Imaging the Flare Response With FDG PET/CT in Patients With Advanced Metastatic Melanoma on Pembrolizumab.
Conditions: Metastatic Melanoma
Sponsor: Abramson Cancer Center at Penn Medicine

 
1 
 
 
Title: 
Imaging the flare response with FDG PET/CT in patients with advanced 
metastatic melanoma on pembrolizumab. 
 
 
Principal Investigator: 
Michael Farwell, MD 
 
Assistant Professor of Radiology 
 
University of Pennsylvania 
 
Department of Radiology  
 
3400 Spruce Street 
 
Philadelphia, PA 19104 
 
215-662-7750 
 
 
Funding Sponsor:   
Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc. 
 
One Merck Drive 
 
Whitehouse Station, NJ 08889-0100 
 
USA 
 
 
 
Sub-Investigators: 
Tara Gangadhar, MD 
 
Alexander Huang, MD 
 
E. John Wherry, PhD 
 
Giorgos Karakousis, MD 
 
Lynn M. Schuchter, MD 
 
 
 
 
 
Ravi K. Amaravadi, MD 
 
 
Biostatistician: 
Rosemarie Mick, MS 
 
 
 
 
Current Version Date: October 18, 2017 
Previous Version Date: February 4, 2016 

 
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1.0 TRIAL SUMMARY 
Abbreviated Title 
Imaging the flare response from pembrolizumab with FDG PET/CT. 
Trial Phase 
Correlative  
Clinical Indication 
Patients with advanced metastatic melanoma who are starting 
pembrolizumab. 
Trial Type 
Prospective observational cohort study. 
Type of control 
No control. 
Route of administration 
n/a 
Trial Blinding 
None 
Treatment Groups  
n/a 
Number of trial subjects 
30 evaluable subjects  
Estimated enrollment period 
1 year 
Estimated duration of trial 
2 years 
Duration of Participation 
6 months 
Estimated average length of 
treatment per patient 
n/a 
 
 
 
 
2.0 TRIAL DESIGN 
2.1 
Trial Design 
This is a single institution, prospective, observational cohort study of FDG PET/CT as an early 
measure of response in patients with advanced metastatic melanoma scheduled to start 
pembrolizumab. Patients may participate in this study if they are greater than 18 years of age; most 
participants will be receiving care at the clinical practices of the University of Pennsylvania. Patients 
that may meet eligibility criteria will be approached about study participation regardless of race or 
ethnic background.  
We anticipate enrolling up to 35 participants with metastatic melanoma who meet eligibility 
requirements for this study. The target accrual goal is to reach 30 evaluable patients, defined as those 
who complete two (Baseline and 1st Post-therapy) of the three planned FDG PET/CT scans. Thus, 
accrual is estimated to occur over approximately 1 year. 
 
FDG PET/CT imaging will be used to evaluate glycolytic activity at sites of metastatic disease using 
the 
FDA 
approved 
clinical 
Positron 
Emission 
Tomography 
(PET) 
radiotracer, 
[18F]Fluorodeoxyglucose (FDG). Study imaging will be performed in a manner identical to standard 
clinical FDG PET/CT imaging procedures used at the Hospital of the University of Pennsylvania 
using a dedicated whole-body PET/CT scanner. For each PET scan static images from the vertex of 
the skull to toes will be acquired approximately 60 minutes after injection of FDG. Imaging data will 
be processed as per standard protocol. The study will be performed under the regulatory approval of 
the Penn Institutional Review Board (IRB). 
 
 

 
3 
 
A total of three FDG PET/CT exams will be performed as follows: 
• 
Baseline: A restaging FDG PET/CT will be obtained no more than 4 weeks prior to initiation 
of pembrolizumab to assess baseline, pre-therapy tumor glycolytic activity. This exam will be 
obtained for study purposes, although in some cases it may be obtained as clinical standard of 
care for restaging of disease prior to initiation of a new line of therapy. If this exam is 
obtained as clinical standard of care, it may occur prior to consent and enrollment in this 
study. 
 
• 
1st Post-therapy: at approximately 3 weeks (± 3 weeks) following the start of pembrolizumab, 
a post-therapy FDG PET/CT will be obtained. This exam will be obtained for study purposes, 
to assess glycolytic activity at sites of metastatic disease at an early time-point. 
 
• 
2nd Post-therapy: When clinically indicated, as per the judgment of the patient’s oncologist, a 
restaging FDG PET/CT will be obtained at > 10 weeks following the start of pembrolizumab. 
This exam is clinical standard of care for restaging of disease after initiation of a new line of 
therapy, and would be performed even if the patient was not enrolled in the study. In some 
cases MRI or CT imaging may be performed as standard of care for restaging disease instead 
of FDG PET/CT.  
 
Almost all of the patients treated for melanoma in the University of Pennsylvania Health System 
(UPHS) have consented to participate in the Penn Melanoma Tissue Collection Program (UPCC 
#08607, IRB #703001). For patients participating in this program, blood samples will be collected, 
processed, and stored according to the program’s standard operating procedure. Currently, this 
includes drawing less than 65 mL of blood according to the following schedule (after initiation of 
pembrolizumab): weeks 0, 3, 6, 9, 12, 15, and at 1 year. For the purposes of this study, patients will 
have an additional sample of blood drawn at approximately 1 week after starting pembrolizumab; this 
sample will also be collected, processed, and stored according to the Penn Melanoma Tissue 
Collection Program standard operating procedure. If patients have not consented to participate in the 
Penn Melanoma Tissue Collection Program, then no blood samples will be drawn for the purposes of 
this study. Analysis of blood samples will include, but is not limited to, analysis of serum cytokines, 
chemokines, and T cell markers. This data will be correlated to patient response as well as changes in 
tumor FDG uptake. Some of these studies will be performed upon receipt of additional funding. There 
may be patients for which some or all of these blood draws are not possible, in this case it will be 
documented in the CRF and the blood draws may be omitted and it will not be considered a protocol 
deviation. Experimental pathology results will not be reported to the participant and will have no 
impact on the treatment decisions made by the treating physician. 
 
Patients may undergo tumor biopsy as part of their clinical care prior to initiation of pembrolizumab. 
For patients participating in the Penn Melanoma Tissue Collection Program, these pre-therapy tissue 
samples will be collected, processed, and stored according to the program’s standard operating 
procedure. For the purposes of this study, patients will have a post-therapy tumor biopsy which will 
be scheduled within 7 days after the 1st Post-therapy FDG PET/CT; this tissue will also be collected, 
processed, and stored according to the Penn Melanoma Tissue Collection Program standard operating 
procedure. If patients have not consented to participate in the Penn Melanoma Tissue Collection 
Program, then no tumor biopsies will be performed for the purposes of this study. The biopsy 
techniques preferred for this study include core biopsies or surgical biopsies. The same biopsy 
techniques should be used as routinely used for a given tumor lesion/location. When possible, the 

 
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post-therapy tumor biopsy should target the same lesion that was biopsied pre-therapy; if the original 
lesion cannot be biopsied, then a lesion with a significant change in FDG activity should be targeted 
for biopsy. Analysis of tissue samples will include, but is not limited to, analysis of tumor-infiltrating 
lymphocytes (TILs) and macrophages. This data will be correlated to patient response as well as 
changes in tumor FDG uptake. Some of these studies will be performed upon receipt of additional 
funding. There may be patients for which a pre- or post- therapy tumor biopsy is not performed; those 
cases will be documented in the CRF and will not be considered a protocol deviation. In some cases 
tissue may be collected outside of the 7 day window following the 1st Post-therapy FDG PET/CT; this 
will not be considered a protocol deviation. Experimental pathology results will not be reported to the 
participant and will have no impact on the treatment decisions made by the treating physician. 
 
