Influence of Nutrition and SarcoPenia on Esophageal Cancer ouTcomes (INSPECT Study)
Conditions: Esophageal Cancer, Sarcopenia
Sponsor: Benaroya Research Institute

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Principal Investigator 
Dr Donald E Low MD 
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 98101 
Tel: 2062236164 
 
 
Study Title: 
Influence of Nutrition and SarcoPenia on Esophageal 
Cancer ouTcomes (INSPECT study) 
 
 

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Table of Contents 
 
A. SPECIFIC AIMS .............................................................................................................................3 
B. BACKGROUND AND SIGNIFICANCE ......................................................................................5 
C. PRELIMINARY STUDIES ...........................................................................................................7 
D. RESEARCH DESIGN AND METHODS .....................................................................................9 
E. PROTECTION OF HUMAN SUBJECTS ................................................................................. 13 
F. REFERENCES ............................................................................................................................. 22 
G. CONSULTANTS ......................................................................................................................... 24 
H. APPENDIX.................................................................................................................................. 25 
 
 
 

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A. SPECIFIC AIMS 
 
Hypothesis: Esophageal cancer is associated with changes in patient’s body 
composition that are correlated with biochemical markers of cell metabolism (acetone) 
and systemic inflammation. Knowledge of these changes can influence patients’ care 
through modification of treatments and outcome prediction. 
 
Purpose: An observational, pilot study to assess longitudinal variation in exhaled 
acetone, body composition and other markers of nutritional status and inflammation in 
patients undergoing multimodal therapy for esophageal cancer. Where possible 
variations in these markers will be correlated with changes to early and late treatment 
outcomes as well as nutritional interventions that form part of patients routine care. 
 
 

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B. STUDY OUTCOMES 
 
Primary outcome 
i. Longitudinal assessment of breath acetone in patients undergoing multimodal 
therapy for esophageal cancer 
 
Secondary outcomes 
i. Correlation of breath acetone with alternate measures of nutritional status including 
anthropometric measurements, body composition, resting energy expenditure and 
blood parameters 
ii. Correlation of breath acetone and alternate measures of nutritional status with 
patient reported nutritional intake and quality of life  
iii. Assessment of the repeatability and accuracy of point of care testing of breath 
acetone using the Ketonix device 
 
 
 

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C. BACKGROUND AND SIGNIFICANCE 
 
Rates of cachexia and malnutrition in patients with esophageal cancer are amongst the 
highest of all cancers (1-3) and are a negative prognostic indicator. Explanation for this 
is multifactorial but principally reflects changes in energy metabolism, tumor burden 
and the effects of aggressive multimodal therapy. Current methods of assessing 
cachexia and nutritional status however rely on measures that are often subjective and 
dependent on effective patient recall and co-operation. Assessment of body 
composition (skeletal muscle and adiposity), has found success in a number of other 
areas of medicine and oncology (4-7), supporting the argument that such measures 
should become part of routine clinical practice. The association between body 
composition and biochemical markers of nutritional status, including acetone, has yet 
to be studied in patients with esophageal cancer. 
 
Critical knowledge gap 1: Despite its clinical promise, there remains inconsistency 
and a lack of consensus in regard to the adoption of methods of body composition 
assessment in patients with esophageal cancer. Following a systematic review of the 
current literature, we identified 29 studies that reported methods of body composition 
assessment in esophageal cancer patients. Meta-analysis of outcomes presented within 
these studies indicated that sarcopenia was associated with significantly lower overall 
survival (HR 1.70, 95% CI 1.33 to 2.17, P<0.0001) and higher rates of postoperative 
pneumonia (OR 2.03, 95% CI 1.32 to 3.11, P=0.001) (APPENDIX I). It was also 
noted that the reported incidence of sarcopenia in preoperative esophageal cancer 
patients varied between 16% and 75% and was not associated with global region. The 
strength of conclusions drawn from this review were however tempered by a lack of 
clear guidance regarding optimal methodology and reporting standards.  
 
Critical knowledge gap 2: the association between cachexia and biochemical 
markers of nutritional status and inflammation in patients with esophageal cancer 
remains incompletely understood. Characterization of the relationship between 
changes in body composition and concurrently measured biochemical markers may 
contribute to our understanding and support the development of future assessment 
tools. 
 
