A Pilot Study Combining Pembrolizumab With Locally Delivered Radiation Therapy for the Treatment of Metastatic Esophageal Cancers
Phase: PHASE1
Conditions: Esophageal Cancer, Cancer of the Esophagus
Sponsor: Washington University School of Medicine

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A pilot study combining pembrolizumab with locally delivered radiation therapy for the 
treatment of metastatic esophageal cancers 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Campus Box 8056 
St. Louis, MO  63110 
 
Protocol #: 201603087 
Version Date: 09/09/2022 
 
Principal Investigator: 
. Clifford Robinson, M.D. 
 Phone: (314) 362-8567 
 
 
 
 
 E-mail: crobinson@wustl.edu 
 
 
Sub-Investigators 
 
 
 
Modality 
Olivia Aranha, M.D., Ph.D.  
 
Medical Oncology 
Nusayba Bagegni, M.D. 
 
 
Medical Oncology 
Salman Chaudhry, M.D. 
 
 
Medical Oncology 
Matthew Ciorba, M.D. 
 
 
Medical Oncology 
Tanner Johanns, M.D. , Ph.D.  
 
Medical Oncology 
Eric Knoche, M.D. 
 
 
 
Medical Oncology 
Kian-Huat Lim, M.D., Ph.D.  
 
Medical Oncology 
Arjun Natarajan, M.D. 
 
 
Medical Oncology 
Peter Oppelt, M.D. 
 
 
 
Medical Oncology 
Katrina Pedersen, M.D. 
 
 
Medical Oncology 
Joel Picus, M.D. 
 
 
 
Medical Oncology 
Caron Rigden, M.D.  
 
 
Medical Oncology 
Clifford Robinson, M.D. 
 
 
Radiation Oncology 
Rama Suresh, M.D. 
 
 
 
Medical Oncology 
Benjamin Tan, M.D.  
 
 
Medical Oncology 
Nikolaos Trikalinos, M.D. 
 
 
Medical Oncology 
Ningying Wu, Ph.D.  
 
 
Biostatistics 
 
Study Drug(s): 
 
Pembrolizumab (MK-3475, Keytruda) 
 
IND #: 
129724 
ClinicalTrials.gov #: 
NCT02642809 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to 
obtain informed consent, may not be disclosed to another party unless law or regulations require such 

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disclosure.  Persons to whom the information is disclosed must be informed that the information is 
confidential and may not be further disclosed by them 
 
 
A pilot study combining pembrolizumab with locally delivered radiation therapy for the 
treatment of metastatic esophageal cancers 
 
Protocol Revision History 
 
Initial Approval Version 
 
 
 
 
 
 
03/08/16 
Amendment #1 Version 
 
 
 
 
 
 
06/09/16 
Amendment #2 Version 
 
 
 
 
 
 
08/01/16 
Amendment #3 Version 
 
 
 
 
 
 
01/26/17 
Amendment #4 Version (Merck approved only)  
 
 
07/31/17 
Amendment #4 Version 
 
 
 
 
 
 
10/04/17 
Amendment #5 Version 
 
 
 
 
 
 
11/16/17 
Amendment #6 Version 
 
 
 
 
 
 
12/22/17 
Amendment #7 Version 
 
 
 
 
 
 
03/08/18 
Amendment #8 Version 
 
 
 
 
 
 
03/01/19 
Amendment #9 Version 
 
 
 
 
 
 
06/15/22 
Amendment #10 Version                                                                              09/09/22  

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A pilot study combining pembrolizumab with locally delivered radiation therapy for the 
treatment of metastatic esophageal cancers 
 
 
SCHEMA 
 
 
 
 
Eligible Patients 
N = 18 
Endoscopic Biopsy 
N = 18 
Brachytherapy 8 Gy x 2 
N = 18 
Pembrolizumab 200 mg Q3W 
N = 18 
Endoscopic Biopsy 
N = 8 
Endoscopic Biopsy 
N = 18 
PROGRESSION 
1 WEEK 
8 WEEKS 

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Table of Contents 
SCHEMA ...................................................................................................................................3 
1.0 
BACKGROUND AND RATIONALE .....................................................................6 
1.1 
Esophageal Cancers .................................................................................................6 
1.2 
Immune Therapy ......................................................................................................6 
1.3 
Radiotherapy and the Immune Response ..................................................................7 
1.4 
Pembrolizumab (MK-3475) .....................................................................................8 
1.5 
Study Rationale ...................................................................................................... 11 
1.6 
Correlative Studies Background ............................................................................. 12 
2.0 
OBJECTIVES ........................................................................................................ 14 
2.1 
Primary Objective .................................................................................................. 14 
2.2 
Secondary Objectives ............................................................................................. 14 
2.3 
Exploratory Objective ............................................................................................ 14 
3.0 
PATIENT SELECTION ......................................................................................... 14 
3.1 
Inclusion Criteria.................................................................................................... 14 
3.2 
Exclusion Criteria .................................................................................................. 15 
3.3 
Inclusion of Women and Minorities ....................................................................... 17 
4.0 
REGISTRATION PROCEDURES ......................................................................... 17 
4.1 
Confirmation of Patient Eligibility ......................................................................... 17 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database ...................... 17 
4.3 
Assignment of UPN ............................................................................................... 17 
5.0 
TREATMENT PLAN ............................................................................................ 18 
5.1 
Premedication Administration ................................................................................ 18 
5.2 
Agent Administration ............................................................................................. 18 
5.3 
Evaluability ............................................................................................................ 18 
5.4 
General Concomitant Medication and Supportive Care Guidelines ......................... 18 
5.5 
Women of Childbearing Potential........................................................................... 20 
5.6 
Duration of Therapy ............................................................................................... 23 
5.7 
Duration of Follow-up ............................................................................................ 23 
6.0 
DOSE DELAYS/DOSE MODIFICATIONS .......................................................... 24 
7.0 
REGULATORY AND REPORTING REQUIREMENTS ...................................... 28 
7.1 
Definitions ............................................................................................................. 28 
7.2 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University .............................................................................................................................. 30 
7.3 
Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University .......................................................................................................... 31 
7.4 
Reporting to the FDA ............................................................................................. 31 
7.5 
Reporting to Merck & Co., Inc. .............................................................................. 31 
7.6 
Timeframe for Reporting Required Events ............................................................. 33 
8.0 
PHARMACEUTICAL INFORMATION ............................................................... 33 
8.1 
Pembrolizumab ...................................................................................................... 33 
9.0 
CORRELATIVE STUDIES ................................................................................... 34 
9.1 
Collection of Specimen(s) ...................................................................................... 34 
9.2 
Immune Monitoring ............................................................................................... 36 
10.0 
STUDY CALENDAR ............................................................................................ 38 
11.0 
DATA SUBMISSION SCHEDULE....................................................................... 39 

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12.0 
MEASUREMENT OF EFFECT............................................................................. 39 
12.1 
Antitumor Effect – Solid Tumors ........................................................................... 39 
12.2 
Immune-Related Response Criteria [Ref Wolchok Clin Cancer Res 2009] ............. 39 
12.3 
12.3 Duration of Response ..................................................................................... 41 
13.0 
DATA AND SAFETY MONITORING ................................................................. 42 
14.0 
STATISTICAL CONSIDERATIONS .................................................................... 42 
14.1 
Study Design .......................................................................................................... 42 
14.2 
Study Endpoints ..................................................................................................... 43 
14.3 
Data Analysis ......................................................................................................... 43 
14.4 
Power Analysis and Sample Size ............................................................................ 43 
14.5 
Accrual .................................................................................................................. 43 
14.6 
Toxicity and Plans for Data and Safety Monitoring (Stopping Rule) ....................... 43 
12. 
REFERENCES ...................................................................................................... 45 
APPENDIX A: ECOG Performance Status Scale ...................................................................... 49 
 
 

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1.0 
BACKGROUND AND RATIONALE 
 
1.1 
Esophageal Cancers 
 
In 2014, it is estimated that there will be 18,170 new cases of esophageal cancer diagnosed 
and 15,450 deaths in the United States. Alarmingly, esophageal adenocarcinomas are 
increasing in incidence more rapidly than other malignancies and most notably in patients 
younger than 40 years of age. Unfortunately 80-90% of newly diagnosed patients present 
with regional or distant metastatic disease.  Standard front-line therapy for patients with 
metastatic esophageal cancers is palliative systemic chemotherapy, usually with a 
fluoropyrimidine and a platinum agent, and for patients presenting with profound 
dysphagia at diagnosis, an abbreviated course of radiation therapy followed by the systemic 
chemotherapy or radiation therapy concurrently with the chemotherapy may also be given.  
Despite these treatments, the response rate for first-line therapies is generally about 40%, 
and the median survival is less than one year. Therefore, new therapeutic strategies are 
needed. 
 
