A Pilot Study to Develop Predictive Biomarkers for the Response to Immunotherapy in Lung Cancer
Conditions: Lung Cancer
Sponsor: Abramson Cancer Center at Penn Medicine

PROJECT TITLE: A PILOT STUDY TO DEVELOP PREDICTIVE BIOMARKERS FOR THE RESPONSE TO 
IMMUNOTHERAPY IN LUNG CANCER 
PRINCIPAL INVESTIGATOR/FACULTY SPONSOR: Charu Aggarwal, MD, MPH 
PROTOCOL SUMMARY VERSION DATE: August, 2018 (VERSION 2) 
 
 
INTRODUCTION AND OBJECTIVES 
 
 
1- Abstract 
Several immune checkpoint inhibitors targeting the PD-1 pathway have been developed with clinical 
trials showing an approximately 20% durable response in unselected patients with advanced non-small 
cell lung cancer (NSCLC). At the moment, no clear biomarker exists to accurately predict anti-PD1/PDL1 
tumor responsiveness. The goal of this study is to broadly discover and evaluate the utility of blood based 
biomarkers for use in measuring and predicting response to immunotherapy in patients with lung cancer.  
 
2- Background 
Despite significant advances in detection and therapy, lung cancer remains the number one cause of 
cancer related death in both men and women worldwide (1). Most patients are diagnosed at an advanced 
stage with poor overall survival.  NSCLC is the most common histotype representing 85% of new 
diagnoses (2), and the overall five year survival rate of patients with NSCLC is 16% (3). Improved 
understanding of the interaction between tumors and host immune surveillance has enabled the 
development of several immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway that have 
shown an approximate 20% durable response in unselected patients with advanced NSCLC, and improved 
response rates in select patients with PDL1 expression (Reck, NEJM 2016). Preliminary data suggest that 
detection of tumor PD-L1 protein expression on human cancers using chromogenic-based 
immunohistochemistry (IHC) in formalin-fixed paraffin embedded tissue samples (FFPE) may predict 
clinical response to PD-1/PD-L1 directed therapy (4,5). However, meaningful responses have also been 
observed in PD-L1 negative patients. Moreover, use of IHC may be limited by access to tissue as well as 
assay variability and interpretive subjectivity. In addition, conventional radiographic assessment of 
treatment response using WHO criteria (Response Evaluation Criteria in Solid Tumors [RECIST]) does 
not fully capture the response patterns of immunotherapeutic agents often necessitating frequent repeat 
imaging to assess a therapeutic response. Mutation burden and neoantigen expression have been evaluated 
as potential biomarkers for immunotherapy, however there is no validated biomarker to follow these 
changes yet. As a result, a biomarker that could accurately predict the patients most likely to derive 
benefit from immunotherapy, as well as monitor response to treatment would be of significant clinical 
utility.   
 
One biomarker that is being increasingly utilized for the non-invasive monitoring of patients with various 
malignancies is cell-free DNA (cfDNA). cfDNA consists of short double-stranded DNA fragments shed 
into the blood by tumor cells undergoing apoptosis or necrosis. We and others have shown that cfDNA 
can be readily extracted and sequenced from NSCLC patient blood for mutation detection and disease 
monitoring (Thompson J et al. CCR 2016; Couraud S et al. CCR 2014; Oxnard GR et al. CCR 2014; 
Newman AM et al. Nature Medicine 2013). While surgically resected tumor or fine needle aspirate have 
traditionally been the source of tissue for molecular monitoring, these samples often provide insufficient 
and/or inferior quality DNA for sequencing, and can under-represent the full molecular heterogeneity of 
the patient’s disease, especially in the setting of metastasis. cfDNA thus represents a non-invasive 
approach for patient monitoring and may allow for a “liquid biopsy” for genomic alterations of the 
primary or metastatic tumor, monitoring of the disease status, and/or development of resistance to 
therapy.   
 
