Combining SBRT and Immunotherapy in Early Stage NSCLC Patients Planned for Surgery: Exploring Safety and Immunological Proof of Principle.
Phase: PHASE1
Conditions: NSCLC, Stage I
Sponsor: Amsterdam UMC, location VUmc

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SPONSOR: VU University Medical Center 
 
TITLE: Combining SBRT and immunotherapy in early stage NSCLC patients planned for 
surgery: exploring safety and immunological proof of principle. 
 
IND NUMBER: 
 
EudraCT NUMBER: 2016‐003819‐36 
 
 

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Protocol ID 
MK3475-586 
Short title 
Combining SBRT and immunotherapy in early stage 
NSCLC patients planned for surgery: exploring safety 
and immunological proof of principle  
EudraCT number  
2016‐003819‐36 
Version 
1.4 
Date 
15-12-2017 
 
Principal investigator 
Egbert F. Smit, MD, PhD 
Dept. of Pulmonary Diseases 
VU University Medical Center 
PO Box 7057 
1007 MB Amsterdam 
The Netherlands 
Tel: +31204444782 
Email: ef.smit@vumc.nl 
 
Writing committee 
Adrianus J. de Langen, MD, PhD 
Dept. Pulmonary Diseases 
VU University Medical Center 
PO Box 7057 
1007 MB Amsterdam 
The Netherlands 
Tel: +31204441889 
Email: j.delangen@vumc.nl 
 
Egbert F. Smit, MD, PhD 
Dept. of Pulmonary Diseases 
VU University Medical Center 
PO Box 7057 
1007 MB Amsterdam 
The Netherlands 
Tel: +31204444782 
Email: ef.smit@vumc.nl 
 
Suresh Senan, MD, PhD 
VU University Medical Center 
PO Box 7057 
1007 MB Amsterdam 
The Netherlands 
Tel: +31204444782 
Email: ef.smit@vumc.nl 
 
Guus A.M.S. van Dongen, PhD 
Dept of Radiology and Nuclear Medicine 

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VU University Medical Center 
De Boelelaan 1117 
1081 HV Amsterdam 
The Netherlands 
Tel.: +31204442869 
Email: GAMS.vanDongen@vumc.nl 
 
Bert Windhorst, PhD 
VU University Medical Center  
Dept. of Radiology and Nuclear Medicine  
Location Radionuclide Center  
De Boelelaan 1085c  
1081 HV Amsterdam  
The Netherlands  
Tel: +31204449101  
Email: ad.windhorst@vumc.nl 
 
M.C. Huisman, PhD 
Dept of Radiology and Nuclear Medicine 
VU University Medical Center 
De Boelelaan 1117 
1081 HV Amsterdam 
The Netherlands 
Tel.: +3120444 9638 
Email: m.huisman@vumc.nl 
 
Tanja D. de Gruijl, PhD 
VU University Medical Center – Cancer Center 
Amsterdam 
Dept. of Medical Oncology – Immunotherapy 
Laboratory 
De Boelelaan 1117 – CCA 2.44 
1081 HV Amsterdam 
The Netherlands 
Tel: +31204444063 
Email: td.degruijl@vumc.nl 
 
Erik Thunnissen, MD, PhD 
Dept. of Pathology 
VU University Medical Center 
De Boelelaan 1117 
1081HV Amsterdam 
The Netherlands 
Tel: +31204444048 
Email: e.thunnissen@vumc.nl 

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Independent expert 
A.J.M. van den Eertwegh, MD, PhD 
Dept. of Medical Oncology 
VU University Medical Center 
De Boelelaan 1118 
1081 HZ Amsterdam 
The Netherlands 
Tel: +31204444336 
 
Pharmacy 
Pharmacy visiting address: 
Klara Bruyn, Anneke Admiraal, Marlies Hutters 
Apotheek VUmc  
De Boelelaan 1117  
1081 HV Amsterdam  
tel.:020-4443534  
fax:020-4444627  
e-mail:onderzoeksmedicatie@vumc.nl 
 
Pharmacy delivery address:  
Apotheek VUmc , onderzoeksmedicatie 
ruimte PK 1 X 070 
De Boelelaan 1118 
1081 HZ Amsterdam 
tel:020-4443435 
Fax 020-4444627 
email:onderzoeksmedicatie@vumc.nl 
 
 

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TABLE OF CONTENTS 
 
1.0 
TRIAL SUMMARY.................................................................................................... 9 
2.0 
TRIAL DESIGN.......................................................................................................... 9 
2.1 
Trial Design ............................................................................................................. 9 
2.2 
Trial Diagram ........................................................................................................ 11 
3.0 
Objectives & Hypotheses .......................................................................................... 12 
3.1 
Primary Objectives & Hypotheses ...................................................................... 12 
3.2 
Secondary Objectives & Hypotheses ................................................................... 12 
3.3 
Explorative Objectives ......................................................................................... 12 
4.0 
BACKGROUND & RATIONALE .......................................................................... 12 
4.1 
Background ........................................................................................................... 13 
4.1.1 Stereotactic body radiotherapy for the treatment of early stage NSCLC ........... 13 
4.1.2 Pharmaceutical and therapeutic treatment with pembrolizumab ........................ 13 
4.1.3 Preclinical and Clinical Trial Data of pembrolizumab treatment ....................... 14 
4.1.4 Combination treatment with radiotherapy and immunotherapy ......................... 14 
4.1.5 Time window for the combination of radiotherapy and immunotherapy ........... 15 
4.1.6 Adjuvant Surgical resection ................................................................................ 16 
4.1.7 Safety of the trimodality approach ....................................................................... 16 
4.1.8 Predictive biomarkers of response to radiotherapy +/- pembrolizumab 
treatment ............................................................................................................. 17 
4.2 
Rationale ................................................................................................................ 17 
4.2.1 Rationale of the trimodality approach ................................................................. 17 
4.2.2 Rationale for Dose Selection/Regimen/Modification ......................................... 18 
4.2.3 Translational research ......................................................................................... 19 
4.2.4 Rationale for Endpoints ...................................................................................... 19 
4.2.5 
89Zr-pembrolizumab imaging .............................................................................. 20 
5.0 
METHODOLOGY ................................................................................................... 21 
5.1 
Entry Criteria ........................................................................................................ 21 
5.1.1 Diagnosis/Condition for Entry into the Trial ...................................................... 21 
5.1.2 Subject Inclusion Criteria.................................................................................... 21 
5.1.3 Subject Exclusion Criteria .................................................................................. 22 
5.2 
Sample size ............................................................................................................. 23 
5.3 
Pretreatment imaging ........................................................................................... 24 
5.3.1 Prestudy diagnostic and staging imaging ............................................................ 24 
5.3.2 
89Zr-pembrolizumab PET .................................................................................... 24 
5.3.2.1 Radiolabeling of pembrolizumab and quality controls ................................ 24 
5.3.2.2 Radiation exposure....................................................................................... 25 
5.3.2.3 Injection procedure ...................................................................................... 25 
5.3.2.4 Blood sample analysis.................................................................................. 25 
5.3.2.5 
89Zr-pembrolizumab PET procedure............................................................ 25 
5.3.2.6 Analysis of PET data ................................................................................... 25 
5.4 
On-treatment imaging .......................................................................................... 26 
5.4.1 Quantitative changes in lung density .................................................................. 26 

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5.5 
Ultrasound endoscopy guided cytological lymph node aspiration for 
immunological cell composition (EBUS FACS) ................................................. 26 
5.5.1 Bronchoscopy and EBUS procedure................................................................... 26 
5.5.2 Lymph node analysis .......................................................................................... 26 
5.6 
Treatment plan ...................................................................................................... 27 
5.6.1 Stereotactic body radiotherapy (SBRT) .............................................................. 27 
5.6.2 Pembrolizumab Treatment .................................................................................. 28 
5.6.2.1 Dose Modification ....................................................................................... 28 
5.6.2.2 Timing of Dose Administration ................................................................... 29 
5.6.3 Surgical resection ................................................................................................ 30 
5.7 
Concomitant Medications/Vaccinations (allowed & prohibited) ..................... 30 
5.7.1 Acceptable Concomitant Medications ................................................................ 30 
5.7.2 Prohibited Concomitant Medications.................................................................. 30 
5.8 
Rescue Medications & Supportive Care ............................................................. 31 
5.8.1 Supportive Care Guidelines ................................................................................ 31 
5.9 
Diet/Activity/Other Considerations ..................................................................... 34 
5.9.1 Diet ...................................................................................................................... 34 
5.9.2 Contraception ...................................................................................................... 34 
5.9.3 Use in Pregnancy ................................................................................................ 34 
5.9.4 Use in Nursing Women ....................................................................................... 35 
5.10 
Subject Withdrawal/Discontinuation Criteria ................................................... 35 
5.11 
Subject Replacement Strategy ............................................................................. 35 
5.12 
Beginning and End of the Trial ........................................................................... 36 
5.13 
Clinical Criteria for Early Trial Termination ................................................... 36 
6.0 
TRIAL FLOW CHART ........................................................................................... 37 
6.1 
Study Flow Chart .................................................................................................. 37 
7.0 
TRIAL PROCEDURES ........................................................................................... 39 
7.1 
Trial Procedures ................................................................................................... 39 
7.1.1 Administrative Procedures .................................................................................. 39 
7.1.1.1 Informed Consent......................................................................................... 39 
7.1.1.2 Inclusion/Exclusion Criteria ........................................................................ 39 
7.1.1.3 Medical History ........................................................................................... 40 
7.1.1.4 Prior and Concomitant Medications Review ............................................... 40 
7.1.1.4.1 Prior Medications .................................................................................. 40 
7.1.1.4.2 Concomitant Medications ..................................................................... 40 
7.1.1.5 Disease Details and Treatments ................................................................... 40 
7.1.1.5.1 Disease Details ...................................................................................... 40 
7.1.1.5.2 Prior Treatment Details ......................................................................... 40 
7.1.1.5.3 Subsequent Anti-Cancer Therapy Status .............................................. 40 
7.1.1.6 Assignment of Screening Number ............................................................... 40 
7.1.1.7 Randomization ............................................................................................. 40 
7.1.2 Clinical Procedures/Assessments ........................................................................ 40 
7.1.2.1 Adverse Event (AE) Monitoring .................................................................. 41 
7.1.2.2 Full Physical Exam ...................................................................................... 41 
7.1.2.3 Directed Physical Exam ............................................................................... 41 

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7.1.2.4 Vital Signs .................................................................................................... 41 
7.1.2.5 Eastern Cooperative Oncology Group (ECOG) Performance Scale ........... 41 
7.1.2.6 Tumor Imaging and Assessment of Disease ................................................ 41 
7.1.2.7 Tumor Tissue Collection.............................................................................. 41 
7.1.2.7.1 Pretreatment tumor biopsy .................................................................... 42 
7.1.2.7.2 Post-resection specimen ........................................................................ 42 
7.1.2.8 Laboratory Procedures/Assessments ........................................................... 43 
7.1.2.9 Laboratory Safety Evaluations (Hematology, Chemistry and Urinalysis) .. 43 
7.1.2.10 Immunophenotyping on peripheral blood .................................................... 43 
7.1.2.11 Ultrasound endoscopy guided cytological lymph node aspiration for 
immunological cell composition (EBUS FACS) ......................................... 44 
7.1.3 Withdrawal/Discontinuation ............................................................................... 44 
7.1.4 Visit Requirements .............................................................................................. 44 
7.1.4.1 Screening...................................................................................................... 44 
7.1.4.1.1 Screening Period ................................................................................... 44 
7.1.4.2 Treatment Period .......................................................................................... 45 
7.1.4.3 Post-Treatment Visits................................................................................... 45 
7.2 
Assessing and Recording Adverse Events .......................................................... 45 
7.2.1 Definition of an Overdose for This Protocol and Reporting of Overdose .......... 46 
7.2.2 Reporting of Pregnancy and Lactation................................................................ 46 
7.2.3 Immediate Reporting of Adverse Events ............................................................ 46 
7.2.3.1 Serious Adverse Events ............................................................................... 46 
7.2.3.2 Events of Clinical Interest ............................................................................ 48 
7.2.4 Evaluating Adverse Events ................................................................................. 48 
7.2.5 Sponsor Responsibility for Reporting Adverse Events ...................................... 51 
8.0 
STATISTICAL ANALYSIS PLAN ........................................................................ 51 
8.1 
Safety ...................................................................................................................... 51 
8.2 
Analysis of immune effector subset data ............................................................ 51 
8.2.1 Tumor tissue ........................................................................................................ 51 
8.2.2 Immunophenotyping in peripheral blood ............................................................ 52 
8.2.3 Immunophenotyping on lymph nodes using EBUS-FACS analysis .................. 52 
8.3 
Analysis of PET data ............................................................................................ 52 
8.3.1.1 Heterogeneity analysis ................................................................................. 52 
8.3.1.2 Interpatient heterogeneity ............................................................................ 52 
8.3.1.3 Intrapatient heterogeneity ............................................................................ 52 
8.3.1.4 Intratumor heterogeneity .............................................................................. 52 
8.4 
Analysis of CT data ............................................................................................... 53 
8.5 
Correlation between PET data and Blood and Tissue markers ....................... 53 
8.6 
Correlation between organ SUV and irAEs ....................................................... 53 
9.0 
LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL 
SUPPLIES ................................................................................................................. 53 
9.1 
Investigational Product ........................................................................................ 53 
9.2 
Packaging and Labeling Information ................................................................. 53 
9.3 
Clinical Supplies Disclosure ................................................................................. 53 
9.4 
Storage and Handling Requirements .................................................................. 53 

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9.5 
Returns and Reconciliation .................................................................................. 54 
10.0 
Ethical considerations ............................................................................................... 55 
10.1 
Regulation statement ............................................................................................ 55 
10.2 
Recruitment and consent ...................................................................................... 55 
10.3 
Benefits and risks assessment, group relatedness .............................................. 55 
10.3.1 Benefit the trimodality approach......................................................................... 55 
10.3.2 Benefit from 89Zr-pembrolizumab PET .............................................................. 55 
10.3.3 Safety of  89Zr-pembrolizumab PET ................................................................... 55 
10.3.4 Safety of pembrolizumab treatment .................................................................... 56 
10.3.5 Safety of the trimodality approach ...................................................................... 56 
10.3.6 Radiation exposure .............................................................................................. 56 
10.3.7 Discomfort during scanning ................................................................................ 56 
10.3.8 Additional blood collection ................................................................................. 56 
10.3.9 Additional EBUS ................................................................................................ 57 
10.4 
Compensation for injury ...................................................................................... 57 
11.0 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ......... 58 
11.1 
Data Collection ...................................................................................................... 58 
11.2 
Handling and storage of data and documents .................................................... 58 
11.3 
Monitoring plan .................................................................................................... 58 
11.4 
Amendments .......................................................................................................... 58 
11.5 
Annual progress report ........................................................................................ 58 
11.6 
End of study report ............................................................................................... 59 
11.7 
Public disclosure and publication policy ............................................................ 59 
11.8 
Trial sponsoring and financing ............................................................................ 59 
12.0 
LIST OF REFERENCES ......................................................................................... 60 
 

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1.0 TRIAL SUMMARY 
Rationale: Despite the early stage of disease, a substantial number of patients with early 
stage non-small-cell lung cancer (NSCLC) relapse within five years of treatment. A pooled 
analysis of the ROSEL and STARS trials showed that stereotactic body radiotherapy (SBRT) 
may provide an alternative to surgical treatment for patients with early stage disease. One of 
the reasons for the favorable effects of SBRT may be due to an immunological response 
against tumor cells, elicited by radiotherapy. In this protocol we will explore the addition of 
immunotherapy to SBRT to augment the antitumor T cell response, associated with 
radiotherapy. Because surgical resection is still the current standard of care for patients who 
are fit to undergo surgery, the subjects will undergo a lobectomy with hilar and mediastinal 
lymph node dissection after SBRT +/- pembrolizumab treatment. This is a key feature of this 
study and an unique opportunity to perform histological examination of the entire lung 
tumor, its associated lymph nodes and normal lung tissue after immunotherapy, allowing the 
study of immune response heterogeneity and correlating this with non-invasive imaging and 
blood results. In the surrounding normal lung tissue, radiation and immunotherapy induced 
toxicity can be evaluated.  
Objectives: To evaluate the safety and mechanisms of action of the trimodality treatment 
approach. 
Study design: An open label randomized exploratory study of the safety and mechanisms of 
action of combined treatment with SBRT and immunotherapy (pembrolizumab, anti-PD1) 
for early stage NSCLC. 
Estimated study duration: We expect to enroll one patient per month. Therefore, the study 
can be finished within 2.5 years from the time the first subject signs the Informed Consent 
Form (ICF) through the final contact. 
Study population: Patients with histologically or cytologically confirmed diagnosis of early 
stage (T1N0 and T2aN0) peripherally located NCSLC, eligible for surgical resection. 
Intervention: Patients will be randomized between SBRT with or without 2 cycles of 
pembrolizumab treatment (starting on the first day of radiotherapy). The patients will 
undergo a lobectomy with hilar and mediastinal lymph node dissection after SBRT +/- 
pembrolizumab treatment. Translational research to explore the immune mechanism of 
action will include biological imaging with immuno-PET. Expression rates and activation 
states of immune effector subsets will be assessed in tumor core biopsy specimens, peripheral 
blood and tumor draining lymph nodes (TDLNs) by means of fine needle aspirates of 
TDLNs. Samples will be taken before and after SBRT +/- pembrolizumab treatment and at 
surgery. 
Main study parameters/endpoints: To assess the safety of combined SBRT and 
pembrolizumab treatment in early stage NSCLC and to identify the immunological 
mechanism of action. 
 