1.2 
Immune Therapy 
 
1.2.1 Immunosurveillance 
 
Immunosurveillance is a term used to describe the role of the host immune system 
in controlling tumor development. The role of immunosurveillance in the control 
and/or development of tumors has been described in three steps termed the three 
E’s: Elimination, Equilibrium, and Escape. During the elimination phase, the 
immune system is able to recognize aberrantly expressed antigens or molecules that 
signal to the immune system that the cell has undergone a transformative event and 
should be eliminated. Unfortunately, transformed cells eventually adopt evasive 
properties that allow it to subvert detection or destruction by the immune system 
(Equilibrium) permitting its eventual outgrowth into a clinically evident tumor 
(Escape).2 Thus, immunotherapy refers to strategies that manipulate the immune 
response in an attempt to re-establish an effective immunosurveillance mechanism 
to control tumor growth.  Another benefit of cancer immunity is that the immune 
system represents a relatively tumor-specific effector mechanism that can 
simultaneously target multiple disease sites including the CNS and distant 
metastatic sites of disease, and the response can be long-lived often persisting for 
years.3   
 
One mechanism by which tumors are able to evade immunologic detection is 
through the upregulation of inhibitory signals. For example, PD-L1 and PD-L2 are 
molecules frequently expressed on many tumor types. Their cognate receptor, PD-
1, is expressed on the surface of many activated immune cell populations, including 
tumor-infiltrating T lymphocytes, and binding of PD-1 to either of these inhibitory 
ligands results in an “exhausted” or suppressed effector phenotype of infiltrating T 
cells permitting tumor escape.4 Under normal conditions, these inhibitory 

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molecules serve to control chronic immune activation representing a “checkpoint” 
mechanism to prevent unregulated inflammatory responses that may be potentially 
detrimental to the host.5 In the context of cancer immunity, however, the tumor 
microenvironment has coopted this checkpoint pathway to permit immune 
escape.4,5 Therefore, one approach to bypass this immunosuppressive environment 
to reestablish immunosurveillance is through the use of blocking monoclonal 
antibodies specific to these inhibitory molecules to allow for the re-activation and 
expansion of endogenous anti-tumor T cells.  This approach has been termed 
“checkpoint blockade” and initial phase I studies using either anti-PD-1 or anti-PD-
L1 antibodies have demonstrated significant objective, long-term responses in 
patients with various tumor types.6-10     
 
1.2.2 Immunosuppression in Esophageal Cancer 
 
The immune system is composed of both pro-inflammatory effector cells and anti-
inflammatory suppressive cells. The balance between these two opposing cell 
populations dictates the overall outcome of the immune response. In esophageal 
cancer, a predominance of effector cytotoxic CD8+ Tcells is associated with 
improved patient survival 11, while a significantly higher number of the principle 
suppressive cell populations, myeloid derived suppressor cells (MDSCs) and 
regulatory T cells (Tregs), is associated with a poor prognosis 12,13. Moreover, larger 
tumor burden and more advanced stage disease are correlated with a higher level 
of circulating MDSCs13. These data suggest that the relative balance between 
effector:regulatory immune cells determines the efficacy of immunosurveillance in 
controlling esophageal cancer. 
 
The exact mechanism by which esophageal cancer subverts immune detection is 
presently unknown. Interestingly, MDSCs isolated from esophageal tumors were 
noted to have dramatically upregulated expression of PD-L1 13suggesting that the 
PD-1 pathway may be involved. In further support of this point, PD-L1 is expressed 
in 35-45% of esophageal cancers 14,15, while the other ligand for PD-1, PD-L2, is 
present in over 80% of tumors 15. Furthermore, PD-1+ tumor-infiltrating 
lymphocytes (TILs) were detected in approximately 60% of esophageal tumor 
samples 15. The expression of PD-1 or its ligands, PD-L1 or PD-L2, are also 
associated with more advanced disease and a poorer outcome compared to patients 
that do not have evidence of upregulated expression of this pathway14,15. As such, 
inhibition of the PD-1/PD-L1 pathway may have therapeutic implications in the 
treatment of esophageal cancer. 
 
1.3 
Radiotherapy and the Immune Response 
 
Radiation therapy is a standard treatment modality for many patients with esophageal 
cancer and is often used in the palliative setting to alleviate symptoms of dysphagia 
associated with locally progressive disease.  
 
Intraluminal brachytherapy is a specific means of delivering radiotherapy to the luminal 

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surface of the esophagus with a specially designed catheter, typically with large 
hypofractionated doses delivered over only a few fractions16,17. This can be given at any 
point within an obstructed esophagus due to the small caliber of the introducer.  The main 
advantage of brachytherapy over stent insertion is its potential to provide lasting control of 
tumor-related symptoms, and its main advantage of external beam radiotherapy is its ability 
to safely deliver large doses of radiation with great spatial precision while shortening 
treatment time and sparing surrounding organs-at-risk.  Modern brachytherapy is typically 
delivered with a high-dose rate (HDR) afterloader.  Histologic findings after such 
esophageal brachytherapy include necrosis, keratin formation, fibrosis, and giant cell 
reaction in all layers of the esophageal wall 17. As such, the rapid shrinkage of luminal 
tumor masses results in prompt resolution of symptomatic dysphagia. While the ideal 
dosing of intraluminal brachytherapy in esophageal cancer remains unclear, a dose of 16 
Gy in 2 fractions over 2 weeks gave identical results as 18 Gy in 3 fractions over 3 weeks 
17,18.  
 
Radiotherapy has also been shown to lead to significant alterations in the tumor 
microenvironment, including effects on immune cells. This effect is thought to primarily 
be due to the release of tumor-derived antigens and pro-inflammatory cytokines 19-21, a 
process known as immunologic cell death, which leads to the induction of an antitumor 
immune response 22. Radiation-induced cell death results in release of reactive oxygen 
species and damage-associated molecular patterns (DAMPs) which activate local antigen-
presenting cells to enhance the priming of tumor-specific T cells 23-26. Furthermore, the 
local induction of a radiation-induced tumor-specific T cell response has been shown to 
result in reduction of systemic, non-radiated, tumor burden in a CD8+ T cell dependent 
manner 27. The generation of a systemic anti-tumor immune response associated with local 
tumor therapy is referred to as the abscopal effect 28.  
 
An area of active interest is focused on combining local therapy with immunotherapy to 
optimize the abscopal effect in metastatic cancer 29-33. Preclinical models have supported a 
synergistic effect between local radiotherapy and checkpoint blockade; however, these 
studies have largely been performed using CTLA-4 checkpoint blockade, another T cell 
inhibitory molecule34-38. Interestingly, several recent studies have suggested that PD-1/PD-
L1 expression actually mediates resistance to fractionated radiotherapy with amelioration 
of the abscopal response, and that inhibition of the PD-1/PD-L1 axis can overcome this 
resistance leading to improved systemic immune responses against non-radiated metastatic 
lesions following local fractionated radiotherapy 39,40. Thus, these data support the use of 
local fractionated radiation combined with checkpoint blockade as a rationale therapeutic 
strategy for the treatment of metastatic cancer. 
 
1.4 
Pembrolizumab (MK-3475) 
 
The importance of intact immune surveillance in controlling outgrowth of neoplastic 
transformation has been known for decades.  Accumulating evidence shows a correlation 
between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in 
various malignancies.  In particular, the presence of CD8+ T-cells and the ratio of CD8+ 
effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis 

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and long-term survival in many solid tumors. 
 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress 
immune control.  The normal function of PD-1, expressed on the cell surface of activated 
T-cells under healthy conditions, is to down-modulate unwanted or excessive immune 
responses, including autoimmune reactions.  PD-1 (encoded by the gene Pdcd1) is an Ig 
superfamily member related to CD28 and CTLA-4 that has been shown to negatively 
regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2).  
The structure of murine PD-1 has been resolved.  PD-1 and family members are type I 
transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible 
for ligand binding and a cytoplasmic tail which is responsible for the binding of signaling 
molecules.  The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an 
immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-
based switch motif (ITSM).  Following T-cell stimulation, PD-1 recruits the tyrosine 
phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, leading to 
the dephosphorylation of effector molecules such as CD3ζ, PKCθ and ZAP70 which are 
involved in the CD3 T-cell signaling cascade.  The mechanism by which PD-1 down 
modulates T-cell responses is similar to, but distinct from that of CTLA-4 as both 
molecules regulate an overlapping set of signaling proteins.  PD-1 was shown to be 
expressed on activated lymphocytes including peripheral CD4+ and CD8+ T-cells, B-cells, 
T regs and Natural Killer cells.  Expression has also been shown during thymic 
development on CD4-CD8- (double negative) T-cells as well as subsets of macrophages 
and dendritic cells.  The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed 
or can be induced in a variety of cell types, including non-hematopoietic tissues as well as 
in various tumors.  Both ligands are type I transmembrane receptors containing both IgV- 
and IgC-like domains in the extracellular region and contain short cytoplasmic regions with 
no known signaling motifs.  Binding of either PD-1 ligand to PD-1 inhibits T-cell activation 
triggered through the T-cell receptor.  PD-L1 is expressed at low levels on various non-
hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is 
only detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic 
inflammatory environments.  PD-L2 is thought to control immune T-cell activation in 
lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in 
peripheral tissues.  Although healthy organs express little (if any) PD-L1, a variety of 
cancers were demonstrated to express abundant levels of this T-cell inhibitor.  PD-1 has 
been suggested to regulate tumor-specific T-cell expansion in subjects with melanoma 
(MEL).  This suggests that the PD-1/PD-L1 pathway plays a critical role in tumor immune 
evasion and should be considered as an attractive target for therapeutic intervention. 
 
Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of 
the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its 
ligands, PD-L1 and PD-L2.  KeytrudaTM (pembrolizumab) has recently been approved in 
the United Stated for the treatment of patients with unresectable or metastatic melanoma 
and disease progression following ipilimumab and, if BRAF V600 mutation positive, a 
BRAF inhibitor. 
 
Refer to the Investigator’s Brochure for Preclinical and Clinical data. 