For patients participating in the Penn Melanoma Tissue Collection Program, we will follow the 
standard operating procedure regarding archival tissue access for patients undergoing a biopsy or 
surgery on melanoma or other skin cancer for diagnosing, treating or research purposes, but for which 
there is no applicable left over fresh tissue, or patients who had a biopsy or surgical procedure done 
on melanoma or other skin cancer for diagnosing, treating or research purposes at the University of 
Pennsylvania Health System (UPHS) or at an institution outside of UPHS. If patients have not 
consented to participate in the Penn Melanoma Tissue Collection Program, then no archival tissue 
will be requested. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
5 
Research FDG PET/CT scan 
(3 weeks ± 3 weeks post therapy start) 
 
2.2 
Trial Diagram 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 Pembrolizumab therapy will be given as part of the standard clinical care as determined by the treating 
oncology team. The blood draw at 1 week will be performed only if the patient is participating in this study and 
has also agreed to participate in the Penn Melanoma Tissue Collection Program, and may be omitted if it cannot 
be collected. The patient will remain evaluable for the primary endpoint even if the blood draw is omitted. 
2 Tumor tissue will be collected, processed, and stored according to the Penn Melanoma Tissue Collection 
Program standard operating procedure. If patients have not consented to participate in the Penn Melanoma 
Tissue Collection Program, then a tumor biopsy will not be performed for the purposes of this study. In some 
patients the tumor biopsy may not be performed, or may be performed outside of the 7 day window following 
the Research FDG PET/CT; omission/delay of the tumor biopsy will not be considered a protocol deviation. 
Consent and Enrollment 
FDG PET/CT 
Clinical Restaging Scan3 
(>10 weeks post therapy start) 
Long term follow up:          
progression-free survival and 
overall survival recorded 
FDG PET/CT 
Research FDG PET/CT scan 
(Baseline FDG PET/CT within 4 weeks 
prior to therapy start) 
FDG PET/CT 
Pembrolizumab started1 
(with a research blood draw on week 1) 
Post-therapy tumor tissue collection2 
(within 7 days after the Research FDG PET/CT) 

 
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3 The 2nd Post-Therapy scan will be ordered as a clinical restaging scan. In some cases MRI or CT imaging may 
be performed as standard of care for restaging disease instead of FDG PET/CT.  
3.0 OBJECTIVES & HYPOTHESES 
3.1 
Primary Objectives & Hypotheses 
(1) Objective:  To determine the distribution of changes from baseline in tumor FDG uptake 
on PET/CT at 3 weeks and >10 weeks after initiation of pembrolizumab. 
 
Hypothesis:  None, descriptive statistics. 
(2) Objective:  To evaluate the ability of FDG PET/CT to serve as an early measure of 
objective response to therapy in patients with melanoma on pembrolizumab. 
Hypothesis:  Melanoma metastases demonstrate changes in FDG uptake on a 3 week 
PET/CT scan that are associated with objective response to pembrolizumab. 
3.2 
Secondary Objectives & Hypotheses 
(1) Objective:  Based on changes in tumor FDG uptake at 3 weeks, determine which patients 
have FDG “flare” (an early increase in tumor FDG PET/CT uptake), and assess whether 
FDG “flare” is predictive of progression-free survival and overall survival in patients 
with melanoma on pembrolizumab. 
Hypothesis:  A flare response on FDG PET/CT performed 3 weeks after initiation of 
pembrolizumab is predictive of progression-free survival and overall survival in patients 
with melanoma. 
(2) Objective:  To collect tumor tissue pre- and post- treatment with pembrolizumab for 
correlation with changes in FDG uptake and objective response to therapy, and for 
studies of mechanism of action of PD-1 blockade with pembrolizumab. 
Hypothesis:  Treatment with pembrolizumab will result in a measurable change in tumor 
immune biomarkers, which will correlate with changes in tumor FDG uptake (including a 
“flare” response) and objective response to therapy, and will contribute to a better 
understanding of the mechanism of action of pembrolizumab. 
(3) Objective: To measure immune biomarkers in blood from subjects before and at serial 
time points after treatment with pembrolizumab, for correlation with changes in FDG 
uptake and objective response to therapy, and for studies of mechanism of action of PD-1 
blockade with pembrolizumab. 
Hypothesis:  Treatment with pembrolizumab will result in a measurable change in blood 
immune biomarkers, which will correlate with changes in tumor FDG uptake (including a 
“flare” response) and objective response to therapy, and will contribute to a better 
understanding of the mechanism of action of pembrolizumab. 

 
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(4) Objective:  To correlate changes in tumor FDG uptake (including a “flare” response), 
serum immune biomarkers, and tissue immune biomarkers with objective response to 
therapy, progression-free survival, and overall survival. Changes in FDG uptake within 
immune related tissues (lymph nodes, spleen, bone marrow, and thymus) will also be 
correlated with serum and tissue immune biomarkers, and response. 
Hypothesis:  Serum and tissue immune biomarkers provide additional predictive value 
regarding objective response to therapy, progression-free survival, and overall survival 
when combined with FDG PET/CT imaging data. 
4.0 BACKGROUND & RATIONALE 
4.1 
Rationale 
Recent clinical trials have shown great promise for a range of immunotherapy drugs, 
including anti-PD-1 therapy, in a variety of cancers. These therapies all share the common 
approach of activating the patient’s immune system towards the tumor. This differs from 
conventional cytotoxic systemic therapies which non-specifically target dividing cells, 
including immune cells. For example, anti-PD-1 drugs such as pembrolizumab target PD-1, a 
receptor expressed on immune cells, which mediates an immunosuppressive effect when 
allowed to interact with its ligands PDL1 and/or PDL2. Since many tumors express 
PDL1/PDL2, therapies such as pembrolizumab block this interaction and have the potential 
to unmask the tumor to the immune system. When successful, this leads to an immune 
infiltration of the tumor and ultimately tumor cell death. 
From the radiological perspective, following cancer response to immunotherapy presents 
unique challenges compared to conventional cytotoxic chemotherapies. Studies of anti-
CTLA4 therapy with ipilimumab in melanoma revealed that a significant subset of 
responding tumors may exhibit non-traditional patterns of response that can confound 
interpretation on CT (computed tomography).1 An example of a non-traditional response is 
the initial increase in size of the tumor over the course of several months with a later 
decrease in tumor size. In these cases, the initial increase in size of the tumor is thought to be 
on the basis of tumor infiltration by immune cells and can be mistaken for tumor growth on 
CT.  Another example of a non-traditional tumor response to immunotherapy is a decrease in 
the size of the primary tumor with the development of new small lesions. This pattern has 
also been observed to correspond with favorable outcome in the setting of immunotherapy 
but would be considered disease progression by conventional measures.  
An apparent increase in tumor burden that later resolves or decreases has been called a flare 
response, and is hypothesized to result from a transient immune-cell infiltrate in the tumor 
stroma. Several studies provide support for this hypothesis, including a case study of an 
ipilimumab-treated patient with apparent progressive disease at 12 weeks, in which the 
histological analysis of a lung nodule demonstrated a T-cell infiltrate, extensive necrosis, and 
no residual tumor cells.2 Thus, a different set of response criteria have been developed, 
termed immune-related response criteria (irRC), that incorporate the possibility of a transient 
flare response, as well as a delayed immune response, into the assessment.1,3 While irRC has 

 
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been shown to be an improvement over conventional RECIST (Response Evaluation Criteria 
In Solid Tumors) in the setting of immunotherapy, irRC still does not accurately identify all 
responders and non-responders. This is partly due to the fact that morphological changes to 
the tumor from immunotherapy can take several months to manifest. Thus, a more timely and 
accurate measure of response to immunotherapy is needed. 
FDG PET/CT as a marker of cancer and inflammation:  
2-Deoxy-2-[18F]fluoro-D-glucose (FDG) is an 18F analogue of glucose that serves as a PET 
radiotracer of cellular metabolism. FDG enters the cell through cell surface glucose 
transporters and becomes trapped within the cell following enzymatic phosphorylation by 
hexokinase. For decades, FDG has been employed to detect cancer exploiting a phenomenon 
known as the Warburg effect in which most malignancies preferentially produce ATP 
through a boost in extra-mitochondrial glycolysis, rather than mitochondrial oxidative 
phosphorylation, despite aerobic conditions. This metabolic switch, termed aerobic 
glycolysis, has a greater rate of glucose utilization albeit overall lower ATP yield, and 
provides the generation of key carbon precursors needed for the synthesis of nucleic acids, 
phospholipids, fatty acids, cholesterol and porphyrins.4 As a result of the increased rate of 
glycolysis characteristic of the Warburg effect, most malignancies utilize more glucose (and 
FDG) than their non-transformed neighboring cells, which has led to the use of FDG PET/CT 
as a highly sensitive and moderately specific tool for the detection of occult metastatic 
disease. 
It has recently been recognized that immune activation results in a similar cellular metabolic 
switch from oxidative phosphorylation to aerobic glycolysis. This is occurs with both the 
innate and adaptive immune activation. For example, when activated using TLR ligands or 
pro-inflammatory cytokines, neutrophils, dendritic cells, and macrophages have been 
demonstrated to switch their metabolism from oxidative phosphorylation to aerobic 
glycolysis.5 Similarly, following TCR stimulation, lymphocytes increase glucose utilization 
by several orders of magnitude and switch glucose metabolism from oxidative 
phosphorylation to aerobic glycolysis.6,7 Interestingly anti-inflammatory subpopulations of 
both the innate and adaptive immune system, such as M2 macrophages, regulatory T cells 
and quiescent memory T cells, demonstrate a predominant use of oxidative phosphorylation 
rather than aerobic glycolysis as opposed to their inflammatory counterparts such as activated 
macrophages and T-helper 17 cells.8   
The upregulation of aerobic glycolysis in immune activation is readily visualized with FDG 
PET/CT imaging. Inflammatory conditions such as infection, rheumatoid arthritis and 
sarcoidosis yield intense avidity for FDG that compares with or exceeds that of most 
malignancies.9,10 In fact, a significant limitation to the use of FDG PET/CT in cancer 
restaging has been interference of the tumor FDG signal by post-therapeutic inflammatory 
tissues including surgical changes, talc pleurodesis, and radiation therapy.11  
In this study we propose utilizing FDG PET/CT to assess the immunotherapy response by 
detecting the superimposed increased metabolism within the tumor presented by a therapy-
induced infiltrating population of glucose avid activated immune cells. Of note, a flare 

