Temporal Variation in Exhaled Volatile Organic Compounds in Response to Therapeutic Intervention in Esophageal Cancer Patients
Phase: NA
Conditions: Esophageal Cancer
Sponsor: Benaroya Research Institute

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Proposal: Temporal variation in exhaled volatile organic 
compounds in response to therapeutic intervention in 
esophageal cancer patients 
Principal investigator:  
Donald E Low FACS FRCS (c) FRCSI (Hon) FRCSE (Hon) 
Head Of Thoracic Surgery and Thoracic Oncology 
Director Digestive Disease Institute Esophageal Center of Excellence 
President  Ryan Hill Research Foundation 
Virginia Mason Medical Center 
1100 Ninth Ave 
Seattle, WA, USA 98111 
Telephone: +1 (206) 223-6164 
Email: Donald.Low@virginiamason.org  
Co-investigator: 
George B Hanna FRCS, PhD 
Professor of Surgical Sciences 
Head of Division of Surgery 
Department of Surgery and Cancer, 
Imperial College London,  
St Mary’s Hospital,  
London, W2 1NY, UK 
Telephone: +44 (0)207 886 2125 
E-mail: g.hanna@imperial.ac.uk

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SPECIFIC AIMS 
 
Hypothesis: we hypothesize that cancer-associated Volatile Organic Compounds 
(VOCs) will vary significantly following therapeutic intervention in patients with 
esophageal cancer (EC) and that variation will predict treatment response.  
 
Primary aim: to determine longitudinal variation in exhaled VOC concentrations 
during intended curative therapy for EC cancer. 
 
Secondary aims: 
i. 
To validate the previously published model for a exhaled VOC breath test of EC 
cancer 
ii. To correlate the exhaled VOCs of EC cancer patients to important clinical 
parameters 
iii. To determine the relationship between exhaled VOCs and response to 
neoadjuvant chemoradiotherapy 
iv. To study changes in exhaled VOCs following tumor resection and their association 
with disease recurrence. 
v. 
To correlate the exhaled and urinary VOC concentrations in EC cancer patients 
vi. To longitudinal variation in the upper gastrointestinal microbiome and its 
relationship to exhaled and urinary VOC concentrations.  
 
 
STUDY OUTCOMES 
 
Primary outcome 
i. 
Confirmation of exhaled and urinary VOC biomarkers of EC cancer 
 
Secondary outcomes 
i. 
Verification of changes in VOC concentrations that occur in response to 
therapeutic intervention  
ii. Refine diagnostic model for the detection and monitoring of EC cancer through 
analysis of exhaled VOCs 
iii. Linkage of longitudinal VOC data to predominant upper gastrointestinal bacterial 
species 
iv. Patient acceptability of breath test 
 
 

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BACKGROUND AND SIGNIFICANCE 
 
Background: The global prevalence of esophageal cancer (EC) in 2016 was 2.75 million 
cases with an estimated 1.60 million new cases and 1.25 million deaths during the 
same year (1, 2). Early EC cancer typically has non-specific symptoms that are 
commonly ascribed to benign conditions. Current guidelines recommend referral for 
endoscopy, only in the setting of ‘red flag’ symptoms that are frequently associated 
with inoperable disease. As a consequence, 7 out of 10 new cases of EC cancer 
diagnosed are considered to be at an advanced stage with less that 1 in 3 patients 
eligible for potentially curative therapy and less than 1 in 5 patients alive at 5 years 
(3). 
 
Volatile organic compounds and EC cancer biomarkers:  
Our approach to this clinical challenge is to establish a non-invasive test for the 
detection of EC cancer that is based upon the unique signature of volatile organic 
compounds (VOCs) within exhaled breath. 
 
Volatile organic compounds (VOCs) are carbon-containing molecules that are 
sufficiently volatile to be detectable in a gas form at room temperature. VOCs may 
emanate from numerous sources within the human body as well as our local 
environment. Established uses of VOC measurements include the assessment of 
environmental contamination, the flavor and fragrance industries and in counter-
terrorism. There is now established evidence that VOCs which are released from the 
human body in breath and urine can be used as a non-invasive method of detection 
and monitoring (patho)physiological processes.  
 
