 
6. Section 4.1.17: WOCBP definition added to eligibility criteria regarding negative serum pregnancy 
test 
7. Section 5.1.2: revisions to radiation treatment parameters that clarify total treatment dose 
8. Section 5.1.2.3 – 5.1.2.9: radiation dosing constraints clarified 
9. Section 5.4: Table 2 removed and replaced with table 3 (Table 3 relabeled as table 2).  
10. Section 6.1, 6.2 & 6.3: added directions and timing of cosmesis study procedures 
11. Section 6.1: clarification of screening procedure timing within 14 days prior to C1D1 
12. Section 6.2: clarification of adverse event collection for both pembrolizumab and radiation therapy 
13. Section 6.4: Time and Events Table 
o 
Cosmesis study added to table 
o 
Clarification of adverse event collection  
o 
Clarification of pregnancy evaluations 
o 
Clarification of follow up scheduled, Q6Mo’s post treatment 
14. Section 7.3: clarification of adverse event collection timepoints 
15. Section 7.6.4: addition of IND reporting guidelines 
16. Appendix C: Harvard Four Tier Cosmesis Scale added to accompany breast cosmesis study 
17. Formatting throughout document 
 
 

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Amendment 3 (Changes made in Protocol Version 4 dated 16AUG2018) 
 
1. Formatting throughout document 
2. Section 4.1.6: Clarified eligibility criteria to match study schema and current practices. TNBC 
patients (defined as ER<10%, PR<10%, HER2-neu 0-1+ by IHC or FISH-negative; or as per MD 
discretion). 
Amendment 4 (changes made in Protocol Version 5 dated 10SEP2018) 
1. Formatting changes throughout document 
2. Addition of 10 TNBC subjects throughout document: 
a. Study Schema and Study Summary 
b. Section 3.0 Study Design 
c. Section 9.1 Sample Size 
3. Radiation Therapy window updated to 5 business days throughout document 
4. Section 5.8 Subject Replacement: Clarified subjects who fail to complete the study intervention or 
are not evaluable for the primary endpoint will be replaced. 
5. Section 6.0 Study Procedures: Screening window updated to 28 calendar days 
6. Section 9.0 Statistics: Update to statistics to reflect enlarged sample size.  
 
Amendment 5 Changes made in Protocol Version 6 dated 04DEC2018 
 
1. Formatting changes throughout document 
2. Addition of optional research breast MRI to the following sections: 
a. Study schema and study summary updated 
b. 1.6 Correlative Studies 
c. Secondary Objective 2.3.6 added MRI to assess disease status 
d. Section 4.2.8: exclusion criteria added: For subjects who agree to the research breast 
MRI sub-study: Four or more previous gadolinium contrast scans in the past 12 months 
due to the risk of brain deposits following repeated use of gadolinium-based contrast 
agents. 
e. Study procedures 6.0 updated with correct MRI timepoints 
f. 
Section 6.4 Schedule of events updated to include MRI 
g. Optional MR Breast Imaging section added to 8.0 Correlatives/Special Studies 
 
 
Amendment 6 Changes made in Protocol Version 7 dated 30MAY2019 
 
 
1. Formatting changes throughout document 
2. Study objective 2.2.3 clarified safety/tolerability to feasibility throughout document 
3. Addition of new study objective: To assess the pathological complete response (pCR) rates. 
a. Study summary updated 
b. Secondary Objective 2.3.7 added  
c. New study endpoint added to section 2.4 
4. Clarification to section 3.0 to state that:  
• 
If at least 80% of the first 10 subjects in the TNBC cohort proceed with standard of care 
therapy without delay, the combination will be deemed safe in the cohort, and enrollment 
in the HR+ cohort, as well as continued enrollment in the TNBC cohort (from 10 to 20 
subjects), will occur at that time. 
5. Eligibility updates for inclusion criteria, section 4.1: 
a. Addition of coagulation parameters 
b. Standard of care imaging criterion clarified 
6. Eligibility updates for exclusion criteria, Section 4.2: 
a. Addition of evidence of metastatic disease 
b. Addition of exclusion of immunodeficiencies  

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c. Addition of exclusion of additional known malignancies progressing or receiving active 
treatment in past 3 years.  
d. Revised exclusion of concurrent diseases 
e. Revised exclusion of prohibited treatments and/or therapies 
f. 
Exclusion of WOCBP with positive pregnancy test within 72 hours prior to study start.  
7. Toxicity information updates from Merck added to section 5.4 Dosing 
a. Inclusion of Table 3  Pembrolizumab Infusion Reaction Dose modification and Treatment 
Guidelines; provided by Merck 
8. Changed Section 10.5 to reflect the addition of the Data Safety and Monitoring Committee and 
revised risk categories and safety monitoring.  
 