 
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phenomenon has been described in several cancer types following therapy; in patients with 
breast cancer on endocrine therapy, a clinical flare response was identified in 4 of 17 
responders and 1 of 34 nonresponders, and an FDG PET flare response was seen in 15 of 17 
responders and none of the 34 nonresponders.12 These results suggest that FDG PET is more 
sensitive and specific for detecting a flare response compared to clinical criteria, and the 
results also suggest that a flare response is predictive of response to therapy.   
FDG PET/CT studies are typically performed at approximately 12 weeks following the 
initiation of 
pembrolizumab, and 
the probability of 
seeing a flare 
response on these 
clinical studies is 
very low; this is 
supported by data 
presented at ASCO in 
2014, in which 3.6% 
of patients with 
melanoma 
demonstrated a flare response on imaging (≥25% increase in tumor burden) at 12 weeks. We 
propose looking for a flare response at about 3 weeks following the start of immunotherapy. 
The choice of this time point is considered appropriate for the activation and expansion of 
immune cells and is supported by data on pembrolizumab in non-small cell lung cancer, 
which revealed that circulating cytotoxic T cells specific to tumor neoantigens peak at around 
3 weeks from the initiation of therapy (Figure 1).13 Additional data on pembrolizumab in 
multiple other tumor types also found that activated cytotoxic T cells peak at around 3 
weeks.14  
No significant change is expected in the tumor’s inherent basal glycolytic activity over the 
short interval between the baseline FDG PET/CT and the 3 week PET/CT. Therefore we 
hypothesize that the flare response will be able to be visualized with FDG PET/CT by 
detection of a superimposed burst of tumoral glycolytic activity resulting from 
immunotherapy induced immune infiltration. If successful, this study will help to determine 
the ideal time point for visualization of the flare response, test the potential for the flare 
response to serve as a biomarker for detecting an early response to therapy, and contribute to 
our understanding of pembrolizumab’s mechanism of action.   
In addition, studies in human tumor tissue are needed to define the mechanism of action of 
PD-1 inhibition, and provide an explanation for observed changes in tumor FDG uptake. In 
this study we propose collecting pre- and post- treatment tumor tissue in which the post-
treatment tumor tissue is acquired within 7 days following the 3 week PET/CT. We also 
propose acquiring blood samples pre-therapy, and at serial time-points during therapy 
(including a 1 week sample, which is earlier than other studies have explored to date). 
Analysis of these samples will include, but is not limited to, serum cytokines, chemokines, 
and T cell markers, and tumor-infiltrating lymphocytes (TILs) and macrophages; some of 

 
10 
these studies will be performed upon receipt of additional funding. Of note, some of these 
studies will be informed by, and/or modeled after the current study at Penn titled, “A Phase 
Ib Tissue Collection Study of Pembrolizumab (MK-3475) in Subjects with Resectable 
Advanced Melanoma.” This data will be correlated to patient response as well as changes in 
tumor FDG uptake. Thus, we expect the proposed study to generate mechanism of action 
data, and help identify factors that predict response to treatment with PD-1 inhibition, which 
may allow for more strategic development of combination immune therapy strategies, and 
ultimately may help to increase objective response rates to immune therapy in melanoma and 
other tumor types. 
4.1.1 Dose Rationale and Risk/Benefits Assessment 
2-Deoxy-2-[18F]fluoro-D-glucose (FDG) is FDA approved for imaging in clinical oncology, 
and has been extensively used in oncologic PET imaging with no evidence of 
pharmacological effect. No adverse events (AEs) have been reported in the large number of 
published studies of human experience for FDG at the dose to be used in this study. In this 
study we will administer approximately 15 mCi of FDG according to the standard procedures 
used for clinical FDG PET/CT at the Hospital of the University of Pennsylvania.  
Risk/Benefit 
Participants will undergo up to two additional FDG PET/CT examinations for the purposes 
of this study. This will be in addition to other clinically indicated CT and/or PET/CT 
examinations. FDG is a positron emitting radiopharmaceutical. As such, it poses an intrinsic 
radiation exposure risk. However, when administered in low tracer doses as a PET imaging 
agent, as described in this protocol, this risk is felt to be extremely small. In addition to the 
radioactive FDG tracer, a low-dose CT of the chest, abdomen and pelvis is typically 
performed with the FDG PET/CT for the purpose of anatomical registration and attenuation 
correction of PET images. There are no separate diagnostic CT scans performed as part of 
this scan. FDG PET/CT scans are a standard clinical procedure for patients with metastatic 
melanoma. This research involves exposure to radiation that could affect a fetus or pregnant 
woman, due to this risk a urine pregnancy test will be performed for women of childbearing 
potential at the time of screening. Women who are pregnant at the time of screening will not 
be included in this study. 
The dose will be delivered intravenously by skilled clinical professionals who typically 
administer FDG for clinical purposes; venous cannulation is a routine clinical procedure that 
carries minimal risks when performed by trained personnel. It is possible that bruising, 
dizziness or fainting could occur in some subjects. There is a risk of phlebitis or infection, 
which is very remote. Also, the PET/CT scan takes place in a small, enclosed space and 
therefore can be uncomfortable for some people with claustrophobia or musculoskeletal 
disorders (such as arthritis). Subjects will be made as comfortable as possible and staff will 
be available throughout the imaging to address any discomfort. 
No psychological, social or legal risk is expected. While loss of confidentiality is possible, it 
is felt to be very unlikely due to the small number of professionals involved in the study with 

 
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knowledge of this information. All clinicians and research staff involved are well trained in 
HIPAA practices. There is no anticipated additional financial risk to the patient due to 
participation in this study since the two additional study FDG PET/CT’s, 1 week blood draw 
(if performed), and post-therapy tumor biopsy (if performed) will be paid for through study 
funds. The clinical restaging FDG PET/CT examinations and pre-therapy tumor biopsy (if 
performed) will be paid for through the patient’s insurance as per routine clinical care for 
their cancer. Additional blood draws described in this protocol outside of the 1 week sample 
will be paid for by the Penn Melanoma Tissue Collection Program. 
The risk to the patient is felt to be minimal with only the added radiation considered the 
likely risk posed to the patient. It is not unusual for oncologic patients to receive multiple 
radiological tests, including multiple PET/CT scans, in the course of clinical management 
and the addition of two FDG PET/CT scans is not considered to add a large risk to the 
subject. This study proposes the addition of two PET/CT scans to the patient’s care. This 
study also proposes the addition of one tumor biopsy to the patient’s care; this procedure 
carries a low risk of bleeding or infection at the biopsy site, thus the additional risk to the 
patient from this procedure is felt to be minimal. 
There is no anticipated benefit to study subjects as a result of their participation in this study. 
There is potential benefit to general society if FDG PET/CT proves to be a useful imaging 
agent for detecting an early response to immunotherapy for cancer, or helps in the 
understanding of pembrolizumab’s mechanism of action.  
4.1.2 Rationale for Endpoints 
4.1.2.1 Primary Endpoint 
Objective response to therapy at 6 months, based on imaging and the clinical evaluation of 
the patient, is an acceptable primary endpoint in this prospective, observational cohort study 
of FDG PET/CT as an early measure of response in patients with advanced metastatic 
melanoma. Response will be determined by RECIST version 1.1. While either CT or MRI 
may be utilized, as per RECIST 1.1, CT is the preferred imaging technique in this study. 
Patients will be categorized as responders (i.e., complete response or partial response) or 
nonresponders (stable disease or progressive disease). 
4.1.2.2 Secondary Endpoints 
Progression free survival (PFS), is defined as the time from initiation of Pembrolizumab to 
documented disease progression, death due to any cause or last patient follow-up that 
documented lack of disease progression.  
Overall survival, is defined as start date of Pembrolizumab until death from any cause or last 
patient contact alive. Survival data will be obtained from the electronic medical records.  
 