The analysis of VOCs excreted within exhaled breath is a particularly attractive 
diagnostic prospective as it offers a potentially non-invasive window on to normal 
human physiology and diseases states within the body. Examples of the analysis of 
exhaled VOCs within clinical practice include breathalyzer devices for ethanol 
detection, 13C urea breath testing for H. pylori, exhaled Nitric Oxide in asthma and 
Hydrogen/Methane testing for small bowel bacteria overgrowth (4-7). There is now 
an appreciation that increased production of certain VOCs may be associated with 
cancer. In a recent literature review, we identified 63 publication presenting data for 
target VOC analysis in 12 distinct cancer subtypes. Pooled analysis of these studies 
revealed a combined sensitivity and specificity of 79% (95% CI 77%-81%) and 89% 
(95% CI 88%-90%) respectively for a ‘cancer breath test’. This review included six 
publications concerning the VOC-based breath tests for the diagnosis of EC cancers (8-
13). VOCs released from the urine of EC cancer patients has also been shown to 
contain a unique profile, although no study has directly compared this to breath 
signatures (14). 
 
Our approach to addressing this clinical challenge is to establish a non-invasive breath 
test for early detection of EC cancer based on its unique profile of volatile organic 
compounds (VOCs). Our previous studies have reported higher concentrations of 
specific VOCs, including volatile fatty acids (butanoic-, pentanoic- and hexanoic acid), 
within the breath, gastric content and urine of EC cancer patients (1-4). We have also 

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established standardized and quality controlled protocols for breath sampling, 
transfer and storage that ensure accuracy and reproducibility of VOC analysis by mass 
spectrometry methods (5-9). 
 
Infrastructure for research 
Existing infrastructure at Virginia Mason Medical Center that will support the 
proposed study include: high volume center for the management of esophageal 
cancer; support from the Benaroya Research Institute at Virginia Mason including 
access to study coordinators, and; a track record for conducting high quality research 
in esophageal cancer. 
 
The proposed study will be conducted in collaboration with the volatile biomarker 
research group, based at St Mary’s Hospital (Imperial College London). The primary 
focus of this group’s research has been the non-invasive diagnosis of gastrointestinal 
cancer through the detection of VOCs within exhaled breath. They have previously 
established diagnostic models of gastroesophageal and colorectal cancers (8, 9, 15, 
16). In a recent multicenter validation study they demonstrated a diagnostic 
performance of a breath test for gastroesophageal cancers based on five VOCs, with 
an area under the receiver operator characteristic curve of 0.85 and sensitivity and 
specificity of 80% and 81% respectively (15). Their previous work has alluded to 
importance of volatile fatty acids, aldehydes and phenols as tumor markers. In parallel 
to these clinical trials they have sought to investigate the mechanisms whereby VOCs 
are deregulated in cancer by undertaking carefully designed in-vitro and in-vivo 
studies. 
Specifically 
they 
have 
demonstrated 
an 
association 
between 
gastroesophageal cancer, its predominant associated bacterial species (E.coli, K. 
pneumoniae and S. anginosus) and VOCs production (unpublished data). Whilst these 
findings give support to the hypothesis that cancer has associated bacteria, no study 
has sought to directly compare the intestinal microbiome and exhaled VOC levels. 
 
Critical knowledge gap: Previous studies have established an important link between 
deregulated VOC production and EC. These studies have all assessed exhaled VOCs at 
a single point in time using a cross-section study design. Longitudinal variation in 
exhaled and urinary VOCs, the contribution of the upper gastrointestinal microbiome 
and the effect of therapeutic intervention in EC cancer patients remain unknown. An 
understanding of this variation is critically important in establishing intra-subject 
variability and likely mechanisms of VOC production and release. Furthermore the 
expected decline in target VOCs following chemoradiotherapy and/or tumor resection 
would both verify the link between the tumor and aberrant VOCs production as well 
as offering an opportunity to utilize VOCs as a means to assess treatment response 
including the early detection of recurrence. 
 
 

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RESEARCH DESIGN AND METHODS 
 
Study design: longitudinal cohort study 
 
Sample size: 50 curative tri-modality patients. Study sample size has been determined 
pragmatically based on estimated patient recruitment during the course of 1 year in a 
high volume center for the management of EC 
 
Human subjects: patients undergoing potentially curative therapy for EC cancer at 
Virginia Mason Medical Center who meet the eligibility criteria will be invited to 
participate in this study.  
 
