A Pilot Study of Exhaled Breath Analysis to Predict Risk of Symptomatic Pneumonitis After Chemoradiotherapy For Stage III Non-Small Cell Lung Cancer
Phase: NA
Conditions: Pneumonitis, Nonsmall Cell Lung Cancer Stage III
Sponsor: Wake Forest University Health Sciences

A Pilot Study of Exhaled Breath Analysis to Predict Risk of Symptomatic Pneumonitis 
After Chemoradiotherapy For Stage III Non-Small Cell Lung Cancer 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
 
 
 
Principal Investigator(s):  
Andrew Bishop, PhD 
Department of Medicine, Section of Molecular 
Medicine 
Wake Forest School of Medicine 
  
 
Michael Farris, MD 
 
 
 
 
 
Department of Radiation Oncology 
 
 
 
 
 
Wake Forest School of Medicine 
  
 
Co-Principal Investigator(s): 
Ryan Hughes, MD 
Department of Radiation Oncology 
Wake Forest School of Medicine 
 
Biostatistician:  
 
 
Beverly Levine, Ph.D. 
Biostatistics Core 
Comprehensive Cancer Center 
Wake Forest University School of Medicine  
Medical Center Boulevard  
Winston-Salem, NC 27157 
 
Study Coordinators: 
 
Jeffrey Kettler 
 
 
 
 
 
Comprehensive Cancer Center 
 
Sharon L. Averill, RN CRCC 
 
 
 
 
 
Comprehensive Cancer Center 
 
 
 
Regulatory Contact: 
 
Emily Teal, MA 
 
 
 
 
 
Comprehensive Cancer Center 
 
 
Participating Institution(s): 
Wake Forest Baptist Comprehensive Cancer Center 
 
 
Version Date: 
 
 
05/14/21 
 
 
 
ClinialTrials.gov:  NCT04040244 
 
 
Confidential

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 2 of 31 
 
 
Table of Contents 
Study Schema............................................................................................................................ 4 
1.0 
Introduction and Background .......................................................................................... 5 
1.1 
Summary and Rationale ........................................................................................... 7 
2.0 
Objectives ...................................................................................................................... 7 
2.1 
Primary Objective ....................................................................................................... 8 
2.2 
Secondary Objectives ................................................................................................. 8 
3.0 
Study Population ............................................................................................................ 8 
3.1 
Inclusion Criteria ......................................................................................................... 9 
3.2 
Exclusion Criteria ........................................................................................................ 9 
3.3 
Inclusion of Women and minorities ............................................................................. 9 
4.0 
Methods ........................................................................................................................10 
4.1 
Registration Procedures ............................................................................................10 
4.2 
Data Collection Methods ............................................................................................10 
4.3 
Research Data Collection and Analysis .................................................................13 
5.0 
Study Outcomes and Outcome Measures .....................................................................15 
5.1 
Primary Outcome .......................................................................................................15 
5.2 
Secondary Outcomes ................................................................................................15 
6.0 
Analytic Plan..................................................................................................................16 
6.1 
Analysis of Primary Outcome, Sample Size, and Power ............................................16 
6.2 
Analysis of Secondary Outcomes ..............................................................................17 
6.4 
Accrual Rate ..............................................................................................................18 
6.5 
Length of Study .........................................................................................................18 
7.0 
Treatment Plan ..............................................................................................................19 
7.1 
Treatment Administration .......................................................................................19 
7.2 
Follow-up .................................................................................................................19 
 
Study Calendar .............................................................. Error! Bookmark not defined. 
7.3 .........................................................................................................................................20 
8.0 
Specimen Collection and Storage ..............................................................................21 
9.0 
Data Management .........................................................................................................21 
10.0 
Confidentiality and Privacy ............................................................................................22 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 3 of 31 
 
11.0 
Data Safety and Monitoring ...........................................................................................22 
12.0 
Reporting of Unanticipated Problems, Adverse Events or Deviations ............................22 
References ...............................................................................................................................26 
Appendix A – Subject Eligibility Checklist ............................................................................29 
Appendix B – Protocol Registration Form ............................................................................31 
 
 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 4 of 31 
 
Study Schema 
 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 5 of 31 
 
1.0 
Introduction and Background 
 
Summary of Pneumonitis in Patient with Stage III NSCLC 
 
Definitive chemoradiotherapy (CRT) followed by consolidative immunotherapy is a 
standard treatment for locally advanced non-small cell lung cancer (NSCLC) (1, 2). 
Pneumonitis represents injury to the lungs in the form of acute or chronic inflammation as 
a result from these treatments. This injury can either be asymptomatic or symptomatic, 
and may cause cough, shortness of breath, oxygen requirement, or worse. In patients 
treated with CRT followed by standard of care immunotherapy, symptomatic pneumonitis 
(SP), defined as Common Toxicity Criteria for Adverse Events (CTCAE) grade 2 or higher 
pneumonitis, occurs in approximately 15-20% of patients (3, 4). Current treatment of SP 
is largely dependent on symptom severity. This ranges from observation or anti-tussive 
medications for mildly symptomatic cases, to prolonged corticosteroid use, oxygen 
supplementation, or even hospitalization in more severe cases. Rarely, death may result 
from SP. The most prevalent etiologies of pneumonitis in these patients includes radiation 
pneumonitis and immune-mediated pneumonitis. Radiation-induced lung injury, which 
includes radiation pneumonitis and pulmonary fibrosis, occurs with variable severity (from 
asymptomatic to fatal) in 20% to 50% of patients treated with modern CRT (5, 6). 
Radiation pneumonitis generally refers to a reversible, subacute inflammatory reaction 
that occurs within several weeks to months of CRT (7). Immune-mediated pneumonitis, 
exhibited by focal or diffuse inflammation of the lung parenchyma, occurs in 
approximately 5% of patients receiving immune checkpoint inhibitors (8).  
 
Currently, it is very difficult to identify patients at high risk of SP prior to treatment and 
limited data exist to support individualized prophylactic strategies for those at highest-risk 
of SP (9). Given the relatively sparse tools at hand to predict an individual’s risk of 
developing SP after combined-modality therapy for locally advanced NSCLC, there is a 
dire need for more sensitive, personalized methods of detecting patients at risk. The main 
technique presently available to reduce an individual’s risk of SP is to limit the radiation 
dose to normal lung tissue. Prior studies discussed below have demonstrated correlations 
between serum markers, but these findings have not been routinely employed for risk 
stratification in current practice.  
 