 
Amendment 7 Changes made in Protocol Version 8 dated 15AUG2019 
 
1. Revised study title 
2. Clarification of Principal Investigators 
a. Stephen Shiao, MD, PhD as overall study PI 
b. Reva Basho, MD as PI for ER+ cohort 
3. Revisions to Study Schema for clarification of cohorts 
4. Study Summary 
a.  Number of subjects changed from 30 to 60 
5. Background and Rationale 
a. Section 1.3.2 Clinical Data updated with recent observations to proceed with the phase II 
portion of this trial 
6. Study Objectives 
a. Section 2.1 Primary Objectives revised  
7. Study Design Section 3.0 
a. Updated description of study design to Phase Ib/II study 
b. Expansion of cohorts to 50 TNBC and 10 HR+ women 
c. Clarified safety assessment 
8. Exclusion Criteria 4.2.8 minor correction to incomplete sentence 
9. Exclusion Criteria 
a. Removed pregnancy testing within 72 hours prior to study start and moved as part of 
Study Procedures 
10. Time and Events Table 
a. Added footnote regarding pregnancy testing for clarification on timepoints 
11. Added Section 7.7 for Reporting to US Army Medical Research and Material Command Office 
(USARMC) of Research Protections (ORP) Human Research Protection Office (HRPO)  
12. Statistical Considerations 
a. Updated Section 9.1 Sample Size with description of Phase 1b and Phase II 
b. Minor revision to Section 9.2.1 Efficacy Analysis for clarification and addition of Phase II 
13. Registration Procedures Section 10.3 revised for new CCTO name 
14. Safety Monitoring Section 10.5.2 
a. Added details and reference to the DSMC Charter and appointed DSMC Medical Monitor 
for this study 
 
Amendment 8 Changes made in Protocol Version 9 dated 06NOV2019 
1. Formatting changes throughout document 
2. Inserted Protocol Signature Page 
3. Study Schema: added procurement of fresh surgical tissue collection post neoadjuvant systemic 
therapy 
4. Inclusion Criteria Clarification 
a. 4.1.7: minor revision to include acceptable assessment by clinical exam 
b. 4.1.12: minor revision to clarify that eligibility includes candidacy for breast-conserving 
therapy versus planned 

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5. Section 6.4: Time & Events Table 
a. Revised footnote #10 to add option to procure fresh tissue specimen if surgery is 
after completion of SOC neoadjuvant systemic therapy 
6. Section 6.5: minor corrections to subheading typos 
7. Section 7.6.2: Expedited Reporting 
a. Added DSMC required language regarding expedited reporting for interventional cancer 
IITs 
8. Section 8.1: Assay Methodology 
a. Correction of Appendix label in reference to the Salgado criteria 
b. Added description RCB value as a supplemental methodology for pCR assessment 
9. Section 9.1 Sample Size: revised description to align with Section 3.0. 
10. Addition of language to allow for fresh specimen collection at the time of SOC surgery if subject 
receives neoadjuvant systemic therapy after study treatment. The following sections incorporate 
this change: 
a. Section 6.2.2: Prior to standard of care treatment or at the time of surgery if that is 
the planned standard of care treatment 
b. Section 6.4: Time and Events Table; Footnote #10 
c. Section 8.2: Sample Collection / Specimen Banking  
11. Sections 10.5.2 and 10.7.3 updated with standard institutional language and required language 
by the Department of Defense. Removed references to the DSMC Charter as this is an internal 
document not to be released.  
 
Amendment 9 Changes made in Protocol Version 10 dated 08JAN2020 
1. Section 6.3: Follow-up Procedures 
a. Added 6.3.5 description of endocrine toxicity assessment, including collection of full 
thyroid function panel and cortisol results at one year +/- 2 months post-treatment follow-
up. 
b. Section 6.3.2: AE Assessment revised to clarify that AE collection may be completed via 
phone call for subjects who are receiving standard treatment at their local treating 
physician’s office. 
2. Section 6.4: Time and Events Table 
a. Minor revision for additional timepoint for full thyroid function panel at one year post-
treatment follow-up 
b. Added cortisol results collection at one year post-treatment follow-up as part of standard 
of care endocrine toxicity assessment 
c. Added Footnote #16 for description of cortisol lab results along with full thyroid function 
panel at one year post-treatment follow-up (+/- 2 months). 
3. Section 7.0: Adverse Events 
a. Added note to clarify that AE collection may be done via phone call, usually around 
Weeks 7 – 25, for subjects receiving standard chemotherapy at their treating physician’s 
office. 
 
Amendment 10 Changes made in Protocol Version 10.1 dated 25MAR2020 
1. Section 4.1 Inclusion Criteria 
a. Inclusion criteria 4.1.17: Added note to clarify that in the setting of egg harvesting, 
patients are not required to take a pregnancy test within 72 hours of C1D1 due to the 
high likelihood of false positive results.  
2. Section 6.1 Study Procedures 
a. Study Procedures 6.1.10: Added note to clarify that in the setting of egg harvesting, 
patients are exempt from taking a pregnancy test within 72 hours of C1D1 due to the high 
likelihood of false positive results. 