Inclusion criteria: patients with the following characteristics will be eligible for 
inclusion in this study: 
i. 
aged 18-90 years 
ii. 
newly diagnosed, treatment naïve patients with esophageal and/or 
gastroesophageal junctional cancer 
iii. 
planning to undergo curative treatment, including neoadjuvant 
chemoradiotherapy and surgical resection  
 
Exclusion criteria: patients with the following characteristics will not be eligible for 
inclusion in this study: 
i. 
pregnant females 
ii. 
without malignant esophageal disease 
iii. 
malignancy at a secondary site other than the esophagus 
iv. 
patients undergoing palliative treatment for esophageal cancer 
v. 
patients not receiving neoadjuvant chemoradiotherapy and surgical 
resection for esophageal cancer 
vi. 
inability or unwillingness to provided written informed consent 
 
 
Study time points: subjects will be asked to provide exhaled breath, urine and saliva 
samples at the following time points during their routine care: 
i. 
Before commencing treatment for esophageal cancer* 
ii. Following conclusion of neoadjuvant therapy (at the time of a routine outpatient 
appointment or on the day of surgery) 
iii. Following surgical resection (after the removal of the nasogastric tube, during the 
inpatient hospital stay) 
iv. At the time of routine postoperative follow-up at VM (between six months to one-
year following surgery) 
 
Subjects will also be asked to complete an engagement questionnaire at one of the 
study timepoints. See “Subject Engagement” section of the protocol. 
 
*Where feasible at the time of initial breath sampling subjects will be asked to provide 
breath samples on two separate routine clinical appointments (often on consecutive 
days) in order to assess short-term temporal variation in exhaled VOC levels. At the 

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time of their initial assessment it is common for patients to have routine clinical 
appointments on consecutive days.  
Any data that are not able to be collected with reasonable efforts will not be 
considered protocol deviations. 
 
Exhaled breath collection and analysis: Subjects will be asked to fast for a minimum 
of 1-2 hours  prior to breath sampling, with an ideal fast of 4 hours or longer. Where 
possible sampling will be performed on a day when subjects are fasting as part of their 
routine care (e.g. prior to CT-PET or endoscopy). Before breath sampling, subjects will 
also be asked to rinse their mouth with water. Following a period of relaxed tidal 
breathing subjects will be asked to exhale directly in to double thickness Nalophan® 
bags (Scentroid, Canada).  
 
Breath samples will be immediately (within 30-120 mins of collection, target: 30 
minutes) transferred from Nalophan sample bags to thermal desorption (TD) tubes 
(Markes International, Llantrisant, UK) pre-packed with 200 mg of Tenax and 100 mg 
of Carbograph 5. 500ml aliquots of breath will be transferred to four TD tubes via a 
handheld precision pump (Easy-VOC, Markes International, Llantrisant, UK). TD tubes 
will be securely capped and stored at -80oC prior to being transferred to Imperial 
College London for analysis. 
 
TD tubes containing exhaled VOCs will be analyzed by Gas Chromatography Mass 
Spectrometry (GC-MS). For thermal desorption GC-MS analysis, adsorbent tubes will 
be loaded into a sample tray (TD-100, Markes International). The tube will be dry-
purged for 1 minute to remove excess moisture and air. VOCs will be desorbed from 
the tube by a specific combination of heat and gas flow and will be refocused on the 
cold-trap. The cold-trap is then rapidly heated to 290oC with carrier gas flow so that 
VOCs are transferred to the GC column in a narrow band. For separation, the GC 
column will be ramped. The mass spectrometer will run in electron impact ionisation 
mode, scanning mass ions 20-250 m/z. An initial solvent delay will minimize 
interference from water. Chromatographic data will be analyzed using the Mass 
Hunter package (Agilent Technologies) in combination with the NIST mass spectral 
library (2014, Maryland, USA) for compound identification and semi-quantification. 
 
Urine sampling and analysis: Patients will be asked to provide a clean catch urine 
sample (approximately 10ml) into a sterile sample container. Urine samples will be 
aliquoted and stored at -80oC prior to being transferred on dry ice to Imperial College 
London for analysis. 
 