 
 
2.0 TRIAL DESIGN 
 
2.1 
Trial Design 
This is an open label randomized exploratory study of the safety and mechanisms of action of 
combined treatment with stereotactic body radiotherapy (SBRT) and immunotherapy 
(pembrolizumab, anti-PD1) for early stage NSCLC. Because the mode of action of combined 

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SBRT and anti-PD-1 therapy is unknown, it is necessary to perform translational research to 
identify the mechanisms of action and toxicity before large, more expensive and time 
consuming effect size studies can be initiated with recurrence free and overall survival as 
outcome parameters.  
Patients with early stage (T1bN0 and T2aN0) peripherally located and histologically 
confirmed NSCLC, eligible for surgical resection, will be enrolled in this study. Patients will 
be randomized between SBRT, with or without 2 cycles of pembrolizumab treatment 
(starting on the first day of radiotherapy). Because surgical resection is the current standard 
of care for these patients, the subjects will undergo a lobectomy with hilar and mediastinal 
lymph node dissection after SBRT +/- pembrolizumab treatment.  
Surgical resection after the induction treatment is a unique opportunity to perform 
histological examination of the entire lung tumor and lymph nodes after immunotherapy, 
allowing to examine immune response heterogeneity and to correlate this with the non-
invasive imaging and blood results. In the surrounding normal lung tissue, radiation and 
immunotherapy induced toxicity can be evaluated.  
In week 8-10 after SBRT +/- pembrolizumab treatment, patients will undergo a lobectomy 
with hilar and mediastinal lymph node dissection. Patients randomized to ‘no pembrolizumab 
treatment’ will also undergo a resection 8-10 weeks after SBRT. 
The primary endpoints of this study are safety of the trimodality approach and unraveling the 
immune mechanism of action. Serial invasive and noninvasive measurements of 
immunological biomarkers will be performed for the primary tumor, lymph nodes and 
peripheral blood. A study scheme is provided on page 11 of this protocol. The translational 
research will include biological imaging, immune effector subset examination and 
examination of healthy tissue for radiation induced changes. 
 

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2.2 
Trial Diagram 
 
Study population
Early stage (T1N0 and T2aN0) peripherally located NCSLC, 
eligible for surgical resection.
Treatment
SBRT monotherapy
Pre-treatment assessment
Blood sampling for immunophenotyping
EBUS FACS
Tumor core biopsy
Pre-treatment assessment
3D 89Zr-Pembrolizumab 
PET
Treatment
SBRT + 2 cycles 
pembrolizumab
1: 1 randomization
Post-treatment assessment
Blood sampling for immunophenotyping
EBUS FACS
Lobectomy with hilar and mediastinal lymph 
node dissection
Post-resection assessment
Blood sampling for immunophenotyping
3D-histological tumor analysis for PD-1 and PD-L1 
IHC and immunological cell composition
1:1 3D correlation of 89Zr-Pembrolizumab PET 
images with tumor histopathology
Histological lymph node analysis for 
immunophenotyping
Radiation induced changes to healthy lung tissue
Follow-up
Rate of pneumonitis (CTCAE grade)
Blood sampling for immunophenotyping 
 
 
 

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3.0 OBJECTIVES & HYPOTHESES 
 
3.1 
Primary Objectives & Hypotheses 
Objective 1:  
To assess the safety of combined SBRT and pembrolizumab treatment. 
 
Objective 2: 
To identify the immunological response to combined SBRT and pembrolizumab treatment in 
early stage NSCLC. Expression rates and activation states of immune effector subsets will be 
assessed in tumor core biopsy specimens, peripheral blood and tumor draining lymph nodes 
(TDLNs) by means of EBUS derived fine needle aspirates. Samples will be taken before and 
after SBRT +/- pembrolizumab treatment and at surgery. 
 
Hypothesis 2: 
We anticipate that SBRT and prembrolizumab treatment will alter the immune activation 
status as well as the cellular composition in TDLNs, tumor and blood reflected by local 
and/or systemic switches from a suppressive immune content to a more activated immune 
content. The magnitude of this response is higher in the combination SBRT and 
pembrolizumab group than in the SBRT monotherapy group.    
 
3.2 
Secondary Objectives & Hypotheses 
 
Objective 1:   
To assess uptake (visual and quantitatively, expressed as SUVmax, SUVmean and SUVpeak) of 
radiolabeled pembrolizumab (89Zr-pembrolizumab) in the tumor and lymph nodes by use of 
PET. 
Hypothesis 1:   
89Zr-pembrolizumab tumor uptake heterogeneity between patients is expected and can be 
visualized. We expect that the level of 89Zr-Pembrolizumab uptake will be related to the 
immunohistological results (immune effector subsets with special interest to the expression 
of PD-1 and PD-L1) of the whole tumor as well as regional tumor areas. 
 
3.3 
Explorative Objectives 
Objective 1: 
To evaluate radiation induced changes to healthy lung tissue after combined SBRT and 
pembrolizumab treatment in the resected lung lobe. 
 
Objective 2:  
To assess radiation induced changes in the tumor cell surface glycoproteins.  
 
 
4.0 BACKGROUND & RATIONALE 
 

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4.1 
Background 
 
4.1.1 Stereotactic body radiotherapy for the treatment of early stage NSCLC  
The current standard treatment for early stage NSCLC is an anatomical surgical resection, 
with SBRT being reserved for patients not fit to undergo resection. Despite the early stage of 
disease, 5-year overall survival after surgical resection is only 45-50% due to locoregional as 
well as distant relapse (1). Although none of the planned randomized studies of surgery 
versus SBRT have completed accrual, a pooled analysis of our ROSEL study together with 
the STARS trial from MD Anderson has been published (2). Estimated overall survival at 3 
years was 95% (95% CI 85-100) in the SBRT group compared with 79% (64-97) in the 
surgery group (hazard ratio [HR] 0.14 [95% CI 0.017-1.190], log-rank p=0.037). Recurrence-
free survival at 3 years was 86% (95% CI 74-100) in the SBRT group and 80% (95% CI 65-
97) in the surgery group (HR 0.69 [95% CI 0.21-2.29], log-rank p=0.54). 
This was a very surprising finding because the main presumed advantage of surgery over 
SBRT is the ipsilateral hilar and mediastinal lymph node dissection with unexpected lymph 
node (micro)metastases found in 15% of patients (3). The unexpectedly good results with 
SBRT may be caused by an immunological response against tumor cells, triggered by the 
radiotherapy. High dose radiation has been shown to induce an antitumor immune response 
mediated by T-cell regulation, both in the primary tumor and the TDLNs (4, 5). However, 
despite the promising results of SBRT and this exciting biological mechanism, overall 
survival was not different between the two modalities in this study. In another study, 676 
patients underwent SBRT for early stage lung cancer with a 2-year recurrence rate of 22% 
and a median overall survival of 41 months (6). Furthermore, of the 124 recurrences, 82 
(66%) were distant recurrences.  
These results clearly reveal that disease recurrence rates still remain substantial with both 
modalities, despite their curative intent. 
 
4.1.2 Pharmaceutical and therapeutic treatment with pembrolizumab 
The importance of intact immune surveillance in controlling outgrowth of neoplastic 
transformation has been known for decades (7). Accumulating evidence shows a correlation 
between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in 
various malignancies (8-12). In particular, the presence of CD8+ T-cells and the ratio of 
CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved 
prognosis and long-term survival in many solid tumors. 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress 
immune control. The normal function of PD-1, expressed on the cell surface of activated T-
cells under healthy conditions, is to down-modulate unwanted or excessive immune 
responses, including autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an Ig 
superfamily member related to CD28 and CTLA-4 which has been shown to negatively 
regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2) 
(13, 14). The structure of murine PD-1 has been resolved (15). 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-

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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 (13, 16-18). 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 (19, 20). 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 (21, 22). Expression has also been shown during thymic 
development on CD4-CD8- (double negative) T-cells as well as subsets of macrophages and 
dendritic cells (23). 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 (19, 24-26). 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 (19). 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) (27). 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 (previously known as SCH 900475 and MK-3475) is a potent and highly 
selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to 
directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2.   
 
4.1.3 Preclinical and Clinical Trial Data of pembrolizumab treatment 
Please see the Investigator’s Brochure for Preclinical and Clinical data. 
On October 2, 2015, the U.S. Food and Drug Administration granted accelerated approval for 
pembrolizumab to treat patients with advanced (metastatic) NSCLC whose disease has 
progressed after other treatments and with tumors that express ≥1% PD-L1, as assessed by 
the companion diagnostic, the PD-L1 IHC 22C3 pharmDx test. 
 
4.1.4 Combination treatment with radiotherapy and immunotherapy 
In this protocol we will explore the addition of immunotherapy to SBRT to augment the 
antitumor T cell response, already associated with radiotherapy. 
In different preclinical studies the expression of tumor antigens has been shown to be up-
regulated directly after radiotherapy and might therefore lead to changes in the immune 
response of the body. It is conceivable that radiotherapy treatment acts as an "in situ vaccine" 
to prime the immune response. Nascent preclinical and early clinical findings have supported 
this possibility, suggesting that radiation, through its immune-stimulating properties, may be 
used as a systemic therapy in addition to a means of local tumor control (28-31). 

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Although radiotherapy results in T cell stimulation and upregulation of tumor antigens, T 
cells might be still depressed and tumor tolerable due to upregulation of inhibitory co-signals 
like CTLA-4 and PD-(L)1.  
For this reason, anti–PD-L1 treatment can add to ionizing radiation that increases the 
production and presentation of tumor antigens (32, 33). Other studies showed that the 
combination of radiation and PD-L1 checkpoint blockade synergistically reduce 
immunosuppressive MDSCs (34). Systemic responses have been observed in patients with 
melanoma treated with the combined regimen of anti–CTLA-4 mAb ipilimumab and 
radiation, suggesting that coupling radiotherapy with immunotherapy may hold promise for 
inducing powerful effects in human patients (31, 35). 
Preclinical studies in murine models have demonstrated efficacy of combination therapy with 
immunomodulators and radiotherapy (36). Results of a preclinical study of murine 
intracranial glioma treated with anti–PD-1 mAb plus radiotherapy showed not only long-term 
survival of the treated mice, but also robust systemic immunologic memory in the surviving 
mice, as they were able to reject a secondary challenge of glioma cells injected in the flank 
(37). The combination of radiation plus anti–PD-1 therapy resulted in a median survival to 53 
days (P < 0.05 by log-rank Mantel–Cox test), and 15% to 40% of mice survived more than 
180 days after treatment (37). The combination therapy increased tumor infiltration by CD8+ 
CTLs and decreased the number of CD4+ Tregs. 
On the contrary, median survival periods were similar for control mice (25 days) and mice 
given only anti–PD-1 mAbs (27 days) or radiation (28 days). In the test of immunologic 
memory, naïve and long-term surviving mice were injected in the flanks with GL261-luc 
cells and all 8 naïve mice died from the growth of the challenged glioma cells, whereas mice 
that received prior treatment with the combined regimen rejected the glioma challenge (37). 
In a preclinical study of triple-negative breast cancer, neither anti–PD-1 mAb nor radiation 
when given alone were effective in a murine model of triple-negative breast cancer. 
However, the addition of anti–PD-1 mAbs enhanced the curative capacity of radiotherapy 
and a-CD137 (an agonist antibody for costimulatory molecule 4-1BB) against both 
established tumors and secondary tumor challenge, indicating that the combined regimen 
conferred antitumor immune responses and memory (38). Indeed, the combination of a-
CD137, anti–PD-1, and radiation showed greater efficacy (40% rejection) than anti–PD-1 or 
radiation, given alone or in combination (38).  
On the basis of the results of these preclinical studies, several clinical trials have been 
initiated to assess the efficacy of combining anti–PD-1 immunotherapy with radiotherapy.  
 
4.1.5 Time window for the combination of radiotherapy and immunotherapy 
In recent preclinical studies, immunotherapy has been administered either a few days prior 
to, concomitant with, or post radiotherapy. In mouse colon and breast carcinoma models, 
delaying administration of anti-CTLA-4 mAbs at 2 days after radiotherapy completion can 
reduce the therapeutic efficacy when compared with 2 days before or on the day of 
radiotherapy completion, implying that immunotherapy should not be administered too late 
after RT (39). Dovedi et al. have evaluated three distinct combinatorial schedules where mice 
bearing colon tumors received a conventional fractionated radiotherapy of 2 Gy × 5 with 
administration of anti-PD-L1 mAbs on day 1 of the radiotherapy cycle (schedule A), day 5 of 
the cycle (schedule B), or 7 days after the completion of radiotherapy (schedule C), 

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respectively. No significant difference in overall survival is found between schedules A and 
B. However, sequential treatment with radiotherapy followed by anti-PD-L1 mAbs 7 days 
later is obviously ineffective for improving overall survival when compared with 
radiotherapy alone.  
 
4.1.6 Adjuvant Surgical resection 
Because surgical resection is the standard of care for operable patients, the subjects will 
undergo a lobectomy with hilar and mediastinal lymph node dissection after SBRT +/- 
pembrolizumab treatment. The use of SBRT as neoadjuvant therapy prior to surgery may 
provide a novel therapeutic opportunity. In oncology, the use of neoadjuvant radiotherapy or 
chemoradiotherapy prior to surgery has become widespread for several types of cancer, and in 
many instances improves local control and/or survival compared to surgery alone (40). 
Neoadjuvant radiotherapy provides several theoretical advantages, including potentially 
decreasing the rate of positive margins, decreasing the size of the required resection, or by 
sterilizing the tumor to avoid seeding of circulating tumor cells during surgery (40).  
 