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3. Section 6.4 Time & Events Table 
a. Footnote #8 clarifies pregnancy testing requirements in the setting of egg harvesting. 
Amendment 11 Changes made in Protocol Version 11 dated 22JUL2020 
 
1. Study Summary 
a. For addition of survival status assessment for up to 5 years, study duration changed from 
“Approx. 1 year” to “Approx. 5 years” 
 
2. Section 4.1 Inclusion Criteria 
a. Inclusion criterion 4.1.17: Added note to incorporate language from Protocol Clarification 
Memo dated 30JUN2020 regarding pregnancy testing exemption for egg harvesting 
patients where due to false positive results lasting for at least two weeks (and therefore 
are exempt from pregnancy testing within 14 days of C1D1), these subjects may enroll 
only per investigator discretion. 
 
3. Section 6.1 Study Procedures 
a. Study Procedures 6.1.10: Added note to incorporate language from Protocol Clarification 
Memo dated 30JUN2020 regarding pregnancy testing exemption for egg harvesting 
patients where due to false positive results lasting for at least two weeks (and therefore 
are exempt from pregnancy testing within 14 days of C1D1), these subjects may enroll 
only per investigator discretion. 
b. Section 6.3.6: Added survival status assessment and any evidence of recurrence to be 
conducted every 6 months for up to 5 years 
c. Research blood tubes corrected to 3 CPT, 1 red-top tube, and 1 green-top tube of blood. 
 
4. Section 6.4 Time and Events Table 
a. Added timepoint for survival status assessment and documentation of recurrence for 5 
years 
b. Research blood tubes corrected to 3 CPT, 1 red-top tube, and 1 green-top tube of blood. 
5. Section 8.0 Correlatives/Special Studies 
a. Added description of whole genome/exome sequencing from frozen or FFPE tumor 
samples. 
 
 
 

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APPENDIX B: SALGADO CRITERIA FOR TIL SCORE ASSESSMENT56 
  
 
Recommendations for assessing tumor-infiltrating lymphocytes (TILs) in breast cancer 
1. TILs should be reported for the stromal compartment (=% stromal TILs). The denominator 
used to determine the % stromal TILs is the area of stromal tissue (i.e. area occupied by 
mononuclear inflammatory cells over total intratumoral stromal area), not the number of 
stromal cells (i.e. fraction of total stromal nuclei that represent mononuclear inflammatory cell 
nuclei). 
2. TILs should be evaluated within the borders of the invasive tumor. 
3. Exclude TILs outside of the tumor border and around DCIS and normal lobules. 
4. Exclude TILs in tumor zones with crush artifacts, necrosis, regressive hyalinization as well as 
in the previous core biopsy site. 
5. All mononuclear cells (including lymphocytes and plasma cells) should be scored, but 
polymorphonuclear leukocytes are excluded. 
6. One section (4–5 µm, magnification ×200–400) per patient is currently considered to be 
sufficient. 
7. Full sections are preferred over biopsies whenever possible. Cores can be used in the 
pretherapeutic neoadjuvant setting; currently no validated methodology has been developed to 
score TILs after neoadjuvant treatment. 
8. A full assessment of average TILs in the tumor area by the pathologist should be used. Do not 
focus on hotspots. 
9. The working group's consensus is that TILs may provide more biological relevant information 
when scored as a continuous variable, since this will allow more accurate statistical analyses, 
which can later be categorized around different thresholds. However, in daily practice, most 
pathologists will rarely report for example 13.5% and will round up to the nearest 5%–10%, in 
this example thus 15%. Pathologist should report their scores in as much detail as the 
pathologist feels comfortable with. 
10. TILs should be assessed as a continuous parameter. The percentage of stromal TILs is a 
semi quantitative parameter for this assessment, for example, 80% stromal TILs means that 
80% of the stromal area shows a dense mononuclear infiltrate. For assessment of percentage 
values, the dissociated growth pattern of lymphocytes needs to be taken into account. 

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Lymphocytes typically do not form solid cellular aggregates; therefore, the designation ‘100% 
stromal TILs would still allow some empty tissue space between the individual lymphocytes. 
11. No formal recommendation for a clinically relevant TIL threshold(s) can be given at this stage. 
The consensus was that a valid methodology is currently more important than issues of 
thresholds for clinical use, which will be determined once a solid methodology is in place. 
lymphocyte-predominant breast cancer can be used as a descriptive term for tumors that 
contain more lymphocytes than tumor cells. However, the thresholds vary between 50% and 
60% stromal lymphocytes. 
 
 
 
 
 
 

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APPENDIX C: HARVARD FOUR-TIER COSMESIS SCALE 
 
Score 
 
Excellent 
Treated breast nearly identical to untreated breast 
Good 
Treated breast slightly different from untreated breast 
Fair 
Treated breast clearly different from untreated breast but not 
seriously distorted 
Poor 
Treated breast seriously distorted