 
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4.1.2.3 Biomarker Research 
Almost all of the patients treated for melanoma in the University of Pennsylvania Health 
System (UPHS) have consented to participate in the Penn Melanoma Tissue Collection 
Program (UPCC #08607, IRB #703001). For patients participating in this program, blood 
samples will be collected, processed, and stored according to the program’s standard 
operating procedure. Currently, this includes drawing less than 65 mL of blood according to 
the following schedule (after initiation of pembrolizumab): weeks 0, 3, 6, 9, 12, 15, and at 1 
year. For the purposes of this study, patients will have an additional sample of blood drawn at 
approximately 1 week after starting pembrolizumab; this sample will also be collected, 
processed, and stored according to the Penn Melanoma Tissue Collection Program standard 
operating procedure. If patients have not consented to participate in the Penn Melanoma 
Tissue Collection Program, then no blood samples will be drawn for the purposes of this 
study. Analysis of blood samples will include, but is not limited to, analysis of serum 
cytokines, chemokines, and T cell markers. This data will be correlated to patient response as 
well as changes in tumor FDG uptake. Some of these studies will be performed upon receipt 
of additional funding. There may be patients for which some or all of these blood draws are 
not possible, in this case it will be documented in the CRF and the blood draws may be 
omitted and it will not be considered a protocol deviation. Experimental pathology results 
will not be reported to the participant and will have no impact on the treatment decisions 
made by the treating physician. 
 
Patients may undergo tumor biopsy as part of their clinical care prior to initiation of 
pembrolizumab. For patients participating in the Penn Melanoma Tissue Collection Program, 
these pre-therapy tissue samples will be collected, processed, and stored according to the 
program’s standard operating procedure. For the purposes of this study, patients will have a 
post-therapy tumor biopsy which will be scheduled within 7 days after the 1st Post-therapy 
FDG PET/CT; this tissue will also be collected, processed, and stored according to the Penn 
Melanoma Tissue Collection Program standard operating procedure. If patients have not 
consented to participate in the Penn Melanoma Tissue Collection Program, then no tumor 
biopsies will be performed for the purposes of this study. The biopsy techniques preferred for 
this study include core biopsies or surgical biopsies. The same biopsy techniques should be 
used as routinely used for a given tumor lesion/location. When possible, the post-therapy 
tumor biopsy should target the same lesion that was biopsied pre-therapy; if the original 
lesion cannot be biopsied, then a lesion with a significant change in FDG activity should be 
targeted for biopsy. Alternatively, the largest lesion or most accessible lesion may be 
biopsied. Analysis of tissue samples will include, but is not limited to, analysis of tumor-
infiltrating lymphocytes (TILs) and macrophages. This data will be correlated to patient 
response as well as changes in tumor FDG uptake. Some of these studies will be performed 
upon receipt of additional funding. There may be patients for which a pre- or post- therapy 
tumor biopsy is not performed; those cases will be documented in the CRF and will not be 
considered a protocol deviation. In some cases tissue may be collected outside of the 7 day 
window following the 1st Post-therapy FDG PET/CT; this will not be considered a protocol 
deviation. Experimental pathology results will not be reported to the participant and will have 
no impact on the treatment decisions made by the treating physician. 

 
13 
 
For patients participating in the Penn Melanoma Tissue Collection Program, we will follow 
the standard operating procedure regarding archival tissue access for patients undergoing a 
biopsy or surgery on melanoma or other skin cancer for diagnosing, treating or research 
purposes, but for which there is no applicable left over fresh tissue, or patients who had a 
biopsy or surgical procedure done on melanoma or other skin cancer for diagnosing, treating 
or research purposes at the University of Pennsylvania Health System (UPHS) or at an 
institution outside of UPHS. If patients have not consented to participate in the Penn 
Melanoma Tissue Collection Program, then no archival tissue will be requested. 
 
 
5.0 METHODOLOGY 
5.1 
Entry Criteria 
5.1.1 Diagnosis/Condition for Entry into the Trial 
Melanoma. 
5.1.2 Subject Inclusion Criteria 
In order to be eligible for participation in this trial: 
1. The subject must be informed of the investigational nature of this study and provide 
written informed consent in accordance with institutional and federal guidelines prior 
to study-specific procedures. 
2. The subject must be ≥ 18 years of age on day of signing informed consent. 
3. The subject must have biopsy proven or clinically documented advanced stage 
metastatic melanoma.   
4. The subject must have measurable disease (per RECIST 1.1) that is seen on CT, MRI, 
or FDG PET/CT. 
5. The subject must be recommended to start pembrolizumab. 
5.1.3 Subject Exclusion Criteria 
The subject must be excluded from participating in the trial if: 
1. Females who are pregnant or breast-feeding at the time of screening will not be 
eligible for this study. Female participants of child-bearing potential will have a urine 
pregnancy test at the time of the screening visit. 

 
14 
2. Subject is not able to tolerate imaging procedures in the opinion of the investigator or 
treating physician. 
3. Subject has a history or current evidence of any condition, therapy, or laboratory 
abnormality that might confound the results of the trial, interfere with the subject’s 
participation for the full duration of the trial, or is not in the best interest of the 
subject to participate, in the opinion of the treating investigator.   
4. Subject has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 
agent as assessed by medical record review and/or self-reported. 
5. Subject has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 
antibodies) as determined by medical record review. 
6. Subject has a diagnosis of immunodeficiency or is receiving systemic steroid therapy 
or any other form of immunosuppressive therapy within 7 days prior to the first dose 
of trial treatment. 
 
7. Subject has known active Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., 
HCV RNA [qualitative] is detected) as determined by medical record review. 
8. Subject has known active central nervous system metastases and/or carcinomatous 
meningitis. 
Note: Subjects with previously treated brain metastases will be eligible to 
participate provided they are stable (without evidence of progression by 
imaging for at least four weeks prior to receiving pembrolizumab 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. 
 
5.2 
Subject Recruitment and Screening 
Patients will generally be offered enrollment at the time of their office visit to oncology at the 
Hospital of the University of Pennsylvania. Study personnel or an investigator will present 
the patient with information regarding the study and enrollment. Enrollment into the study 
will be presented as optional and will not impact clinical management or choice of 
therapeutic intervention. It will be emphasized that enrollment in the study is not mandatory 
and that failure to enroll will not result in prejudice by the treating clinicians and will not 
affect patient care. 
All patients being considered for the study and eligible for screening must sign an informed 
consent for the study prior to any study specific procedures. Patients will generally have their 
baseline FDG PET/CT performed as a research scan. Occasionally, the baseline FDG 
PET/CT may be ordered by a treating physician as part of standard medical care, in which 
case the scan may occur before the patient is consented and enrolled in this study. 

 
15 
Information from this baseline FDG PET/CT scan will be compared to the post-therapy FDG 
PET/CT scans. FDG PET/CT scans performed at an institution outside of University of 
Pennsylvania health system will not be able to be used in this study. 
5.3 
Subject Withdrawal/Discontinuation Criteria 
The criteria for enrollment must be followed explicitly.  If a subject who does not meet 
enrollment criteria is inadvertently enrolled, that subject should be discontinued from the 
study.  A subject may voluntarily discontinue participation in this study at any time.  The 
investigator may also, at his or her discretion, discontinue the subject from participating in 
this study at any time.  If a subject is prematurely discontinued from participation in the 
study for any reason, at any time, at either the investigator’s discretion or the subject’s 
request, an effort must be made to document the reason(s) why a subject fails to return to the 
study clinic for necessary visits or is discontinued from the study. 
The primary reason for discontinuing participation in the study may include, but is not 
limited to, one of the following: 
• The subject or legal representative (such as a parent or legal guardian) withdraws 
consent. 
• Development of an intercurrent illness, injury, or medical condition likely to interfere 
with subject safety or the overall assessment. 
• Noncompliance with protocol, e.g., the patient fails to appear at one or more imaging 
procedures. 
• Development of any condition for which the investigator feels study withdrawal is 
justified.  
• The subject has a confirmed positive serum pregnancy test 
• The subject is lost to follow-up. 
• Termination of the study.  
Follow-up information will be obtained for subjects who discontinue the study. Additional 
details are listed in Section 7.1.4 (Visit Requirements).  
Subjects may withdraw from the study at any time at their own request, or they may be 
withdrawn at any time at the discretion of the investigator or sponsor for safety, behavioral, 
or administrative reasons. If the subject withdraws from the study, and also withdraws 
consent for disclosure of future information, no further evaluations should be performed, and 
no additional data should be collected. The investigator may retain and continue to use any 
data collected before such withdrawal of consent. 