Personalized Strategies to Mitigate Pneumonitis Risk Are Lacking 
 
There are guidelines in place for minimizing radiation dose to normal lung tissue with the 
goal of decreasing the risks of pneumonitis (10). However, the most appropriate 
dosimetric thresholds are controversial and are rather crude in their ability to predict SP 
after radiotherapy. Current practice is to apply generic lung constraints to all patients 
receiving CRT, generally ensuring that the amount of bilateral normal lung tissue 
receiving at least 20 Gy is less than 30-35%. Such constraints were devised prior to 
common use of immunotherapy in this patient population. Given that immunotherapy 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 6 of 31 
 
alone can induce symptomatic pneumonitis, it is unclear if these constraints remain 
appropriate. Development of biomarker-based risk prediction models of SP may facilitate 
further investigation of customized prophylactic strategies (i.e. inhaled corticosteroids or 
short courses of systemic steroids during CRT). 
 
Serum biomarkers including transforming growth factor-beta 1 (TGF-β1), interleukin (IL)-
6, IL-1α, and IL-10 have been associated with development of SP (11-14). Previous 
studies have combined both dosimetric and serum TGF- β1 to estimate the risk of SP 
(15). However, inconsistent reports and the use of frequent blood draws during CRT has 
made this approach difficult for patients and clinicians to implement in practice. 
Additionally, these studies were performed prior to the adoption of consolidative 
immunotherapy after CRT. Due to these limitations and inconsistencies, better predictive 
metrics, collected non-invasively, are needed to identify patients at high risk for SP who 
may benefit from personalized radiation therapy or prophylactic medical therapy.  
 
Because of the inconsistencies described above, we plan to include serum analyses 
(Collected during routine labs) for each of the target biomarkers that will be analyzed in 
exhaled breath samples. Serum and exhaled breath are two different sources of biological 
sampling and will provide different metabolic information which may be useful as we 
elucidate the association between our targeted biomarkers and SP in the era of 
immunotherapy. Serum biomarkers can be physiologic references but given the proximity 
of the breath analyses to the localization of injury, breath biomarkers may provide a 
clearer window in which to correlate predictive markers with SP development. 
 
Exhaled Breath Analyses for Risk Stratification 
 
Exhaled Breath Condensate (EBC) and Exhaled Breath Volatiles (EBV) can be non-
invasively captured by collecting air as the patient exhales over the course of 5-10 
minutes into a commercially available R-tube and ReCIVA device, respectively (Figure 
1a and 1b). These collections provide biological samples that are a rich source of 
metabolic and genetic information and have the potential to result in novel biomarkers 
predictive of SP. Prior studies have detected 
various cytokines, proteins, and other molecules 
in the EBC of lung cancer patients and TGF-β1 in 
EBC of patients with sarcoidosis (16). Exhaled 
octane has the potential to predict development of 
acute respiratory distress syndrome (17). These 
findings suggest that we will be able to identify 
these and potentially other molecules in our 
cohort of NSCLC patients to correlate with SP. 
One previous study has demonstrated the 
feasibility of using exhaled breath analyses to 
predict radiation pneumonitis in patients treated 
Figure 1a: EBC 
collection device 
Figure 1b: EBV collection device 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 7 of 31 
 
with stereotactic body radiotherapy (18), but this has not been attempted in locally 
advanced lung cancer patients receiving CRT. 
 
EBV analyses as described have not been performed using unbiased metabolomics. A 
metabolomics approach is able to obtain a snapshot of biological and molecular 
mechanisms underlying a disease process by capturing metabolic profiles that are 
indicative of cellular metabolism. Current profiling technology results in the identification 
of hundreds to thousands of metabolites, which can be used as an unbiased analysis of 
biochemical pathways and potentially target biological dysfunction (19-21). 
 
Recent studies have also focused on the link between cancer and the human microbiota, 
with special interest in determining how the microbiota contributes to the pathogenesis of 
various cancer types, including lung cancer (22). Similarly, other studies have illustrated 
that alterations in the rodent gut microbiome are capable of potentiating radiation-induced 
bowel injury (23). Mechanistically, the gut microbiome is thought to directly impact 
gastrointestinal immune cells (24). It is well-established that the lungs are not sterile, and 
scientists have recently demonstrated that the lung microbiome is very similar to the oral 
microbiome (25-27). An informative approach to assessing the human microbiota is 
through sequencing, which allows the characterization of microorganisms and provides 
information on the putative function of the microbiota. Microbial 16S rRNA is also detected 
in exhaled breath condensate (EBC), but its impact regarding SP has not been explored 
(28). Collecting EBC before CRT offers a noninvasive approach for studying the role the 
normal pre-treatment microbiota plays in SP, which has not previously been performed. 
 
1.1 
Summary and Rationale 
In this proposed pilot study, we seek to measure exhaled breath-derived 
biomarkers at different points in the CRT timeline (baseline, at 2 weeks, 6 weeks, 
and 1 month post-treatment). We hypothesize that these measurements will have 
measurable differences between each time point, and, further, that differences 
relative to baseline will be associated with the development of SP in a group (n=50) 
of stage III NSCLC patients treated with CRT followed by immunotherapy. As part 
of this goal, we will also test the same biomarkers in serum to evaluate the 
association between serum and breath-derived biomarker concentrations and to 
determine the association between serum measurements and SP. We also will 
evaluate whether constituents of the lung microbiome prior to CRT are related to 
the development of SP. 
2.0 
Objectives 
This is a prospective pilot study investigating exhaled breath condensate analyses 
to quantify the variability over time of various biomarkers associated with SP. 
Compounds of interest have been chosen due to previous studies demonstrating 
association with pneumonitis in analyses of serum. Our primary hypothesis is that 
biomarker concentrations will change over the course of treatment (pre-treatment, 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 8 of 31 
 
2-weeks on treatment, 6-weeks on treatment, and at 1-month post-treatment, 
Section 2.1.1). Our secondary hypotheses are that these changes in biomarker 
concentrations will correlate with SP (Section 2.2.1), which we expect to occur in 
15-20% of our patient sample, and that these changes will also occur in serum, 
which will also correlate with SP (Section 2.2.2). Finally, we hypothesize that pre-
treatment EBC-derived microbial signatures will be different in patients who 
develop SP versus those who do not (Section 2.2.3). 
2.1 
Primary Objective 
 
2.1.1 To quantify the intra-person variability of concentrations of TGF-β1, 
IL-6, IL-1α, and IL-10 measured in exhaled breath condensate. 
2.2 
Secondary Objectives 
 
2.2.1 To examine the associations between differences in pre-treatment 
and post-treatment EBC concentrations of TGF-β1, IL-6, IL-1α, and 
IL-10 and the development of CTCAE grade 2+ SP. 
 
2.2.2 To examine the associations between serum measures of TGF-β1, 
IL-6, IL-1α, and IL-10 and: 
 
2.2.2.1 
EBC measures of the same biomarkers, and 
2.2.2.2 
The development of CTCAE grade 2+ SP. 
 
2.2.3 To examine the association between microbiome signatures found 
in pre-treatment EBC and the development of CTCAE grade 2+ SP. 
 