The measurement of acetone (a ketone body) within exhaled breath may offer a novel 
approach to the assessment of cancer cachexia.  Ketosis occurs in all humans and is a 
critical part of energy metabolism, influenced by dietary intake, body composition and 
the presence of disease states including cancer. Ketosis relies on the breakdown of 
adipose tissue to produce free fatty acids that are metabolized by the liver to acetyl-
CoA and subsequently to ketone bodies (APPENDIX II). Due to its specific chemical 
properties, acetone is capable of diffusing into alveolar airspaces permitting detection 
within exhaled breath. Several authors have demonstrated exhaled acetones potential 

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as a marker of fat loss (8). Our own recent studies, in patients undergoing treatment 
for obesity, identified that exhaled acetone increased significantly in patients following 
both a low carbohydrate diet and bariatric surgery compared to patients prior to these 
interventions (APPENDIX III). Exhaled acetone levels were also found to be inversely 
proportional to BMI and excess body weight. 
 
C-reactive protein (CRP) is the most widely accepted index of systemic inflammation 
and is recognized as a robust biochemical marker for inflammation in consensus 
statements for malnutrition (9, 10). Accordingly CRP has been incorporated in to 
several clinical indices, including the Glasgow Prognostic Score (GPS), owning to its 
powerful association with mortality (11). The GPS has been validated as a prognostic 
tool in multiple cancers, predicting tumor progression, survival and symptom burden 
(11, 12). Other inflammatory mediators have also been linked to the development of 
cancer cachexia and associated poor outcomes. Macrophage inhibitory cytokine 1 (MIC-
1) is elevated in cachectic patients with levels correlating to weight loss (13). Cytokines 
including IL-1, IL-6, IL-10 and TNFα are also believed to increase catabolism in cancer 
patients (14). 
 
Significance: The accurate assessment and prevention of cachexia and malnutrition 
in patients with esophageal cancer continues to present a major unmet clinical need.  
 
It is intended that this study should provide pilot data showing how variation in breath 
acetone is correlated to changes in body composition (particularly adipose tissue) and 
other markers of nutritional status and inflammation. It is hoped that based on the 
findings of this study we will be well placed to apply for future funding for a prospective 
interventional trial to assess the influence of supplementary nutrition in patients with 
esophageal cancer. Ultimately we hope to change clinical practice within the field 
through validation of the routine assessment of body composition and point of care 
testing of breath acetone. 
 
 
 

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D. PRELIMINARY STUDIES 
 
Expertise, Experience and Resources: Based on his leadership and established 
collaborations Dr Low is internationally recognized for his ongoing contribution to the 
field of esophageal surgery. He has established the international Esophagectomy 
Complications Consensus Group (ECCG) that serves as a foundation for ongoing 
research within the field. Professor Baracos is a world leader in the field of cancer 
cachexia and is recognized for her role in developing CT methods of body composition 
assessment and in establishing consensus definitions for cancer-associated 
malnutrition. Professor Hanna is an esophageal surgeon with expertise in translational 
research including exhaled breath analysis. Mr Boshier is a surgical resident and 2017 
Ryan Hill Research Fellow with experience in translational and outcomes research. The 
applicant and co-applicants have extensive experience in clinical research that is 
evidenced by their publications and ability to secure competitive funding. The University 
of Alberta and Imperial College London are world renowned academic institutions with 
the capacity to support the proposed research including an established methodology 
for body composition assessment and one of the world’s largest research laboratory 
for clinical breath research. 
 
Patient recruitment: Virginia Mason Medical Center and St Mary’s Hospital are both 
high volume expert centers for the surgical treatment of esophageal cancer. Both 
centers have the required patient numbers and infrastructure to support prospective 
patient accrual. 
 
CT body composition (University of Alberta): a leading research group in the field 
of cancer cachexia and malnutrition led by one of the sub-investigators (VB) will 
support prospective CT assessment of body composition in a consulting capacity. The 
group have published extensively on standardized methods of body composition 
assessment. The group has a proven track record in this field as evidenced by peer 
reviewed publications and successful funding applications. Actual CT analysis will be 
performed on site at Virginia Mason Medical Center, with instruction and guidance 
provided by University of Alberta’s sub-investigator (VB). No images or subject data 
will be sent to University of Alberta. 
 
Volatile organic compound (VOC) lab (Imperial College London): an 
established laboratory led by one of the sub-investigators (GH) will support 
investigations in to the role of acetone as potentially non-invasive markers of nutritional 
status. Over the last 10 years the group has developed reliable methods for the high 
throughput analysis of VOCs utilizing a combination of complementary mass-
spectrometry based platforms for gas phase VOC analysis. The group has a proven 
track record in this field as evidence by peer reviewed publications and successful 
funding applications. 

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Immunology lab (Benaroya Research Institute at Virginia Mason): an 
established laboratory led by Dr Alice Long PhD will support the analysis of 
inflammatory markers in collected blood samples using enzyme-linked immunosorbent 
assays (ELISA). This group has extensive experience in performing and interpreting the 
results of this analysis. 
 