VOCs within the headspace gas above urine samples sealed in air-tight vials will be 
analyzed by GC-MS. Urine samples (2ml) will be diluted in 6ml deionized pure water, 
acidified using HCl to pH 2.0. Urine osmolality (OsmoPRO®, Advanced Instruments, 
Norwoor, UK) will be analyzed for the purpose of subsequent standardization. 
Samples will be placed in air tight headspace vials into which a HiSorb probe (Markes 
International, Llantrisant, UK) will be inserted. Headspace vials including the HiSorb 
probe will be heated and agitated at 60oC for 30 minutes. Headspace VOCs extracted 

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onto HiSorb probes will be analyzed using an Agilent 7890B GC with 5977 MSD (Agilent 
Technologies, Cheshire, UK) coupled to a Markes TD-100 device. 
 
Saliva sampling and analysis: Subjects will be asked to fast for a minimum of 1-2 hours 
(Target: 4 hours) prior to saliva sampling. Where possible saliva sampling will be 
performed on a day when subjects are fasting as part of their routine care (e.g. prior 
to CT-PET or endoscopy). Saliva samples (1-3ml) will be collected by asking subjects to 
spit into a sample pot. Saliva samples will be aliquoted and stored at -80oC prior to 
being transferred on dry ice to Imperial College London for analysis.  
 
Bacterial RNA will be extracted from saliva samples and analyzed by 16S rRNA gene 
amplicon sequencing to identify the species composition. Bacterial community types 
derived from tissues biopsies will be identified by cluster analysis of compositional 
data from 16S rRNA sequencing. Bacterial interaction networks will be constructed to 
identify any keystone species.  
 
Patient demographics and metadata: essential accompanying patient demographics 
and metadata will include: age; sex; comorbidities; height; weight; tumor histology 
and stage; details of (neo)adjuvant therapy; details of surgery; duration of pre-test 
fasting, and; radiological parameters (tumor dimensions, nodal burden and PET-
standardized uptake value). Where possible, data will be obtained via the IRB 
approved institutional database (IRB18-076).  
 
Statistical analysis: Both univariate and multivariate data analysis techniques will be 
applied to identify breath and urinary VOCs with the best discriminating ability and to 
develop a multivariate discriminant analysis model. Appropriate univariate statistical 
techniques will be used to compare intra- and inter- individual variability in exhaled 
VOCs in response to therapeutic intervention. Associations between predominant 
bacterial species and VOCs with be determined through a univariate and multivariate 
data analysis techniques. 
 
Using the data collected, several machine learning algorithms will be adapted, trained 
and evaluated for the purpose of diagnostic inference based on the breath sample 
chemical analysis. We will start with an exploratory analysis of the VOC concentration 
data, including Principal Component Analysis  (PCA) and Multi-dimensional Scaling 
(MDS), to reduce the feature space and develop intuitions about the data. Next, 
Machine Learning Algorithms (MLA) such as Support Vector Machines  (SVM) and k-
Nearest Neighbors (kNN) will be trained and validated to reliably separate cancer from 
non-cancer cases. An Information Theory based MLA will also be developed. Cross-
validation algorithms such as k -fold training will be used to rigorously assess the 
reliability of the learning algorithms. Models will then be compared through suitable 
accuracy scores and ROC curves will be produced to aid the selection of appropriate 
clinical diagnostic measure, which balances specificity against sensitivity. 
 
 
Sample storage 

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Breath, urine and saliva samples will be stored in a -80oC freezer within the Benaroya 
Research Institute. Samples will be deidentified and only labeled using a unique study 
identifier composed of the patient’s study ID and the date the sample was collected.  
 
 
Data storage 
Virginia Mason Medical Center subject data will be stored in a pseudonymized form 
within Microsoft Excel. Data will be stored on a password protected institutional 
(Virginia Mason Medical) computer. Any paper records, including patient consent 
forms, will be stored in locked filing cabinet within the department of Thoracic Surgery 
Virginia Mason Medical Center as well as in the Clinical Research Program offices at 
Benaroya Research Institute. Access to the data will be limited to the principal 
investigator, nominated members of the research team and any regulatory or 
institutional compliance team member in case of review or audit only. Any data from 
samples shared with Imperial College London (UK) will be in a linked anonymized form. 
 