 
16 
 
 
 
6.0 TRIAL FLOW CHART 
6.1 
Study Flow Chart 
 
Screening Phase 
Baseline FDG 
PET/CT4 
1st Post-therapy FDG 
PET/CT 
Tumor Tissue 
Collection5 
2nd Post-therapy FDG 
PET/CT6 
Scheduling window relative to 
pembrolizumab initiation: 
 
< 4 weeks prior  
3 weeks after (± 3 weeks) 
 
> 10 weeks after 
Administrative Procedures 
Informed Consent 
X 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
 
Demographics and Medical History 
X 
 
 
 
 
Clinical Procedures/Assessments 
Review Adverse Events1 
 
 
X 
X 
 
Laboratory Procedures/Assessments: analysis performed on site 
Pregnancy Test – Urine or Serum β-HCG 
X 
 
 
 
 
Tumor Imaging and Assessment of Disease 
FDG PET/CT2 
 
X 
X 
 
X 
Tumor Biopsies/Archival Tissue Collection/Correlative Studies Blood 
Archival or Newly Obtained Tissue 
Collection 
X 
 
 
X 
 
Correlative Studies Blood Collection3 
 
 
 
 
 
 
1Adverse events related to the research PET/CT will only be assessed for the period of time from the injection of FDG to the completion of the imaging exam. AEs related to the 
clinical treatment will not be collected or reported as part of this study. 
2All FDG PET/CT scans will be performed as per routine clinical procedure. 
3Less than 65 mL of blood will be collected after initiation of pembrolizumab according to the following schedule: weeks 0, 1, 3, 6, 9, 12, 15, and at 1 year; specimens will be 
processed and stored according to the Penn Melanoma Program tissue collection Standard Operating Procedure. The week 1 blood sample will be collected only for the purposes 
of this study. If blood samples cannot be collected they will be omitted and not considered a protocol deviation. 
4This scan will usually be performed as a research exam. Occasionally, it may be performed as a clinically indicated restaging exam, which may occur prior to consent and 
enrollment in this study. 
5Tumor tissue is to be collected within 7 days after the 1st Post-therapy FDG PET/CT. The biopsy techniques preferred for this study include core biopsies or surgical biopsies. The 
same biopsy techniques should be used as routinely used for a given tumor lesion/location and specimens will be processed and stored according to the Penn Melanoma Program 

 
17 
 
 
 
tissue collection Standard Operating Procedure. When possible, the post-therapy tumor biopsy should target the same lesion that was biopsied pre-therapy; if the original lesion 
cannot be biopsied, then a lesion with a significant change in FDG activity should be targeted for biopsy. If patients have not consented to participate in the Penn Melanoma Tissue 
Collection Program, then a tumor biopsy will not be performed for the purposes of this study. In some patients the tumor biopsy may not be performed, or may be performed 
outside of the 7 day window following the 1st Post-therapy FDG PET/CT; omission/delay of the tumor biopsy will not be considered a protocol deviation. 
6This scan is a clinically indicated restaging exam. In some cases MRI or CT imaging may be performed as standard of care for restaging disease instead of FDG PET/CT. 
 
 
 

 
 
 
 
 
18 
 
7.0 TRIAL PROCEDURES 
7.1 
Trial Procedures 
The Trial Flow Chart - Section 6.0 summarizes the trial procedures to be performed at each 
visit.  Individual trial procedures are described in detail below.  It may be necessary to 
perform these procedures at unscheduled time points if deemed clinically necessary by the 
investigator. 
Furthermore, additional evaluations/testing may be deemed necessary by the Sponsor and/or 
Merck for reasons related to subject safety. In some cases, such evaluation/testing may be 
potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may 
require that additional informed consent be obtained from the subject.  In these cases, such 
evaluations/testing will be performed in accordance with those regulations. 
7.1.1 Administrative Procedures 
7.1.1.1 Informed Consent 
The Investigator or delegate must obtain documented consent from each potential subject 
prior to participating in a clinical trial. 
7.1.1.1.1  General Informed Consent 
Consent must be documented by the subject’s dated signature or by the subject’s legally 
acceptable representative’s dated signature on a consent form along with the dated signature 
of the person conducting the consent discussion.  
A copy of the signed and dated consent form should be given to the subject before 
participation in the trial. 
The initial informed consent form, any subsequent revised written informed consent form 
and any written information provided to the subject must receive the IRB/ERC’s 
approval/favorable opinion in advance of use.  The subject or his/her legally acceptable 
representative should be informed in a timely manner if new information becomes available 
that may be relevant to the subject’s willingness to continue participation in the trial.  The 
communication of this information will be provided and documented via a revised consent 
form or addendum to the original consent form that captures the subject’s dated signature or 
by the subject’s legally acceptable representative’s dated signature. 
Specifics about a trial and the trial population will be added to the consent form template at 
the protocol level.   
The informed consent will adhere to IRB/ERC requirements, applicable laws and regulations 
and Sponsor requirements. 
 

 
 
 
 
 
19 
 
7.1.1.2 Inclusion/Exclusion Criteria 
All inclusion and exclusion criteria will be reviewed by the investigator or qualified designee 
to ensure that the subject qualifies for the trial.  
7.1.1.3 Medical History 
A baseline medical history and physical exam will be collected from the medical record from 
standard clinical visits with the treating medical oncologist. 
7.1.2 Clinical Procedures/Assessments 
7.1.2.1 Adverse Event (AE) Monitoring 
The investigator or qualified designee will assess each subject to evaluate for potential new 
or worsening AEs as specified in the Trial Flow Chart. Adverse events that are grade 3 or 
higher will be recorded. Adverse events related to the research PET/CT will only be assessed 
for the period of time from the injection of FDG to the completion of the imaging exam. AEs 
related to the clinical treatment will not be collected or reported as part of this study. 
Please refer to section 7.2 for detailed information regarding the assessment and recording of 
AEs.   
7.1.2.2 Tumor Imaging and Assessment of Disease 
The following procedures will be done at each imaging session, as per routine clinical 
practice for FDG PET/CT imaging. 
• 
Injection of FDG performed as per routine clinical procedure. 
All women of child-bearing potential will be asked on the day of the PET/CT scan if they 
might be pregnant; this is a standard question for all patients who will be undergoing 
PET/CT scans due to the radiation exposure associated with the scan. If the patient is unsure 
about whether she might be pregnant then a urine pregnancy test will be performed prior to 
the injection of FDG.  
The patient will be made comfortable in a preparatory room. Approximately 15 mCi of FDG 
will be administered according to the standard procedures used for clinical FDG PET/CT at 
the Hospital of the University of Pennsylvania. All patients will undergo a vertex of skull to 
toes PET/CT scan starting at approximately 60 minutes after FDG injection. A brief low-dose 
CT scan will be acquired according to standard PET/CT imaging procedures; this is used for 
attenuation correction and anatomical localization of findings in the PET scan. This can be 
performed either before or after the PET transmission scan. There are no separate diagnostic 
CT scans performed as part of this research.  
A total of three FDG PET/CT exams will be performed as follows: 

 
 
 
 
 
20 
 
• Baseline: A restaging FDG PET/CT will be obtained no more than 4 weeks prior to 
initiation of pembrolizumab to assess baseline, pre-therapy tumor glycolytic activity. 
This exam will be obtained for study purposes, although in some cases it may be 
obtained as clinical standard of care for restaging of disease prior to initiation of a 
new line of therapy. If this exam is obtained as clinical standard of care, it may occur 
prior to consent and enrollment in this study. 
 
• 1st Post-therapy: at approximately 3 weeks (± 3 weeks) following the start of 
pembrolizumab, a post-therapy FDG PET/CT will be obtained. This exam will be 
obtained for study purposes, to assess glycolytic activity at sites of metastatic disease 
at an early time-point. 
 
• 2nd Post-therapy: When clinically indicated, as per the judgment of the patient’s 
oncologist, a restaging FDG PET/CT will be obtained at > 10 weeks following the 
start of pembrolizumab. This exam is clinical standard of care for restaging of disease 
after initiation of a new line of therapy, and would be performed even if the patient 
was not enrolled in the study. In some cases MRI or CT imaging may be performed 
as standard of care for restaging disease instead of FDG PET/CT.  
 
7.1.2.3 Tumor Tissue Collection and Correlative Studies Blood Sampling 
For patients participating in the Penn Melanoma Tissue Collection Program (UPCC #08607, 
IRB #703001), blood samples will be collected, processed, and stored according to the 
program’s standard operating procedure. Currently, this includes drawing less than 65 mL of 
blood according to the following schedule (after initiation of pembrolizumab): weeks 0, 3, 6, 
9, 12, 15, and at 1 year. For the purposes of this study, patients will have an additional 
sample of blood drawn at approximately 1 week after starting pembrolizumab; this sample 
will also be collected, processed, and stored according to the Penn Melanoma Tissue 
Collection Program standard operating procedure. If patients have not consented to 
participate in the Penn Melanoma Tissue Collection Program, then no blood samples will be 
drawn for the purposes of this study. Analysis of blood samples will include, but is not 
limited to, analysis of serum cytokines, chemokines, and T cell markers; proposed cytokines 
and chemokines are listed in Table 1, and proposed T cell markers are listed in Table 2. This 
data will be correlated to patient response as well as changes in tumor FDG uptake. Some of 
these studies will be performed upon receipt of additional funding. There may be patients for 
which some or all of these blood draws are not possible, in this case it will be documented in 
the CRF and the blood draws may be omitted and it will not be considered a protocol 
deviation. Experimental pathology results will not be reported to the participant and will have 
no impact on the treatment decisions made by the treating physician. 
 