 

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E. RESEARCH DESIGN AND METHODS 
 
Design: prospective multicenter longitudinal (observational) study recruiting from 
tertiary centers for the surgical management of esophageal cancer; Virginia Mason 
Medical Center (Seattle, USA) and St Mary’s Hospital (Imperial College Healthcare NHS 
Trust, London, UK). This is intended to be a pilot study.  
 
Sample size: 35 patients (15 from St Mary’s Hospital Imperial College Healthcare NHS 
Trust, London, UK recruitment center and 20 from Virginia Mason Medical center, 
Seattle, USA). Sample size has been determined pragmatically based on the predicted 
number of patients undergoing treatment for esophageal cancer in recruitment centers 
during the study period. It is noted that even in high volume centers such as Virginia 
Mason Medical Center and St Mary’s Hospital the longitudinal accrual of patients at the 
time of diagnosis and definitive surgery seldom exceeds 20-30 patients per year. 
Accordingly number of patients decided from each site during the intended 8 month 
study period is a conservative estimate of study recruitment. It is intended that data 
obtained from this pilot study will support formal power calculations for future studies.   
 
Aspects of recruitment, consenting, data collection, and data storage will be the 
responsibility of each of the recruiting center. Separate ethical approval for this study 
will be sought at both Virginia Mason Medical Center and St Mary’s Hospital where the 
conduct of the research will be required to meet local standards. Responsibility for the 
conduct of research at each site will be assigned to the local lead (Dr Low at Virginia 
Mason Medical Center and Professor G Hanna at St Mary’s Hospital). 
 
 
Inclusion criteria: 
Patients with the following characteristics will be eligible for inclusion in this study: 
i. aged 18-90 years 
ii. newly diagnosed (prior to treatment) with esophageal and/or gastroesophageal 
junctional cancer (adeno- or squamous cell carcinoma) 
iii. planning to undergo curative treatment, including surgical resection with 
neoadjuvant therapy 
 
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. 
inability or unwillingness to provided informed written consent 
 
Methodology: patients undergoing curative treatment for esophageal cancer will be 
recruited at the time of routine clinical assessment shortly following initial diagnosis 

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and will undergo clinical evaluation at two study time points: (1) staging investigation 
(e.g. laparoscopy or endoscopy) and (2) definitive surgical resection. Study time points 
have been judiciously chosen to coincide with interventions that form part of patient’s 
routine clinical care. It is intended that sampling will occur on the day of each 
intervention/surgery following a routine period of fasting.  
 
Breath sample collection: 
Analysis of breath acetone from samples collected from Virginia Mason Medical Center 
and St Mary’s Hospital will be performed using a KETONIX® point of care device. 
Patients will be asked to exhale directly into this handheld device, which provides an 
on-line and real-time measurement of breath acetone concentration in parts per million. 
  
At St Mary’s Hospital breath acetone analysis will also be performed using the Proton 
Transfer Reaction Mass Spectrometry (PTR-MS) in order to provide cross platform 
validation of results.  
 
Blood sampling: 
At each study time point a single sample of venous blood (15ml) will be collected for 
the purpose of determining serum concentrations of a number of metabolic and 
inflammatory markers, e.g. C-reactive protein (CRP), albumin, lipid profile, glucose, 
insulin, acetone, insulin-like growth factor (IGF), macrophage inhibitory cytokine-1 
(MIC-1), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), and; tumor 
necrosis factor alpha (TNFα). Once collected, samples will be centrifuged and the 
supernatant (serum) will be aliquoted for storage at -80oC. 
 
Analysis of serum CRP, albumin, lipid profile, glucose and insulin will be performed 
within the biochemical pathology laboratories of the individual recruitment centers. The 
remaining biochemical parameters will be analyzed using commercial available enzyme-
linked immunosorbent assays (ELISA) kits. An agreement has been reached with the 
Benaroya Research Institute at Virginia Mason to conduct the ELISA analysis for all 
samples. 
 
Indirect calorimetry: 
At the time of sample collection, patients will also undergo assessment of resting 
energy expenditure by indirect calorimetry (Fitmate, COSMED Inc, Chicago, USA). This 
non-invasive measurement is made by asking patients to exhale at rest into a sterile 
facemask that is placed over their mouth and nose. Subjects will be asked to exhale 
into this facemask at a normal rate and tidal volume for approximately 5 minutes while 
measurement of their resting energy expenditure is determined. Prior to taking 
measurements patients will be required to remain seated at rest for 20 minutes (in the 
majority of cases patients will have already been waiting for longer than this duration 
in department wherein they are undergoing either their surgery or other intervention). 
Patient will be assessed on the day of surgery or other intervention and hence will be 

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fasting as part of their routine care. Patients will be requested to abstain from smoking 
and exercise for four hours prior to testing. We will also record their food consumption 
in the preceding 3 days. 
 