2.3 
Exploratory Objective 
 
2.3.1 To examine the association between pre- and post-treatment 
exhaled breath volatile-based metabolites and the development of 
SP 
3.0 
Study Population 
 
Patients with biopsy-proven non-small cell lung cancer who are planned to 
undergo standard of care treatment will be enrolled in this study. Treatment will 
include definitive chemoradiotherapy followed by consolidative immunotherapy. 
Patients who are not able or unwilling to provide breath samples will not be 
enrolled. Patients treated with any prior radiotherapy directed at the chest, or those 
taking corticosteroids at baseline will be excluded. 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 9 of 31 
 
3.1 
Inclusion Criteria 
3.1.1 Age greater than or equal to 18 years old. 
 
3.1.2 Clinically diagnosed or suspected Stage III non-small cell lung 
cancer to be treated with chemoradiotherapy as part of cancer 
treatment, as determined by the treating clinician 
 
3.1.3 Plan for treatment with definitive radiotherapy (≥60 Gy) with 
concurrent chemotherapy at the discretion of the treating radiation 
and medical oncologists. 
 
3.1.4 Willing and able to tolerate exhaled breath collection. 
 
3.1.5 Able to provide informed consent.  
 
3.2 
Exclusion Criteria   
3.2.1 Systemic (oral, intravenous or intramuscular) corticosteroid use for 
any reason within 5 days of registration. 
 
3.2.2 Prior radiotherapy directed at the chest (thoracic inlet superiorly to 
diaphragm inferiorly). 
 
3.2.3 Any systemic antibiotic use within 2 weeks of registration.  
 
3.3 
Inclusion of Women and minorities 
Men and women of all races and ethnicities who meet the above-described 
eligibility criteria are eligible to participate in this study. The study consent 
form will also be provided in Spanish for Spanish-speaking participants.  
 
Based 
on 
WFBCCC 
population 
estimates, 
we 
may 
expect 
approximately 44% of participants to be women. Translating this to our 
sample size estimate of 50 patients, we may enroll approximately 
22 women. We may enroll approximately 10-13% Black or African American 
patients. Based on our catchment area and hospital demographics we do 
not expect high accruals of individuals of Hispanic/ Latino, American 
Indian/Alaska Native or Asian ancestry; however, no individual will be 
excluded from the study if they satisfy the above inclusion/exclusion criteria. 
Should we not meet or exceed these estimates, the PI will engage the Office 
of Cancer Health Equity to discuss strategies to enhance recruitment in 
these target populations. 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 10 of 31 
 
4.0 
Methods 
 
4.1 
Registration Procedures 
All patients entered on any WFBCCC trial, whether treatment, companion, 
or cancer control trial, must be linked with a study protocol in EPIC within 
24 hours of Informed Consent. Patients must be registered prior to the 
initiation of the study.   
 
You must perform the following steps in order to ensure prompt registration 
of your patient: 
 
1.0 
Complete the Eligibility Checklist (Appendix A) 
2.0 
Complete the Protocol Registration Form (Appendix B) 
3.0 
Alert the Cancer Center registrar by phone, and then send the signed 
Informed Consent Form, Eligibility Checklist and Protocol 
Registration Form to the registrar, either by fax or e-mail. 
 
Contact Information: 
Protocol Registrar PHONE (336) 713-6767 
Protocol Registrar FAX (336) 713-6772 
Protocol Registrar E-MAIL (registra@wakehealth.edu) 
            
*Protocol Registration is open from 8:30 AM - 4:00 PM, Monday-
Friday. 
 
4.0 
Fax/e-mail ALL eligibility source documents with registration. 
Patients will not be registered without all required supporting 
documents. 
Note: If labs were performed at an outside institution, provide a printout of 
the results. Ensure that the most recent lab values are sent. 
 
To complete the registration process, the Registrar will:   
 
• assign a patient study number 
• register the patient on the study 
 
4.2 
Data Collection Methods 
 
4.2.1 Clinical Data Collection 
Implementation of this prospective observational pilot study has the full 
support of the Wake Forest Thoracic Multidisciplinary Research Committee. 
We plan to enroll 50 patients with newly diagnosed locally advanced non-

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 11 of 31 
 
small cell lung cancer undergoing standard of care chemoradiotherapy. 
Baseline characteristics including demographics (age, sex, ethnicity, and 
race), tobacco use history, past medical history (COPD, interstitial lung 
disease, asthma, etc.), antibiotic and steroid use, and oncologic history 
(TNM stage, tumor size, location, histology and date of diagnosis) will be 
recorded. Baseline pulmonary function, tested as a part of routine pre-
treatment workup, will be collected if available. Treatment-related factors 
will be recorded including radiotherapeutic modality (3D conformal 
radiotherapy, intensity-modulated radiotherapy, or volumetric modulated 
radiotherapy), detailed volumetric and dosimetric parameters for each 
patient’s individualized radiation plan, as well as type of chemotherapy and 
immunotherapy used.  
 
4.2.2 Exhaled Breath Sample Collection 
At the baseline visit prior to the initiation of CRT, pre-treatment sample 
collection will occur within the Radiation Oncology Clinic. This may occur 
on the day of the radiation treatment planning CT simulation scan. Patients 
will be brought to a clinic room or a holding area depending on room 
availability. Each of these areas has access to medical air valves located 
on the wall. (Figure 2) 
 
For exhaled breath condensate, we will utilize the R-tube collection device 
(Respiratory Research Inc., Figure 3). The R-tube is a one-time use 
apparatus that utilizes a metal cooling sleeve, placed over the plastic tube, 
to cool the patient’s breath, creating condensation build up within the 
device. The patient will breathe normally through the mouth piece for 10 
minutes. Following the collection, a metal plunger will be used to 
consolidate the condensate within the collection tube and the EBC sample 
can be transferred to a fresh sterile tube. All biofluids will be aliquoted, 
frozen and stored at -80° C until analysis. 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 12 of 31 
 