Patients may undergo tumor biopsy as part of their clinical care prior to initiation of 
pembrolizumab. For patients participating in the Penn Melanoma Tissue Collection Program, 
these pre-therapy tissue samples will be collected, processed, and stored according to the 
program’s standard operating procedure. For the purposes of this study, patients will have a 
post-therapy tumor biopsy which will be scheduled within 7 days after the 1st Post-therapy 

 
 
 
 
 
21 
 
FDG PET/CT; this tissue will also be collected, processed, and stored according to the Penn 
Melanoma Tissue Collection Program standard operating procedure. If patients have not 
consented to participate in the Penn Melanoma Tissue Collection Program, then no tumor 
biopsies will be performed for the purposes of this study. The biopsy techniques preferred for 
this study include core biopsies or surgical biopsies. The same biopsy techniques should be 
used as routinely used for a given tumor lesion/location. When possible, the post-therapy 
tumor biopsy should target the same lesion that was biopsied pre-therapy; if the original 
lesion cannot be biopsied, then a lesion with a significant change in FDG activity should be 
targeted for biopsy. Analysis of tissue samples will include, but is not limited to, analysis of 
tumor-infiltrating lymphocytes (TILs), macrophages, and other myeloid subsets; changes in 
tumor cells will also be evaluated, and the fraction of necrotic cells will be calculated. TILs 
will be analyzed using multiparameter flow cytometry; proposed phenotypic markers are 
listed in Table 2. This data will be correlated to patient response as well as changes in tumor 
FDG uptake. Some of these studies will be performed upon receipt of additional funding. 
There may be patients for which a pre- or post- therapy tumor biopsy is not performed; those 
cases will be documented in the CRF and will not be considered a protocol deviation. In 
some cases tissue may be collected outside of the 7 day window following the 1st Post-
therapy FDG PET/CT; this will not be considered a protocol deviation. Experimental 
pathology results will not be reported to the participant and will have no impact on the 
treatment decisions made by the treating physician. 
 
For patients participating in the Penn Melanoma Tissue Collection Program, we will follow 
the standard operating procedure regarding archival tissue access for patients undergoing a 
biopsy or surgery on melanoma or other skin cancer for diagnosing, treating or research 
purposes, but for which there is no applicable left over fresh tissue, or patients who had a 
biopsy or surgical procedure done on melanoma or other skin cancer for diagnosing, treating 
or research purposes at the University of Pennsylvania Health System (UPHS) or at an 
institution outside of UPHS. If patients have not consented to participate in the Penn 
Melanoma Tissue Collection Program, then no archival tissue will be requested. 
 
Chemokines 
Inflammatory Cytokines 
Inhibitory Cytokines 
Mip1a 
IFNg 
IL4 
Mip1b 
TNF 
IL10 
Rantes 
IL1 
TGFbeta 
IP10 
IL2 
 
ITAC 
IL6 
 
Fractalkine 
IL12 
 
 
IL17 
 
 
IL23 
 
  Table 1. Proposed cytokine / chemokine analysis. 
 
 

 
 
 
 
 
22 
 
 
Lineage 
Markers 
Differentiation 
Transcription Factors 
Inhibitory Receptors 
Function 
CD3 
CD27 
Tbet 
PD-1 
Granzyme B 
CD4 
CD45RA 
Eomes 
Tim3 
Ki67 
CD8 
 
 
LAG-3 
Perforin 
FoxP3 
 
 
CD160 
 
CD14 
 
 
CTLA4 
 
CD16 
 
 
KLRG1 
 
CD19 
 
 
SAP 
 
 
 
 
CD244 
 
 
 
 
TIGIT 
 
Table 2. Proposed flow cytometric analysis. 
 
 
7.1.3 Laboratory Procedures/Assessments 
A urine pregnancy test will be performed on women of childbearing potential only at 
screening.  
 
7.1.4 Visit Requirements 
Visit requirements are outlined in Section 6.0 - Trial Flow Chart.   Specific procedure-related 
details are provided above in Section 7.1 - Trial Procedures. 
7.1.4.1 Screening 
The investigator will provide for the protection of the subjects by following all applicable 
regulations.  These regulations are available upon request from the sponsor.  The sponsor and 
IRB must review the informed consent form used during the informed consent process, and it 
must be available for inspection.   
Before any procedures specified in this protocol are performed, a subject must: 
• 
Be informed of all pertinent aspects of the study and all elements of informed 
consent.  
• 
Be given time to ask questions and time to consider the decision to participate. 
• 
Voluntarily agree to participate in the study.  
• 
Sign and date an IRB-approved informed consent form.    

 
 
 
 
 
23 
 
Patients will usually have their baseline FDG PET/CT performed as a research scan. 
Occasionally, the baseline FDG PET/CT may be ordered by a treating physician as part of 
standard medical care, in which case the scan may occur before the patient is consented and 
enrolled in this study. Information from this baseline FDG PET/CT scan will be compared to 
the post-therapy FDG PET/CT scans. FDG PET/CT scans performed at an institution outside 
of University of Pennsylvania health system will not be able to be used in this study. 
The following additional patient data will be obtained:  demographics (including gender, date 
of birth, height, weight, and race), histologic diagnosis, and year of diagnosis. Correlative 
pertinent imaging including CT, MRI, x-ray, ultrasound, FDG PET/CT, or bone scan may be 
reviewed if necessary to confirm eligibility. For patients referred from outside the University 
of Pennsylvania health system, patient records, biopsy material, and imaging will be 
reviewed to determine eligibility for the study.  For women of childbearing potential a urine 
pregnancy test will be performed at the screening visit. 
7.1.4.2 Clinical Follow-up 
Clinical follow-up may include standard of care imaging, tumor markers and standard visits 
with a treating physician at intervals standard for metastatic melanoma follow-up after the 
start of new treatment, and usually at the time of symptomatic progression. The timing of 
clinical visits will be determined by the treating physician and is not mandated by this study. 
Information will be collected through medical record review to assess for status of disease 
and the date of progression will be documented at the time of clinical progression. Clinical 
follow up for overall survival, by medical record review, will continue until loss to follow up 
or death. For those whose are cared for by a non-UPHS clinician during this period, this 
clinician will be contacted by phone for follow-up. The patients may also be contacted by 
phone for follow-up. 
7.1.5 Image Interpretation 
Static images will be reconstructed using standard procedure and analyzed by visual 
inspection and standardized uptake value (SUV) analysis. The tumor avidity, as estimated by 
the SUVmax, of the primary tumor and up to 5 metastatic lesions will be measured on each 
FDG PET/CT scan. Patients will be grouped into responders (partial/complete response) and 
nonresponders (stable disease or progressive disease) based on RECIST. Patients will also be 
grouped based on a positive flare response (greater than 20% increase in SUVmax) or 
negative flare response (less than 20% increase in SUVmax. An exploratory tumor-by-tumor 
FDG PET/CT response analysis will be performed for the primary tumor and up to 5 
metastatic lesions in each patient. Changes in FDG uptake within immune related tissues 
(lymph nodes, spleen, bone marrow, and thymus) will also be measured as an exploratory 
objective using SUVmax and SUVmean. 

 
 
 
 
 
24 
 
7.2 
Assessing and Recording Adverse Events 
An adverse event is defined as any untoward medical occurrence in a patient or clinical 
investigation subject that develops or worsens in severity during the course of the study. An 
adverse event can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding, for example), symptom, or disease temporally associated with the use of a 
protocol-specified procedure, whether or not considered related to the protocol-specified 
procedure.  Any worsening (i.e., any clinically significant adverse change in frequency 
and/or intensity) of a preexisting condition that is temporally associated with the study 
period, is also an adverse event. 
Progression of the cancer under study is not considered an adverse event.  
Adverse events will not be collected for subjects during the pre-screening period (for 
determination of archival tissue status) as long as that subject has not undergone any 
protocol-specified procedure or intervention.  If the subject requires a blood draw, fresh 
tumor biopsy etc., the subject is first required to provide consent to the study and AEs will be 
captured according to guidelines for standard AE reporting. 
 
7.2.1 Immediate Reporting of Adverse Events to the Sponsor and to Merck 
7.2.1.1 Serious Adverse Events 
A serious adverse event is any adverse event occurring during participation in this study that: 
• Results in death; 
• Is life threatening; 
• Results in persistent or significant disability/incapacity; 
• Results in or prolongs an existing inpatient hospitalization; 
• Is a congenital anomaly/birth defect; 
• Is an other important medical event  
Refer to Table 6 for additional details regarding each of the above criteria. 
For the time period beginning when the consent form is signed until the completion of the 
post-therapy tumor tissue biopsy any serious adverse event, or follow up to a serious adverse 
event, including death due to any cause other than progression of the cancer under study that 
occurs to any subject must be reported within 24 hours to the Sponsor and within 2 working 
days to Merck Global Safety if it causes the subject to be excluded from the trial. 
Additionally, any serious adverse event, considered by an investigator who is a qualified 
physician to be related to the research FDG PET/CT scan procedure or post-therapy tumor 
tissue biopsy that is brought to the attention of the investigator at any time following consent 
through the end of the specified safety follow-up period specified in the paragraph above, or 
at any time outside of the time period specified in the previous paragraph also must be 
reported immediately to the Sponsor and to Merck Global Safety. 