4.1.7 Safety of the trimodality approach 
To our knowledge, no study has employed the combination of SBRT, immunotherapy and 
resection with the goal of maximizing local and distant tumor control.  
In modern radiotherapy, image-derived SBRT delivery has improved treatment precision and 
the radiation-induced toxicity to normal tissues. The toxicity of radiotherapy is generally 
limited to the irradiated target organ. For example, esophagitis is a complication seen in 
radiation of locally advanced central lung tumors and mediastinal lymph nodes. Pneumonitis 
depends on the target volume and is rarely seen with treatment of early stage lung cancer. In 
current clinical trials, immune-checkpoint blockade therapy presents acceptable toxicity. 
Even occasional severe toxicity can be managed through treatment interruption or 
involvement of other immunosuppressors. Compared with CTLA-4, the blockade of PD-1/L1 
has less severe ir-AEs. In the ongoing phase I trial with pembrolizumab in NSCLC, 495 
patients were treated. Drug-related grades 3–4 adverse events were found in 9.5% of patients 
with 1.8% developing pneumonitis (41). Considering the acceptable adverse events, the 
combination of radiotherapy and immune checkpoint inhibitors appears to be feasible in 
patients.  
Combining SBRT with planned surgery appears safe: at least 4 small studies have reported on 
patients who have undergone surgery for salvage in patients who have recurred after SBRT (42-
45). Such surgery is generally well tolerated with a favorable toxicity profile, with only one 
patient sustaining a major toxicity (fistula requiring further surgery for correction) (44). An 
ongoing Phase II Canadian trial is accruing patients with early stage NSCLC for treatment with 
SBRT plus adjuvant surgical resection with a treatment interval of 10 weeks (NCT02136355). 
Currently there are five patients who underwent a resection, all without complications. In two 
patients, viable tumor was found in the resection specimen (personal communication, 
unpublished data).  
As only patients with peripheral tumors are eligible for this trial and patients receive a 
lobectomy according to national guidelines on the treatment for NSCLC, the resection margins 
(i.e. central bronchial and vascular structures) will not be involved in the radiation target 
volume. Therefore we do not foresee problems with healing of these structures and do not 

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anticipate increased risks for a fistula or other surgical complications, beyond those seen with a 
primary surgical resection. Furhtermore, because the irradiated tumor and surrounding lung 
tissue are resected with the lobectomy, we do not foresee increased toxicity from combined 
SBRT and pembrolizumab treatment.  
 
4.1.8 Predictive biomarkers of response to radiotherapy +/- pembrolizumab treatment 
Because benefit of the addition of pembrolizumab to radiotherapy is expected to occur in a 
subset of patients, it is of great importance to identify biomarkers that correlate with clinical 
activity and that can be used to select patients that will benefit from the addition of 
pembrolizumab treatment.  
Selecting those patients that benefit the most is challenging. Until now companion biomarker 
development has focused on the level of PD-L1 expression by immunohistochemistry. 
Among 204 patients with NSCLC treated on an expansion cohort phase I trial, tumor PD-L1 
expression (defined in IHC as ≥1% tumor membrane PD-L1 expression) was present in 86% 
of the tested patients. For patients with a proportion score (PS, percentage of cells with 
membranous PD-L1 staining of any intensity) ≥50%, the objective response rate (ORR) was 
45%, while the ORR was 17% and 11% for patients with PS of 1-49% and <1%, respectively 
(41). In the phase III KEYNOTE-010 trial evaluating pembrolizumab versus docetaxel for 
previously treated PD-L1 (≥1%) positive advanced NSCLC patients, ORR was 18%, while 
this was 30% in patients with a proportion score of ≥50% (46).  
On October 2, 2015, the U.S. Food and Drug Administration granted accelerated approval for 
pembrolizumab to treat patients with advanced (metastatic) NSCLC whose disease has 
progressed after other treatments and with tumors that express ≥1% PD-L1, as assessed by 
the companion diagnostic, the PD-L1 IHC 22C3 pharmDx test. 
In conclusion, tumor PD-L1 expression seems to be related to response but the signal is not 
straightforward since responses are seen across the level of PD-L1 expression and even in 
patients that are PD-L1 negative. Adding to the complexity, the level of tumor PD-L1 
expression is variable over time and influenced by several host and environmental factors 
such as TNM stage, chemotherapy and cytokines like IFN-α (47, 48). PD-L1– tumors can 
become positive and vice versa (49). Temporal and spatial variation of tumor PD-L1 
expression (within and between tumor lesions) might be responsible for its suboptimal 
predictive value for treatment benefit. Currently, some study protocols require positive tumor 
PD-L1 staining for inclusion. Looking at the above there seems to be an urgent need to 
further validate tumor PD-L1 IHC as predictive biomarker, as well as looking at alternatives. 
 
4.2 
Rationale 
 
4.2.1 Rationale of the trimodality approach 
As stated in section 4.1.1, despite the promising results of SBRT, overall survival was not 
different between the two modalities and the relapse rate is still substantial. In a study by 
Senthi et al, 676 patients underwent SBRT for early stage lung cancer with a 2-year 
recurrence rate of 22% (6). Furthermore, of the 124 recurrences, 82 (66%) were distant 
recurrences. Looking at the preclinical and clinical data on the synergistic systemic antitumor 
effect of combination radiotherapy and immunotherapy, there is a strong biological rationale 

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to study these two modalities together as induction therapy for patients with operable early 
stage NSCLC.   
In this protocol we will explore the addition of immunotherapy to SBRT to augment the 
antitumor T cell response, already associated with radiotherapy. This exploratory study aims 
primarily to assess the safety of the trimodality treatment strategy and to identify and 
characterize the immunological mechanisms of action of combined SBRT and anti-PD-1 
treatment. Because the SBRT technique allows to irradiate small tumor volumes with 
maximal sparing of healthy lung tissue, this is the ideal and safest setting to introduce the 
combination of pulmonary radiotherapy and immunotherapy in lung cancer. The results of 
this study can serve as a proof of principle for the immunological effect of thoracic 
radiotherapy in general and might serve as a vehicle for further development of the 
combination of radiotherapy and immunotherapy in early stage lung cancer and stage III 
disease, a setting where radiation pneumonitis is a well-known and feared complication.  
Because surgical resection is still the current standard of care for these patients, the subjects 
will undergo a lobectomy with hilar and mediastinal lymph node dissection after SBRT and 
pembrolizumab treatment. This is a key feature of this study and an unique opportunity to 
perform histological examination of the entire lung tumor and lymph nodes after 
immunotherapy, allowing to examine immune response heterogeneity and to correlate this 
with the non-invasive imaging and blood results. In the surrounding normal lung tissue, 
radiation and immunotherapy induced toxicity can be evaluated.  
The goal of this study is to evaluate the safety and mechanisms of action of the novel 
trimodality treatment approach.  
 
4.2.2 Rationale for Dose Selection/Regimen/Modification 
Pembrolizumab will be administered intravenously 200 mg Q3W in this trial. The rationale 
for further exploration of 2 mg/kg and comparable doses of pembrolizumab in solid tumors is 
based on: 1) similar efficacy and safety of pembrolizumab when dosed at either 2 mg/kg or 
10 mg/kg Q3W in melanoma patients, 2) the flat exposure-response relationships of 
pembrolizumab for both efficacy and safety in the dose ranges of 2 mg/kg Q3W to 10 mg/kg 
Q3W, 3) the lack of effect of tumor burden or indication on distribution behavior of 
pembrolizumab (as assessed by the population PK model) and 4) the assumption that the 
dynamics of pembrolizumab target engagement will not vary meaningfully with tumor type. 
The choice of the 200 mg Q3W as an appropriate dose for the switch to fixed dosing is based 
on simulations performed using the population PK model of pembrolizumab showing that the 
fixed dose of 200 mg every 3 weeks will provide exposures that 1) are optimally consistent 
with those obtained with the 2 mg/kg dose every 3 weeks, 2) will maintain individual patient 
exposures in the exposure range established in melanoma as associated with maximal 
efficacy response and 3) will maintain individual patients exposure in the exposure range 
established in melanoma that are well tolerated and safe.  
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. 
 

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4.2.3 Translational research 
Because the mode of action of combined SBRT and anti-PD-1 therapy is unknown, it is 
essential to perform translational research to identify the mechanisms of action and toxicity 
before effect size studies can be initiated with recurrence free and overall survival as 
outcome parameters.  
Serial invasive and noninvasive measurements of immune biomarkers will be performed for 
the primary tumor, lymph nodes and peripheral blood. A study schedule is provided in 
section 2.2. In short, the translational research will include: 
• Biological imaging of tumor PD-1 expression in patients randomized to combination 
radiotherapy and pembrolizumab treatment with 89Zr-pembrolizumab immuno-PET 
prior to treatment initiation. Biological imaging of PD-1 expression allows non-
invasive in-vivo monitoring of PD-1 binding. To visualize PD-1 engagement by 
pembrolizumab we use a novel technique called immuno-PET in which positron 
emission tomography (PET) is combined with a radiolabeled monoclonal antibody. 
Imaging with radiolabelled pembrolizumab (89Zr-pembrolizumab) allows for non-
invasive quantification of its direct target, the PD-1 receptor on tumor infiltrating 
lymphocytes. Because the technique is non-invasive and whole body, it allows to 
evaluate tumor heterogeneity (volumetric analysis of tumor PD-1 expression). 
• Immunohistochemical examination of immune effector subsets in a tumor core biopsy 
specimen at baseline. The following markers will be evaluated for expression: PD-1, 
PD-L1, CD4, CD8 and FoxP3, combined with Ki67 double staining. 
• Multicolor flowcytometric assessment of immune effector subset rates and activation 
state in peripheral blood and TDLNs by means of endoscopic ultrasound guided fine 
needle aspiration (EBUS-FNA) at baseline and after SBRT +/- pembrolizumab 
treatment. The following subsets/populations will be included in this analysis: 
(memory/effector) CD4+ and CD8+ T cells, Tregs, granulocytic and monocytic 
MDSCs, conventional and plasmacytoid DCs, and macrophages.   
• Three-dimensional tumor and lymph node histological examination of immune 
effector subsets in the resection specimen after SBRT +/- pembrolizumab treatment. 
This enables to   
 Explore intratumor heterogeneity of immune effector subsets after 
SBRT +/- pembrolizumab treatment, possibly associated with 
therapeutic resistance. 
 Correlate the 3D 89Zr-pembrolizumab PET image results with the 3D 
primary tumor histopathology results and the lymph node 
histopathology results. 
• In the lobectomy specimen the tumor surrounding healthy tissue will be examined for 
radiation induced changes.   
 
4.2.4 Rationale for Endpoints 
This an exploratory study that aims to evaluate the safety of the trimodality approach and 
characterize the immunological mechanisms of action of combination radiotherapy and 
immunotherapy. Therefore the primary endpoints of this study are the tolerability and 
toxicity of combined SBRT +/- pembrolizumab treatment and surgery and to analyze the 
immunological antitumor response. The endpoints will be assessed by clinical signs of 

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radiation pneumonitis and examination of tumor core biopsies and resection material, FACS 
analysis on EBUS-FNA of TDLNs and peripheral blood to detect immunomodulation.  
Secondary endpoints are the correlations of in-vivo PD-1 expression, quantified by immune-
PET, with tissue and blood based immune-parameters.  
The levels of PD-1 and PD-L1 expression are variable over time (see section 4.1.8) and 
expression has been found to be heterogeneous between and within tumor lesions of the same 
patient. The biomarker PD-L1 IHC is currently being validated in multiple clinical studies to 
stratify patients based on the intensity and percentage of positive staining. This trial is an 
unique opportunity to study the 3-dimensional uptake of 89Zr-pembrolizumab in the tumor 
and correlate the imaging result with the 3D immunohistochemistry results of the surgical 
tumor specimen.    
Although pneumonitis will be assessed as a symptom, an exploratory objective is to evaluate 
radiation induced changes to healthy lung tissue after SBRT +/- pembrolizumab treatment in 
the resected lung lobe. 
The number of patients (n=10 in each arm) is too low to correlate study parameters to clinical 
outcome in terms of progression-free or overall survival. 
 
4.2.5 
89Zr-pembrolizumab imaging 
To identify patients that will benefit the most from treatment with mAbs like pembrolizumab, 
better knowledge of the in vivo behavior of the drug is warranted. For this, positron emission 
tomography (PET) imaging with radiolabeled mAbs (immuno-PET) is an attractive option.  
The ability of PET to quantitatively image the distribution of radiolabeled drugs within the 
body makes this technique a valuable tool for drug development and application. Immuno-
PET allows to learn about the ideal drug dosing for optimal tumor targeting (e.g., saturation 
of receptors), the uptake in critical normal organs to anticipate toxicity, and the variation in 
pharmacokinetics and tumor targeting. Drug imaging is able to provide this information in an 
efficient and safe way. Pretreatment imaging with the drug of interest can be used for patient 
selection because it can show target expression and drug accumulation in tumor lesions and 
normal tissues noninvasively, quantitatively, and over time. This information might be 
particularly relevant for heterogeneous target expression or when targeted drugs are 
combined with other treatment modalities like parallel pathway inhibition, to find routes of 
synergism. In this way, immuno-PET can be used for better understanding of in vivo biology 
(“immunohistochemistry in vivo”) (50). 
To enable visualization of a targeted drug with a PET camera, the drug should be labeled 
with a positron emitter in an inert way, i.e., that neither binding nor pharmacokinetic 
characteristics of the drug become altered. Moreover, the physical half-life of the positron 
emitter should be compatible with the residence time of the targeted drug in the body, which 
is typically several days for slow kinetic intact mAbs. Universal procedures exist for 
radiolabeling of intact mAbs with the long-lived positron emitter Zirconium-89 (89Zr, t½ = 
78.4 h) (51).  
During the past years, several preclinical and clinical 89Zr-immuno-PET studies have been 
performed with intact mAbs, for example with mAbs against cMET, EGFR, VEGF and 
HER2 (52-55). 
The first clinical 89Zr-immuno-PET study ever was reported in 2006 by Börjesson et al. (56). 
This feasibility study with 20 head and neck cancer patients showed that 89Zr-cmAb, directed 

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against CD44v6, can be safely applied in patients and that it is a promising tool for PET 
detection of primary tumors as well as of metastases in the neck. There was good agreement 
between tumor uptake derived by PET and that derived by tumor biopsy. This suggests that 
patients with high and low mAb uptake can be differentiated, which might be important for 
the selection of patients with the highest chance of benefit from mAb therapy. High quality 
images were obtained in an immuno-PET study with 89Zr-trastuzumab in breast cancer 
patients (57). Excellent visualization of mAb uptake in HER2-positive lesions as well as in 
metastatic liver, lung, bone, and even brain HER2-positive lesions was observed. 89Zr-
trastuzumab PET allowed the quantification of conjugate uptake in HER2-positive lesions, 
and it became clear that for some patients with extensive tumor load, no HER2 saturation 
occurred during trastuzumab therapy. There was a good correlation between 89Zr-
trastuzumab tumor uptake and response as assessed by CT, MRI, and bone scans.  
 
Biological imaging of the PD-1 pathway with 89Zr-pembrolizumab allows to visualize the 
PD-1/PD-L1 pathway and non-invasive quantification of its direct target, the PD-1 receptor 
on tumor infiltrating lymphocytes. Because the technique is non-invasive and whole body, it 
allows for serial measurements of tumor uptake as well as looking at heterogeneity within 
and between tumor lesions.  
We hypothesize that 89Zr-pembrolizumab tumor uptake is heterogeneous within the tumor of 
individual patients and between tumor lesions of different patients and that uptake is related 
to PD-1 expression, PD-L1 expression. When a good correlation is found, 89Zr-
pembrolizumab PET might be used in other clinical settings to validate PD-1 and PD-L1 IHC 
as predictive biomarker of treatment benefit and to elucidate why some patients with positive 
PD-L1 IHC do not achieve a favorable tumor response and some patients with negative PD-
L1 IHC do.  
 
5.0 METHODOLOGY 
 
5.1 
Entry Criteria 
 
5.1.1 Diagnosis/Condition for Entry into the Trial 
Patients with early stage (T1bN0 and T2aN0) peripherally located lesions that are cytologically 
or histologically proven and eligible for surgical resection. Although varying definitions for 
centrally located tumors have been used, the International Association for the Study of Lung 
Cancer has recommended the following: a tumor within 2 cm in all directions of any 
mediastinal critical structure, including the bronchial tree, esophagus, heart, brachial plexus, 
major vessels, spinal cord, phrenic nerve, and recurrent laryngeal nerve (58).  
 