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1.4.1 Rationale for Dosing 
 
An open-label Phase I trial (Protocol 001) is being conducted to evaluate the safety 
and clinical activity of single agent MK-3475.  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 testing a Q2W and Q3W dosing schedule.   
 
A population pharmacokinetic analysis has been performed using serum 
concentration time data from 476 patients. Within the resulting population PK 
model, clearance and volume parameters of MK-3475 were found to be dependent 
on body weight. The relationship between clearance and body weight, with an 
allometric exponent of 0.59, is within the range observed for other antibodies and 
would support both body weight normalized dosing or a fixed dose across all body 
weights.  MK-3475 has been found to have a wide therapeutic range based on the 
melanoma indication.  The differences in exposure for a 200 mg fixed dose regimen 
relative to a 2 mg/kg Q3W body weight based regimen are anticipated to remain 
well within the established exposure margins of 0.5 – 5.0 for MK-3475 in the 
melanoma indication. The exposure margins are based on the notion of similar 
efficacy and safety in melanoma at 10 mg/kg Q3W vs. the proposed dose regimen 
of 2 mg/kg Q3W (i.e. 5-fold higher dose and exposure). The population PK 
evaluation revealed that there was no significant impact of tumor burden on 
exposure. In addition, exposure was similar between the NSCLC and melanoma 
indications. Therefore, there are no anticipated changes in exposure between 
different indication settings. 
 
The rationale for further exploration of 2 mg/kg and comparable doses of 
pembrolizumab in solid tumors is based on: 1) similar efficacy and safety of 
pembrolizumab when dosed at either 2 mg/kg or 10 mg/kg Q3W in melanoma 
patients, 2) the flat exposure-response relationships of pembrolizumab for both 
efficacy and safety in the dose ranges of 2 mg/kg Q3W to 10 mg/kg Q3W, 3) the 
lack of effect of tumor burden or indication on distribution behavior of 
pembrolizumab (as assessed by the population PK model) and 4) the assumption 
that the dynamics of pembrolizumab target engagement will not vary meaningfully 

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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.  
 
A fixed dose regimen will simplify the dosing regimen to be more convenient for 
physicians and to reduce potential for dosing errors.  A fixed dosing scheme will 
also reduce complexity in the logistical chain at treatment facilities and reduce 
wastage. 
 
1.5 
Study Rationale 
 
The abscopal effect is a phenomenon in which local radiotherapy is associated with the 
regression of metastatic lesions distant to the irradiated site, and is thought to be mediated 
by a systemic anti-tumor immune response generated by local immune activation41. The 
intensity of the radiotherapy-elicited immune response has been shown to be positively 
associated with the local response 42. Therefore, hypofractionated radiotherapy would be 
expected to more effectively elicit an anti-tumor immune response than standard radiation, 
and this has been supported in preclinical studies 43. Furthermore, increased expression of 
PD-1 and PD-L1 have been shown to abate the generation of an abscopal response 
following fractionated radiotherapy in preclinical mouse models but regression of non-
radiated metastases is restored when hypofractionated radiotherapy is combined with PD-
1/PD-L1 blockade 39,40 supporting the rationale for combining hypofractionated local 
radiation with checkpoint blockade in the treatment of metastatic cancer. 
 
Unfortunately, there is a paucity of data evaluating the effect of this combinatorial 
approach in esophageal cancer. 
 
High dose-rate intraluminal brachytherapy is a well-established tool for palliation of 
dysphagia in patients with metastatic esophageal cancer 44. Compared with external beam 
radiotherapy, brachytherapy has the appeal of delivering highly localized, intense doses of 
hypofractionated radiation to the tumor tissue with minimal exposure of surrounding 
normal tissues to radiation. Limiting exposure to surrounding normal tissue has additional 
appeal in the use of immune stimulatory approaches, as recent reports have demonstrated 
a correlation between radiation dose to normal tissues (such as lung) and lymphopenia with 
a resultant decrease in overall survival 45. Furthermore, esophageal cancers have been 
shown to have high expression of PD-L1 and an immunosuppressive microenvironment. 
Therefore, it is our hypothesis that a short course of intense radiation therapy will generate 
a systemic tumor-specific T cell response that will be enhanced by PD-1 inhibition 
resulting in systemic antitumor efficacy. 

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We therefore propose to treat patients with metastatic esophageal cancers and dysphagia 
with two fractions of brachytherapy followed by pembrolizumab. The brachytherapy is 
hypofractionated and will provide a radiation dose of sufficient intensity to induce the 
release of tumor-derived antigens and trigger an antitumor immune response. The 
simplicity of the design should maximize the chance to examine the hypothesis that 
radiotherapy can induce an immune response, which can then be augmented by 
pembrolizumab treatment. Success in this study would provide the impetus to conduct 
further trials aimed at developing this unique strategy as a more broadly applicable 
therapeutic option in the treatment of patients suffering from these deadly cancers, and will 
provide important mechanistic insights into the relationship between radiation treatment 
and immune therapy augmentation.  
 
Taken together, these data indicate that targeting the PD-1/PD-L1 axis in esophageal 
cancers in combination with radiation therapy may be a rational treatment strategy for these 
cancers. 
 
1.6 
Correlative Studies Background 
 
The clinical correlates of this study will have two aims: 
• 
Aim 1: Identify tumor-associated and systemic biomarkers that predict response to the 
combination of pembrolizumab and brachytherapy in patients with esophageal cancer. 
• 
Aim 2: Characterize the immune response at baseline (pre-brachytherapy), post-
brachytherapy, and post-pembrolizumab to identify immune parameters associated 
with protection and the development of an abscopal effect. 
 
Peripheral blood samples will be drawn at baseline (pre-brachytherapy), Day 1 (pre-
pembrolizumab), and post-pembrolizumab on Day 22, 3 months, 6 months, 12 months, 
and/or at time of progression (no further blood samples will be drawn after progression if 
occurs before 12 months). Peripheral blood samples will be used to characterize and 
quantify the immune response following brachytherapy and pembrolizumab.  
 
Endoscopic evaluation with biopsies will be performed prior to brachytherapy (all 
patients), after brachytherapy/pre-pembrolizumab (8 of 18 patients), and at 2-6 months 
post-pembrolizumab (all patients) to assess the local immune environment. 
 
1.6.1 Aim 1: Biomarker assessment 
 
 
PD-L1 expression on tumor cells has been used as a clinical biomarker to correlate 
with response to PD-1 checkpoint blockade. However, there remains debate 
regarding an appropriate cut-off to define positivity. Moreover, there are a 
substantial percentage of patients who benefit from therapy who are considered 
negative for PD-L1 expression, which may be associated with uninhibited 
interactions between PD-1/PD-L2 or PD-L1/CD80, which also induce immune 
regulation. Furthermore, it is not clear what should be considered an appropriate 
definition of positivity. Lastly, there have been no studies to date that have 

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demonstrated concordance/discordance in checkpoint inhibitor expression between 
primary and metastatic lesions. Therefore, Aim 1 will address two primary goals: 
• 
Quantify expression of PD-1, PD-L1, PD-L2, and CTLA-4 (an alternative 
checkpoint pathway) by IHC on tumor cells and tumor infiltrating lymphocytes 
(TILs) to determine if there is an association between response or resistance to 
brachytherapy + pembrolizumab. If an association is detected, the level of 
expression on either tumor and/or TIL that adequately defines positivity of 
associated molecule will be defined to predict patients that would most likely 
benefit from brachytherapy and pembrolizumab therapy.  
• 
Compare checkpoint inhibitor expression levels of PD-1, PD-L1, PD-L2, and 
CTLA-4 in paired primary and metastatic biopsies. This data will allow 
determination of frequency of concordant or discrepant expression of various 
mechanisms of checkpoint inhibition between primary and metastatic disease 
sites. This information can help clarify potential etiology of any mixed clinical 
responses (primary response and metastatic lesion progresses, or vise versa). It 
would also help determine associations that would predict the potential 
development of an abscopal effect.  
 
1.6.2 Aim 2: Immune characterization 
 
The tumor infiltrating lymphocytes (TIL) have been studied in esophageal cancer 
and are generally felt to be associated with good prognosis.46-49 The presence of 
TILs has been used as justification for the presence of immunosurveillance and thus 
the rationale for the use of immunotherapy.  While the presence of CD8+ cytotoxic 
T cells within the TIL have predominantly been associated with protection, there is 
often the presence of immunosuppressive subsets such as regulatory T cells (Tregs) 
and myeloid derived suppressor cells (MDSCs) that are associated with poor 
prognosis.50,51 The interplay between these effector and suppressive subsets in 
determining the response to checkpoint inhibitor therapy has yet to be 
elucidated.  Therefore, Aim 2 will characterize the tumor immune infiltrate and 
circulating immune cells using the following approaches: 
• 
TCR profiling by spectrotyping and flow cytometry to assess changes in relative 
frequency of TCR gene utilization as a surrogate of clonal expansion and 
selection of the CD4 and CD8 T cell repertoire (i.e. tumor-specific T cell 
response) following brachytherapy and pembrolizumab. 
• 
Flow cytometry and CyTOF to identify and quantify the immunophenotype of 
various effector immune subsets to determine changes in immune profile 
between baseline, post-brachytherapy, and post-pembrolizumab. Correlations 
between treatment response and resistance will be determined to predict 
potential patients that will benefit from combined brachytherapy and 
pembrolizumab. The following immune subsets will be assessed:  
o T cell: CD3, CD8, CD4, Foxp3 (Tregs), t-bet (Th1), ROR-γ (Th17), 
effector memory T cells (Tem), central memory T cells (Tcm); 
intracellular staining for IFN-γ, TNF-α, IL-2, IL-17, IL-4, IL-10 will be 
determined to assess functional capacity of T cells before and after 
brachytherapy and pembrolizumab. 