 
 
 
 
 
25 
 
All subjects with serious adverse events must be followed up for outcome. 
SAE reports and any other relevant safety information are to be forwarded to the Merck 
Global Safety facsimile number:  +1-215-993-1220 
A copy of all 15 Day Reports and Annual Progress Reports is submitted as required by FDA, 
European Union (EU), Pharmaceutical and Medical Devices agency (PMDA) or other local 
regulators. Investigators will cross reference this submission according to local regulations to 
the Merck Investigational Compound Number (IND, CSA, etc.) at the time of submission.  
Additionally investigators will submit a copy of these reports to Merck & Co., Inc. (Attn: 
Worldwide Product Safety; FAX 215 993-1220) at the time of submission to FDA. 
 
7.2.2 Evaluating Adverse Events 
An investigator who is a qualified physician will evaluate all adverse events according to the 
NCI Common Terminology for Adverse Events (CTCAE), version 4.0. Any adverse event 
which changes CTCAE grade over the course of a given episode will have each change of 
grade recorded on the adverse event case report forms/worksheets. 
All adverse events regardless of CTCAE grade must also be evaluated for seriousness. 

 
 
 
  
 
 
26 
Table 6  Evaluating Adverse Events 
An investigator who is a qualified physician, will evaluate all adverse events as to: 
V4.0 CTCAE 
Grading 
Grade 1 
Mild; asymptomatic or mid symptoms; clinical or diagnostic observations only; intervention not indicated. 
 
Grade 2 
Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate instrumental ADL. 
 
Grade 3 
Severe or medically significant but not immediately life-threatening; hospitalization or prolongation or hospitalization indicated; 
disabling; limiting self-care ADL. 
 
Grade 4 
Life threatening consequences; urgent intervention indicated. 
 
Grade 5 
Death related to AE 
Seriousness 
A serious adverse event is any adverse event that: 
 
†Results in death; or 
 
†Is life threatening; or places the subject, in the view of the investigator, at immediate risk of death from the event as it occurred (Note: This does not include an 
adverse event that, had it occurred in a more severe form, might have caused death.); or 
 
†Results in a persistent or significant disability/incapacity (substantial disruption of one’s ability to conduct normal life functions); or 
 
†Results in or prolongs an existing inpatient hospitalization (hospitalization is defined as an inpatient admission, regardless of length of stay, even if the 
hospitalization is a precautionary measure for continued observation. (Note: Hospitalization for an elective procedure to treat a pre-existing condition that has not 
worsened is not a serious adverse event.  A pre-existing condition is a clinical condition that is diagnosed prior to the use of a Merck product and is documented in 
the patient’s medical history.); or 
 
†Is a congenital anomaly/birth defect (in offspring of subject taking the product regardless of time to diagnosis);or 
 
Other important medical events that may not result in death, not be life threatening, or not require hospitalization may be considered a serious adverse event when, 
based upon appropriate medical judgment, the event may jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes 
listed previously (designated above by a †). 
Duration 
Record the start and stop dates of the adverse event. If less than 1 day, indicate the appropriate length of time and units 
Action taken  
Did the adverse event cause Merck product to be discontinued? 

 
 
 
  
 
 
27 
Relationship to 
Merck Product  
Did Merck product cause the adverse event? The determination of the likelihood that Merck product caused the adverse event will be provided by an investigator 
who is a qualified physician. The investigator’s signed/dated initials on the source document or worksheet that supports the causality noted on the AE form, ensures 
that a medically qualified assessment of causality was done. This initialed document must be retained for the required regulatory time frame. The criteria below are 
intended as reference guidelines to assist the investigator in assessing the likelihood of a relationship between the test drug and the adverse event based upon the 
available information. 
The following components are to be used to assess the relationship between Merck product and the AE; the greater the correlation with the components and 
their respective elements (in number and/or intensity), the more likely Merck product caused the adverse event (AE): 
 
Exposure 
 
Time Course 
 
Likely Cause 
Is there evidence that the subject was actually exposed to Merck product such as: reliable history, acceptable compliance assessment (pill count, 
diary, etc.), expected pharmacologic effect, or measurement of drug/metabolite in bodily specimen? 
 
 
 
 
 
 
 

 
 
 
  
 
 
28 
Relationship 
The following components are to be used to assess the relationship between the test drug and the AE: (continued) 
to Merck 
Product 
(continued)  
Dechallenge 
Was Merck product discontinued or dose/exposure/frequency reduced? 
If yes, did the AE resolve or improve? 
If yes, this is a positive dechallenge. If no, this is a negative dechallenge. 
(Note: This criterion is not applicable if: (1) the AE resulted in death or permanent disability; (2) the AE resolved/improved despite continuation of 
the Sponsor’s product; or (3) the trial is a single-dose drug trial); or (4) Sponsor’s product(s) is/are only used one time.) 
 
Rechallenge 
Was the subject re-exposed to Merck product in this study? 
If yes, did the AE recur or worsen? 
If yes, this is a positive rechallenge. If no, this is a negative rechallenge. 
(Note: This criterion is not applicable if: (1) the initial AE resulted in death or permanent disability, or (2) the trial is a single-dose drug trial); or 
(3) Sponsor’s product(s) is/are used only one time). 
NOTE: IF A RECHALLENGE IS PLANNED FOR AN ADVERSE EVENT WHICH WAS SERIOUS AND WHICH MAY HAVE BEEN 
CAUSED BY MERCK PRODUCT, OR IF REEXPOSURE TO MERCK PRODUCT POSES ADDITIONAL POTENTIAL SIGNIFICANT RISK 
TO THE SUBJECT, THEN THE RECHALLENGE MUST BE APPROVED IN ADVANCE BY THE SPONSOR AS PER DOSE 
MODIFICATION GUIDELINES IN THE PROTOCOL. 
 
Consistency 
with Trial 
Treatment 
Profile 
Is the clinical/pathological presentation of the AE consistent with previous knowledge regarding Merck product or drug class pharmacology or 
toxicology? 
The assessment of relationship will be reported on the case report forms /worksheets by an investigator who is a qualified physician according to his/her best clinical judgment, including 
consideration of the above elements. 
Record one of the following 
Use the following scale of criteria as guidance (not all criteria must be present to be indicative of Merck product relationship). 
Yes, there is a reasonable 
possibility of Merck product 
relationship. 
There is evidence of exposure to Merck product.  The temporal sequence of the AE onset relative to the administration of Merck product is 
reasonable.  The AE is more likely explained by Merck product than by another cause. 
No, there is not a reasonable 
possibility of Merck product 
relationship 
Subject did not receive the Merck  product OR temporal sequence of the AE onset relative to administration of Merck product is not reasonable 
OR the AE is more likely explained by another cause than the Merck product.  (Also entered for a subject with overdose without an associated 

 
 
 
  
 
 
29 
AE.) 

 
 
 
  
 
30 
 
 
7.2.3 Sponsor Responsibility for Reporting Adverse Events 
All Adverse Events will be reported to regulatory authorities, IRB/IECs and investigators in 
accordance with all applicable global laws and regulations.  
 
8.0 STATISTICAL CONSIDERATIONS 
8.1 Design: 
This is a pilot correlative study for patients with metastatic melanoma receiving 
pembrolizumab on a standard regimen. The intent of this study is to evaluate FDG PET/CT 
as an early measure of response to immunotherapy. Tumor FDG uptake will be measured at 
baseline and at 3 and >10 weeks after initiation of pembrolizumab. 30 evaluable patients will 
be enrolled; evaluable implies that the patient had baseline and week 3 FDG PET/CT scans. 
This study will also include serum and tissue sampling for immune biomarkers.  
 
8.2 Objectives:  
The primary objectives are to: 
1. Determine the distribution of changes from baseline in tumor FDG uptake on PET/CT 
at 3 weeks and >10 weeks after initiation of pembrolizumab. 
2. Test for the association between change in tumor FDG uptake on PET/CT at 3 weeks 
and objective response (RECIST). 
 
The secondary objectives are to: 
1. Based on change in FDG uptake at 3 weeks, determine which patients have FDG 
“flare.” 
2. Test for the association between FDG flare (yes/no) and objective response.  
3. Test for the association between FDG flare and progression-free and overall survival. 
4. Test for correlation between change in FDG uptake and levels of immune biomarkers 
in serum and tissue. 
5. Test for association between changes in immune biomarkers in serum and tissue with 
objective response, progression-free survival, and overall survival. 
 