5.1.2 Subject Inclusion Criteria 
In order to be eligible for participation in this trial, the subject must: 
1. Have a histologically or cytologically confirmed diagnosis of early stage (T1bN0 and 
T2aN0) peripherally located NCSLC, eligible for surgical resection.  
2. Be willing and able to provide written informed consent/assent for the trial. 
3. Be ≥ 18 years of age on day of signing informed consent. 
4. Have measurable disease based on RECIST 1.1.   

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5. Must provide tissue from a core or excisional biopsy of the primary tumor lesion. 
6. Have a performance status of 0-1 on the ECOG Performance Scale. 
7. Demonstrate adequate organ function as defined in Table 1, all screening labs should 
be performed within 10 days of treatment initiation. 
   
Table 1.  Adequate Organ Function Laboratory Values 
System 
Laboratory Value 
Hematological 
 
Absolute neutrophil count (ANC) 
≥1,500 /mcL 
Platelets 
≥100,000 / mcL 
Hemoglobin 
≥9 g/dL or ≥5.6 mmol/L  
Renal 
 
Serum creatinine OR 
Measured or calculated creatinine 
clearance 
(GFR can also be used in place of 
creatinine or CrCl) 
≤1.5 X upper limit of normal (ULN) OR 
 
≥45 mL/min for subject with creatinine levels > 1.5 X 
institutional ULN 
Hepatic 
 
Serum total bilirubin 
≤ 1.5 X ULN OR 
 
Direct bilirubin ≤ ULN for subjects with total bilirubin 
levels > 1.5 ULN 
AST (SGOT) and ALT (SGPT) 
≤ 2.5 X ULN  OR 
≤ 5 X ULN for subjects with liver metastases 
 
8. Female subject of childbearing potential should have a negative urine or serum 
pregnancy within 72 hours prior to receiving the first dose of study medication. If the 
urine test is positive or cannot be confirmed as negative, a serum pregnancy test will 
be required.   
9. Female subjects of childbearing potential should be willing to use 2 methods of birth 
control or be surgically sterile, or abstain from heterosexual activity for the course of 
the study through 120 days after the last dose of study medication. Subjects of 
childbearing potential are those who have not been surgically sterilized or have not 
been free from menses for > 1 year.  
10. Male subjects should agree to use an adequate method of contraception starting with 
the first dose of study therapy through 120 days after the last dose of study therapy. 
 
5.1.3 Subject Exclusion Criteria 
The subject must be excluded from participating in the trial if the subject: 
1. Is currently participating in or has participated in a study of an investigational agent 
or using an investigational device within 4 weeks of the first dose of treatment. 
2. 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. 
 
3. Has had a prior monoclonal antibody within 4 weeks prior to study Day 1 or who has 
not recovered (i.e., ≤ Grade 1 or at baseline) from adverse events due to agents 
administered more than 4 weeks earlier.  

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4. Has had prior chemotherapy, targeted small molecule therapy, or radiation therapy 
within 2 weeks prior to study Day 1 or who has not recovered (i.e., ≤ Grade 1 or at 
baseline) from adverse events due to a previously administered agent. 
- 
Note: Subjects with ≤ Grade 2 neuropathy are an exception to this criterion 
and may qualify for the study. 
- 
Note: If subject received major surgery, they must have recovered adequately 
from the toxicity and/or complications from the intervention prior to starting 
therapy.   
5. Has a known additional malignancy that is progressing or requires active treatment.  
Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the 
skin, or in situ cervical cancer that has undergone potentially curative therapy.   
6. Has an active autoimmune disease requiring systemic treatment within the past 3 
months or a documented history of clinically severe autoimmune disease, or a 
syndrome that requires systemic steroids or immunosuppressive agents. Subjects with 
vitiligo or resolved childhood asthma/atopy would be an exception to this rule.  
Subjects that require intermittent use of bronchodilators or local steroid injections 
would not be excluded from the study. Subjects with hypothyroidism stable on 
hormone replacement or Sjorgen’s syndrome will not be excluded from the study. 
7. Has a history of (non-infectious) pneumonitis that required steroids, evidence of 
interstitial lung disease or active, non-infectious pneumonitis. 
8. Has an active infection requiring systemic therapy. 
9. 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.   
10. Has known psychiatric or substance abuse disorders that would interfere with 
cooperation with the requirements of the trial. 
11. Is pregnant or breastfeeding, or expecting to conceive or father children within the 
projected duration of the trial, starting with the pre-screening or screening visit 
through 120 days after the last dose of trial treatment. 
12. Has received prior therapy with an anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, 
or anti-Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody (including 
ipilimumab or any other antibody or drug specifically targeting T-cell co-stimulation 
or checkpoint pathways). 
13. Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies). 
14. Has known active Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA 
[qualitative] is detected). 
15. Has received a live vaccine within 30 days prior to the first dose of trial treatment. 
 
5.2 
Sample size 
Twenty patients will be included in this study. A formal power analysis to calculate a sample 
size is not feasible due to the explorative nature of this trial. The sample size of 10 patients in 
each arm is based on two previous analyses that we performed. In a cohort of 9 head and 
neck cancer patients we were able to find significant differences in the expression of 
activation markers between different dendritic cell subsets (data on file). In a cohort of 9 

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melanoma patients we were able to find significant differences in the expression of activation 
markers between dendritic cell subsets of patients with a Breslow thickness more or less than 
1.5 mm (60). 
 
5.3 
Pretreatment imaging 
 
5.3.1 Prestudy diagnostic and staging imaging 
Standard of care pre-treatment staging includes a diagnostic CT chest and a whole-body static 
PET-CT.  
 
5.3.2 
89Zr-pembrolizumab PET 
 
5.3.2.1 Radiolabeling of pembrolizumab and quality controls 
Using standard procedures for the production of 89Zr-labeled monoclonal antibodies, several 
clinical 89Zr-immuno-PET studies have been performed, are ongoing, or will be started 
shortly. In these ongoing studies safety was confirmed. 89Zr is coupled to pembrolizumab via 
the bifunctional chelator desferal. 89Zr will be produced at the VU University Medical Center 
by Cyclotron BV according to procedures essentially as described before (51). 
Radiochemical purity of the purified 89Zr used for labeling will be according to the 
specification as described in the same paper. 89Zr-mAb conjugates produced in this way were 
found to be fully stable in vitro and in vivo as shown in several preclinical and clinical 
studies (61). Using the same procedures, the following conjugates have been produced and 
administered to patients without any adverse event: 89Zr-cMAb U36, 89Zr-ibritumomab 
tiuxetan (ZevalinTM), 89Zr-rituximab (RituxanTM), 89Zr-trastuzumab (HerceptinTM), 89Zr-
bevacizumab (AvastinTM) and 89Zr-cetuximab (ErbituxTM) (56, 62). 
Radiolabeling of pembrolizumab with 89Zr will be performed according to Standard 
Operating Procedures (SOPs) within the premises of the Radionuclide Center (RNC) of the 
Department of Nuclear Medicine & PET research. The whole process will be performed 
according to state-of-the-art Good Manufacturing Practice (GMP) standards. This production 
site was recently inspected by the Dutch Health Care Inspectorate and is licensed to 
manufacture tracers for human use according to the latest EU guidelines. This site has also 
been audited by the largest EU pharma-companies.  
 
The procedures for radiolabeling of mAbs with 89Zr have been validated with respect to the 
synthesis procedure and the final quality of the prepared conjugates. The radiochemical 
purity, immunoreactivity and endotoxin content of every batch will be assessed prior to 
administration to a patient. The radiochemical purity as assessed by ITLC (instant thin layer 
chromatography) and HPLC (high performance liquid chromatography) should always be 
>90%, preferably >95%, while the immunoreactive fraction as assessed by cell binding 
assay, should always exceed 70%. This analysis will be performed according to a procedure 
essentially as described by Lindmo et al (63). The endotoxin content is determined according 
to pharmacopeia using an endosafe PTS reader. Sterility of each 89Zr-pembrolizumab batch 
will be assured by performing a media fill immediately after final sterilization of each batch. 
The expiration time of the conjugates will be defined in validations. 
 

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5.3.2.2 Radiation exposure 
For the patient the radiation dose of administering 37 MBq of 89Zr-pembrolizumab is 
expected to be around 18 mSv. Low dose CT scans used for attenuation correction will give 
an additional dose of 3 mSv per CT. The effective dose is therefore expected to be around 24 
mSv for the immune-PET scan. After injection no shielding is required and the patient can 
immediately go home with instructions.  
 
5.3.2.3 Injection procedure 
The injection will take place at the department of Nuclear Medicine & PET research. A tracer 
dose (2 mg) of 89Zr-pembrolizumab will be injected as a bolus of 20 mL with flushing (10 
mL physiologic saline).  
During administration of 89Zr-pembrolizumab the patient will be monitored closely and 
appropriate measurements will be performed whenever judged necessary. In case of an 
allergic reaction to 89Zr-pembrolizumab the patient will be treated according to the standard 
clinical protocol. In this situation, the patient will be eliminated from the study.  
 
5.3.2.4 Blood sample analysis 
Blood samples (7 mL) will be withdrawn at 10 min post-injection, 72 hours and 144 hours 
post-injection of 89Zr-pembrolizumab to determine 89Zr-kinetics based on measurements of 
radioactivity. 
 
5.3.2.5 89Zr-pembrolizumab PET procedure  
All PET scans will be performed on a Philips Ingenuity TF PET/CT scanner.  
All patients will undergo two whole body PET scans at t = 72 and t = 144 hours post-
injection. The PET scans will be preceded by a 30 mAs low-dose CT, used for attenuation 
correction and anatomical localization of the PET-signal. Following CT, a total body PET 
scan will be acquired. PET scans consist of 10-12 bed positions, depending on the length of 
the patient, of 5 minutes each. Total acquisition time per scan, including low dose CT will be 
around 60 minutes.  
PET data will be normalized and corrected for randoms, tissue attenuation, decay, scatter and 
dead time. PET-CT data will be reconstructed with TF-OSEM, resulting in a transaxial 
spatial resolution of ~7 mm in the centre of the field of view. Regions of interest (ROIs) will 
be defined using the CT images which are co-registered to the PET-CT data. ROIs around the 
organs which accumulate 89Zr (e.g. liver, lung and other metastatic lesions) will be drawn 
manually directly on the PET images. All tissue concentrations will be related to the blood 
concentration (venous samples). The radioactivity concentrations in all regions for all 
imaging time points will be recorded digitally in a spread sheet.  
Quantitative accuracy of 89Zr-PET scans was previously confirmed in our center using 
phantom studies and comparisons between PET-derived and biopsy tumor radioactivity 
concentrations (64, 65).  
 
5.3.2.6 Analysis of PET data 
Tumor volumes of interest will be delineated using semi-automated in-house developed 
software. SUVmax, SUVmean and SUVpeak will be calculated for the tumor and visible lymph 
nodes (67-69).  

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5.4 
On-treatment imaging 
The diagnostic CT chest will be repeated within two weeks before surgical resection.  
 
5.4.1 Quantitative changes in lung density 
The aim of these measurements is to determine whether a relationship exists between SBRT 
dose and radiographic lung injury, as measured by CT density changes (70). 
Contours of selected isodose levels (0.5 Gy, 3 Gy, 6 Gy, 12 Gy, 18 Gy, 24 Gy, 36 Gy, and 50 
Gy) will be exported from the planning system along with the contours of the lung and 
internal target volume, and the average-intensity CT dataset. These isodose lines are chosen 
to provide a wide range of doses and large enough volumes between lines to allow for 
meaningful density measurements. This results in a total of eight evaluable dose-density 
regions in the ipsilateral lung. In the contralateral lung, only one dose-density region 
(receiving ≥3 Gy) will be analyzed, because it is uncommon for the contralateral lung to 
receive >5 Gy. The contralateral lung receiving <3 Gy will be considered unirradiated and as 
such used to correct for baseline differences between scanners. The regions receiving >50 Gy 
are not analyzed because these volumes are small and because small errors in registration can 
place a few millimeters of regressing or stable tumor within that volume, artificially 
increasing density measurements. After deformation of the follow-up scans, isodoses from 
the endinspiratory phase of the planning CT scan will then be overlaid on the deformed 
follow-up scan, and changes in Hounsfield unit (HU) density assessed.  
The air-filled fraction (fair) will be calculated from HU density using the formula fair = _0.001 
_ NCT, where NCT is the CT number in HU (71). fair represents the percentage of lung tissue 
that contains air, and it decreases with increasing CT density. 
   
 
5.5 
Ultrasound 
endoscopy 
guided 
cytological 
lymph 
node 
aspiration 
for 
immunological cell composition (EBUS FACS) 
 
5.5.1 
Bronchoscopy and EBUS procedure 
EBUS-FNA of the ipsilateral and contralateral hilar and mediastinal lymph nodes will be 
performed within 2 weeks before and between weeks 2 and 3 after SBRT. Patient preparation 
and sedation will be performed according to regular institutional practice. Needle aspirations 
will be performed with a 22-gauge needle and 15 passages of the needle per lymph node. The 
ipsilateral hilar and mediastinal TDLNs will be sampled. 
 
5.5.2 
Lymph node analysis 
Cells obtained though needle aspirations will be collected in 7.5ml of culture medium 
(Iscove’s Modified Dulbecco’s Medium (IMDM)). Since these aspirates contain small tissue 
components, the specimen will be enzymatically digested for 45 minutes at 37°C in a sterile 
glass flask by adding 30ml of IMDM with 0.02% DNAse and 0.1% Collagenase (60). The 
cell suspension will then be filtered through a 100μm sterile filter and the flask is rinsed with 
20ml IMDM. Cells will be pelleted by centrifugation (5 minutes, 530xg) and counted using 
trypan blue staining to identify dead cells. Depending on the cell yield, the following immune 
subsets will be analyzed by flow cytometry using subset-specifying markers (in order of 

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interest): 1). CD4, CD8 and T regulatory cells (FoxP3), 2). MDSC, 3). T-cell activation and 
memory/effector phenotypes, 4). myeloid DC subsets, 5). plasmacytoid DC, 6). NK/NKT 
cells, 7). γδT cells and 8) B cells.  
For flow cytometry, surface marker staining will be performed by incubating the cells with 
specific antibodies for 30 minutes at 4°C in PBS supplemented with 1% BSA and 0.02% 
NaN3 (FACS buffer). If erythrocytes are abundant in the specimen, these will be lysed by 
adding BD lysis buffer after 15 minutes of surface antibody staining, after which cells will be 
stained for an additional 15 minutes. Cells will be washed in FACS buffer and if no 
intracellular markers need to be stained (FoxP3), cells are ready for analysis. For intracellular 
staining of FoxP3 and CTLA-4, cells will be permeabilized and fixated using the eBioscience 
FoxP3 fix/perm kit according to manufacturer’s guidelines: cells are first incubated with 
perm/fix solution for 30 minutes at 4°C and washed with perm/wash solution before blocking 
with normal rabbit serum (for FoxP3 stainings) and addition of FoxP3 or CTLA-4 specific 
antibodies. Antibody staining is performed for 30 minutes at 4°C, after which cells are 
washed once in perm/wash solution and once in FACS buffer prior to analysis on a flow 
cytometer.  
 
5.6 
Treatment plan 
 
5.6.1 Stereotactic body radiotherapy (SBRT) 
SBRT delivery will be performed using techniques that are in accordance with the 2017 
guidelines of European Society for Radiotherapy and Oncology (ESTRO)-ACROP An 
intensity-modulated radiotherapy technique will be used to deliver  a risk-adapted protocol, with 
the dose and number of fractions dependent on the size and location of the tumor (72, 73). 
Standard SBRT doses will be used in accordance with the previous ROSEL study in operable 
patients [reference] 
 
5.6.1.1 Immobilization, Imaging and Registration 
Patients will be set-up using reproducible positioning, verified using an on-line protocol. 
Immobilization may include a custom immobilization device, such as a vac-loc bag. All 
patients treated on a linear accelerator will undergo 4-D planning CT simulation as described 
previously (72). A 4-D planning scan will not be mandatory in patients who undergo MR-
guided SBRT [reference].  
 