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o NK cell: CD56, CD16 
o MDSC: CD14, HLA-DR 
o Activation markers: ICOS, OX40, CD137, CD25, GITR 
o Inhibitory markers: PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3 
• 
Multiplex analysis and QPCR to assess changes in cytokine profile: IFN-α, 
IFN-β, IFN-γ, IL-2, TNF-α, IL-2, IL-4, IL-5, IL-13, IL-10, IL-6, TGF-β. 
• 
Tumor cells will be microdissected from tissue biopsies and sent for whole 
exome sequencing and RNAseq to quantify the mutational landscape as a 
surrogate of antigenic burden. As antigenic burden has been correlated with 
response to checkpoint inhibitor therapy, we will determine if response to 
brachytherapy and pembrolizumab is associated with mutational burden.52-54 
 
 
2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine the tolerability of localized esophageal hypofractionated brachytherapy 
administered in two fractions when combined with pembrolizumab in patients with 
metastatic esophageal carcinoma. 
 
2.2 
Secondary Objectives 
 
1. To assess the local antitumor effect of hypofractionated brachytherapy followed by 
pembrolizumab as determined by endoscopic measurements of change in tumor length 
and esophageal lumen diameter and by grade of dysphagia per CTCAE criteria. 
2. To assess the systemic efficacy by irRC-based criteria of hypofractionated 
brachytherapy to the esophagus combined with systemic pembrolizumab on non-
radiated metastatic lesions. 
3. To determine the preliminary antitumor efficacy of pembrolizumab combined with 
local hypofractionated brachytherapy in patients with metastatic esophageal 
carcinoma. 
 
2.3 
Exploratory Objective 
 
1. To determine the expression profile of activating and inhibitory molecules in tumor 
samples after hypofractionated brachytherapy and pembrolizumab therapy to identify 
biomarkers correlated with response or resistance. 
2. To annotate the local and distant immune response after hypofractionated 
brachytherapy and pembrolizumab to determine mediators of protection and 
development of an abscopal effect. 
 
3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 

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1. Any patient with metastatic esophageal cancer that is deemed a candidate for 
brachytherapy for local control or treatment of dysphagia as determined by treating 
physician.  
 
2. Presence of an evaluable metastatic lesion (locoregional lymph nodes are acceptable). 
 
3. At least 18 years of age. 
 
4. ECOG performance status 0-2 (see Appendix A) 
 
5. Adequate bone marrow and organ function as defined below: 
a. Absolute neutrophil count ≥ 1,500/mcL 
b. Platelets ≥ 100,000/mcL 
c. Hemoglobin ≥ 9 g/dL 
d. Total bilirubin ≤ 1.5 x IULN 
OR 
Direct bilirubin ≤ IULN for patients with total bilirubin > 1.5 x IULN 
e. AST(SGOT)/ALT(SGPT) ≤ 2.5 x IULN (or ≤ 5 x IULN for patients with liver 
metastases) 
f. Serum creatinine ≤ 1.5 x IULN 
OR 
Creatinine clearance by Cockcroft-Gault ≥ 60 mL/min/1.73 m2 for patients with 
creatinine levels > 1.5 x IULN 
g. INR or PT ≤ 1.5 x IULN unless patient is receiving anticoagulant therapy as long 
as INR or PTT is within therapeutic range of intended use of anticoagulants 
h. aPTT ≤ 1.5 x IULN unless patient is receiving anticoagulant therapy as long as INR 
or PTT is within therapeutic range of intended use of a anticoagulants 
 
6. Sexually active women of childbearing potential and men must agree to use 
contraceptive methods as described in Section 5.5 prior to study entry, for the duration 
of study participation, and for 120 days after the last dose of pembrolizumab.  Should 
a woman become pregnant or suspect she is pregnant while participating in this study, 
she must inform her treating physician immediately. 
 
7. Either enrolled in HRPO# 201107221 (“Tissue and Blood Acquisition for Genomic 
Analysis and Collection of Health Information for Patients with Gastrointestinal 
Cancers”), which facilitates the collection of specimens for correlative studies, or 
consenting to collection of blood and tissue as part of this protocol for research testing. 
 
8. Ability to understand and willingness to sign an IRB approved written informed 
consent document (or that of legally authorized representative, if applicable). 
 
3.2 
Exclusion Criteria 
 
1. Prior treatment with an anti-PD-1, anti-PD-L1 or  anti-PD-L2 agent. 
 

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2. Received a live vaccine within 30 days prior to the first dose of pembrolizumab. 
Examples of live vaccines include, but are not limited to, the following: measles, 
mumps, rubella, varicella/zoster (chicken pox), yellow fever, rabies, Bacillus Calmette-
Guérin (BCG), and typhoid vaccine.  Seasonal influenza vaccines for injection are 
generally killed virus vaccines and are allowed; however, intranasal influenza vaccines 
(e.g. FluMist) are live attenuated vaccines and are not allowed. 
 
3. Presence of a concurrent active, incurable malignancy that may alter the outcome of 
the treatment for esophageal cancer as determined by the treating physician. 
 
4. Has a 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 pembrolizumab. 
 
5. Currently receiving any other investigational agents, has participated in a study of an 
investigational agent, or use of an investigational device within 4 weeks of the first 
dose of pembrolizumab. 
 
6. Has received systemic therapy within 4 weeks of the first dose of pembrolizumab. 
 
7. Known active central nervous system metastases and/or carcinomatous meningitis.  
Subjects with previously treated brain metastases may participate provided they are 
stable (without evidence of progression by imaging for at least 4 weeks prior to the first 
dose of MK-3475 and any neurologic symptoms have returned to baseline), have no 
evidence of new or enlarging brain metastases, and are not using steroids for at least 7 
days prior to trial treatment.  This exception does not include carcinomatous meningitis 
which is excluded regardless of clinical stability. 
 
8. A history of allergic reactions attributed to compounds of similar chemical or biologic 
composition to pembrolizumab or other agents used in the study. 
 
9. Uncontrolled intercurrent illness that would limit compliance with study requirements. 
This would include, but is not limited to: ongoing or active infection, symptomatic 
congestive 
heart 
failure, 
unstable 
angina 
pectoris, 
cardiac 
arrhythmia, 
immunosuppression, autoimmune conditions, underlying pulmonary disease, or 
psychiatric illness/social situations. 
 
10. Has an active autoimmune disease requiring systemic treatment within the past 2 years 
(i.e. with use of disease modifying agents, corticosteroids, or immunosuppressive 
drugs).  Replacement therapy (e.g. thyroxine, insulin, or physiologic corticosteroid 
replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form 
of systemic treatment. 
 
11. History of (non-infectious) pneumonitis that required steroids or current pneumonitis. 
 

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12. Pregnant and/or breastfeeding.  Women of childbearing potential must have a negative 
pregnancy test within 72 hours of study entry. 
 
13. Known history of hepatitis B (defined as hepatitis B surface antigen [HBsAg] reactive) 
or known active hepatitis C virus (defined as HCV RNA [qualitative] is detected). 
 
14. Known history of active TB. 
 
15. Known history of HIV (HIV 1/2 antibodies). 
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 
 
 
4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center OnCore database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility by collecting the information listed below: 
 
1. Registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 
All patients must be registered through the Siteman Cancer Center OnCore database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  All data 
will be recorded with this identification number on the appropriate CRFs. 
 

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5.0 
TREATMENT PLAN 
 
This is a single institution, open-label, single-dose-level, pilot study combining hypofractionated 
brachytherapy with standard dose pembrolizumab. 
 
5.1 
Premedication Administration 
 
No premedications are required, but antiemetics may be given as per institutional practice 
if needed. 
 
5.2 
Agent Administration 
 
Brachytherapy can be given at any point within the esophagus.  It will be delivered using 
a high-dose-rate Ir-192 afterloader via a dedicated esophageal applicator.  The prescription 
dose will be 16 Gy delivered in 2 fractions of 8 Gy per fraction, separated by a 7 to 10 day 
interval between fractions.  The dose will be prescribed at 1 cm from the source axis, with 
a 1 to 2 cm proximal and distal margin at the discretion of the treating radiation oncologists.  
The maximum total active length is not to be > 10 cm.  CT simulation is preferred, though 
fluoroscopic simulation is acceptable. 
 
Pembrolizumab will be started within 1 week (± 3 days) after completion of brachytherapy 
at a standard dose of 200 mg administered as an intravenous infusion over 30 minutes (-5 
minutes/+10 minutes).  It will be given every 3 weeks. 
 
5.3 
Evaluability 
 
All patients who receive any study treatment are evaluable for toxicity.  Patients are 
evaluated from first receiving study treatment until a 30-day follow up after the conclusion 
of treatment or death. 
 
All patients are evaluable for disease response unless they discontinue treatment due to 
treatment related adverse events(s) prior to completion of Cycle 2 and have not had any 
disease assessment. 
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
All treatments that the investigator considers necessary for a patient’s welfare may be 
administered at the discretion of the investigator in keeping with the community standards 
of medical care. 
 