After the initial exhaled breath condensate sample is obtained, the patient 
will be invited to provide a second breath sample for exhaled breath 
volatiles. For this, we will utilize the ReCIVA (Figure 1b, Owlstone medical), 
breath collection device. The device will be attached to the in-house medical 
air supply (yellow) with airflow set to 40L/min per the company’s 
recommendation (Figure 2). This medical air is between 19-22% oxygen 
(similar to room air) and is readily available in the Radiation Oncology clinic 
rooms/holding area where breath samples will be obtained (Figure 2). One-
time use masks will be placed over the patient’s mouth and nose.  The 
patient will be allowed to breathe normally to acclimate themselves to the 
mask and airflow. The patient will be allowed to hold the device, if desired, 
and a head strap will be placed around their head for additional support and 
comfort. Once the patient is acclimated (<1 min), they will be prompted to 
start the collection. Pre-loaded collection parameters will be set with the 
device software to simultaneously collect four tubes of exhaled breath from 
patient’s expired breath. Each collection tube (pictured in Figure 1b) is 
packed with material to bind volatile chemicals. Four (4) tubes constitute 
one sample at each time point. Once collection begins, the system will learn 
the breathing pattern of the patient (25 sec) and will adjust the collection 
pumps accordingly. Breath collection will take between 5-7 min, depending 
on the patient’s breathing rate and total collection volume. This study will 
collection 500 mL of exhaled breath on each of the four tubes. All exhaled 
breath samples will be stored at 4°C until analysis. Due to the nature of the 
EBV, processing and analysis will occur in batches every 1-2 weeks. This 
essentially uses the entirety of the sample; the EBV samples will be spent 
after completion.  
 
 
4.2.3 Blood Collection 
Patients treated with CRT have blood counts checked prior to CRT and 
weekly during CRT to ensure their eligibility for concurrent chemotherapy. 
Figure 2: Clinic breath collection setup 
Figure 3: R-tube collection 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 13 of 31 
 
This is part of routine standard of care in medical oncology. Additionally, 
routine blood count testing is performed approximately 1 month after 
completion of CRT, to ensure eligibility for consolidative immunotherapy as 
per the standard of care. In order to minimize the number of blood draws 
the patients are subjected to, we will plan to obtain research blood samples 
simultaneously with clinical blood draws at baseline (visit prior to CRT which 
will occur at the time of the RT planning CT simulation), the last blood draw 
within 1 week of the last fraction of RT, and at follow-up approximately 1 
month after completion of CRT. With the patient’s consent, we will obtain 1 
x purple-top vacutainer EDTA tube and 1 x red-top tube in conjunction with 
these standard blood draws.  
All samples collected from this study will be banked and will have the 
potential to be used for future scientific experimentation, which may include 
additional genetic, proteomic, and metabolomic analyses not described in 
the data collection section below. 
 
4.2.4 Follow-up 
Patients will be followed clinically and with routine CT imaging at baseline, 
1 month, and 3-4 months post-treatment. Appointments will take place in 
the morning in Dr. Farris’ clinic in the Radiation Oncology Department, 1st 
Floor Cancer Center. Upon entering the clinic, they will be taken to a room 
where the breath samples will be obtained as described above. Blood 
samples at follow-up will coincide with follow-up blood draws as part of 
routine care as described above. All samples collected from this study will 
be banked and will have the potential to be used for future scientific 
experimentation, which may include additional genetic, proteomic, and 
metabolomic analyses not described in the data collection section below. 
After four months of follow-up, the study will end and the patients will no 
longer be followed.  
 
4.3 
Research Data Collection and Analysis 
 
4.3.1 Breath and Blood Biomarker Concentration Analysis 
State-of-the-art AlphaLISA immunoassay kits (Perkin Elmer, Waltham, MA) 
will be used to measure the concentration of TGF-β1, IL-6, IL-1a and IL-10 
in EBC and serum at baseline, 2-week after CRT start, 6-week after CRT 
start (end of RT) and 1-month post-CRT end. The AlphaLISA platform is a 
no wash immunoassay which has increased sensitivity and selectivity 
compared to the conventional ELISA assay. Assays will be analyzed on an 
EnVision 2105 Multimode plate reader (Perkin Elmer, Waltham, MA).  
Measurements for known standards and samples at each time point will be 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 14 of 31 
 
performed in triplicate. Concentration curves will be plotted in Excel 
(Microsoft) and used to determine sample concentrations. 
 
4.3.2 Breath Microbiome Analysis 
DNA will be extracted from each EBC sample using the QIAamp® UCP 
Pathogen Mini Kit (Qiagen) with extended homogenization of the breath 
samples using the Mini-Beadbeater-96 cell disrupter (Biospec Products). 
The quantity and quality of the DNA extracts will be determined using a 
DeNovix DS-11 FX+ Spectrophotometer/Fluorometer and Agilent 4200 
TapeStation, respectively. The DNA will then be prepared for shotgun 
metagenomic sequencing using the Nextera® DNA Flex Library Preparation 
Kit (Illumina). DNA libraries will be normalized and quantified prior to pooling 
to ensure equal library representation. Pooled DNA libraries will then be 
sequenced using the Illumina NovaSeq 6000 S1 Reagent Kit and 
sequencer, generating 2 x 150 bp paired-end reads. Raw sequence reads 
(FASTQ files) will be imported into the Microbial Genomics Pro Suite within 
the 
CLC 
Genomics 
Workbench 
(version 
11.0.1; 
https://www.qiagenbioinformatics.com) for QC, adapter trimming, and 
analysis. High-quality (≥Q30) reads for each sample will be aligned to those 
in a blank extraction sample (negative control) that will be processed from 
DNA extraction through library preparation to identify and remove 
contaminating sequences. The filtered reads will then be aligned to a 
reference database consisting of complete genomes in the RefSeq 
database for prokaryotes (bacteria and archaea), eukaryotes (fungi and 
protozoa) and viruses for taxonomic profiling. The filtered reads will also be 
assembled into contigs de novo, and MetaGeneMark (29) will be used to 
identify protein-coding regions within the reads to predict functional diversity 
of the microbiota identified in the samples. The coding regions will be 
annotated with Pfam domains and GO terms, and the abundance of 
functional categories will be estimated to build a functional profile for each 
sample.  
 
The results from the taxonomic and functional profiling will each be merged 
to estimate similarities and differences among the samples. Heat maps will 
be generated using Euclidean distance and clustering based on average 
linkage to assess functional similarities between samples, and principal 
component analysis (PCA) ordination of Bray-Curtis similarity between 
samples will be used to visualize the microbiota diversity across the 
samples. 
 