In this study, we will collect blood and urine specimens from patients receiving immunotherapy as part of 
their standard of care for the treatment of NSCLC. We will use various platforms for measurement of 
new biomarkers, and these biomarkers will be assessed for their ability to predict responsiveness 

PROJECT TITLE: A PILOT STUDY TO DEVELOP PREDICTIVE BIOMARKERS FOR THE RESPONSE TO 
IMMUNOTHERAPY IN LUNG CANCER 
PRINCIPAL INVESTIGATOR/FACULTY SPONSOR: Charu Aggarwal, MD, MPH 
PROTOCOL SUMMARY VERSION DATE: August, 2018 (VERSION 2) 
 
to immunotherapy, correlation with radiographic response, ability to detect lung cancer specific 
genetic changes present in the primary tumor (e.g analysis of cfDNA for EGFR mutations), and impact 
on prognosis (response to therapy, progression free survival and overall survival).  
  
Portions of the blood and urine samples drawn will also be stored for future research. 
 
3- Study Objectives 
The overall objective is to develop a comprehensive system to collect blood and urine samples from 
patients with NSCLC receiving immunotherapy 
 
Primary Outcome Variable 
1. Discovery and validation of new blood based biomarkers that predict response to immunotherapy 
in NSCLC 
 
Secondary Outcome Variable 
      1.    Storage of samples for future research endeavors 
 
4- Location 
Abrahamson Cancer Center at the Hospital of the University of Pennsylvania and Penn Presbyterian 
Medical Center 
 
STUDY DESIGN 
 
 
5- General Design 
 
Single center, longitudinal cohort study of NSCLC patients receiving immunotherapy with 
biomarker evaluation.  
 
This is an observational study. Biomarkers that are detected in CLIA certified laboratories and 
are already part of standard of care in the management of lung cancer patients (e.g. circulating 
tumor DNA)  may be disclosed to the treating physician and care may be altered based on 
routine practice. Biomarkers that are in research development and/or not performed in CLIA 
certified laboratories will not be disclosed to treating physician and no treatment decisions will 
be based on these results. 
 
The organizational structure will support the collection of peripheral blood and urine samples 
from subjects with NSCLC receiving immunotherapy for evaluation of circulating biomarkers. 
Physicians, nurse practitioners, and physician assistants will identify subjects with NSCLC who 
are scheduled to receive an immune checkpoint inhibitor as part of their routine clinical care at 
the University of Pennsylvania. The subjects will be consented for the collection of blood and 
urine samples. Given the exploratory nature of this study and that the hypotheses for circulating 
tumor material in NSCLC patients receiving immunotherapy will adapt as information from this 
analysis proceeds, the timeline and frequency of blood collection are not defined. The frequency 
of research blood collection will not exceed the lesser of 50 mL or 3 mL/kg in an 8 week period 
and will not occur more frequently than 2 times per week. Blood will generally be collected at 
the time of routine phlebotomy for the management of their disease, but in rare instances the 

PROJECT TITLE: A PILOT STUDY TO DEVELOP PREDICTIVE BIOMARKERS FOR THE RESPONSE TO 
IMMUNOTHERAPY IN LUNG CANCER 
PRINCIPAL INVESTIGATOR/FACULTY SPONSOR: Charu Aggarwal, MD, MPH 
PROTOCOL SUMMARY VERSION DATE: August, 2018 (VERSION 2) 
 
subject may be asked to provide only a research blood sample if the timing of the routine 
phlebotomy does not correlate with the timing necessary for the study. Urine specimens will be 
obtained in a sterile specimen cup.  
 
 
 
Duration 
The total study duration will be 5 years. Subjects will be considered active participants on days of blood 
acquisition. Baseline data will be collected by chart review. Cancer outcome data may be collected 
longitudinally by chart review only until the end of the study. 
  
 
SUBJECT SELECTION & WITHDRAWAL 
 
 
6- Inclusion and Exclusion Criteria 
 
A. Inclusion Criteria 
1. Advanced Metastatic NSCLC 
2. Scheduled to initiate an immune checkpoint inhibitor 
3. Age >= 18 years 
4. Able to provide informed consent  
 
 
B. Exclusion Criteria 
 
1. Other, unrelated, concomitant active, invasive malignancy 
 
7- Vulnerable Populations 
Although vulnerable populations are not specifically excluded from this study, there will be no specific 
efforts to target these populations.  
 