5.6.1.2 Volume Definitions and Prescription 
The gross tumor volume (GTV) will be defined as the visible tumor on CT or MR-imaging 
(after verification on planning CT)Planning Target Volume (PTV)  will be generated in 
accordance with ESTRA-ACROP guidelines, and the margins used will depend on the choice of 
imaging and delivery techniques used. All relevant organs at risk in the proximity of the PTV 
will be contoured. Dose fractionation schemes to be used are listed in Table 2.  
 
Table 2. Dose fractionation schemes according to tumor location 
Tumor Size and Location 
Total Dose 
(Gy) 
Number of 
fractions  
Dose 
per 
fraction (Gy) 
Frequency 
Tumors 3 cm or less 
surrounded 
by 
lung 
54 
3  
18 
Every 
second 
day 

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parenchyma 
Abutting chest wall or >3 
cm 
55 
5 
11 
Every 
second 
day 
 
Organ-at-risk  constraints will be those used in the protocol for a phase III trial in patients who 
were fit to undergo surgery (74). Doses are prescribed to the 80% isodose line encompassing the 
PTV, resulting in a hotspot of 120-140% in the ITV. 95% of the PTV should be encompassed 
by the prescription dose, and 99% of the PTV should be covered by 90% of the prescription 
dose.  
 
5.6.1.3 Quality Assurance 
In order to ensure patient safety and effective treatment delivery, a robust quality assurance 
protocol is incorporated. The following requirements must be completed for each patient: 
• Prior to treatment, each patient must be discussed at departmental quality assurance 
(QA) rounds. 
• All radiotherapy plans must meet protocoldose criteria for organs at riskand plans must 
be approved by physics and verified  by the  treating physician. Dose constraints should 
not be exceeded without prior discussion with the responsible radiation oncologist in the 
study team. 
• Institutional treatment verification procedures may vary based upon equipment 
available, but compliance with ESTRO-ACROP guidelines must be ensured. On linear 
accelerators cone-beam CT will generally be used to verify tumor localization  prior to 
treatment, and, if indicated, verification can also be performed during and/or after 
treatmentFor MR-guided adaptive SBRT, real-time imaging using gated delivery can be 
performed.  
 
5.6.2 Pembrolizumab Treatment 
Patients randomized to the pembrolizumab arm will be treated with 200 mg pembrolizumab 
Q3W for 2 cycles or until unacceptable side effects, starting on the first day of radiotherapy. 
 
5.6.2.1 Dose Modification 
Pembrolizumab will be withheld for drug-related Grade 4 hematologic toxicities, non-
hematological toxicity ≥ Grade 3 including laboratory abnormalities, and severe or life-
threatening AEs as per Table 3 below. 
 
Table 3: Dose modification guidelines for drug-related adverse events. 
 
Toxicity 
Hold 
Treatment 
For Grade 
Timing for Restarting Treatment 
Treatment Discontinuation 
Diarrhea/Colitis 
2-3 
 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks 
4 
Permanently discontinue 
Permanently discontinue 
AST, ALT, or 
Increased Bilirubin 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose 
3-4 
Permanently discontinue 
(see exception below)a 
Permanently discontinue 

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Toxicity 
Hold 
Treatment 
For Grade 
Timing for Restarting Treatment 
Treatment Discontinuation 
Type 1 diabetes 
mellitus (if new 
onset) or 
Hyperglycemia 
T1DM or  
3-4 
Hold pembrolizumab for new onset Type 
1 diabetes mellitus or Grade 3-4 
hyperglycemia associated with evidence of 
beta cell failure 
Resume pembrolizumab when patients are clinically and 
metabolically stable 
Hypophysitis 
2-4 
Toxicity resolves to Grade 0-1. Therapy 
with pembrolizumab can be continued 
while endocrine replacement therapy is 
instituted 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks 
Hyperthyroidism  
3 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks 
4 
Permanently discontinue 
Permanently discontinue 
Hypothyroidism 
 
Therapy with pembrolizumab can be 
continued while thyroid replacement 
therapy is instituted 
Therapy with pembrolizumab can be continued while thyroid 
replacement therapy is instituted 
 
Infusion Reaction 
2b 
Toxicity resolves to Grade 0-1  
Permanently discontinue if toxicity develops despite adequate 
premedication  
 
3-4 
Permanently discontinue 
Permanently discontinue 
Pneumonitis 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks 
3-4 
Permanently discontinue 
Permanently discontinue 
Renal Failure or 
Nephritis 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks 
3-4 
Permanently discontinue 
Permanently discontinue 
All Other Drug-
Related Toxicityc 
3 or Severe 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks 
4 
Permanently discontinue 
Permanently discontinue 
Note: Permanently discontinue for any severe or Grade 3 drug-related AE that recurs or any life-threatening event. 
a For patients with liver metastasis who begin treatment with Grade 2 AST or ALT, if AST or ALT increases by greater than or equal to 50% relative to baseline 
and lasts for at least 1 week then patients should be discontinued. 
b 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; Refer to Table 2 – Infusion 
Treatment Guidelines for further management details. 
c Patients with intolerable or persistent Grade 2 drug-related AE may hold study medication at physician discretion.  Permanently discontinue study drug for 
persistent Grade 2 adverse reactions for which treatment with study drug has been held, that do not recover to Grade 0-1 within 12 weeks of the last dose. 
 
 
In case toxicity does not resolve to Grade 0-1 within 12 weeks after last infusion, trial 
treatment should be discontinued. Subjects with a laboratory adverse event still at Grade 2 
after 12 weeks may continue treatment in the trial only if asymptomatic and controlled. For 
information on the management of adverse events, see Section 5.7.1.   
Subjects who experience a recurrence of the same severe or life-threatening event at the same 
grade or greater with re-challenge of pembrolizumab should be discontinued from trial 
treatment. 
 
5.6.2.2 Timing of Dose Administration 
Trial treatment should be administered on Day 1 of each cycle after all 
procedures/assessments have been completed as detailed on the Trial Flow Chart (Section 
6.0). Trial treatment may be administered up to 3 days before or after the scheduled Day 1 of 

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each cycle due to administrative reasons. All trial treatments will be administered on an 
outpatient basis. 
Pembrolizumab will be administered as a 30 minute IV infusion (treatment cycle intervals 
may be increased due to toxicity as described in Section 5.8.1.2). Effort must be made to 
target infusion timing to be as close to 30 minutes as possible. However, a window of -5 
minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). 
 
5.6.3 Surgical resection 
Surgery will occur 8-10 weeks following SBRT +/- pembrolizumab treatment. Surgery will 
consist of a lobectomy and may employ either an open approach or a video-assisted 
thoracoscopic approach. Surgical sampling of ipsilateral hilar and mediastinal lymph nodes at 
the time of resection is mandatory.  
 
5.7 
Concomitant Medications/Vaccinations (allowed & prohibited) 
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed 
during the ongoing trial. If there is a clinical indication for one of these or other medications 
or vaccinations specifically prohibited during the trial, discontinuation from trial therapy or 
vaccination may be required. The final decision on any supportive therapy or vaccination 
rests with the investigator and/or the subject's primary physician.  
  
5.7.1 Acceptable Concomitant Medications 
All treatments that the investigator considers necessary for a subject’s welfare may be 
administered at the discretion of the investigator in keeping with the community standards of 
medical care.  
 
5.7.2 Prohibited Concomitant Medications 
Subjects are prohibited from receiving the following therapies during the Screening and 
Treatment Phase of this trial: 
• Anti-cancer systemic chemotherapy or biological therapy  
• Immunotherapy not specified in this protocol 
• Investigational agents other than pembrolizumab 
• Live vaccines within 30 days prior to the first dose of trial treatment and while 
participating in the trial. Examples of live vaccines include, but are not limited to, the 
following: measles, mumps, rubella, chicken pox, yellow fever, rabies, BCG, and 
typhoid (oral) vaccine. Seasonal influenza vaccines for injection are generally killed 
virus vaccines and are allowed; however intranasal influenza vaccines (e.g. Flu-
Mist®) are live attenuated vaccines, and are not allowed.  
• 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 is allowed.  
Subjects who, in the assessment by the investigator, require the use of any of the 
aforementioned treatments for clinical management should be removed from the trial.  
Subjects may receive other medications that the investigator deems to be medically 
necessary. The Exclusion Criteria describe other medications which are prohibited in this 
trial. There are no prohibited therapies during the Post-Treatment Follow-up Phase. 

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5.8 
Rescue Medications & Supportive Care 
 
5.8.1 Supportive Care Guidelines 
Subjects should receive appropriate supportive care measures as deemed necessary by the 
treating investigator.  Suggested supportive care measures for the management of adverse 
events with potential immunologic etiology are outlined below. Where appropriate, these 
guidelines include the use of oral or intravenous treatment with corticosteroids as well as 
additional anti-inflammatory agents if symptoms do not improve with administration of 
corticosteroids.   Note that several courses of steroid tapering may be necessary as symptoms 
may worsen when the steroid dose is decreased. For each disorder, attempts should be made 
to rule out other causes such as metastatic disease or bacterial or viral infection, which might 
require additional supportive care. The treatment guidelines are intended to be applied when 
the investigator determines the events to be related to pembrolizumab.  
Note: if after the evaluation the event is determined not to be related, the investigator does 
not need to follow the treatment guidance (as outlined below). Refer to Section 5.2.1 for dose 
modification.  
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or 
skin photography as part of evaluation of the event.  
• Pneumonitis:  
o For Grade 2 events, treat with systemic corticosteroids. When symptoms 
improve to Grade 1 or less, steroid taper should be started and continued over 
no less than 4 weeks. 
o For Grade 3-4 events, immediately treat with intravenous steroids.  
Administer additional anti-inflammatory measures, as needed. 
o Add prophylactic antibiotics for opportunistic infections in the case of 
prolonged steroid administration. 
• Diarrhea/Colitis:  
Subjects should be carefully monitored for signs and symptoms of enterocolitis (such 
as diarrhea, abdominal pain, blood or mucus in stool, with or without fever) and of 
bowel perforation (such as peritoneal signs and ileus).   
o All subjects who experience diarrhea/colitis should be advised to drink liberal 
quantities of clear fluids.  If sufficient oral fluid intake is not feasible, fluid 
and electrolytes should be substituted via IV infusion.  For Grade 2 or higher 
diarrhea, consider GI consultation and endoscopy to confirm or rule out 
colitis. 
o For Grade 2 diarrhea/colitis,  administer oral corticosteroids.  
o For Grade 3 or 4 diarrhea/colitis, treat with intravenous steroids followed by 
high dose oral steroids.   
o When symptoms improve to Grade 1 or less, steroid taper should be started 
and continued over no less than 4 weeks. 
• Type 1 diabetes mellitus (if new onset, including diabetic ketoacidosis [DKA]) or 
≥ Grade 3 Hyperglycemia, if associated with ketosis (ketonuria) or metabolic 
acidosis (DKA) 
o For T1DM or Grade 3-4 Hyperglycemia 

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 Insulin replacement therapy is recommended for Type I diabetes 
mellitus and for Grade 3-4 hyperglycemia associated with metabolic 
acidosis or ketonuria.  
 Evaluate patients with serum glucose and a metabolic panel, urine 
ketones, glycosylated hemoglobin, and C-peptide.  
• Hypophysitis: 
o For Grade 2 events, treat with corticosteroids. When symptoms improve to 
Grade 1 or less, steroid taper should be started and continued over no less than 
4 weeks. Replacement of appropriate hormones may be required as the steroid 
dose is tapered. 
o For Grade 3-4 events, treat with an initial dose of IV corticosteroids followed 
by oral corticosteroids. When symptoms improve to Grade 1 or less, steroid 
taper should be started and continued over no less than 4 weeks. Replacement 
of appropriate hormones may be required as the steroid dose is tapered. 
• Hyperthyroidism or Hypothyroidism:  
Thyroid disorders can occur at any time during treatment. Monitor patients for 
changes in thyroid function (at the start of treatment, periodically during treatment, 
and as indicated based on clinical evaluation) and for clinical signs and symptoms of 
thyroid disorders. 
o Grade 2 hyperthyroidism events (and Grade 2-4 hypothyroidism): 
 In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are 
suggested as initial therapy. 
 In hypothyroidism, thyroid hormone replacement therapy, with 
levothyroxine or liothyroinine, is indicated per standard of care. 
o Grade 3-4 hyperthyroidism  
 Treat with an initial dose of IV corticosteroid followed by oral 
corticosteroids. When symptoms improve to Grade 1 or less, steroid 
taper should be started and continued over no less than 4 weeks. 
Replacement of appropriate hormones may be required as the steroid 
dose is tapered. 
• Hepatic: 
o For Grade 2 events, monitor liver function tests more frequently until 
returned to baseline values (consider weekly). 
 Treat with IV or oral corticosteroids 
o For Grade 3-4 events, treat with intravenous corticosteroids for 24 to 48 
hours.  
o When symptoms improve to Grade 1 or less, a steroid taper should be started 
and continued over no less than 4 weeks. 
• Renal Failure or Nephritis: 
o For Grade 2 events, treat with corticosteroids. 
o For Grade 3-4 events, treat with systemic corticosteroids. 
o When symptoms improve to Grade 1 or less, steroid taper should be started 
and continued over no less than 4 weeks.  

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• Management of Infusion Reactions: Signs and symptoms usually develop during or 
shortly after drug infusion and generally resolve completely within 24 hours of 
completion of infusion.  
Table 2 below shows treatment guidelines for subjects who experience an infusion 
reaction associated with administration of pembrolizumab (MK-3475). 
Table 5 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) 
Stop Infusion. 
Additional appropriate medical therapy 
may include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Epinephrine 
No subsequent dosing 

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NCI CTCAE Grade 
Treatment 
Premedication at 
subsequent dosing 
Grade 4: 
Life-threatening; pressor or 
ventilatory support indicated 
 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion 
of the investigator. 
Hospitalization may be indicated. 
Subject is permanently discontinued 
from further trial treatment 
administration. 
Appropriate resuscitation equipment should be available in the room and a physician readily available 
during the period of drug administration. 
 
 
5.9 
Diet/Activity/Other Considerations 
 
5.9.1 Diet 
Subjects should maintain a normal diet unless modifications are required to manage an AE 
such as diarrhea, nausea or vomiting. 
 
5.9.2 Contraception 
Pembrolizumab may have adverse effects on a fetus in utero. Furthermore, it is not known if 
pembrolizumab has transient adverse effects on the composition of sperm. Non-pregnant, 
non-breast-feeding women may be enrolled if they are willing to use 2 methods of birth 
control or are considered highly unlikely to conceive. Highly unlikely to conceive is defined 
as 1) surgically sterilized, or 2) postmenopausal (a woman who is ≥45 years of age and has 
not had menses for greater than 1 year will be considered postmenopausal), or 3) not 
heterosexually active for the duration of the study. The two birth control methods can be 
either two barrier methods or a barrier method plus a hormonal method to prevent pregnancy. 
Subjects should start using birth control from study Visit 1 throughout the study period up to 
120 days after the last dose of study therapy.  
The following are considered adequate barrier methods of contraception: diaphragm, condom 
(by the partner), copper intrauterine device, sponge, or spermicide. Appropriate hormonal 
contraceptives will include any registered and marketed contraceptive agent that contains an 
estrogen and/or a progestational agent (including oral, subcutaneous, intrauterine, or 
intramuscular agents). 
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 they must adhere to the contraception requirement (described above) for the 
duration of the study and during the follow-up period defined in section 7.2.2 – Reporting of 
Pregnancy and Lactation. If there is any question that a subject will not reliably comply with 
the requirements for contraception, that subject should not be entered into the study. 
 