4.3.3 Exhaled Breath Volatile Data Generation and Metabolomic Analysis 
Unbiased metabolomics analysis of bio-volatiles and biofluids has the 
potential to identify 100-1000’s of metabolites in any single sample. Our 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 15 of 31 
 
group has successfully assessed bio-volatiles and biofluids by unbiased 
metabolomics (30, 31). Although there is no single method that allows for 
the complete analysis of the metabolite, utilizing multiple modes of sample 
preparation and analysis will potentially identify a number of classes of 
compounds relevant to this study. The bio-volatile that will potentially be 
identified are various hydrocarbons, aldehydes, ketones, and alcohols.  
Exhaled breath samples will be analyzed using a Markes thermal desorber, 
TD100-xr, to inject breath samples onto an Agilent two-dimensional GC 
system 7890B in tandem with LECO’s Pegasus 4D Time-of-Flight 
instrument system (TD-GCxGC-TOFMS). The collected spectra will be 
processed using LECO’s ChromaTOF version 4.72 software. Initial data 
processing includes peak deconvolution, 1st and 2nd dimension retention 
time recordings, signal to noise ratio calculation, unique mass detection, 
peak area determination, and compound identification based on the 
spectral pattern similarity match against established mass spectral 
reference libraries (NIST17, 2017 National Institute of Standard 
Technology, NIH, USA). Identified spectral matches will be aligned using R-
program R2DGC (32) to determine common metabolites in patients prior to 
and after CRT. MetaboAnalyst 4.0 will be employed to normalize and 
transform the data for univariate and multivariate statistical analysis (33). 
Additional statistical analysis may be performed using Graphpad Prism 8 
(Graphpad Software, Inc). 
 
4.3.4 Data Storage 
 
All research data collected on this study will be stored on Wake Forest 
Baptist Medical Center password protected computers and servers. 
5.0 
Study Outcomes and Outcome Measures 
5.1 
Primary Outcome 
 
5.1.1 Concentrations of TGF-β1, IL-6, IL-1α, and IL-10 (ng/mL) measured 
in exhaled breath condensate prior to CRT (baseline), at 2 weeks 
after CRT start, 6-weeks after CRT start (the end of CRT), and 1 
month after completion of CRT. 
 
5.2 
Secondary Outcomes 
 
5.2.1 Concentrations of TGF-β1, IL-6, IL-1α, and IL-10 (ng/mL) measured 
in exhaled breath condensate prior to CRT (baseline), at 2 weeks 
after CRT start, 6-weeks after CRT start (the end of CRT), and 1 

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month after completion of CRT compared among patients who 
develop SP versus those who do not. SP will be assessed at each 
time point and will be defined as presence of CTCAE version 5.0 
grade 2 or higher SP (Appendix C). 
 
5.2.2 Concentration of TGF-β1, IL-6, IL-1α, and IL-10 (ng/mL) measured 
in serum prior to CRT (baseline), at 2 weeks after CRT start, 6-weeks 
after CRT start (the end of CRT), and 1 month after completion of 
CRT: 
 
5.2.2.1 
Compared to concentrations of same biomarkers 
measured in exhaled breath condensate at same time 
points (see 5.2.1);  
5.2.2.2 
and compared among patients who develop SP versus 
those who do not. 
 
5.2.3  Microbial signatures measured in exhaled breath condensate prior 
to CRT, and presence/absence of SP at study end. 
 
 
5.3 
Exploratory Outcome 
 
5.3.1 Metabolomic signatures associated with the development of SP. This 
will include quantitative assessments of metabolic characteristics 
measured at prior to CRT (baseline), at 2 weeks after CRT start, 6-
weeks after CRT start (the end of CRT), and 1 month after 
completion of CRT. Quantitative assessments of metabolomic 
assessments measured prior to CRT. Using these three sets of 
quantitative assessments (at each of the three time points), 
“signatures” will be developed (described in statistical analysis 
section) and compared to see whether there are characteristics that 
remain similar or change when comparing SP and no-SP measures. 
6.0 
Analytic Plan 
6.1 
Analysis of Primary Outcome, Sample Size, and Power 
 
This is a pilot study; therefore, our objectives are exploratory in nature, and we 
have little to no pre-existing findings reported in the literature to guide our 
expectations.  With this in mind, we plan to recruit a single cohort of 50 patients.  
In order to be able to examine the questions concerning intra-person variability in 
biomarker measures over time, and the possible relationships of breath and 
microbial biomarker signatures with lung cancer outcomes, both pre- and post- 
radiation treatment measures are needed on each participant. Our goal is to 

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collect from each patient one pre- (T0) and two post-treatment (T1, immediately 
post treatment and T2, one month following completion of treatment) samples of 
breath and serum to be examined. Our target sample size of 50 patients is the 
target for patients with pre- and post- samples collected. Our main interest is in 
differences between biomarker levels at T1-T0, where we expect to see the 
largest differences. 
 
First, for the primary objective of detecting intra-person variability from T1 to T0 
in EBC measures, the table below shows different power levels for n=50 patients, 
using a 2-tailed alpha of 0.05.   
 
 
 
 
 
 
6.2 
Analysis of Secondary Outcomes 
 
To examine the association between differences in pre-treatment and post-
treatment EBC concentrations of biomarkers and development of SP, we will 
conduct 2-group independent t-tests comparing mean change levels (from T0 to 
T1) in each marker among those who developed SP (expected to be around 10-
15 patients) to those who remained free of SP at the study end. We will also 
conduct various exploratory logistic regression models, if numbers permit, to 
predict the outcome of SP, using various biomarker values (e.g., T0, difference 
between T0 and T1, etc.) as key predictors. If numbers permit, we will also 
examine results from repeated measures logistic regression models, using the 
three repeated measures of the biomarkers as predictors, and/or using at least 
one delta score as a predictor. Our goal in such exploratory modeling will be to 
discern what time points or what differences in EBC biomarkers appear to be 
most highly predictive of occurrence of SP.  We will conduct most of this 
modeling on a single biomarker at a time; based on biological rationale, bivariate 
findings, and numbers, we may also explore some modeling where more than 
one biomarker is included.   
 
We also wish to determine to what degree serum levels of biomarkers correlate 
with levels of the same biomarkers measured in exhaled breath condensate, and 
how well these serum markers predict SP. For this latter goal, we will use a 
similar plan as described in the preceding paragraph. 
 
For the former objective (examining how well serum and breath levels of 
biomarkers correspond), because we do not hypothesize a priori that these 2 
different sources of a biomarker are actually measuring (estimating) the same 
quantity for any of the markers, we do not expect absolute agreement.  We will 
Detectable standard deviation units 
Power 
0.3 
0.55 
0.4 
0.79 
0.5 
0.93 

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thus first examine scatterplots of the serum vs breath measure of each 
biomarker, and if the association appears monotonic in the scatterplot, we will 
compute Spearman rank correlations. (If the association appears linear and 
monotonic, we will compute Pearson correlations.) If an association between a 
serum and breath measure of a biomarker is non-monotonic, we will attempt to fit 
a curve describing the association most accurately. 
 
For our final secondary objective (examining the association between 
microbiome signatures found in pre-treatment EBC and development of SP) we 
will use cluster analysis methods to determine whether SP and non-SP patients 
share similar marker profiles. As stated above, this is a pilot study, therefore 
these cluster analyses will be considered exploratory and hypothesis generating. 
 