Patients are prohibited from receiving the following therapies while in screening for and 
enrolled in this trial: 
 
• Anti-cancer systemic chemotherapy or biological therapy  
• Immunotherapy not specified in this protocol 

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• Chemotherapy not specified in this protocol 
• Investigational agents other than pembrolizumab 
• Radiation therapy other than brachytherapy as dictated in this protocol 
• Live vaccines within 30 days prior to the first dose of treatment and while 
participating in the trial.  Examples of live vaccines include, but are not limited to, 
the following: measles, mumps, rubella, varicella/zoster, yellow fever, rabies, 
BCG, and typhoid vaccine. 
• Systemic glucocorticoids for any purpose other than to modulate symptoms from 
an event of clinical interest of suspected immunologic etiology.  The use of 
physiologic doses of corticosteroids may be approved. 
 
5.4.1 Management of Infusion Reactions 
 
Pembrolizumab may cause severe or life-threatening reactions including severe 
hypersensitivity or anaphylaxis.  Signs and symptoms usually develop during or 
shortly after drug infusion and generally resolve completely within 24 hours of 
completion of infusion.  
 
The table below shows treatment guidelines for subjects who experience an 
infusion reaction associated with administration of pembrolizumab (MK-3475). 
 
 

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Infusion Reaction 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 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 medication and/or 
brief interruption of infusion); 
recurrence of symptoms following 
initial improvement; hospitalization 
indicated for other clinical sequelae 
(e.g., renal impairment, pulmonary 
infiltrates) 
Grade 4: 
Life-threatening; pressor or 
ventilatory support indicated 
Stop Infusion. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Epinephrine 
 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
Hospitalization may be indicated. 
Subject is permanently discontinued from 
further trial treatment administration. 
No subsequent dosing 
Appropriate resuscitation equipment should be available in the room and a physician readily available during the period of 
drug administration. 
 
5.5 
Women of Childbearing Potential 
 
Women of childbearing potential (defined as women with regular menses, women with 
amenorrhea, women with irregular cycles, women using a contraceptive method that 
precludes withdrawal bleeding, and women who have had a tubal ligation) are required to 
have a negative serum or urine pregnancy test within 72 hours prior to study entry and 
again within 72 hours prior to the first dose of pembrolizumab.   
 
Pembrolizumab may have adverse effects on a fetus in utero. 
 
For this trial, male subjects will be considered to be of non-reproductive potential if they 

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have azoospermia (whether due to having had a vasectomy or due to an underlying medical 
condition).   
 
A woman is considered fertile following menarche and until becoming post-menopausal 
unless permanently sterile (see below).  Women in the following categories are not 
considered of childbearing potential: 
 
• Premenarchal 
• Premenopausal female with 1 of the following: 
o Documented hysterectomy 
o Documented bilateral salpingectomy 
o Documented bilateral oophorectomy 
Note:  Documentation can come from the site personnel’s review of the 
participant’s medical records, medical examination, or medical history interview. 
 
• Postmenopausal female 
o A postmenopausal state is defined as no menses for 12 months without an 
alternative medical cause.  
 A high follicle stimulating hormone (FSH) level in the postmenopausal 
range may be used to confirm a postmenopausal state in women not using 
hormonal contraception or hormonal replacement therapy (HRT). However, 
in the absence of 12 months of amenorrhea, confirmation with two FSH 
measurements in the postmenopausal range is required.  
o Females on HRT and whose menopausal status is in doubt will be required to use 
one of the non-hormonal highly effective contraception methods if they wish to 
continue their HRT during the study. Otherwise, they must discontinue HRT to 
allow confirmation of postmenopausal status before study enrollment. 
 
Contraception Requirements 
Subjects should be informed that taking the study medication may involve unknown risks 
to the fetus (unborn baby) if pregnancy were to occur during the study.  In order to 
participate in the study subjects of childbearing potential must adhere to the contraception 
requirement (described below) from the day of study medication initiation (or 14 days prior 
to the initiation of study medication for oral contraception) throughout the study period up 
to 120 days after the last dose of trial therapy.  If there is any question that a subject of 
childbearing potential will not reliably comply with the requirements for contraception, 
that subject should not be entered into the study. 
 
Male Participants: 
Male participants with female partners of childbearing potential are eligible to participate 
if they agree to one of the following during the protocol defined timeframe:  
• Be abstinent from penile-vaginal intercourse as their usual and preferred lifestyle 
(abstinent on a long term and persistent basis) and agree to remain abstinent 
• Use a male condom plus partner use of a contraceptive method with a failure rate 
of <1% per year as described in the table below when having penile-vaginal 
intercourse with a woman of childbearing potential who is not currently pregnant. 

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o Note: Men with a pregnant or breastfeeding partner must agree to remain 
abstinent from penile-vaginal intercourse or use a male condom during each 
episode of penile penetration. 
 
Female Participants 
Female participants of childbearing potential are eligible to participate if they agree to use 
a highly effective method of contraception consistently and correctly as described in the 
table below during the protocol-defined timeframe.  
 
Highly Effective Contraceptive Methods That Are User Dependent a  
Failure rate of < 1% per year when used consistently and correctly. 
● Combined (estrogen- and progestogen-containing ) hormonal contraception b, c 
○ Oral  
○ Intravaginal  
○ Transdermal  
○ Injectable 
● Progestogen-only hormonal contraception b, c 
○ Oral  
○ Injectable  
Highly 
Effective 
Methods 
That 
Have 
Low 
User 
Dependency 
Failure rate of <1% per year when used consistently and correctly. 
● Progestogen- only contraceptive implant b, c  
● Intrauterine hormone-releasing system (IUS) b  
● Intrauterine device (IUD) 
● Bilateral tubal occlusion 
● Vasectomized partner  
A vasectomized partner is a highly effective contraception method provided that the
partner is the sole male sexual partner of the WOCBP and the absence of sperm has been 
confirmed.  If not, an additional highly effective method of contraception should be used.
● Sexual abstinence  
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the study
treatment.  The reliability of sexual abstinence needs to be evaluated in relation to the 
duration of the study and the preferred and usual lifestyle of the participant.)  
Notes:  
Use should be consistent with local regulations regarding the use of contraceptive methods for participants 
of clinical studies.  
 
a) Typical use failure rates are lower than perfect-use failure rates (i.e. when used consistently and 
correctly).  
b) If hormonal contraception efficacy is potentially decreased due to interaction with study treatment,
condoms must be used in addition to the hormonal contraception during the treatment period and for at 
least [X days, corresponding to time needed to eliminate study treatment plus 30 days for study treatments 
with genotoxic potential] after the last dose of study treatment. 
c) If locally required, in accordance with Clinical Trial Facilitation Group (CTFG) guidelines, acceptable
hormonal contraceptives are limited to those which inhibit ovulation.  
 

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Pregnancy Testing 
WOCBP should only be included after a negative highly sensitive urine or serum 
pregnancy test.  
 
Following initiation of treatment, pregnancy testing will be performed whenever an 
expected menstrual cycle is missed or when pregnancy is otherwise suspected, after the 
last dose of study treatment, and as required locally. 
 
Pregnancy testing will be performed whenever an expected menstrual cycle is missed or 
when pregnancy is otherwise suspected. 
 
5.6 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
• Documented and confirmed disease progression 
• Death 
• Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
• General or specific changes in the patient’s condition render the patient unable to 
receive further treatment in the judgment of the investigator 
• Suspected pregnancy 
• Serious non-compliance with the study protocol 
• Lost to follow-up 
• Patient withdraws consent 
• Investigator removes the patient from study 
• The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.7 
Duration of Follow-up 
 
Patients will be followed every 3 weeks while receiving pembrolizumab. After progression, 
patients will be followed every 3 months for 12 months or until death, whichever occurs 
first.  Patients removed from study for unacceptable adverse events will be followed until 
resolution or stabilization of the adverse event. 
 

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6.0 
DOSE DELAYS/DOSE MODIFICATIONS 
 
AEs associated with pembrolizumab exposure may represent an immunologic etiology. These 
immune-related AEs (irAEs) may occur shortly after the first dose or several months after the last 
dose of pembrolizumab treatment and may affect more than on body system simultaneously. 
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 the table below. 
 
 

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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 1-2mg/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 1-2mg/kg prednisone or 
equivalent) followed by taper 
 
• 
Monitor participants for signs and symptoms 
of enterocolitis (i.e. diarrhea, abdominal 
pain, blood or mucus in stool with or 
without fever) and of bowel perforation (i.e. 
peritoneal signs and ileus). 
• 
Participants with ≥ Grade 2 diarrhea 
suspecting colitis should consider GI 
consultation and performing endoscopy to 
rule out colitis.  
• 
Participants with diarrhea/colitis should be 
advised to drink liberal quantities of clear 
fluids.  If sufficient oral fluid intake is not 
feasible, fluid and electrolytes should be 
substituted via IV infusion. 
Grade 4 
Permanently 
discontinue 
AST / ALT 
elevation or 
Increased 
Bilirubin 
Grade 2 
Withhold 
• Administer corticosteroids (initial 
dose of 0.5- 1mg/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 1-2mg/kg prednisone or 
equivalent) followed by taper 

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Type 1 diabetes 
mellitus (T1DM) 
or 
Hyperglycemia 
 
Newly onset 
T1DM or  
Grade 3 or 4 
hyperglycemia 
associated with 
evidence of β-cell 
failure 
Withhold 
• Initiate insulin replacement 
therapy for participants with 
T1DM  
• Administer anti-hyperglycemic in 
participants with hyperglycemia  
 
• 
Monitor participants for hyperglycemia or 
other signs and symptoms of diabetes. 
Hypophysitis 
Grade 2 
Withhold 
• Administer corticosteroids and 
initiate hormonal replacements as 
clinically indicated.   
 