5.9.3 Use in Pregnancy 
If a subject inadvertently becomes pregnant while on treatment with pembrolizumab, the 
subject will immediately be removed from the study. The site will contact the subject at least 

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monthly and document the subject’s status until the pregnancy has been completed or 
terminated. The outcome of the pregnancy will be reported to the PI and to Merck without 
delay and within 24 hours if the outcome is a serious adverse experience (e.g., death, 
abortion, congenital anomaly, or other disabling or life-threatening complication to the 
mother or newborn). The study investigator will make every effort to obtain permission to 
follow the outcome of the pregnancy and report the condition of the fetus or newborn to the 
PI and Merck. If a male subject impregnates his female partner the study personnel at the site 
must be informed immediately and the pregnancy reported to the PI and to Merck and 
followed as described above and in Section 7.2.2. 
 
5.9.4 Use in Nursing Women 
It is unknown whether pembrolizumab is excreted in human milk. Since many drugs are 
excreted in human milk, and because of the potential for serious adverse reactions in the 
nursing infant, subjects who are breast-feeding are not eligible for enrolment. 
 
5.10 Subject Withdrawal/Discontinuation Criteria 
Subjects may withdraw consent at any time for any reason or be dropped from the trial at the 
discretion of the investigator should any untoward effect occur. In addition, a subject may be 
withdrawn by the investigator or the PI if enrollment into the trial is inappropriate, the trial 
plan is violated, or for administrative and/or other safety reasons. Specific details regarding 
discontinuation or withdrawal are provided in Section 7.1.3. 
 
A subject must be discontinued from the trial for any of the following reasons: 
• The subject or legal representative (such as a parent or legal guardian) withdraws 
consent. 
• Confirmed radiographic disease progression 
• Unacceptable adverse experiences 
• Intercurrent illness that prevents further administration of treatment 
• Investigator’s decision to withdraw the subject 
• The subject has a confirmed positive serum pregnancy test 
• Noncompliance with trial treatment or procedure requirements 
• The subject is lost to follow-up 
• Administrative reasons 
 
The End of Treatment and Follow-up visit procedures are listed in Section 6 (Trial Flow 
Chart) and Section 7.1.4 (Visit Requirements). After the end of treatment, each subject will 
be followed for 30 days for adverse event monitoring. Subjects who discontinue for reasons 
other than progressive disease will have post-treatment follow-up for disease status until 
disease progression, initiating a non-study cancer treatment, withdrawing consent or 
becoming lost to follow-up.  
 
5.11 Subject Replacement Strategy 
Subjects that fail to undergo the trimodality treatment will be replaced. 
 

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5.12 Beginning and End of the Trial 
The overall trial begins when the first subject signs the informed consent form. The trial ends 
when the last subject completes the last trial visit, discontinues from the trial or is 
lost to follow-up (i.e. the subject is unable to be contacted by the investigator). 
We expect to enroll one patient per month. This means that study can be finished within 2.5 
years from the time the first subject signs the Informed Consent Form (ICF) through the final 
contact. 
 
5.13 Clinical Criteria for Early Trial Termination 
Early trial termination will be the result of the criteria specified below: 
1. Quality or quantity of data recording is inaccurate or incomplete 
2. Poor adherence to the protocol and regulatory requirements 
3. Incidence or severity of adverse drug reaction in this or other studies indicates a potential 
health hazard to subjects 

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6.0 TRIAL FLOW CHART 
6.1 
Study Flow Chart 
Trial period 
Screening Phase 
Imaging 
Induction treatment 
phase 
Surgery 
Follow-up 
Scheduling Window (Days) 
-30 to -1 
-14 to 0 
From day 1 to day 56 
(2 weeks after 2nd 
pembrolizumab cycle) 
From day 56 to 
day 70 
From surgery to 6 
months thereafter 
 
 
 
 
 
 
Administrative Procedures 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
Elegibility Criteria 
X 
 
 
 
 
Demographics and Medical 
History 
X 
 
 
 
 
Prior and Concomitant 
Medication Review 
X 
 
 
 
 
 
 
 
 
 
 
PET Imaging 
 
 
 
 
 
89Zr-pembrolizumab 
 
Xa 
 
 
 
 
 
 
 
 
 
Clinical 
Procedures/Assessments 
 
 
 
 
 
Review Adverse Events 
X 
 
Xb 
 
 
Full Physical Examination 
X 
 
 
 
 
Directed Physical Examination 
X 
 
Xb 
 
Xc 
Vital Signs and Weight 
X 
 
Xb 
 
 
ECOG Performance Status 
X 
 
Xb 
 
 
12-lead ECG 
X 
 
 
 
 
18F-FDG PET-CT 
 
X 
 
X 
 
 
 
 
 
 
 
Laboratory 
Procedures/Assessments 
 
 
 
 
 
Pregnancy Test 
X 
 
 
 
 
Complete Blood Count (CBC) 
X 
Xi 
Xb 
 
 
Comprehensive Serum Chemistry 
Panel 
X 
 
Xb 
 
 
Urinalysis 
X 
 
 
 
 
T3, FT4 and TSH 
X 
 
Xd 
 
 
 
 
 
 
 
 
Treatment 
 
 
 
 
 
SBRT 
 
 
X 
 
 
Pembrolizumab 
 
 
Xa 
 
 
Surgery 
 
 
 
X 
 
 
 
 
 
 
 
Efficacy Measurements 
 
 
 
 
 
Tumor Imaging (CT thorax +/- 
abdomen and MRI brain) 
 
X 
Xe 
 
Xf 
 
 
 
 
 
 
Tumor Tissue and Blood 
analysis 
 
 
 
 
 
Core tumor biopsy 
X 
 
 
 
 
EBUS FACS 
 
X 
Xg 
 
 
Surgical resection specimen  
 
 
 
X 
 
Immunophenotyping 
 
Xh 
Xh 
Xh 
Xh 
 
 
 
 
 
 

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Xa: only for patients randomized to the SBRT + pembrolizumab arm; Xb: every cycle;  every 6 weeks; Xc: if indicated; Xd: after 
2nd pembrolizumab cycle; Xe: within 2 weeks of surgery; Xf: following resection every 3 months for the first 2 years and every 6 
months for the 3 years thereafter; Xf:; Xg:between week 2 and 3 after SBRT; . Xh: see section 7.1.2.10 for timepoints; Xi : 72 and 144 
hours post-injection for patients undergoing 18F-FGD-PET-CT  
 

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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 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 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 and applicable laws and 
regulations. 
 
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.  
 

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7.1.1.3 Medical History 
A medical history will be obtained by the investigator or qualified designee. Medical history 
will include all active conditions, and any condition diagnosed within the prior 10 years that 
are considered to be clinically significant by the Investigator.  
 
7.1.1.4 Prior and Concomitant Medications Review 
 
7.1.1.4.1 Prior Medications 
The investigator or qualified designee will review prior medication use, including any 
protocol-specified washout requirement, and record prior medication taken by the subject 
within 28 days before starting the trial.  
  
7.1.1.4.2 Concomitant Medications 
The investigator or qualified designee will record medication, if any, taken by the subject 
during the trial. All medications related to reportable SAEs and ECIs should be recorded as 
defined in Section 7.2. 
 
7.1.1.5 Disease Details and Treatments 
 
7.1.1.5.1 Disease Details 
The investigator or qualified designee will obtain prior and current details regarding disease 
status. 
 
7.1.1.5.2 Prior Treatment Details 
The investigator or qualified designee will review all prior cancer treatments including 
systemic treatments, radiation and surgeries. 
 
7.1.1.5.3 Subsequent Anti-Cancer Therapy Status 
If a subject initiates a new anti-cancer therapy within 30 days after the last dose of trial 
treatment, the 30 day Safety Follow-up visit must occur before the first dose of the new 
therapy.  
  
7.1.1.6 Assignment of Screening Number 
All consented subjects will be given a unique screening number that will be used to identify 
the subject for all procedures that occur prior to the PET scans and pembrolizumab treatment. 
The screening assignment will start with the term PembroRTSurgery followed by the number 
of inclusion (e.g. PembroRTSurgery001).  
 
7.1.1.7 Randomization 
Patients will be randomized 1:1 by an automated program.   
 
7.1.2 Clinical Procedures/Assessments 
 

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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 and more frequently if clinically 
indicated. Adverse experiences will be graded and recorded throughout the study according 
to NCI CTCAE Version 4.0. Toxicities will be characterized in terms regarding seriousness, 
causality, toxicity grading, and action taken with regard to trial treatment.  
All AEs of unknown etiology associated with pembrolizumab exposure should be evaluated 
to determine if it is possibly an event of clinical interest (ECI) of a potentially immunologic 
etiology (irAE). See Section 5.8.1 regarding the identification, evaluation and management 
of AEs of a potential immunological etiology.   
Detailed information regarding the assessment and recording of AEs can be found in section 
7.2. 
 
7.1.2.2 Full Physical Exam 
The investigator or qualified designee will perform a complete physical exam during the 
screening period. Clinically significant abnormal findings should be recorded as medical 
history.  
  
7.1.2.3 Directed Physical Exam 
For cycles that do not require a full physical exam per the Trial Flow Chart, the investigator 
or qualified designee will perform a directed physical exam as clinically indicated prior to 
trial treatment administration.  
  
7.1.2.4 Vital Signs 
The investigator or qualified designee will take vital signs at screening, prior to the 
administration of each dose of trial treatment and at treatment discontinuation as specified in 
the Trial Flow Chart (Section 6.0). Vital signs should include temperature, pulse, respiratory 
rate, weight and blood pressure. Height will be measured at screening only. 
 
7.1.2.5 Eastern Cooperative Oncology Group (ECOG) Performance Scale 
The investigator or qualified designee will assess ECOG status at screening, prior to the 
administration of each dose of trial treatment and discontinuation of trial treatment as 
specified in the Trial Flow Chart. 
 
7.1.2.6 Tumor Imaging and Assessment of Disease 
Besides the baseline CT, 18F-FDG PET and 89Zr-pembrolizumab PET imaging (described in 
section 5.3), the diagnostic CT chest will be repeated within two weeks before surgical 
resection (described in section 5.4). Follow-up following resection will be done with a 
diagnostic CT chest every 3 months for the first 2 years and every 6 months for the 3 years 
thereafter, according to institutional standards.  
 
7.1.2.7 Tumor Tissue Collection 
 

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7.1.2.7.1 Pretreatment tumor biopsy 
Tumor tissue for biomarker analysis from a newly obtained formalin fixed biopsy of the  
tumor must be provided in the form of a tissue block or at least ten unstained slides. A fine 
needle aspirate or cytologic specimen will not be acceptable. Needle or excisional biopsies 
are required. IHC analyses will include PD-1, PD-L1, CD4, CD8 and FoxP3, combined with 
Ki67 double staining to ascertain effector T cell activation and proliferation in the tumor 
microenvironment as reported by Tumeh et al. (Nature 2014), also taking into account the 
effect of quantitative IHC. 
 
7.1.2.7.2 Post-resection specimen 
Primary tumor 
After resection, the resection specimen will be marked intraoperatively by the surgeon. To 
enable PET and pathology correlation, the resected specimen and the PET/CT scan are 
carefully inspected and tissue samples analyzed from the appropriate area of the specimen, 
according to institutional protocol (75). Margins of specimens will be inked, formalin fixed 
and sectioned into approximately 1 cm thick slices. Photographs of serial sections will be 
made. Subsequently, several tissue blocks will be sampled and embedded in paraffin. This 
facilitates orientation and localization of anatomical structures corresponding to different 
parts of the tumor on the PET/CT scans. Histological sections of the formalin fixed paraffin 
embedded blocks from the resected specimens will be examined blinded for the PET data. 
An estimation of the visually assessed vital tumor percentage will be given for the whole of 
the tumor. Multiple slides taken from all tumor segments (approximately 1 cm2 each) will be 
scored by the same IHC panel as for the baseline biopsy, which is then used for comparison 
with SUV values. The presence of tumor cells that may influence the uptake of FDG will 
also be determined. All IHC parameters will be scored semi-quantitatively (absent = 0, few = 
1, moderate = 2, extensive = 3). Tumor segment areas were evaluated per 1 cm2, as  his 
approximates the PET resolution and the dimension of the spherical ROI used to determine 
SUVpeak. 
 
Lymph nodes 
Immune cells will be isolated from the hilar and mediastinal lymph nodes in the resection 
specimen by the use of a cytologic scraping method as previously described (60). In short, 
lymph nodes will be bisected lengthwise with a surgical scalpel. From one half, 10 scrapes 
are made with a surgical blade (no. 22). The scrape material will be the be phenotypically 
analyzed by flow cytometry as described in section 5.5.2. 
 
Tumor surrounding normal lobar lung tissue 
The tumor surrounding non-tumor lobar lung tissue will be examined for radiation induced 
(fibrotic) changes. The percentage of fibrosis and immune cell infiltrate surrounding the 
tumor will be assessed semi-quantitatively (absent = 0, few = 1, moderate = 2, extensive = 3) 
(75). 
 

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7.1.2.8 Laboratory Procedures/Assessments 
The total amount of blood/tissue to be drawn/collected over the course of the trial (from pre-
trial to post-trial visits), including approximate blood/tissue volumes drawn/collected by visit 
and by sample type per subject can be found in Table 8.  
 
Table 8 
Volume of blood to be drawn from each subject 
Assessment 
Sample volume 
(mL) 
No. of samples 
Total volume 
(mL) 
Safety 
Clinical chemistry 
4 
4 
16 
 
 
Pharmacodynamic 
Hematology 
 
PBMC 
 
89Zr-pembrolizumab 
PET 
4 
 
40 
 
7 
 
 
4 
 
5 
 
2 
 
 
16 
 
200 
 
14 
Total 
 
 
246 
  
7.1.2.9 Laboratory Safety Evaluations (Hematology, Chemistry and Urinalysis) 
Laboratory tests for hematology, chemistry, urinalysis, and others are specified in Table 9.   
Laboratory tests for screening should be performed within 10 days prior to the first dose of 
treatment. After Cycle 1, pre-dose laboratory procedures can be conducted up to 72 hours 
prior to dosing. Results must be reviewed by the investigator or qualified designee and found 
to be acceptable prior to each dose of trial treatment. 
 
Table 9  Laboratory Tests 
Hematology 
Chemistry 
Other 
Hematocrit 
Albumin 
Free tyroxine (T4) 
Hemoglobin 
Alkaline phosphatase 
Thyroid stimulating hormone (TSH) 
Platelet count 
Alanine aminotransferase (ALT) 
 
Total WBC 
Aspartate aminotransferase (AST) 
Blood for correlative studies 
 
Lactate dehydrogenase (LDH) 
 
 
Creatinine 
 
 
Calcium 
 
 
Glucose 
 
 
Phosphorus 
 
 
Potassium   
 
 
Sodium 
 
 
Magnesium 
 
 
Total Bilirubin 
 
7.1.2.10 Immunophenotyping on peripheral blood 
To evaluate the pre- and on-treament immune status, polychromatic flowcytometric analyses 
of lymphoid and myeloid subsets in peripheral blood will be performed at t = 0, 21 days 
(immediately prior to 2nd therapeutic dose), just before surgery, one week after surgery and 
after 6 months. Heparinized blood (40 ml) will be taken at each of these time points and 
PBMC isolated. The following 8-12 marker panels will be run on a BD LSRFortessa (with 
appropriate FMO controls): 1) Treg/effector T cell activation panel: CD3, CD4, CD8, CD25, 
CD127, CD45RA, Ki67, FoxP3, HLA-DR, CTLA-4, PD-1, CD27; 2) Dendritic cell panel: 

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CD1c/BDCA-1, CD202/BDCA-2, CD141/BDCA-3, M-DC8 (Sulph-LacNac), CD14, 
CD11c, CD16, CD19, CD40, CD80, PD-L1; 3) Myeloid-derived suppressor cell panel: 
CD11b, CD14, CD33, CD3/19/56 (Lin), HLA-DR, CD15, CD16, PD-L1. By performing 
these analyses at the indicated time points, we will gain unique insight in the kinetics of T 
cell activation following SBRT and PD-1 blockade and pre-, on- and post-treatment immune 
effector/suppressor subset profiles that may show differences between the two treatment 
arms, as we previously showed in a trial of combined ipilimumab and Prostate GVAX 
immunotherapy (76-78). 
 