6.3 
Analysis of Exploratory Outcome 
 
For the analysis of metabolic measures, we will examine the association of 
metabolic signatures with development of SP. We hypothesize that metabolic 
markers in patients who develop SP will be different than those who do not. 
Therefore, a first analysis will be to examine a series of paired t-tests to 
determine which markers significantly change between the two time points for 
patients.  
 
In addition to this analysis, we wish to determine whether there are any marker 
signatures that appear in groups of patients. To do this we will use cluster 
analysis methods to determine whether there are any groups of patients who 
share similar marker profiles at baseline and post-CRT, or change from pre- to 
post- for metabolic measures.  If we are able to identify clusters of patients with 
common marker signatures, we will then determine whether there are other 
disease characteristics that are shared by these patients (i.e., tumor stage, lung 
function, etc.).  
 
6.4 
Accrual Rate 
 
Due to the large volume of NSCLC patients treated at this institution, we 
generally see 4-5 patients per month that would meet inclusion criteria. 
Considering those that meet eligibility but do not enroll, we expect an accrual of 
approximately 2 patients per month.  
6.5 
Length of Study 
 
In order to accrue 50 patients at a rate specified above, the length of the accrual 
will be approximately 2 years. The study will complete after the 4-month follow-up 
of the last patient and we estimate the total duration at no longer than 2.5 years.  

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7.0 
Treatment Plan 
 
7.1 
Treatment Administration 
 
This study is observational in nature and does not impact the treatment 
received by enrolled patients in any way. Patients will be treated according 
to the standard of care for stage III NSCLC. They will be seen in consultation 
by a radiation oncologist. When the decision to proceed with definitive CRT 
is made, patients will sign informed consent for their radiation treatment. 
They will also sign informed consent for concurrent chemotherapy at the 
discretion of the medical oncologist.  
 
Treatment planning will comprise a CT simulation with or without contrast 
using appropriate immobilization at the discretion of the treating radiation 
oncologist. Methods to account for respiratory motion that may be utilized 
include 4-dimensional CT imaging and slow CT scan techniques. Target 
and normal tissue delineation as well as treatment planning will be 
performed at the discretion of the treating radiation oncologist. Specific 
radiotherapy dose/fractionation are not mandated by the protocol, but 60-
66 Gy in 1.8-2 Gy daily fractions is recommended. Weekly on-treatment 
visits will be performed according to the standard of care.  
 
Concurrent chemotherapy may be delivered at the discretion of the treating 
medical oncologist. There is not a mandated time frame for administration 
of concurrent chemotherapy with regard to the first fraction of radiotherapy, 
but it is recommended that first dose of concurrent chemotherapy occur 
within 72 hours of the first radiotherapy fraction – ideally on the same day, 
if possible. 
 
7.2 
Follow-up 
 
After completion of CRT, patients will be followed with routine clinical visits 
and radiographic examinations according to the standard of care. This 
observational study utilizes clinical and CT imaging surveillance obtained 
as part of routine follow-up as outcome measures for SP. Patients will return 
for post-treatment follow-up visit at 1-month post-treatment for clinical 
assessment. Serum/EBC samples will be collected at this visit. Three 
months thereafter, at approximately 4 months post-CRT, a routine 
surveillance CT examination of the chest (with or without contrast) will be 
performed. Clinical assessment/follow-up will be performed at this time 
point as well, and the final sample collection will occur at this visit.  
 
 
 
 

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7.3 
Study Calendar 
a Approximately 1-1.5 mL of EBC will be generated in 10 min of breathing 
b 500 ml of EBV in each of 4 collection tubes will be collected in 5-7 min of breathing 
c Blood sample in each redtop (x1) and purpletop (x1) vacutainer blood collection tube 
d Pre-CRT sample collections to occur within 14 days of the date of RT start. It is recommended but not 
required that this occurs on the date of CT simulation. 
e May occur within +/- 7 days of the scheduled time point measured from the date of RT start. The 2-week 
after CRT start, 6-week after CRT start, and 1-month after CRT time points will be coordinated with 
routine blood draws through medical oncology by the study research nurse. 
f To be completed after completion of treatment planning. This may also be completed at any time after 
CRT has started. 
g May occur within +/- 14 days of the scheduled time point measured from the date of RT end. 
h EBV collection is optional, as defined in Section 4.2.2. 
i Pulmonary function tests must have occurred within 1 year of CRT Start. Results of testing performed at 
Wake Forest sites or other outside facilities is acceptable. 
 
 
 
Prior to 
CRT Start 
2-week 
after CRT 
Starte 
6-week 
after CRT 
Starte 
1 Month 
after CRT 
Ende 
4 Months 
after CRT 
Ende 
Informed consent 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Medical history 
(Appendix E) 
X 
 
 
 
 
 
Physical Exam 
X 
 
 
 
 
Pulmonary Function 
Tests (Appendix H) 
Xi 
 
 
 
 
 Antibiotic and Steroid 
Use 
(Appendix D) 
X 
Xe 
Xe 
Xg  
Xg 
Smoking Status 
(Appendix G) 
X 
Xe 
Xe 
Xg  
Xg 
Breath Collection 
(EBCa/EBVb,h) 
Xd 
Xe 
Xe 
Xg  
 
Blood collectionc 
Xd 
Xe 
Xe 
Xg 
 
CT chest with or without 
contrast 
X 
 
 
 
Xg 
Pneumonitis Evaluation 
(Appendix C) 
X 
 
 
Xg 
Xg 
Dietary Evaluation 
(Appendix M) 
Xd 
Xe 
Xe 
Xg 
 
Radiotherapy Detail 
Form (Appendix I) 
Xf 
 
 
 
 

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8.0 
Specimen Collection and Storage 
 
Detailed descriptions of sample collection, aliquoting and storage are described 
in Section 4.0. Exhaled breath condensate samples will be aliquoted, stored and 
secured within a locked freezer at -80° C until analysis.  
 
Exhaled breath volatiles require immediate processing and analysis due to the 
volatile nature of the sample. As such, these will be batched and processed 
within 1-2 weeks after collection. Subsequent to this process, no sample will 
remain in existence. The data generated will be kept in a secure location 
according to Section 4.3.3 for future exploratory analyses.  
 
Blood samples will be obtained in coordination with routine blood draws that 
occur as a part of standard of care for patients receiving CRT. Baseline blood will 
be obtained at the time of CT simulation requiring an additional phlebotomy 
procedure. The 2-week and 6-week blood samples will be obtained at the time of 
routine weekly labs (as part of standard monitoring during CRT). The 1-month 
blood will be obtained at the post-CRT visit, which occurs routinely as part of 
workup and confirmation of eligibility for consolidation immunotherapy.  
 