• 
Monitor for signs and symptoms of 
hypophysitis (including hypopituitarism and 
adrenal insufficiency)  
Grade 3 or 4  
Withhold or 
permanently 
discontinue1  
Hyperthyroidism  
Grade 2 
Continue  
• Treat with non-selective beta-
blockers (e.g. 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 (e.g. 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 1-2mg/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 
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 or exclude other causes 
 
Grade 3  
Withhold or 
discontinue based 
on the type of 
event.  Events that 
require 
discontinuation 

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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).   
 
For information on the management of infusion reactions, see Section 5.4. 
 

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7.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outline below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 7.2. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined in Section 
7.4.  In addition, any fatal or life-threatening adverse experiences where there is a reasonable 
possibility of relationship to study intervention must be reported. 
 
Merck & Co, Inc. requires that all serious adverse events, suspected unexpected serious adverse 
reactions, pregnancies, incidences of lactation, and events of clinical interest (refer to Section 
7.5.2) be reported as outlined in Section 7.5. 
 
7.1 
Definitions 
 
7.1.1 Adverse Events (AEs) 
 
Definition: any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading: the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.03 will be utilized 
for all toxicity reporting.  A copy of the CTCAE version 4.03 can be downloaded 
from the CTEP website. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP).  A copy of 
this guidance can be found on OHRP’s website: 
http://www.hhs.gov/ohrp/policy/advevntguid.html 
 
Study team will only collect those lab results that are clinically significant as 
determined by the investigator. 
 
 
7.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
o Death 

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o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o A new cancer 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
7.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
 
Events that are both serious AND unexpected must be reported to the FDA.  
 
7.1.4 Life-Threatening Adverse Experience  
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 
Life-threatening adverse experiences must be reported to the FDA. 
 
7.1.5 Unanticipated Problems 
 
Definition: 
 
• unexpected (in terms of nature, severity, or 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; 
• 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 or recognized. 
 

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7.1.6 Suspected Unexpected Serious Adverse Reaction 
 
Definition: any serious adverse event, the nature, severity or frequency of which is 
not consistent with information in the most current investigator’s brochure, or with 
respect to a marketed product the most current Summary of Product Characteristics 
(SPC) or Package Insert. 
 
 
7.1.7 Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that 
govern human subjects research or failure to follow the determinations of the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate 
choice to ignore regulations, institutional policies, or determinations of the IRB. 
 
7.1.8 Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial 
harm to subjects or others, or that materially compromises the rights or welfare of 
participants. 
 
7.1.9 Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the 
research team’s control. Exceptions apply only to a single participant or a singular 
situation. 
 
Pre-approval of all protocol exceptions must be obtained prior to the event. 
 
7.2 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University 
 
The PI is required to promptly notify the IRB of the following events: 
 
• Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
• Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
• Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 

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as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
7.3 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
a QASMC auditor. 
 
7.4 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
• Report any unexpected fatal or life-threatening adverse experiences (Section 7.1.4) 
associated with use of the drug (i.e., there is a reasonable possibility that the 
experience may have been caused by the drug) by telephone or fax no later than 7 
calendar days after initial receipt of the information.   
• Report any serious, unexpected adverse experiences (Section 7.1.2), as well as 
results from animal studies that suggest significant clinical risk within 15 calendar 
days after initial receipt of this information.   
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
7.5 
Reporting to Merck & Co., Inc. 
 
The PI shall forward to Merck’s Global Safety group (FAX 215-661-6229) any SAE 
(Section 7.1.2) and SUSAR (Section 7.1.6) information, including, but not limited to, all 

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initial and follow-up information involving any study subject in the study within 2 business 
days or 3 calendar days (whichever comes first) of learning of the SAE or SUSAR. 
 
7.5.1 Reporting Pregnancy and Lactation 
 
Although pregnancy and infant exposure during breastfeeding are not considered 
adverse events, it is the responsibility of investigators or their designees to report 
any pregnancy or lactation in a subject (spontaneously reported to them) that occurs 
during the trial. 
 
Pregnancies and infant exposures during breastfeeding  that occur after the consent 
form is signed but before treatment allocation/randomization must be reported by 
the investigator if they cause the subject to be excluded from the trial, or are the 
result of a protocol-specified intervention, including but not limited to washout or 
discontinuation of usual therapy, diet, placebo treatment or a procedure.   
 
Pregnancies and infant exposures during breastfeeding  that occur from the time of 
treatment allocation/randomization through 120 days following cessation of 
Sponsor’s product, or 30 days following cessation of treatment if the subject 
initiates new anticancer therapy, whichever is earlier, must be reported by the 
investigator. 
 
All 
reported 
pregnancies 
must 
be 
followed 
to 
the 
completion/termination of the pregnancy. Pregnancy outcomes of spontaneous 
abortion, missed abortion, benign hydatidiform mole, blighted ovum, fetal death, 
intrauterine death, miscarriage and stillbirth must be reported as serious events 
(Important Medical Events). If the pregnancy continues to term, the outcome 
(health of infant) must also be reported. 
 
Such events must be reported within 24 hours to the Sponsor and within 2 working 
days to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 661-
6229) 
 
7.5.2 Reporting Events of Clinical Interest 
 
Selected non-serious and serious adverse events are also known as Events of 
Clinical Interest (ECI) and must be reported within 2 working days to Merck Global 
Safety. (Attn: Worldwide Product Safety; FAX 215 661-6229). 
 
Events of clinical interest for this trial include: 
• an overdose of Merck product, that is not associated with clinical symptoms 
or abnormal laboratory results.  
• an elevated AST or ALT lab value that is greater than or equal to 3X the 
upper limit of normal and an elevated total bilirubin lab value that is greater 
than or equal to 2X the upper limit of normal and, at the same time, an 
alkaline phosphatase lab value that is less than 2X the upper limit of normal, 

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as determined by way of protocol-specified laboratory testing or 
unscheduled laboratory testing.* 
 
*Note: These criteria are based upon available regulatory guidance 
documents. The purpose of the criteria is to specify a threshold of abnormal 
hepatic tests that may require an additional evaluation for an underlying 
etiology. 
 
7.5.3 Reporting Overdose 
 
For purposes of this trial, an overdose of pembrolizumab will be defined as any 
dose of 1,000 mg or greater (≥5 times the indicated dose).  No specific information 
is available on the treatment of overdose of pembrolizumab. Appropriate 
supportive treatment should be provided if clinically indicated. In the event of 
overdose, the subject should be observed closely for signs of toxicity.  Appropriate 
supportive treatment should be provided if clinically indicated. 
 
If an adverse event(s) is associated with (“results from”) the overdose of a Merck 
product, the adverse event(s) is reported as a serious adverse event, even if no other 
seriousness criteria are met. 
 
If a dose of Merck’s product meeting the protocol definition of overdose is taken 
without any associated clinical symptoms or abnormal laboratory results, the 
overdose is reported as a non-serious Event of Clinical Interest (ECI), using the 
terminology “accidental or intentional overdose without adverse effect.” 
 
All reports of overdose with and without an adverse event must be reported within 
24 hours to the Sponsor and within 2 working days to Merck Global Safety. (Attn: 
Worldwide Product Safety; FAX 215 661-6229) 
 
7.6 
Timeframe for Reporting Required Events  
 
Adverse events will be tracked for 30 days following the last day of study treatment. 
 
Study team will only collect those lab results that are clinically significant as determined 
by the investigator. 
 
 
 
8.0 
PHARMACEUTICAL INFORMATION 
 
8.1 
Pembrolizumab 
 
8.1.1 Pembrolizumab Description 
 

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Pembrolizumab is a potent humanized IgG4 mAb with high specificity of binding 
to the PD-1 receptor, thus inhibiting its interaction with PD-L1 and PD-L2. Based 
on preclinical in vitro data, pembrolizumab has high affinity and potent receptor 
blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety 
profile and is being advanced for clinical development as an IV immunotherapy for 
advanced malignancies. 
 
8.1.2 Clinical Pharmacology 
 
Refer to Section 5.2 of the IB. 
 
8.1.3 Pharmacokinetics and Drug Metabolism 
 
Refer to Section 5.2 of the IB. 
 
8.1.4 Supplier 
 
Pembrolizumab will be provided free of charge by Merck & Co., Inc. 
 
8.1.5 Dosage Form and Preparation 
 
Merck will provide pembrolizumab as a liquid drug product. 
 
8.1.6 Storage and Stability 
 
Pembrolizumab should be stored under refrigerated conditions (2°C - 8°C). 
 
If not used immediately, vials and/or IV bags may be stored at 2-8 °C for up to a 
cumulative time of 20 hours. If refrigerated, the vials and/or IV bags should be 
allowed to equilibrate to room temperature prior to subsequent use. Pembrolizumab 
solutions may be stored at room temperature for a cumulative time of up to 4 hours. 
 
8.1.7 Administration 
 
Pembrolizumab will be given intravenously over the course of 30 minutes (-5 
min/+10 min) on an outpatient basis.   
 
 
9.0 
CORRELATIVE STUDIES 
 
9.1 
Collection of Specimen(s) 
 
For patients who have consented to HRPO# 201107221, all tumor and peripheral blood 
samples will be collected under that consent, as it is a tissue bank which authorizes 
permission for cellular, molecular, and genomic characterization and return of information. 