7.1.2.11 Ultrasound endoscopy guided cytological lymph node aspiration for 
immunological cell composition (EBUS FACS) 
Patients will undergo an EBUS-FNA of the ipsilateral and contralateral hilar and mediastinal 
lymph nodes within 2 weeks before and between week 2 and 3 after SBRT (see section 5.4). 
 
7.1.3 Withdrawal/Discontinuation 
When a subject discontinues/withdraws prior to trial completion, all applicable activities 
scheduled for the final trial visit should be performed at the time of discontinuation. Any 
adverse events which are present at the time of discontinuation/withdrawal should be 
followed in accordance with the safety requirements outlined in Section 7.2 - Assessing and 
Recording Adverse Events. 
 
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 
 
7.1.4.1.1 Screening Period 
Visit requirements are outlined in Section 6.0 - Trial Flow Chart. Specific procedure-related 
details are provided above in Section 7.1 - Trial Procedures. 
Potential subjects will be evaluated to determine that they fulfill the entry requirements as set 
forth in Section 5.1. Written consent must be obtained prior to performing any protocol 
specific procedure. Results of a test performed prior to the subject signing consent as part of 
routine clinical management are acceptable in lieu of a screening test if performed within the 
specified time frame. Screening procedures are to be completed within 30 days prior to the 
first dose of trial treatment except for the following: 
• Laboratory tests are to be performed within 10 days prior to the first dose of trial 
treatment. 
• Tumor imaging must be performed within 14 days prior to the first dose of trial 
treatment. 
Subjects may be rescreened after initially failing to meet the inclusion/exclusion criteria.  
Results from assessments performed during the initial screening period are acceptable in lieu 
of repeating a screening test if performed within the specified time frame and the results meet 
the inclusion/exclusion criteria. 
 

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7.1.4.2 Treatment Period 
Visit requirements are outlined in Section 6.0 - Trial Flow Chart. Specific procedure-related 
details are provided above in Section 7.1 - Trial Procedures. 
 
7.1.4.3 Post-Treatment Visits 
Visit requirements are outlined in Section 6.0 - Trial Flow Chart. Specific procedure-related 
details are provided above in Section 7.1 - Trial Procedures. Subjects will have post-
treatment follow-up for experiencing disease progression, until death, withdrawing consent, 
becoming lost to follow-up or the start of new anti-neoplastic therapy.  
 
7.1.5.3.1  Safety Follow-Up Visit 
The mandatory Safety Follow-Up Visit should be conducted approximately 30 days after the 
last dose of trial treatment or before the initiation of a new anti-cancer treatment, whichever 
comes first. All AEs that occur prior to the Safety Follow-Up Visit should be recorded.  
Subjects with an AE of Grade > 1 will be followed until the resolution of the AE to Grade 0-
1 or until the beginning of a new anti-neoplastic therapy, whichever occurs first. 
 
7.2 
Assessing and Recording Adverse Events 
An adverse event is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and which does not necessarily 
have to have a causal relationship with this treatment. 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 medicinal product or protocol-
specified procedure, whether or not considered related to the medicinal product or 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 
use of (89Zr-)pembrolizumab, is also an adverse event. 
Changes resulting from normal growth and development that do not vary significantly in 
frequency or severity from expected levels are not to be considered adverse events.  
Examples of this may include, but are not limited to, teething, typical crying in infants and 
children and onset of menses or menopause occurring at a physiologically appropriate time. 
Adverse events may occur during the course of the use of (89Zr-)pembrolizumab in clinical 
trials or within the follow-up period specified by the protocol, or prescribed in clinical 
practice, from overdose (whether accidental or intentional), from abuse and from withdrawal. 
Adverse events may also occur in screened subjects during any pre-allocation baseline period 
as a result of a protocol-specified intervention, including washout or discontinuation of usual 
therapy, diet, placebo treatment or a procedure. 
Progression of the cancer under study is not considered an adverse event unless it is 
considered to be drug related by the investigator. 
All adverse events will be recorded from the time the consent form is signed through 90 days 
following cessation of treatment or the initiation of new anti-cancer therapy, whichever is 
earlier and at each examination on the Adverse Event case report forms/worksheets. The 
reporting timeframe for adverse events meeting any serious criteria is described in section 
7.2.3.1. 
 

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7.2.1 Definition of an Overdose for This Protocol and Reporting of 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 pembrolizumab, the 
adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria 
are met. 
If a dose of pembrolizumab 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 principal investigator (PI) and within 2 working days hours to Merck Global Safety. 
(Attn: Worldwide Product Safety; FAX 215 993-1220) 
 
7.2.2 Reporting of Pregnancy and Lactation 
Although pregnancy and lactation are not considered adverse events, it is the responsibility 
of investigators or their designees to report any pregnancy or lactation in a subject 
(spontaneously reported to them) that occurs during the trial. 
Pregnancies and lactations that occur after the consent form is signed but before treatment 
allocation/randomization must be reported by the investigator if they cause the subject to be 
excluded from the trial, or are the result of a protocol-specified intervention, including but 
not limited to washout or discontinuation of usual therapy, diet, placebo treatment or a 
procedure.   
Pregnancies and lactations that occur from the time of treatment allocation/randomization 
through 120 days following cessation of pembrolizumab, 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 PI and within 2 working days to Merck 
Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220) 
 
7.2.3 Immediate Reporting of Adverse Events 
 
7.2.3.1 Serious Adverse Events 
A serious adverse event is any adverse event occurring at any dose or during any use of 
(89Zr-)pembrolizumab that: 
• Results in death; 
• Is life threatening; 

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• Results in persistent or significant disability/incapacity; 
• Results in or prolongs an existing inpatient hospitalization; 
• Is a congenital anomaly/birth defect; 
• Is a new cancer (that is not a condition of the study); 
• Is associated with an overdose; 
• Is another important medical event  
Details regarding the above criteria can be found in Table 6. 
Progression of the cancer under study is not considered an adverse event unless it results in 
hospitalization or death. 
Any serious adverse event, or follow up to a serious adverse event, including death due to 
any cause other than progression of the cancer under study that occurs to any subject from 
the time the consent is signed through 90 days following cessation of treatment, or the 
initiation of new anti-cancer therapy, whichever is earlier, whether or not related to (89Zr-
)pembrolizumab, must be reported within 24 hours to the PI. 
 
In the event of the occurrence of any clinical AE or abnormal laboratory test value that is 
serious or medically important during the course of the study or the post-treatment period, 
irrespective of the treatment received by the subject, the investigator is obliged to 
immediately inform the PI. The immediate report by the investigator to the PI shall be 
followed by detailed, written, reports using the SAE report form (for an “initial” SAE or for 
“follow-up” information on a previous SAE).  
All SAEs will be reported by the PI through the web portal ToetsingOnline to the accredited 
METC that approved the protocol, within 15 days after the PI has first knowledge of the 
serious adverse reactions.  
SAEs that result in death or are life threatening should be reported expedited. The expedited 
reporting will occur not later than 7 days after the responsible investigator has first 
knowledge of the adverse reaction. This is for a preliminary report with another 8 days for 
completion of the report. 
 
Additionally, any serious adverse event, considered by an investigator who is a qualified 
physician to be related to (89Zr-)pembrolizumab that is brought to the attention of the 
investigator at any time outside of the time period specified in the previous paragraph also 
must be reported immediately to the PI and Merck. 
   
All subjects with serious adverse events must be followed up for outcome. 
 
7.2.3.2 Suspected Unexpected Serious Adverse Reactions (SUSARs) 
SUSARs will be reported through the web portal ToetsingOnline to the accredited METC 
that approved the protocol and to EudraVigilance, within 15 days after the PI has first 
knowledge of the SUSAR. SUSARs within this study and SUSARs of pembrolizumab in 
other studies will be reported separately to the METC.   
 

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7.2.3.3 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 24 hours to the PI and within 2 working days to Merck 
Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220) 
For the time period beginning when the consent form is signed until treatment 
allocation/randomization, any ECI, or follow up to an ECI, that occurs to any subject must be 
reported within 24 hours to the PI and within 2 working days to Merck Global Safety if it 
causes the subject to be excluded from the trial, or is the result of a protocol-specified 
intervention, including but not limited to washout or discontinuation of usual therapy, diet, 
placebo treatment or a procedure. 
For the time period beginning at treatment allocation/randomization through 90 days 
following cessation of treatment, or 30 days following cessation of treatment if the subject 
initiates new anticancer therapy, whichever is earlier, any ECI, or follow up to an ECI, 
whether or not related to pembrolizumab, must be reported within 24 hours to the PI and 
within 24 hours to Merck Global Safety. 
Events of clinical interest for this trial include: 
 
1.  an overdose of pembrolizumab, as defined in Section 7.2.1 - Definition of an Overdose for 
This Protocol and Reporting of Overdose, that is not associated with clinical symptoms or 
abnormal laboratory results.  
2.  an elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of 
normal and an elevated total bilirubin lab value that is greater than or equal to 2X the upper 
limit of normal and, at the same time, an alkaline phosphatase lab value that is less than 2X 
the upper limit of normal, as determined by way of protocol-specified laboratory testing or 
unscheduled laboratory testing.* 
 
*Note:  These criteria are based upon available regulatory guidance documents. The purpose 
of the criteria is to specify a threshold of abnormal hepatic tests that may require an 
additional evaluation for an underlying etiology. 
 
7.2.4 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. 

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Table 10. 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 occurring at any dose or during any use of Merck product 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 [including hospitalization for an 
elective procedure] for a preexisting condition which has not worsened does not constitute a serious adverse event.); or 
 
†Is a congenital anomaly/birth defect (in offspring of subject taking the product regardless of time to diagnosis);or 
 
Is a new cancer; (that is not a condition of the study) or 
 
Is an overdose (whether accidental or intentional).  Any adverse event associated with an overdose is considered a serious adverse event. An 
overdose that is not associated with an adverse event is considered a non-serious event of clinical interest and must be reported within 24 
hours. 
 
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 the Merck product to be discontinued? 
Relationship 
to test drug  
Did the Merck product cause the adverse event? The determination of the likelihood that the 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 the Merck product and the AE; the greater the correlation 
with the components and their respective elements (in number and/or intensity), the more likely the Merck product caused the adverse event 
(AE): 
 
Exposure 
Is there evidence that the subject was actually exposed to the Merck product such as: reliable history, acceptable 
compliance assessment (pill count, diary, etc.), expected pharmacologic effect, or measurement of drug/metabolite in 
bodily specimen? 

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Time Course 
Did the AE follow in a reasonable temporal sequence from administration of the Merck product?  
Is the time of onset of the AE compatible with a drug-induced effect (applies to trials with investigational medicinal 
product)? 
 
Likely Cause 
Is the AE not reasonably explained by another etiology such as underlying disease, other drug(s)/vaccine(s), or other host 
or environmental factors 
 
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 the 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 Merck product; or (3) the trial is a single-dose drug trial); or (4) Merck product(s) is/are only used one time.) 
 
Rechallenge 
Was the subject re-exposed to the 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) Merck 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 THE MERCK PRODUCT, OR IF REEXPOSURE TO THE MERCK PRODUCTPOSES ADDITIONAL POTENTIAL 
SIGNIFICANT RISK TO THE SUBJECT, THEN THE RECHALLENGE MUST BE APPROVED IN ADVANCE BY THE U.S. CLINICAL 
MONITOR 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 the 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 a Merck product relationship). 
Yes, there is a reasonable 
possibility of Merck product 
relationship. 
There is evidence of exposure to the Merck product.  The temporal sequence of the AE onset relative to the administration of the Merck product 
is reasonable.  The AE is more likely explained by the Merck product than by another cause. 
No, there is not a reasonable 
possibility Merck product 
relationship 
Subject did not receive the Merck product OR temporal sequence of the AE onset relative to administration of the Merck product is not 
reasonable OR there is another obvious cause of the AE.  (Also entered for a subject with overdose without an associated AE.) 

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7.2.5 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. The Sponsor will inform Merck 
Global Safety in the appropriate timelines for reporting Adverse Events.  
 
The PI will submit, once a year throughout the clinical trial, a safety report to the accredited 
METC.  
This safety report consists of:  
- 
a list of all suspected (unexpected or expected) serious adverse reactions, 
along with an aggregated summary table of all reported serious adverse 
reactions, ordered by organ system, per study. 
a report concerning the safety of the patients, consisting of a complete safety analysis and an 
evaluation of the balance between the efficacy and the harmfulness of the medicine under 
investigation.  
 
8.0 STATISTICAL ANALYSIS PLAN 
 
8.1 
Safety 
Safety of the combination of SBRT and pembrolizumab will be assessed by the percentage of 
≥3 pneumonitis. When combined SBRT and pembrolizumab treatment results in NCIC-CTC 
grade ≥3 pneumonitis in ≤10% of patients, the combination is regarded as safe. 
 
8.2 
Analysis of immune effector subset data 
Immune effector subset expression, expression rate and activation state will be examined in 
tumor tissue, TDLNs and peripheral blood.  
 
8.2.1 Tumor tissue 
IHC analyses will be done on the pretreatment tumor biopsy and postinduction resection 
specimen of the primary tumor. The analyses include PD-1, PD-L1, CD4, CD8, FoxP3 and 
Ki67. Values will be quantified semi-quantitatively and compared within each patient 
(postinduction vs baseline) and between the treatment arms (SBRT vs SBRT + 
pembrolizumab) with the use of the Wilcoxon signed ranks test and the Wilcoxon-Mann 
Whitney test, respectively. 
 
Immune cells will be isolated from the hilar and mediastinal lymph nodes in the resection 
specimen by the use of a cytologic scraping method as described in section 7.1.2.7.2. Values 
will be quantified on a linear scale and compared between the treatment arms (SBRT vs 
SBRT + pembrolizumab) with the use of the independent samples t-test.     
 
The tumor surrounding non-tumor lobar lung tissue will be examined for radiation induced 
(fibrotic) changes. The percentage of fibrosis and immune cell infiltrate surrounding the 
tumor will be assessed semi-quantitatively and compared between the treatment arms (SBRT 
vs SBRT + pembrolizumab) with the use of the Wilcoxon-Mann Whitney test. 
 

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8.2.2 Immunophenotyping in peripheral blood 
All parameters will be quantified on a linear scale and compared within each patient 
(baseline vs on-treatment vs post-induction vs post-surgery) and between the treatment arms 
(SBRT vs SBRT + pembrolizumab) with the use of the paired t-test and the independent 
samples t-test, respectively. 
 
8.2.3 Immunophenotyping on lymph nodes using EBUS-FACS analysis 
Absolute values of the individual immune cells will be calculated for TDLNs and NTDLNs. 
 
The percentage difference between TDLNs, NTDLNs, peripheral blood and immune cells 
isolates from the resection specimen will be calculated and values will be quantified on a 
linear scale and compared within each patient (postinduction vs baseline) and between the 
treatment arms (SBRT vs SBRT + pembrolizumab) with the use of the paired t-test and the 
independent samples t-test, respectively. 
 
8.3 
Analysis of PET data 
SUVmax, SUVmean and SUVpeak will be measured in all tumor lesions, enlarged lymph nodes 
and liver, kidneys, lungs, spleen and left ventricle of the heart.  
 
8.3.1 Heterogeneity analysis 
 
8.3.1.1 Interpatient heterogeneity 
Test-retest variability of PET using modern machines is ~15% (79-81). Therefore, uptake 
difference between patients (interpatient heterogeneity) is defined as SUV differences of 
15% or greater. 
 