To facilitate coordination between breath and blood collection, breath samples 
will be obtained on the same day as the blood samples, on the day of CT 
simulation (pre-CRT baseline), on the day of 2-week and 6-week routine on-
treatment blood draws, and at the 1-month visit post-CRT. 
 
All samples will be de-identified and stored securely at -80° C in the laboratory of 
Dr. Andrew Bishop. Samples (non-EBV) will be stored until analysis and 
remaining samples will be retained indefinitely for future study.  
 
Blood samples will be stored indefinitely in the laboratory of Dr. Andrew Bishop 
until analyzed. These samples will be de-identified by the unique study ID code 
given at the time of collection. 
9.0 
Data Management 
 
Informed consent document 
EPIC 
Protocol registration form 
WISER/OnCore 
Medical History 
WISER/OnCore 
Specimen Collection Form 
WISER/OnCore 
Antibiotic and Steroid Use 
(Medications Form) 
WISER/OnCore 
Tobacco Use Form 
WISER/OnCore 

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Radiotherapy Form 
WISER/OnCore 
Pneumonitis Form 
WISER/OnCore 
Research Data 
Secure research lab computers and servers 
10.0 Confidentiality and Privacy 
 
Confidentiality will be protected by collecting only information needed to assess study 
outcomes, minimizing to the fullest extent possible the collection of any information that 
could directly identify subjects, and maintaining all study information in a secure 
manner. To help ensure subject privacy and confidentiality, only a unique study 
identifier will appear on the data collection form. Any collected patient identifying 
information corresponding to the unique study identifier will be maintained on a linkage 
file, store separately from the data. The linkage file will be kept secure, with access 
limited to designated study personnel. Following data collection, subject identifying 
information will be destroyed.  Samples will be de-identified and stored indefinitely in a 
secured wake forest laboratory.  Consistent with data validation and study design, 
producing an anonymous analytical data set. Data access will be limited to study staff. 
Data and records will be kept locked and secured, with any computer data password 
protected. No reference to any individual participant will appear in reports, 
presentations, or publications that may arise from the study. 
11.0 Data Safety and Monitoring 
 
The principal investigator will be responsible for the overall monitoring of the data and 
safety of study participants. The principal investigator will be assisted by other members 
of the study staff. 
12.0 Reporting of Unanticipated Problems, Adverse Events or 
Deviations 
 
Due to the observational nature of this study, adverse events of interest are limited to 
those that occur in the immediate duration of the non-invasive exhaled breath collection 
itself. Should an AE related to breath collection occur, the investigators will notify study 
staff for logging in the Adverse Event Log (Appendix L). 
 
Any unanticipated problems, deviations or protocol changes will be promptly reported by 
the principal investigator or designated member of the research team to the IRB. 
 
12.1 Adverse Event Definitions 
 
12.1.1 Adverse Event (AE) 

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An adverse event (AE) is defined as any unexpected, unfavorable 
or unintended condition that occurs immediately during the exhaled 
breath condensate or exhaled breath volatile collection (while the 
collection device is applied to the patient). 
 
12.1.2 Serious Adverse Event (SAE) 
 
A serious adverse event (SAE) is any untoward medical occurrence 
that occurs during the exhaled breath condensate or exhaled 
breath volatile collection (while the collection device is applied to 
the patient) that satisfies any of these criteria: results in death; is 
life-threatening; requires inpatient hospitalization or prolongs 
existing hospitalization; results in persistent or significant disability 
or incapacity or substantial disruption of the ability to conduct 
normal life functions; or if the event results in a congenital anomaly 
or birth defect. 
 
12.2 Recording Adverse Events 
 
When an AE occurs, it is the responsibility of the Investigator to review all 
documentation (e.g., medical progress notes, laboratory, and diagnostics 
reports) relative to the event.  The study staff will then record all relevant 
information regarding an AE in the AE Log (Appendix L).  
 
12.3 Adverse Event Characteristics 
 
• 
CTCAE term (AE description) and grade:  The descriptions and 
grading scales found in the revised NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 5.0 will be utilized for AE 
reporting. All appropriate treatment areas should have access to a 
copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can 
be downloaded from the CTEP web site (http://ctep.cancer.gov). 
 
• 
‘Expectedness’: AEs can be ‘Unexpected’ or ‘Expected’ (see Section 
7.1 above) for expedited reporting purposes only.   
 
• 
Attribution of the AE: 
 
- 
Definite – The AE is clearly related to the study collection. 
- 
Probable – The AE is likely related to the study collection. 
- 
Possible – The AE may be related to the study collection. 
- 
Unlikely – The AE is doubtfully related to the study collection. 
- 
Unrelated – The AE is clearly NOT related to the study collection. 

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12.4 STRC SAE Reporting Requirements 
 
The Data Safety Monitoring Committee (DSMC) is responsible for reviewing 
SAEs for WFBCCC Institutional studies as outlined in Appendix N. All Adverse 
Events that occur during protocol intervention and are coded as either 1) 
unexpected grade 4, 2) unplanned inpatient hospitalization ≥ 24 hours 
(regardless of grade), or grade 5 (death) must be reported to the DSMC using 
the using the SAE console in WISER.  
 
All WFBCCC Clinical Protocol and Data Management (CPDM) staff members 
assisting a Principal Investigator in investigating, documenting and reporting an 
SAE qualifying for DSMC reporting are responsible for informing a clinical 
member of the DSMC as well as the entire committee via the email notification 
procedure of the occurrence of an SAE.  
12.5 WFUHS IRB AE Reporting Requirements 
 
Any unanticipated problems involving risks to subjects or others and adverse 
events shall be promptly reported to the IRB, according to institutional policy. 
Reporting to the IRB is required regardless of the funding source, study sponsor, 
or whether the event involves an investigational or marketed drug, biologic or 
device. Reportable events are not limited to physical injury, but include 
psychological, economic and social harm. Reportable events may arise as a 
result of drugs, biological agents, devices, procedures or other interventions, or 
as a result of questionnaires, surveys, observations or other interactions with 
research subjects. 
 
All members of the research team are responsible for the appropriate reporting to 
the IRB and other applicable parties of unanticipated problems involving risk to 
subjects or others. The Principal Investigator, however, is ultimately responsible 
for ensuring the prompt reporting of unanticipated problems involving risk to 
subjects or others to the IRB. The Principal Investigator is also responsible for 
ensuring that all reported unanticipated risks to subjects and others which they 
receive are reviewed to determine whether the report represents a change in the 
risks and/or benefits to study participants, and whether any changes in the 
informed consent, protocol or other study-related documents are required. 
 
Any unanticipated problems involving risks to subjects or others occurring at a 
site where the study has been approved by the WFUHS IRB (internal events) 
must be reported to the WFUHS IRB within 7 calendar days of the investigator or 
other members of the study team becoming aware of the event. 
 