Subject engagement: Subjects who are recruited to this study will be asked to 
complete a short survey, ideally at the first study time point (but acceptable to be 
collected at any study time point), designed to assess their understanding and 
opinions regarding breath testing.  
 
 

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PROTECTION OF HUMAN SUBJECTS 
 
Human Subject involvement and characteristics 
Human subjects will be enrolled into this study. The purpose of recruiting human 
subjects is to determine the influence of EC and neoadjuvant therapy on VOC 
biomarkers and the upper gastrointestinal microbiome. Recruitment of human 
subjects is critical to achieving the intended outcomes of this study. 
 
Patients with newly diagnosed esophageal and/or gastroesophageal junctional 
adenocarcinoma will be recruited from Virginia Mason Medical Center (n=50). 
Patients will be asked to donate an exhaled breath, urine and saliva sample at four 
study time points. With the exception of a subject engagement questionnaire 
(completed at the time of the first study visit) all other data that are utilized as part of 
this study originates from patients’ routine clinical care and their electronic medical 
record.  
 
Patients who appear to meet study eligibility may sign consent for this study during 
the diagnosis and staging phase of esophageal and/or gastroesophageal junctional 
adenocarcinoma. Initial samples will be collected for these subjects after consent is 
obtained. Upon completion of diagnosis and staging, it is possible that the subject may 
not be recommended for curative tri-modality treatment (surgical resection and 
chemoradiation therapy). Although these subjects are not the focal population for this 
study, their samples at the applicable study time points are valuable for comparison 
purposes. As such, these subjects will continue to have samples captured at the 
protocol defined study time points whenever possible, but any samples not collected 
(generally due to the subject not returning to VM within the appropriate timeframe) 
will not be considered a protocol deviation. These subjects will not count toward the 
goal of 50 enrolled curative tri-modality subjects. Their data will be comparative only. 
 
We do not plan to enroll vulnerable subject groups (children, persons with mental 
disability, prisoners) where ability to give voluntary informed consent may be 
questioned or compromised. 
 
 
Collaborating sites 
This work will be conducted as part of a collaboration between Virginia Mason Medical 
Center (Seattle, USA) and Imperial College London (London, UK).  
 
 
Sources of Materials 
 
Patients will be required to provide written informed consent prior to enrollment in 
this study. Participants will be provided with an approved informed consent form and 
will receive careful explanation by the investigator or approved delegate of the study 
team consisting of what is involved by taking part in the study, including potential risks 
and benefits.  

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i. 
Patient demographics and meta-data will be recorded for all patients (please see 
data collection section above). There is very little risk of breach of confidentiality. 
ii. 
Breath samples: breath sampling is entirely non-invasive and poses negligible risk 
of harm to patients. Patients will be asked to provide a breath sample by blowing 
directly into a disposable (single use) sample bag. The sample is then transferred 
and stored within a Thermal Desorption (TD) tube. Samples will be analyzed at 
Imperial College London. Breath samples are destroyed during the analysis 
process. 
iii. Urine samples: urine sampling is entirely non-invasive and poses negligible risk of 
harm to patients. Patients will be asked to provide a urine sample (10ml) by 
urinating into a sample pot that they will be provided with. The sample will be 
analyzed at Imperial College London. Urine samples will be destroyed following 
the analysis process. 
iv. Saliva samples: saliva sampling is entirely non-invasive and poses negligible risk 
of harm to patients. Patients will be asked to provide a saliva sample (1-3ml) by 
expectorating into a sample pot that they will be provided with. The sample will 
be analyzed at Imperial College London. Saliva samples will be destroyed 
following the analysis process. 
 
 
 

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Potential Risks 
 
Risks and benefits 
The principal risks of this study relate to the preservation of patient confidentiality in 
a multicenter study. 
 
All study related documents and patient information for subjects enrolled in this study 
will be stored securely within a password protected networked computer at Virginia 
Mason. Hard copies of consent forms and paper questionnaires will be stored in a 
locked filing cabinet within the Benaroya Research Institute at Virginia Mason. The 
same standards regarding data and sample storage are practiced at Imperial College 
London.   
 