8- Subject Accrual  
Eligible subjects will be identified by physicians, nurse practitioners, and physician assistants at the 
Abramson Cancer Center and PPMC. A total of 200 patients will be included in this study.   
 
9- Subject Recruitment & Screening 
All adult patients being evaluated by the Penn Thoracic Oncology group meeting inclusion/exclusion 
criteria will be screened for admission into the study. All prospective research subjects will meet with a 
study team member, the study will be explained, questions answered and the Informed Consent Process 
will be initiated 
 
10- Data 
This study will collect the following specific personal health identifiers (PHI) and other data elements for 
use at Penn.  These data elements will be stored electronically in REDCAP, and will only be accessible to 
Penn study members.  See section 12 for specific data elements 
 
 
INVESTIGATIONAL AGENTS 
 
 
11. Drugs/devices (if applicable) 

PROJECT TITLE: A PILOT STUDY TO DEVELOP PREDICTIVE BIOMARKERS FOR THE RESPONSE TO 
IMMUNOTHERAPY IN LUNG CANCER 
PRINCIPAL INVESTIGATOR/FACULTY SPONSOR: Charu Aggarwal, MD, MPH 
PROTOCOL SUMMARY VERSION DATE: August, 2018 (VERSION 2) 
 
N/A 
 
 
 
STUDY PROCEDURES 
 
 
12. Procedures 
1. Informed consent 
2. Collection of baseline clinical information (chart review) 
a. Baseline demographic data (sex, race, ethnicity) 
b. Tobacco history 
c. Medical history 
d. Performance status 
e. Baseline tumor related variables, including histology, immunohistochemistry results, 
TNM stage, sites of tumor involvement 
f. Results of staging CT scans 
g. Mutation status as determined by clinical testing of tumor material 
h. Treatment regimens, including chemotherapy regimen, doses 
3. Blood will be drawn once at baseline prior (up to 50 ml), and the frequency of research blood 
collection will not exceed the lesser of 50 mL or 3 mL/kg in an 8 week period and will not 
occur more frequently than 8 times per year. When possible, research blood samples will be 
drawn at the same time as the clinical sample to minimize discomfort and inconvenience to 
the patient. The listed time points below are approximations and will depend upon when the 
patient is having blood drawn for clinical purposes 
1. Baseline –  up to 30 ml 
2. 2-4 weeks -  up to 20 ml 
3. 8-12 weeks – up to 30 ml 
4. 14-16 weeks – up to 20 ml 
5. 18-24weeks – up to 30 ml  
4. Subjects will be asked if they are willing to provide a single urine specimen in a sterile 
specimen cup at the time of enrollment 
5. Although the vast majority of patients will have had tissue sequencing and 
immunohistochemical analysis to determine PD-L1 expression, if these analyses have not 
been performed, we may request that tumor tissue undergo this testing provided sufficient 
material is available. The result of this information may be disclosed to the patient and 
primary oncologist.  
6. Longitudinal Data collection 
a. CT imaging to monitor treatment response 
b. Date of progression, and sites of progression.  
c. Date of death, if prior to end of study period 
7. Analysis of blood and tissue samples. Samples may undergo the following analyses:  
a. Blood samples will undergo isolation of circulating free DNA and identification of 
specific mutations using either a PCR or sequencing approach  
b. Identification of specific proteins or cell surface markers using various protein 
measurement methods (e.g. mass spectroscopy, flow cytometry)  
 
 
STATISTICAL PLAN 

PROJECT TITLE: A PILOT STUDY TO DEVELOP PREDICTIVE BIOMARKERS FOR THE RESPONSE TO 
IMMUNOTHERAPY IN LUNG CANCER 
PRINCIPAL INVESTIGATOR/FACULTY SPONSOR: Charu Aggarwal, MD, MPH 
PROTOCOL SUMMARY VERSION DATE: August, 2018 (VERSION 2) 
 
 
 
13. Sample Size Determination 
This is a pilot analysis to determine the role of novel biomarkers for use as predictive markers in patients 
with lung cancer receiving immunotherapy. We are unable to conduct formal sample size analysis given 
the pilot nature of this study.  
 