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Any unanticipated problems involving risks to subjects or others occurring at 
another site conducting the same study that has been approved by the WFUHS 
IRB (external events) must be reported to the WFUHS IRB within 7 calendar 
days of the investigator or other members of the study team becoming aware of 
the event. 
 
Any event, incident, experience, or outcome that alters the risk versus potential 
benefit of the research and as a result warrants a substantive change in the 
research protocol or informed consent process/document in order to insure the 
safety, rights or welfare of research subjects. 
 
 
 

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Glendinning L, Wright S, Tennant P, Gill AC, Collie D, McLachlan G. Microbiota 
in Exhaled Breath Condensate and the Lung. Appl Environ Microbiol. 2017;83(12). 
29. 
Zhu W, Lomsadze A, Borodovsky M. Ab initio gene identification in metagenomic 
sequences. Nucleic Acids Res. 2010;38(12):e132. 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Page 28 of 31 
 
30. 
Bishop AC, Libardoni M, Choudary A, Misra B, Lange K, Bernal J, et al. 
Nonhuman primate breath volatile organic compounds associate with developmental 
programming and cardio-metabolic status. J Breath Res. 2018;12(3):036016. 
31. 
Misra BB, Bassey E, Bishop AC, Kusel DT, Cox LA, Olivier M. High Resolution 
GC/MS Metabolomics of Non-Human Primate Serum. Rapid Commun Mass Spectrom. 
2018. 
32. 
Ramaker RC, Gordon ER, Cooper SJ. R2DGC: threshold-free peak alignment 
and identification for 2D gas chromatography-mass spectrometry in R. Bioinformatics. 
2018;34(10):1789-91. 
33. 
Chong J, Soufan O, Li C, Caraus I, Li S, Bourque G, et al. MetaboAnalyst 4.0: 
towards more transparent and integrative metabolomics analysis. Nucleic Acids Res. 
2018;46(W1):W486-W94. 
 
 
 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Protocol version date 05/14/21 
 
 
 
 
Page 29 of 31 
Appendix A – Subject Eligibility Checklist 
 
IRB Protocol No. IRB00059924 
WFBCCC Protocol No. 98119 
Study Title: A Pilot Study of Exhaled Breath Analysis to Predict Risk of Symptomatic Pneumonitis After 
Chemoradiotherapy For Stage III Non-Small Cell Lung Cancer 
Principal Investigator: Michael Farris, MD 
 
Inclusion Criteria 
(as outlined in study protocol) 
Criteria 
is met 
Criteria 
is NOT 
met 
Source Used to Confirm * 
(Please document dates and lab 
results) 
Age greater than or equal to 18 years old 
□ 
□ 
 
Clinically diagnosed or suspected Stage 
III non-small cell lung cancer to be 
treated with chemo-radiotherapy as part 
of cancer treatment, as determined by 
the treating clinician 
□ 
□ 
 
Plan for treatment with definitive 
radiotherapy (≥60 Gy) with concurrent 
chemotherapy at the discretion of the 
treating radiation and medical 
oncologists. 
□ 
□ 
 
Willing and able to tolerate exhaled 
breath collection 
□ 
□ 
 
Able to provide informed consent 
□ 
□ 
 
Exclusion Criteria 
(as outlined in study protocol) 
Criteria 
NOT 
present 
Criteria 
is 
present 
Source Used to Confirm * 
(Please document dates and lab 
results) 
Systemic (oral, intravenous or 
intramuscular) corticosteroid use for any 
reason within 5 days of registration. 
□ 
□ 
 
Prior radiotherapy directed at the chest 
(thoracic inlet superiorly to diaphragm 
inferiorly). 
□ 
□ 
 
Any antibiotic use within 2 weeks of 
registration 
□ 
□ 
 
 
This subject is      
 eligible /  ineligible     for participation in this study. 
 
OnCore Assigned PID: ___ ___ ___ ___ ___ ___ ___ ___  
 
 
 
 
Signature of research professional confirming eligibility: _______________________  
 
Date: ___ ___/___ ___/___ ___ 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Protocol version date 05/14/21 
 
 
 
 
Page 30 of 31 
Signature of Treating Physician**:  _____________________________________  
 
Date: ___ ___/___ ___/___ ___ 
 
 
* Examples of source documents include clinic note, pathology report, laboratory results, etc. When listing 
the source, specifically state which document in the medical record was used to assess eligibility. Also 
include the date on the document. Example: “Pathology report, 01/01/14” or “Clinic note, 01/01/14” 
 
**Principal Investigator signature can be obtained following registration if needed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

A PILOT STUDY OF EXHALED BREATH ANALYSIS TO ASSESS PNEUMONITIS 
DEVELOPMENT AFTER CHEMORADIOTHERAPY FOR STAGE III NSCLC 
Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) 
WFBCCC 98119 
Protocol version date 05/14/21 
 
 
 
 
Page 31 of 31 
Appendix B – Protocol Registration Form 
 
DEMOGRAPHICS 
Patient: Last Name: _____________________ First Name: ___________________________ 
MRN:          ___ ___ ___ ___ ___ ___ ___ ___ DOB (mm/dd/yy): ___ ___ / ___ ___ / ___ ___ 
ZIPCODE:  ___ ___ ___ ___ ___ 
 
 
SEX:   
  Male      Female 
Ethnicity (choose one):   Hispanic 
Non-Hispanic 
Race (choose all that 
apply): 
 WHITE           BLACK            ASIAN 
 PACIFIC ISLANDER                 NATIVE AMERICAN 
Height:   ___ ___.___ inches 
Weight: ___ ___ ___.___lbs.(actual) 
Surface Area: ___ ___.___m2   
 
Primary Diagnosis: ____________________________________________________________ 
Date of Diagnosis: ___ ___ /___ ___/___ ___ 
 
Performance Status: ___ ___ ___    ECOG   Karnofsky 
 
Protocol Registrar can be contact by calling 336-713-6767 between 8:30 AM and 4:00 PM, Monday – Friday. 
 
Completed Eligibility Checklist and Protocol Registration Form must be hand delivered, faxed or e-mailed to 
the registrar at 336-7136772 or registra@wakehealth.edu. 
 
 
 
 
 
 
PROTOCOL INFORMATION 
Date of Registration: 
___ ___ / ___ ___ / ___ ___ 
MD Name (last) : 
______________________       
Informed written consent: 
(consent must be signed prior to 
registration) 
 YES  NO 
 
Date Consent Signed: 
___ ___ / ___ ___ / ___ ___ 
 
PID # (to be assigned by OnCore): 
___ ___ ___ ___ ___ ___ ___ ___