Patients recruited to this study are being considered for curative treatment of 
esophageal cancer involving chemoradiotherapy and surgery. During the course of 
their treatment patients will be regularly required to fast and undergo invasive 
investigations and treatments. The risk of additional non-invasive interventions 
including breath, urine and saliva sampling are therefore considered to be negligible. 
Breath, urine and saliva sampling will be performed at the time of a routine clinical 
visit to avoid patients having to make additional visits to the hospital.  
 
It is intended that the current study should have minimal impact on patient’s routine 
clinical care. No extra financial costs will be passed to patients or their insurance 
provider.  
 
Whilst no direct benefit is intended for individuals who agree to take part in this study, 
some patients may receive altruistic reward through participation in medical research 
that seeks to help other patients in the future. 
 
Benefit is intended for future patients by establishing a greater understanding of 
methods of assessing and monitoring nutritional status in the setting of EC and 
surgery.  
 
 

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Adequacy of Protection Against Risks 
 
Recruitment and Informed Consent 
 
Informed written consent will be sought from all patients recruited to this study. 
 
Patient identification: patients eligible for inclusion will be identified from the surgical 
practice of the local Principal Investigator (Dr Low, Virginia Mason Medical Center). 
Members of the research team will meet regularly with the multidisciplinary clinical 
team to ensure suitable identification and recruitment of patients.  
 
Patients will be first approached by an appropriately trained member of the research 
team prior to receiving any treatment for EC either at the time of a routine outpatient 
appointment or by telephone. Patients will be provided with details of the study in an 
Informed Consent Form (ICF). When patients are initially approached, the details and 
requirements of the study will be explained to them and all questions answered. They 
will be allowed adequate time to decide whether they wish to take part in this study. 
If initial contact is via telephone, patients will be offered the opportunity to have the 
ICF sent to them either in hard copy or electronic form. Written informed consent will 
be obtained in person prior to the completion of any study specific procedures at 
either a routine outpatient appointment or on the day of sample collection. 
 
In case of non English speaking patients, a certified, impartial translator will be 
included in the informed consent process to ensure the patient has a good 
understanding of the study and his/her requirements and that all questions are 
answered. 
 
Patients will be made aware that any personal and confidential data related to their 
participation in this study will not be shared with anyone outside of their routine 
clinical care team, designated members of the research team or compliance oversight 
entities as required. Similarly it will be explained to patients that data from collected 
questionnaires will be pseudonymized before being analyzed. 
  
It will be explained to the patient that a decision not to take part will have no influence 
on the standard of care they receive. Likewise patients will be informed that their 
consent for participation can be withdrawn at any time without need for explanation 
and without affecting the standard of care they receive. In the event of a patient 
withdrawing from the study, any samples that have been collected will be retained for 
analysis. 
  
 
 

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Protection against Risk 
 
Risk of breach of confidentially 
This study will involve the handling, storage and transportation of sensitive patient 
data including: consent forms; demographic data; meta-data; questionnaire 
responses, and results of physiological and biochemical analysis. 
 
With the exception of consent forms, all data will be stored in a pseudonymized form 
using patients’ unique study ID. Electronic data will be stored within password 
protected institutional computers. Paper records and clinical samples will be stored 
within appropriate institutional facilities protected by mechanical and/or electronic 
security controls. Only the principal investigator will have access to the code linking 
patient identities to pseudonymized data and the link will not be broken. 
 
Patient demographics and meta-data, will be collected either in paper or electronic 
(Microsoft Office Excel) form. Where data are collected in paper form they will be 
subsequently transcribed to the electronic database. 
 
Breath, urine and saliva samples will be labelled using a unique study identifier 
composed of patient’s study ID and date sample was collected. It will not contain PHI. 
 
All samples transferred to Imperial College London for the purpose of analysis will 
done so in a linked anonymized from, such that patients cannot be identified by 
outside parties. The link will never be provided in the future.  
 
Once data analysis is complete, the data obtained in this study will be added to the 
IRB approved institutional database (IRB18-076). All research specific paper records 
associated with this protocol will be securely archived and stored for a minimum of 10 
years after the study closes. The data will be stored long-term under IRB18-076 and 
any archival will follow as indicated in that protocol. 
 
Risk of physical harm 
It is considered that the risk of physical harm associated with this study is negligible. 
All interventions are non-invasive. 