8.3.1.2 Intrapatient heterogeneity 
Tracer uptake differences between lesions of the same patient depend on multiple aspects. 
These are both biological (e.g. the level of perfusion and target expression), as well as 
technical (e.g. partial volume and tumor motion effects). True biological differences between 
lesions that correlate with tumor biology are therefore difficult to define. Therefore, 
intrapatient 89Zr-pembrolizumab uptake differences will be assessed qualitatively.     
 
8.3.1.3 Intratumor heterogeneity 
Uptake heterogeneity within a tumor also depends on multiple aspects. It can be the result of 
biological differences, such as perfusion (the tracer is not able to reach some parts of the 
tumor), true target expression or tumor necrosis. Tumor motion effects also influence 
intratumor heterogeneity since PET acquisition is not respiratory gated. 
Intratumor heterogeneity is defined as the ratio of SUVpeak : SUVmean. The level will be 
calculated on a linear scale.    
 

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8.4 
Analysis of CT data 
The pre- and post SBRT +/- pembrolizumab CT scans will be assessed for HU density and 
fair values as outlined in section 5.4.1. Baseline and post induction treatment values within 
patients will be assessed by the paired t-test. Comparison of the baseline and posttreatment 
values between the radiotherapy and radiotherapy + pembrolizumab arms will be assessed 
per time-point by the independent samples t-test. 
 
8.5 
Correlation between PET data and Blood and Tissue markers 
The correlation between the continuous variable SUV and the categorical variables tissue 
PD-1 and PD-L1 IHC will be assessed with the ANOVA test. The correlation between the 
continuous SUV and blood variables will assessed with the paired t-test. 
 
8.6 
Correlation between organ SUV and irAEs 
Organ SUV (higher than background uptake vs. equal to or lower than background uptake) 
will be correlated to irAE (yes or no) using the Chi Square test. 
 
9.0 LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL 
SUPPLIES 
 
9.1 
Investigational Product 
The investigator shall take responsibility for and shall take all steps to maintain appropriate 
records and ensure appropriate supply, storage, handling, distribution and usage of 
investigational product in accordance with the protocol and any applicable laws and 
regulations. 
Clinical Supplies will be provided by Merck as summarized in Table 11. 
 
Table 11.  Product Descriptions 
Product Name & Potency 
Dosage Form 
pembrolizumab 50 mg 
Lyophilized Powder for Injection 
pembrolizumab 100 mg/ 4mL 
Solution for Injection 
 
9.2 
Packaging and Labeling Information 
Clinical supplies will be affixed with a clinical label in accordance with regulatory 
requirements. 
 
9.3 
Clinical Supplies Disclosure 
This trial is open-label; therefore, the subject, the trial site personnel, the Sponsor and/or 
designee are not blinded to treatment. Drug identity (name, strength) is included in the label 
text; random code/disclosure envelopes or lists are not provided. 
 
9.4 
Storage and Handling Requirements 
Clinical supplies must be stored in a secure, limited-access location under the storage 
conditions specified on the label. Receipt and dispensing of trial medication must be recorded 
by an authorized person at the trial site. Clinical supplies may not be used for any purpose 
other than that stated in the protocol. 
 

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9.5 
Returns and Reconciliation 
The investigator is responsible for keeping accurate records of the clinical supplies received 
from Merck or designee, the amount dispensed to and returned by the subjects and the 
amount remaining at the conclusion of the trial. Upon completion or termination of the study, 
all unused and / or partially used investigational product will be destroyed at the site per 
institutional policy. It is the Investigator’s responsibility to arrange for disposal of all empty 
containers, provided that procedures for proper disposal have been established according to 
applicable federal, state, local and institutional guidelines and procedures, and provided that 
appropriate records of disposal are kept. 
 

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10.0 ETHICAL CONSIDERATIONS 
 
10.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, 
Finland, June 1964 and last amended by the 64th WMA General Assembly, Fortaleza, Brazil, 
October 2013) and in accordance with the Medical Research Involving Human Subjects Act 
(in Dutch: Wet Medisch-wetenschappelijk Onderzoek met Mensen (WMO)). 
 
10.2 Recruitment and consent 
Patients eligible for this pilot study will be informed and asked for participation in our 
outpatient clinic. The informed consent document will be used to explain the risks and 
benefits of study participation to the patient in simple terms before the patient is entered into 
the study. It will be emphasized that participation is voluntarily and consent can be retracted 
at any time. 
The investigator is responsible to see that informed consent is obtained from each patient and 
to obtain the appropriate signatures and dates on the informed consent document prior to the 
performance of any protocol procedures and prior to the administration of study drug. 
 
10.3 Benefits and risks assessment, group relatedness 
 
10.3.1 Benefit the trimodality approach 
Pooled analysis of our ROSEL study together with the STARS trial from MD Anderson 
showed that the estimated overall survival at 3 years with SBRT group compares favorably 
with that of surgery (2). However, despite the promising results of SBRT and this exciting 
biological mechanism of in-vivo vaccination, overall survival was not different between the 
two modalities. Senthi et al. showed that 22% of patients relapse within 2 years of SBRT 
treatment. Of these recurrences, 66% were distant (6). The addition of surgery to SBRT 
enables patients to profit from the in-vivo vaccination mechanism and maximizes local 
control. Preclinical data shows that the addition of anti-PD-1 mAb might synergize with 
radiotherapy to control distant micrometastases.  
 
10.3.2 Benefit from 89Zr-pembrolizumab PET 
This is a pilot imaging study. The imaging results will not be used for treatment decision 
making. Therefore no benefit can be derived from the imaging part of this study. 
Visualization of tumor uptake and dosimetry of 89Zr-pembrolizumab will provide insight in 
the mechanism of action of pembrolizumab and we hope this study will lead to improved 
patient selection in future clinical trials to identify which patients benefit the most from 
treatment with pembrolizumab. We aim to develop immuno-PET in general to provide 
individualized therapy with monoclonal antibodies in the future (by selection of responders 
and non-responders). We hope this will contribute to the overall survival of patients and 
prevention of unnecessary side effects. 
 
10.3.3 Safety of  89Zr-pembrolizumab PET 
PET imaging with radiolabeled mAbs is safe as outlined in section 5.2.1. 

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10.3.4 Safety of pembrolizumab treatment 
We refer to the Investigator’s Brochure for safety analyses in the preclinical and clinical 
studies. In summary, pembrolizumab has an excellent safety profile. 
 
10.3.5 Safety of the trimodality approach 
Combining SBRT with surgery appears safe. At least 4 small studies have reported on 
patients who have undergone surgery for salvage in patients who have recurred after SBRT 
(42-45). Since the results of SBRT seem to be equivalent to those of surgery we believe that 
the delay to surgical resection is not harmful and might even be beneficial for patients. The 
addition of pembrolizumab to SBRT might lead to a higher rate of pneumonitis. However, 
the incidence of pneumonitis depends on the target volume and is rarely seen with treatment 
of early stage lung cancer. Because only patients with peripheral tumors are eligible for this 
trial and patients receive a lobectomy according to national guidelines on the treatment for 
NSCLC, the irradiated lung lobe will be resected and resection margins (i.e. central bronchial 
and vascular structures) are not involved in the radiation target volume.  
10.3.6 Radiation exposure 
PET 
Use of positron emitting radionuclides means exposure to ionizing radiation. The effective 
dose administration of 37 MBq of 89Zr-pembrolizumab is expected to be around 18 mSv, 
based on data of 89Zr-ibritumomab tiuxetan (effective dose 0.55 ± 0.07 mSv/MBq) and 89Zr-
MAb-U36 (effective dose 0.53 ± 0.03 mSv/MBq in men, 0.66 ± 0.03 mSv/MBq in women) 
(64, 82). Low dose CT scans used for attenuation correction will give an additional dose of 3 
mSv per CT.  
When considering the justification of application, a risk to benefit analysis has to be 
performed. The ‘International Commission on Radiological Protection’ (ICRP)-62 provides a 
model for this [International Commission on Radiological Protection. Radiological protection 
in biomedical research. ICRP Publication 62. Ann ICRP 1993; 22(3)]. The risk level 
according to this model is stated as Category II “moderate” (effective dosage greater than 10 
mSv (adults)) while the social benefit is regarded as “substantial”. After injection, no 
shielding is required and the patient can go home directly with instructions. 
Radiotherapy 
Radiotherapy can increase the risk of developing a secondary (radiotherapy induced) 
malignancy. However, because of the stereotactic technique and the low target volume, this 
risk is expected to be negligible.   
 
10.3.7 Discomfort during scanning 
Many patients will be familiar with the PET-CT or CT-scanner from earlier treatment 
evaluations. To reduce the amount of anxiety and discomfort caused by scanning as much as 
possible, patients will be informed about the procedure and staff will be present during 
scanning. If necessary / requested the patient can be removed from the scanner. 
 
10.3.8 Additional blood collection 
The total amount of blood to be drawn over the course of the trial is 246 mL.  
 

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10.3.9 Additional EBUS 
EBUS is a safe procedure that is performed on a weekly basis in our institution. The baseline 
EBUS procedure is a regular staging procedure and would also be done outside this study 
protocol. The second EBUS after SBRT +/- pembrolizumab is not part of regular care and 
will be performed for the purpose of this study. The EBUS procedures will be done under 
midazolam or propofol sedation according to national guidelines and no harm is expected 
from this.  
    
10.4 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the 
WMO. 
  
The sponsor (also) has an insurance which is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO). This insurance provides cover for damage to research subjects 
through injury or death caused by the study. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study.  

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11.0 ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 
11.1 Data Collection 
Data collection will be performed by the PI and/or the subinvestigator. Data collection and 
analysis will be monitored according to good clinical practice and to the Dutch Personal Data 
Protection Act (De Wet Bescherming Persoonsgegevens). 
Case Report Forms (CRFs) for the recording of all data will be developed by the PI. The 
forms will be printed on normal paper and the original CRFs will be retained by the 
investigator. Data should be recorded legibly onto the CRF in permanent ink, ballpoint pen. 
Corrections should be made legibly and initially and dated by approved personnel, the 
reasons for significant changes must be provided. Correction fluid or covering labels must 
not be used. Data collected on the CRF are derived from the protocol. 
 
11.2 Handling and storage of data and documents 
The investigators will maintain adequate records, including signed patients informed consent 
forms and information on adverse events. These documents will be kept in a secured area 
with limited access. All records will be signed and dated by the investigators. All records will 
be retained for a period of 15 years following the date the entire clinical investigation is 
completed, terminated or discontinued. The confidentiality will be guaranteed and patients’ 
identification will be coded. The screening assignment will start with the term 
PembroRTSurgery followed by the number of inclusion (e.g. PembroRTSurgery001). Patient 
data will be centralized by the coordinating investigator and kept under strict confidentiality. 
CT data will be stored at the IMS-server for at least 15 years after the last patient of the study 
has been evaluated or longer if necessary. 
Immuno-PET/CT data will be stored in regular diagnostic databases of the nuclear medicine 
department for at least 15 years after the last patient of the study has been evaluated or longer 
if necessary.  
 
11.3 Monitoring plan 
Representatives of the VUmc will perform on site monitoring visits to verify that trial 
conduct at the site is in compliance with the Good Clinical Practice (GCP) and the applicable 
regulatory requirements. Before the study is initiated all aspects of the study are reviewed 
with the investigators and the staff. 
 
11.4 Amendments 
Amendments are changes made to the research after a favourable opinion by the accredited 
METC has been given. All amendments will be notified to the METC. 
All substantial amendments will be notified to the METC and to the competent authority. 
 
11.5 Start of study 
The investigator will inform the METc, when the first patient is included.  

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11.6 Annual progress report 
The investigator will submit a summary of the progress of the trial to the accredited METC 
once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial. 
 
11.7 End of study report 
The investigator will notify the accredited METC of the end of the study within a period of 8 
weeks. The end of the study is defined as the last patient’s last visit. 
In case the study is ended prematurely, the investigator will notify the accredited METC 
within 
15 
days, 
including 
the 
reasons 
for 
the 
premature 
termination. 
Within one year after the end of the study, the investigator will submit a final study report 
with the results of the study, including any publications/abstracts of the study, to the 
accredited METC. 
 
11.8 Public disclosure and publication policy 
With the exception of personal and confidential medical records, all data generated under the 
trial shall be property of the investigator. 
After the last patient entered the study and the results have been obtained and analysed, a 
study report will be written. The aim is to report the study findings in international peer-
reviewed journals and to present the data at international meetings. 
 
11.9 Trial sponsoring and financing 
This is an investigator-initiated study. Financial support for this study was received from 
MSD. 

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12.0 LIST OF REFERENCES 
Reference List 
 
 
1.  Groome PA, Bolejack V, Crowley JJ, Kennedy C, Krasnik M, Sobin LH, Goldstraw 
P. The IASLC Lung Cancer Staging Project: validation of the proposals for revision 
of the T, N, and M descriptors and consequent stage groupings in the forthcoming 
(seventh) edition of the TNM classification of malignant tumours. J Thorac Oncol 
2007;2(8):694-705. 
 
2.  Chang JY, Senan S, Paul MA, Mehran RJ, Louie AV, Balter P, Groen HJ, McRae SE, 
Widder J, Feng L, van den Borne BE, Munsell MF, Hurkmans C, Berry DA, van WE, 
Kresl JJ, Dingemans AM, Dawood O, Haasbeek CJ, Carpenter LS, De JK, Komaki R, 
Slotman BJ, Smit EF, Roth JA. Stereotactic ablative radiotherapy versus lobectomy 
for operable stage I non-small-cell lung cancer: a pooled analysis of two randomised 
trials. Lancet Oncol 2015;16(6):630-637. 
 
3.  Bille A, Pelosi E, Skanjeti A, Arena V, Errico L, Borasio P, Mancini M, Ardissone F. 
Preoperative intrathoracic lymph node staging in patients with non-small-cell lung 
cancer: accuracy of integrated positron emission tomography and computed 
tomography. Eur J Cardiothorac Surg 2009;36(3):440-445. 
 
4.  Schaue D, Ratikan JA, Iwamoto KS, McBride WH. Maximizing tumor immunity 
with fractionated radiation. Int J Radiat Oncol Biol Phys 2012;83(4):1306-1310. 
 
5.  Lee Y, Auh SL, Wang Y, Burnette B, Wang Y, Meng Y, Beckett M, Sharma R, Chin 
R, Tu T, Weichselbaum RR, Fu YX. Therapeutic effects of ablative radiation on local 
tumor require CD8+ T cells: changing strategies for cancer treatment. Blood 
2009;114(3):589-595. 
 
6.  Senthi S, Lagerwaard FJ, Haasbeek CJ, Slotman BJ, Senan S. Patterns of disease 
recurrence after stereotactic ablative radiotherapy for early stage non-small-cell lung 
cancer: a retrospective analysis. Lancet Oncol 2012;13(8):802-809. 
 
7.  Disis ML. Immune regulation of cancer. J Clin Oncol 2010;28(29):4531-4538. 
 
8.  Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, 
Zhu G, Tamada K, Lennon VA, Celis E, Chen L. Tumor-associated B7-H1 promotes 
T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002;8(8):793-
800. 
 
9.  Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 
2002;2(2):116-126. 

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 10.  Brown JA, Dorfman DM, Ma FR, Sullivan EL, Munoz O, Wood CR, Greenfield EA, 
Freeman GJ. Blockade of programmed death-1 ligands on dendritic cells enhances T 
cell activation and cytokine production. J Immunol 2003;170(3):1257-1266. 
 11.  Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and 
autoimmunity. Immunol Rev 2010;236:219-242. 
 12.  Thompson RH, Dong H, Lohse CM, Leibovich BC, Blute ML, Cheville JC, Kwon 
ED. PD-1 is expressed by tumor-infiltrating immune cells and is associated with poor 
outcome for patients with renal cell carcinoma. Clin Cancer Res 2007;13(6):1757-
1761. 
 13.  Talmadge JE, Donkor M, Scholar E. Inflammatory cell infiltration of tumors: Jekyll 
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