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For patients who have not consented to HRPO# 201107221, all specimens will be collected 
as part of this protocol.  All samples will be transported and stored at the Tissue 
Procurement Core unless otherwise indicated. 
 
9.1.1 Endoscopic biopsy 
 
All enrolled patients will undergo endoscopic biopsy per standard of care protocol 
at time of initial diagnosis/time of enrollment and 2-6 months after initiation of 
pembrolizumab.  At this time, further passes will be made so that 4 to 8 additional 
cores will be taken for research purposes. At 1-2 weeks after initiation of 
brachytherapy, eight consenting patients will undergo a research-related optional 
endoscopic biopsy (not coinciding with a SOC endoscopy) consisting of 4 to 8 
cores. These biopsies will be used to assess changes in the local tumor 
microenvironment following brachytherapy. 
 
All specimens will be taken to the Tissue Procurement Core fresh and then 
processed according to the instructions in HRPO# 201107221 (frozen in OCT). 
 
9.1.2 Peripheral blood 
 
Peripheral blood samples will be collected at the following time points: 
• Pre-brachytherapy 
• Post-brachytherapy but pre-pembrolizumab (on day 1) 
• Day 22 after the start of pembrolizumab 
• 3, 6, and 12 months (+/- 2 weeks) after the start of pembrolizumab 
• Time of progression 
 
Six collection tubes (BD Vacutainer® sodium heparin (green top), REF 367874, 
10 mL each for a total of approximately 60 mL) are filled by venipuncture at each 
time point. Blood samples will be transported to the Tissue Procurement Core 
within one hour of collection. PBMC will be obtained by Ficoll-Hypaque gradient 
centrifugation and cryopreserved in 10% DMSO according to standard procedures. 
 
Nucleic acid isolation: DNA will be isolated from PBMC by the LTP for exome 
sequencing at The Genome Institute to serve as reference for germline mutations. 
To identify somatic mutations, DNA and RNA will be extracted from OCT-
embedded banked tissue. The OCT block will be delivered to the LTP where the 
block will be sectioned and stained in order to confirm the presence of tumor, 
determine tumor/normal ratio, and guide isolation of tumor cells by, for instance, 
laser capture microdissection (LCM). All tissue selected for sequencing will be 
processed into a single-cell suspension by mechanical and enzymatic digestion, and 
used to extract nucleic acids. Tumor DNA + RNA will then undergo tumor exome 
and tumor cDNA-capture sequencing, respectively, at The Genome Institute.  
 

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Plasma and lymphocyte separation: PBMC will undergo centrifugation with Ficoll 
to separate the plasma and cellular (lymphocyte) layers from RBC. Both the plasma 
and cellular components will be frozen and stored at -80°C for further studies. 
 
9.2 
Immune Monitoring 
 
 
9.2.1 Immune profiling 
 
Patient-derived PBMC will be used to quantify and characterize the immunologic 
landscape at baseline and at various time points post-brachytherapy and 
pembrolizumab. Single cell suspensions will be isolated as described above. All 
correlative studies will performed in the Immune Monitoring Laboratory at the 
Center of Human Immunology and Immunotherapy Programs at Washington 
University.  
 
The T cell repertoire pre- and post- brachytherapy and pembrolizumab will be 
further characterized by analyzing the TCR gene utilization of circulating PBMCs. 
This will be done by multi-parametric flow cytometry, spectratyping, and 
sequencing. MHC class I and II tetramers will be generated either in the Immune 
Monitoring Laboratory at the Center of Human Immunology and Immunotherapy 
Programs at Washington University, or the NIH Tetramer Core Facility.  
 
Phenotypic and polyfunctional characterization of tumor-specific T cells will be 
performed by ELISPOT, multi-parametric flow cytometry, and mass cytometry 
(CyTOF) in the Immune Monitoring Laboratory at the Center of Human 
Immunology and Immunotherapy Programs at Washington University. 
 
Serum will be analyzed for changes in cytokine expression following 
brachytherapy and pembrolizumab. Cytokine levels will be quantified using 
ELISA, QPCR, and multiplex analysis and performed in the Immune Monitoring 
Laboratory at the Center of Human Immunology and Immunotherapy Programs at 
Washington University. 
 
9.2.2 Microenvironment characterization 
 
Tumor samples obtained from primary and metastatic biopsies will be analyzed for 
biomarkers (PD-1, PD-L1, PD-L2, and CTLA-4) that are associated with response 
to brachytherapy and pembrolizumab to identify individuals who may benefit most 
from this therapeutic approach. These will be sent-out to contracted pathology 
laboratories for analysis. 
 
PD-L1 staining will be performed by QualTek Molecular Laboratories.  Five 
unstained slides from FFPE tissue samples should be shipped cold (2-8°C) using 
the materials provided by QualTek to: 
MISP Receiving 

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QualTek Molecular Laboratories 
300 Pheasant Run 
Newtown, PA 18940 
 
IHC staining of OCT embedded tissue will characterize the location of various 
infiltrating lymphocytes relative to tumor. Furthermore, single cell suspensions can 
be made from fresh frozen tissue to characterize the phenotype of various 
populations 
of 
infiltrating 
leukocytes 
(T 
cells, 
B 
cells, 
NK 
cells, 
monocytes/macrophages, dendritic cells) by multi-parametric flow cytometry and 
mass cytometry. Expression of various activation and inhibitory markers on the TIL 
will also be assessed.  
  
Mutation burden will be determined by whole exome sequencing. PBMC will be 
used to obtain germline sequences for comparison to isolated tumor from tissue 
biopsies (somatic mutations). Isolated tumor DNA + RNA will then undergo tumor 
exome and tumor cDNA-capture sequencing, respectively, at The Genome Institute 
or through a commercially-available, CLIA/CLEP-certified assay.  

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10.0 
STUDY CALENDAR 
 
Screening/baseline evaluations are to be conducted no more than 4 weeks prior to start of protocol therapy. Cycles of pembrolizumab 
are 21 days.  There is a +/- 3 day window for each visit. 
 
Screening / 
baseline 
Pre- 
brachy 
Brachy 
Post- 
brachy 
C1D1 
C2D1 
Day 1 of each 
subsequent 
cycle 
End of 
every 3rd 
cycle 
3 mos1 
6 mos1 
12 mos1 
EOT 
F/U12 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
H&P, ECOG PS 
X 
 
 
 
X 
X 
X 
 
 
 
 
 
 
CBC 
X 
 
 
 
X 
X 
X 
 
 
 
 
 
 
CMP 
X 
 
 
 
X 
X 
X 
 
 
 
 
 
 
aPTT, PT/INR 
X 
 
 
 
 
 
 
 
 
 
 
 
 
β-hCG2 
X3 
 
 
 
X5 
 
 
 
 
 
 
 
 
T3, FT4, TSH 
X 
 
 
 
X 
 
X13 
 
 
 
 
 
 
Urinalysis 
X 
 
 
 
X 
 
X13 
 
 
 
 
 
 
PET/CT or CT or MRI 
X 
 
 
 
 
 
 
X 
 
 
 
X 
 
Brachytherapy 
 
 
X4 
 
 
 
 
 
 
 
 
 
 
Pembrolizumab 
 
 
 
 
X6 
X 
X 
 
 
 
 
 
 
Endoscopic biopsy 
 
X 
 
X7, 14 
 
 
 
 
X8,9 
 
 
 
Blood for PBMCs 
 
X 
 
X7 
 
X10 
 
 
X 
X 
X 
X11 
 
Survivor Follow-up 
 
 
 
 
 
 
 
 
 
 
 
 
X15 
AE assessment 
Patients will be followed for AEs for 30 days following the last dose of pembrolizumab. 
1. 
After the start of pembrolizumab. 
2. 
Women of childbearing potential only. 
3. 
No more than 72 hours before start of brachytherapy. 
4. 
2 fractions with 7-10 days between fractions. 
5. 
No more than 72 hours before start of pembrolizumab. 
6. 
Treatment with pembrolizumab will start approximately 1 week following the end of brachytherapy. 
7. 
Prior to first dose of pembrolizumab. 
8. 
8 patients only. 
9. 
2 to 6 months after the start of pembrolizumab. 
10. Prior to that day’s dose of pembrolizumab. 
11. Time of progression. 
12. Every 3 months for 12 months after patient comes off study. 
13. Every odd-numbered cycle. 
14. Optional 
15. Follow-up can be done via med review in the electronic medical record, phone calls, or medical request from an outside facility. 
 

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11.0 
DATA SUBMISSION SCHEDULE 
 
Case report forms with appropriate source documentation will be completed according to the 
schedule listed in this section. 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
On-Study Form 
Prior to starting treatment 
Brachytherapy Form 
With each fraction of brachytherapy 
Treatment Form 
End of each cycle 
Toxicity Form 
Continuous 
Correlatives Form 
Prior to brachytherapy 
Post-brachytherapy 
End of Cycle 2 
Beginning of Cycle 4 
3 Months 
6 Months 
12 Months 
Treatment Summary Form Completion of treatment 
Follow Up Form 
Every 3 months for 12 months 
MedWatch Form 
See Section 7.0 for reporting requirements 
*We are not capturing abnormal labs unless they are clinically significant as deemed by the investigator 
 
12.0 
MEASUREMENT OF EFFECT 
 
12.1 
Antitumor Effect – Solid Tumors 
 
For the purposes of this study, response and progression will be evaluated in this study 
using the Immune-Related Response Criteria (irRC) guidelines.   
 