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Identifying and reporting adverse events 
 
Reporting Procedures 
All adverse events (serious or non-serious) that are directly related to study activities 
will be reported. Adverse events (serious or non-serious) that occur as a result of 
patient’s disease process or routine care, and any AEs that are not directly related to 
study procedures, will not be reported. 
 
Depending on the nature of the event the reporting procedures below should be 
followed.  Any questions concerning adverse event reporting should be directed to 
the local Principal Investigator.   
 
Non serious adverse events 
All unexpected non-serious adverse events which are related to study procedures 
should be recorded.   
 
Serious adverse events 
All serious adverse events related to a study procedure will be considered unexpected 
and thus will be reported. A report including all pertinent details related to a serious 
adverse event (including but not limited to: start date, stop date, thorough description 
of the event, treatment, and outcome) should be reported to the local Principal 
Investigator (Virginia Mason Medical Center, Dr Low) within 24 hours. Additionally, all 
such serious adverse events will be reported to the IRB.  
 
 
Contact details for reporting SAEs 
USA 
Dr Donald Low MD 
Donald.Low@virginiamason.org 
Tel: (206) 223-6164 
 
 
Data Monitoring 
 
The Clinical Research Program will assess the risk of this trial and will devise and 
implement an internal monitoring and/or auditing plan for this trial. This plan will be 
revised as necessary during the life of the trial based upon a variety of factors, 
including but not limited to: protocol amendments, staff turnover, enrolment metrics, 
and compliance issues noted.  
 
 

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Potential Benefits Of The Proposed Research To The Subjects and Others. 
 
There are no immediate benefits for the research participants recruited in to this 
study. Benefit is intended for future patients through aiding in surgical research. 
Specifically this will be through an improved understanding of the assessment of 
nutritional status in patients with EC. Patients may nevertheless gain altruistic reward 
through participation.  
 
It is considered that the proposed study does not pose unreasonable risk to patients. 
Participation in this study is intended to have no adverse impact of patient’s routine 
standard of care and quality of life. Enrollment to the study will be conducted in 
accordance with Good Clinical Practice and patients will be requested to provide 
informed written consent.   
 
 

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Potential impact: This research is intended to verify VOC biomarkers of EC cancer and 
to establish new understanding regarding response to therapeutic intervention. It is 
intended that this work will be conducted in parallel with other planned studies that 
are designed to establish the both the underlying mechanism for deregulate VOC 
production in EC cancer and methods for optimization diagnostic performance. The 
collective knowledge drawn from these studies will build a platform for future 
research by establishing: (i) proof of principal; (ii) mechanism; (iii) methodology for 
use in clinical practice, and; (iii) acceptability and feasibility. Using this evidence we 
will seek further funding to conduct a phase 4 biomarker development study (17). If 
we are successful in achieving these outcomes, we will have a clear line-of-sight to the 
introduction of our innovation into clinical practice. This work will benefit from our 
existing links to industry and the NIHR London in vitro diagnostic co-operative. 
 
Research team: 
Dr Donald Low is an expert EC cancer surgeon practicing at Virginia Mason Medical 
Center, a high volume center for the management of this disease. Virginia Mason has 
a long history of collaborative and innovative translational research designed to 
enhance patient experience. Existing infrastructure within Virginia Mason will support 
the conduct of this study through a dedicated team of research coordinators.  
 
Professor George Hanna is the head of the VOC research laboratory based at St Mary’s 
Hospital (Imperial College London). The group has over a decade of experience in the 
field of clinical breath testing and VOC research that is evidenced by their high impact 
publications within the field.  
 
Dr Piers Boshier, a visiting scholar at Virginia Mason and NIHR clinical lecturer at 
Imperial College London, has extensive experience in clinical breath analysis. He is 
ideally placed to coordinate activities between US and UK centers. 
 
Existing funds 
This study is supported by funds from the Salgi Foundation. 

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Gantt chart  
 
Year 
2018 
2019 
2020 
Months 
Sep 
Oct 
Nov 
Dec 
Jan 
Feb 
Mar 
Apr 
May 
Jun 
Jul 
Aug 
Sep 
Oct 
Nov 
Dec 
Jan 
Feb 
Mar 
Apr 
May 
Jun 
Jul 
Aug 
Sep 
Oct 
Nov 
Dec 
Application for funding 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IRB application 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study recruitment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drafting manuscript 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Application for further funding 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
    

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