8.3 Endpoints:  
1. Objective response is defined by RECIST. The clinical evaluation takes place at 3 
months per standard pembrolizumab regimens. Patients will be categorized as 
responders (complete response or partial response) or nonresponders (stable disease 
or progressive disease). 

 
 
 
  
 
31 
2. The maximum standardized uptake value (SUVmax) will be analyzed for all tumor 
sites. Immune-related tissues will be analyzed with SUVmax and SUVmean. 
3. Studies have shown that the baseline test/re-test variability is 15-20%. Thus, FDG 
“flare” will likely be defined by >20% increase in tumor FDG uptake at 3 weeks. This 
definition may be modified for the observed distribution of changes in tumor FDG 
uptake on our study. 
4. Progression-free and overall survival are defined in Section 4.1.2. 
5. Immune biomarkers (see Section 7.1.2.3). 
 
8.4 Plans for Data Analysis:  
This is a pilot correlative study of FDG PET/CT imaging to evaluate early response to 
immunotherapy. We will first begin with a descriptive analysis. For continuous variables, we 
will generate graphs and descriptive statistics (e.g., means, standard deviations, medians and 
inter-quartile range). Categorical variables will be described by frequencies, proportions and 
95% exact confidence intervals. Change in tumor FDG uptake will be closely examined. It is 
likely that change in tumor SUVmax >20% will be classified as a FDG “flare” and the 
number of patients with a flare will be computed.  
 
Next, associations between change in tumor FDG uptake with clinical outcomes will be 
conducted. Change in tumor FDG uptake at 3 weeks will be compared between responders 
and nonresponders by a two sample t-test. Also, objective response rates will be compared 
between patients with or without FDG “flare” by Fisher’s exact test.  
 
PFS and OS will be estimated by the method of Kaplan and Meier. Median values and 95% 
confidence intervals will be calculated. PFS and OS will be compared between patients with 
or without FDG “flare” by the log rank test. Cox regression may be used to assess the effect 
of continuously scaled measurements of FDG change. For all survival analyses, a landmark 
analysis will be performed. Here, PFS or OS are defined from a landmark, which is 3 weeks 
after initiation of pembrolizumab. The landmark method is necessary because the change in 
tumor FDG uptake is known at 3 weeks. Serum and tissue biomarkers will be correlated with 
change tumor FDG uptake, using Pearson correlation or Spearman’s correlation, as 
appropriate. Within-patient pre-/post-treatment changes in biomarkers will be evaluated by 
paired Student’s t-test or Wilcoxon signed ranks test, as appropriate. We will test for an 
association between post-/pre-treatment fold change in immune biomarkers in serum and 
tissue and objective response, progression-free survival, and overall survival using logistic 
regression (for response) and Cox regression (for time-to-event outcomes) using landmark 
survival analysis. 
 
8.5 Sample Size Justification:  
The target is 30 evaluable patients, which may require up to 35 enrolled patients. Based on 
our clinical experience, we assume a 50% objective response rate, thus there will be 15 

 
 
 
  
 
32 
responders and 15 nonresponders. A comparison of change in FDG SUV at 3 weeks between 
responders and nonresponders, will have 84% power to detect a 1.0SD difference in mean 
values, using 1-sided two sample t test at a 5% significance level.  The 1-sided test is 
justified, since responders are expected to have greater changes in FDG SUV.  
 
8.6 Study Duration:  
This study will enroll 30 evaluable patients over 1 year. 
 
 
9.0 ADMINISTRATIVE AND REGULATORY DETAILS 
9.1 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Those regulations require a signed subject authorization informing the subject of the 
following:  
• 
What protected health information (PHI) will be collected from subjects in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of their PHI.  
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of 
subject authorization.  For subjects that have revoked authorization to collect or use PHI, 
attempts should be made to obtain permission to collect at least vital status (i.e. that the 
subject is alive). 
All research personnel associated with this study have completed the University of 
Pennsylvania’s Patient Oriented Research Training Program as well as HIPAA Compliance 
Training.  Trained staff will assess eligibility, introduce the study rationale, procedures, study 
risks, and collect the combined informed consent/HIPAA authorization form.  The study 
team will work to uphold the privacy of the participants in several ways.  Communications 
made among study staff regarding participants will use ID numbers whenever possible and 
minimize the use of patient name or other identifying information except when necessary for 
conduct of the study. Paper-based records will be kept in a secure location and only be 
accessible to personnel involved in the study. Computer-based files will only be made 
available to personnel involved in the study through the use of access privileges and 

 
 
 
  
 
33 
passwords. Whenever feasible, identifiers will be removed from study-related information. In 
data analysis sets, we will use ID numbers and/or patient initials only.   
Precautions will be applied to protecting subject privacy and the protected health information 
detailed below: 
1. 
Name 
2. 
Address 
3. 
Date of Birth 
4. 
Telephone Number 
5. 
Email address 
6. 
Emergency contact number, name, and relationship 
7. 
Medical Record Number 
8. 
Health Plan ID numbers 
Data will be accessible to the study investigators, all study staff, Department of Radiology 
IND office representatives, Radiation Research Safety Committee members, UPenn IRB and 
Office of Clinical Research, and the FDA (if desired). 
9.2 
Compliance with Trial Registration and Results Posting Requirements 
Under the terms of the Food and Drug Administration Modernization Act (FDAMA) and the 
Food and Drug Administration Amendments Act (FDAAA), the Sponsor of the trial is solely 
responsible for determining whether the trial and its results are subject to the requirements 
for submission to the Clinical Trials Data Bank, http://www.clinicaltrials.gov.  Information 
posted will allow subjects to identify potentially appropriate trials for their disease conditions 
and pursue participation by calling a central contact number for further information on 
appropriate trial locations and trial site contact information.     
9.3 
Records Retention 
It is the investigator’s responsibility to retain study essential documents for at least 2 years 
after the last approval of a marketing application in their country and until there are no 
pending or contemplated marketing applications in their country or at least 2 years have 
elapsed since the formal discontinuation of clinical development of the investigational 
product. These documents should be retained for a longer period if required by an agreement 
with the sponsor. In such an instance, it is the responsibility of the sponsor to inform the 
investigator/institution as to when these documents no longer need to be retained. 
9.4 
Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
study sponsor, government regulatory bodies, and University compliance and quality 
assurance groups of all study related documents (e.g. source documents, regulatory 
documents, data collection instruments, study data etc.).  The investigator will ensure the 
capability for inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.). 

 
 
 
  
 
34 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable University compliance and quality 
assurance offices. 
9.5 
Ethical Considerations 
This study is to be conducted accordance with applicable US government regulations and 
international standards of Good Clinical Practice, and applicable institutional research 
policies and procedures. 
This protocol and any amendments will be submitted to a properly constituted independent 
Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study conduct.  The decision of the IRB concerning the conduct of the study 
will be made in writing to the investigator and a copy of this decision will be provided to the 
sponsor before commencement of this study.   
All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study.  This consent form will be submitted with the protocol for review 
and approval by the IRB for the study.  The formal consent of a subject, using the IRB-
approved consent form, must be obtained before that subject undergoes any study procedure.  
The consent form must be signed by the subject or legally acceptable surrogate, and the 
investigator-designated research professional obtaining the consent. 
 
10.0 STUDY FINANCES 
10.1 Subject Stipends or Payments 
We will pay for the cost of parking, if not already covered through the hospital, on the day of 
the baseline PET/CT scan and on the day of the 1st post-therapy PET/CT scan. In addition, 
the patient will receive $100 after completion of the 1st post-therapy PET/CT scan, to cover 
the inconvenience of time and effort spent in participation of this study.  
 

 
 
 
  
 
35 
11.0 APPENDICES 
 
11.1 Common Terminology Criteria for Adverse Events V4.0 (CTCAE) 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 4.0 will be utilized for adverse event reporting. 
(http://ctep.cancer.gov/reporting/ctc.html) 
11.2 Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 Criteria for 
Evaluating Response in Solid Tumors 
RECIST version 1.1* will be used in this study for assessment of tumor response. While 
either CT or MRI may be utilized, as per RECIST 1.1, CT is the preferred imaging technique 
in this study. 
* As published in the European Journal of Cancer: 
 
E.A. Eisenhauer, P. Therasse, J. Bogaerts, L.H. Schwartz, D. Sargent, R. Ford, J. 
Dancey, S. Arbuck, S. Gwyther, M. Mooney, L. Rubinstein, L. Shankar, L. Dodd, R. Kaplan, 
D. Lacombe, J. Verweij. New response evaluation criteria in solid tumors: Revised RECIST 
guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47. 
In addition, volumetric analysis will be explored by central review for response assessment. 
 
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