12.2 
Immune-Related Response Criteria [Ref Wolchok Clin Cancer Res 2009] 
 
For the purposes of this study, patients should be re-evaluated for response/progression 
every 9 weeks. A confirmatory scan should also be obtained not less than 4 weeks 
following initial documentation of objective response. 
 
Response and progression will be evaluated in this study using immune-related response 
criteria. For the immune-related response criteria, only index and measurable new lesions 
are taken into account.  At the baseline tumor assessment, the sum of the products of the 
two largest perpendicular diameters of all index lesions (5 lesions per organ, up to 10 
visceral lesions and 5 cutaneous index lesions) is calculated.  At each subsequent tumor 
assessment, the sum of the products of the two largest perpendicular diameters of the index 
lesions and of new, measurable lesions (≥ 5 x 5 mm; up to 5 new lesions per organ: 5 

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cutaneous lesions and 10 visceral lesions) are added together to provide the total tumor 
burden: 
 
 
Tumor Burden = SPDindex lesions + SPDnew, measurable lesions 
 
Percentage changes in tumor burden per assessment time point describe the size and growth 
kinetics of both conventional and new, measurable lesions as they appear.  At each tumor 
assessment, the response in index and new, measurable lesions is defined based on the 
change in tumor burden (after ruling out immune-related progressive disease).  Decreases 
in tumor burden must be assessed relative to baseline measurements.  The immune-related 
response criteria were derived from WHO criteria and, therefore, the thresholds of response 
remain the same. 
 
The overall response according to the immune-related response criteria is derived from 
time-point response assessments (based on tumor burden) as follows: 
• irCR, complete disappearance of all lesions (whether measurable or not, and no 
new lesions) – confirmation by a repeat, consecutive assessment no less than 4 
weeks from the date first documented 
• irPR, decrease in tumor burden ≥ 50% relative to baseline – confirmed by a 
consecutive assessment at least 4 weeks after first documentation 
• irSD, not meeting criteria for irCR or irPR, in absence of irPD 
• irPD, increase in tumor burden ≥ 25% relative to nadir (minimum recorded tumor 
burden) – confirmation by a repeat, consecutive assessment no less than 4 weeks 
from the date first documented 
 
Patients are considered to have irPR or irSD even if new lesions are present as long as 
they meet the respective thresholds of response as described above.  Furthermore, 
patients are not considered to have irPD if new lesions are present and the tumor burden 
of all lesions does not increase by at least 25%. 
 
12.2.1 Treatment Beyond Progression 
Accumulating evidence indicates that a minority of patients treated with 
immunotherapy may derive clinical benefit despite initial evidence of 
radiographic PD. 
 
Patients will be permitted to continue on study treatment beyond initial 
radiographic PD, as long as they meet the following criteria: 
 
• Patient had investigator-assessed clinical benefit. 
• Patient has stable performance status. 
• Patient is tolerating study treatment. 
• Treatment beyond progression will not delay an imminent intervention to 
prevent serious complications of disease progression (eg, CNS metastases). 
 
The decision to continue study treatment beyond initial investigator-assessed 
progression should be clearly documented in the study records. A follow-up scan 

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should be performed at the next scheduled imaging evaluation (but no sooner than 
6 weeks) to determine whether there has been a decrease in the tumor size or 
continued PD. The assessment of clinical benefit should be balanced by clinical 
judgment as to whether the patient is clinically deteriorating and unlikely to 
receive any benefit from continued study treatment. If the investigator feels that 
the patient continues to achieve clinical benefit by continuing study treatment, the 
patient should remain on the study. 
 
12.3 
12.3 
Duration of Response 
 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
 
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that progressive disease is objectively documented. 
 
 
Duration of stable disease:  Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since the 
treatment started, including the baseline measurements.  
 
 
 

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13.0 
DATA AND SAFETY MONITORING 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months after accrual has opened (if at least five patients have been enrolled) or one 
year after accrual has opened (if fewer than five patients have been enrolled at the six-month mark). 
 
The Principal Investigator will review all patient data at least monthly (or before each dose-
escalation if occurring sooner than monthly), and provide a semi-annual report to the Quality 
Assurance and Safety Monitoring Committee (QASMC).  This report will include: 
• HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
• Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
• History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
• Study-wide target accrual and study-wide actual accrual 
• Protocol activation date 
• Average rate of accrual observed in year 1, year 2, and subsequent years 
• Expected accrual end date 
• Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
• Measures of efficacy 
• Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
• Summary of toxicities 
• Abstract submissions/publications 
• Summary of any recent literature that may affect the safety or ethics of the study 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
 
14.0 
STATISTICAL CONSIDERATIONS 
 
14.1 
Study Design 
 
This single arm, phase I study will primarily investigate the tolerability of combining 
pembrolizumab with locally delivered radiation therapy for the treatment of patients with 
metastatic esophageal carcinoma.  
 

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14.2 
Study Endpoints 
 
The primary endpoint is the safety as measured by treatment related adverse events. 
Secondary endpoints are progression-free survival (PFS) and overall survival (OS) for all 
the metastatic esophageal carcinoma patients, and the evaluation of antitumor efficacy 
including the total tumor size of all the target lesions based on irRC criteria, the length of 
esophageal tumor, the lumen size, and the dysphagia grade measured prior to 
brachytherapy, after brachytherapy/pre-pembrolizumab, and at 8 weeks post-
pembrolizumab. Exploratory endpoints are the quantified tumor-associated and systemic 
biomarkers and immune responses obtained through tumor tissue biopsies and peripheral 
blood at baseline (pre-brachytherapy), post-brachytherapy, and post-pembrolizumab.  
 
14.3 
Data Analysis 
 
Demographic, clinical characteristics and treatment related adverse events will be 
summarized using descriptive statistics. PFS and OS will be analyzed by Kaplan-Meier 
(KM) method. Paired t-test and/or paired-sample Wilcoxon Signed Rank test will used to 
compare the total tumor size of all the target lesions, tumor length, lumen size, dysphagia 
grade, and antitumor immune responses before and after hypofractionated brachytherapy 
and pembrolizumab therapy.  
 
14.4 
Power Analysis and Sample Size 
 
The primary objective of this study will be to evaluate the safety/tolerability of this novel 
radiation plus drug combination. A design has been chosen which will provide a reasonable 
ability to detect serious adverse events (SAE) and overall tolerability associated with the 
treatment. Sample size calculations for safety are expressed in terms of the ability to detect 
SAEs. The ability of the study to identify SAE/MTD is best expressed by the maximum 
true rate of events that would be unlikely to be observed and the minimum true rate of 
events that would very likely be observed. Specifically, for the sample size in the study 
(n=18 patients), there is at least 85% chance of observing at least 1 SAE/MTD if the true 
rate of such an event is at least 0.10. Conversely, there is at least 90% chance that we would 
not observe at least 1 SAE if the true rate is less than 0.006. The secondary objective of 
this trial is to evaluate the systemic response rate of the novel therapeutic approach. We 
have chosen to power the study to provide a reasonably reliable estimate of the ability of 
this approach to induce a positive outcome as measured by irRC criteria (please note that 
stable disease denotes efficacy by irRC criteria). As an example if we observe 5 responses 
out of 18 trial subjects, our 95% exact binomial confidence interval for the true rate will 
range from 0.10 - 0.53. If we observe 9 responses (50%), our 95% exact binomial 
confidence interval for the true rate will range from 0.26 to 0.74. 
 
14.5 
Accrual 
 
It is estimated 18 patients will be enrolled within 2 and a half years’ time.  
 
14.6 
Toxicity and Plans for Data and Safety Monitoring (Stopping Rule) 

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Toxicity will be reviewed on a continuous basis. Early stopping of this trial will be based 
on the excessive treatment related adverse events. By assuming that a toxicity rate of 1/6 
or less is acceptable and that a toxicity rate of 1/3 or more would definitely be unacceptable, 
based on the sequential probability ratio test (SPRT) with 80% power and 0.05 significance 
level, the study will be halted if 5 of the first 8, or 6 of the first 12 patients experience 
unacceptable toxicities, or if the 7th unacceptable toxicity is observed before the 15th patient 
has completed the trial. 
 
 
 

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We would like to thank the Alvin J. Siteman Cancer Center at Washington University School of 
Medicine and Barnes-Jewish Hospital in St. Louis, Missouri, for the use of the Clinical Trials 
Core which provided protocol development and study coordination services. The Siteman Cancer 
Center is supported in part by an NCI Cancer Center Support Grant #P30 CA91842. 
 
 
 

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APPENDIX A: ECOG Performance Status Scale 
 
 
Grade 
 
 
Description 
0 
Normal activity.  Fully active, able to carry on all pre-disease 
performance without restriction. 
1 
Symptoms, but ambulatory.  Restricted in physically strenuous 
activity, but ambulatory and able to carry out work of a light or 
sedentary nature (e.g., light housework, office work). 
2 
In bed <50% of the time.  Ambulatory and capable of all self-care, but 
unable to carry out any work activities.  Up and about more than 50% 
of waking hours. 
3 
 
In bed >50% of the time.  Capable of only limited self-care, confined 
to bed or chair more than 50% of waking hours. 
 
4 
100% bedridden.  Completely disabled.  Cannot carry on any self-care.  
Totally confined to bed or chair. 
5 
 
Dead.