14. Statistical Methods 
We will use descriptive statistics for variables collected. We will describe the demographic characteristics 
of subjects using frequencies and percentages for categorical variables and medians and ranges for 
quantitative variables. Data analysis will focus on the following analyses (based on our primary objective) 
1. Prognostic markers:  The analysis will determine the association with response rate, PFS, and 
OS. 
2. When feasible, we will calculate the correlation between markers identified in the blood and 
in the primary tumor. 
 
SAFETY & ADVERSE EVENTS 
 
 
15. Potential Risks 
The risk of study participation is minimal. The two main risks are loss of confidentiality and the risk of 
venipuncture. A number of mechanisms are in place to protect subject confidentiality (see above). The 
risk of venipuncture is minimal. There may be bruising or bleeding, and rarely infection. The overall 
amount of blood drawn for this study is not to exceed 50 ml not to exceed in an 8 week period and will 
not occur more frequently than 8 times per year and should not have any clinical implications for study 
participants. 
 
16. Potential Benefits 
Discovery of a minimally invasive biomarker to predict response to immunotherapy has the potential to 
significantly alter the current management of patients with lung cancer. This study may lead to the 
development of new methods for the management of patients with lung cancer receiving immunotherapy. 
 
17. Confidentiality & Data Storage 
Precautions will be taken to ensure that strict confidentiality is maintained. All source documents will be 
identified by a unique study ID number (unique for this project). No results will be reported in a 
personally identifiable manner. Study data will be maintained either on a study-dedicated computer whose 
files are password protected or in a locked filing cabinet to which only the investigators and their 
designated staff have access. Data that will be disclosed outside of Penn: 
a. Baseline demographic data (sex, race, ethnicity) 
b. Tobacco history 
c. Performance status 
d. Baseline tumor related variables, including histology, immunohistochemistry results, TNM 
stage, sites of tumor involvement 
e. Results of staging CT scans 
f. Mutation status as determined by clinical testing of tumor material 
g. Treatment regimens, including chemotherapy regimen, doses 
 
18. Data Safety & Monitoring 

PROJECT TITLE: A PILOT STUDY TO DEVELOP PREDICTIVE BIOMARKERS FOR THE RESPONSE TO 
IMMUNOTHERAPY IN LUNG CANCER 
PRINCIPAL INVESTIGATOR/FACULTY SPONSOR: Charu Aggarwal, MD, MPH 
PROTOCOL SUMMARY VERSION DATE: August, 2018 (VERSION 2) 
 
Safety data will be monitored by the principal investigator formally on a quarterly basis in the course of 
study conduct. We plan to have our data reviewed manually by the study coordinator who will bring any 
discrepancies to the attention of the study team for investigation and resolution.  
 
19. Risk/Benefit Ratio 
Although there is no immediate, direct benefit to the individual participants of this study, there is 
considerable potential benefit to future subjects, including participants in this study, and to the 
community as a whole. Physicians may be able to better predict the patients most likely to benefit from 
receiving immunotherapy for the treatment of lung cancer. In view of the minimal risks to study 
participants and considerable benefits of the study, the risk/benefit ratio is extremely favorable. 
 
 
REGULATORY & ETHICAL CONSIDERATIONS 
 
 
21. Informed Consent 
Informed consent will be obtained during the patient's in-patient or out-patient visit at UPHS. Only 
competent patients will be approached to participate. This will be judged by an informal assessment by 
the investigator, but a mini-mental status exam will be performed if necessary. The goals of the study will 
be described by a study coordinator. The consent document will be reviewed and provided to prospective 
subjects. Comprehension of the consent document will be assessed by the study coordinator and 
investigators. 
 
 
STUDY FINANCES 
 
 
22. Funding Source 
None 
 
23. Compensation to Subjects for Participation 
No compensation will be provided for the participants. 
 
24. Conflict of Interest 
All University of Pennsylvania investigators will follow the University conflict of interest policy.  There 
is no conflict of interest for the principal investigator or any member of the study staff. 
 
 
PUBLICATION PLAN 
 
 
25. Publication Plan 
Results of the proposed research may be presented at intramural conference sessions within the Abramson 
Cancer Center, and in various oral, print and electronic publications. The principal investigator holds 
primary responsibility for presentation of the results of the study. 
 
References