Assessment of body composition: 
CT images that are routinely obtained at the time of diagnosis and following 
neoadjuvant therapy will be used to determine body composition parameters. 
Pseudonymized images at the anatomical level of the midpoint of the body of the third 
lumbar vertebra will be obtained and exported to a Digital Imaging and 
Communications in Medicine (DICOM) format. Images are pseudonymized at the time 
of retrieval from the hospital picture archiving and communication system (PACS, 
which removes all patient related information from the saved DICOM image. Images 
will be subject to segmentation to determine cross sectional muscle and adipose area 
using commercially available software (SlicOmatic Ver 5.0, Tomovision, Magog, 
Canada). Analysis of CT images will be performed at each recruiting institution. 
Definitions of skeletal muscle index, sarcopenia and visceral obesity have been 
previously (APPENDIX IV)(15, 16). 
 
Data collection  
The following data will be retrieved from the patient’s electronic medical record (EMR) 
for inclusion into the database: medical record number; date of birth; sex; ethnic origin; 
comorbidities; details of neoadjuvant and adjuvant therapy; details of pre-operative 
nutritional supplementation; pathological stage and tumor characteristics. Additional 
data capture at the time of sampling will include: height; weight; any recent antibiotics 
(≤4 weeks); time of last oral intake; three day dietary report (to be completed 
retrospectively on the day of sampling); health-related quality of life questionnaires 
(QLQ-OES18, QLQ-C30, QLQ-CAX24); Patient generated subjective global assessment 
(PG-SGA) food intake category; performance status (ECOG, determined by either Dr 
Low or Professor Hanna); GPS, and; patient reported weight loss in last 6 months at 
study time point 1. The CTCAE (17) and ECCG (18) definitions will be used when 
reporting outcomes of chemotherapy and surgery respectively.  
 
Sample storage 
Blood samples will be stored in a -80oC freezer within the Benaroya Research Institute. 
Samples will be 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 a suitable platform (e.g Microsoft Office Excel or REDCap). Data will be stored 
on a password protected institutional (Virginia Mason Medical) computer. Paper records 
(including completed questionnaires) will be stored in locked filing cabinet with the 
department of Thoracic Surgery Virginia Mason Medical Center. Access to the data will 

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be limited to the principal investigator and nominated members of the research team 
only. Any data from samples shared with external collaborators in the United Kingdom 
or Canada will be in a linked anonymized form. 
 
Data collected at St Mary’s Hospital will be stored and protected as per local IRB 
requirements and sent to Virginia Mason Medical for inclusion in the data analysis via 
a secure system (e.g., Box). 
 
Data analysis 
Univariable and linear regression analysis will be used to compare exhaled acetone 
concentrations with body composition parameters. The coefficient of variation will be 
determined for repeated measurements recorded using the Ketonix device. Acetone 
concentrations measures using the Ketonix device and PTR-MS (St Mary’s Hospital 
only) will be compared using Bland Altman plots. Comparisons between continuous 
data will be made using either the Mann-Whitney U test or Student t test, whilst 
categorical variables will be compared by 2 or Fischer exact test. Statistical significance 
will be assigned to 2-sided P values <0.05. 

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F. PROTECTION OF HUMAN SUBJECTS 
 
Risks to 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 esophageal cancer and neoadjuvant therapy 
on nutritional status and inflammatory markers. Recruitment of human subjects is 
critical to achieving the intended outcomes of this study. 
 
Patients with newly diagnosed esophageal and/or gastroesophageal junctional cancer 
(adeno- or squamous cell carcinoma will be recruited from Virginia Mason Medical 
Center (n=20) and St Mary’s Hospital (n=15). Patients will be asked to donate an 
exhaled breath sample and blood sample (15ml) at two study time points. Patients’ 
resting energy expenditure will also be determined and patients will be asked to 
complete a three day dietary diary, PG-SGA food intake and quality of life 
questionnaires at each study time point. Any other data that are utilised as part of this 
study originates from patients’ routine clinical care and EMR.  
 
We do not plan to enroll vulnerable subject groups (children, persons with mental 
disability, prisoners) where ability to give voluntary informed consent maybe 
questioned or compromised. 

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Enrollment: 
 
Collaborating sites: 
This work will be conducted as part of a collaboration between Virginia Mason Medical 
Centre (Seattle, USA) and St Mary’s Hospital (Imperial College Healthcare NHS Trust, 
London, UK).  
 
Patient recruitment has been divided equally between the two sites and each site will 
be responsible for local recruitment, consenting, and data collection. All biological 
samples will be analyzed at the local recruiting hospital, with the exception of the 
analysis of specific mediators (i.e. acetone, IL-1, IL-6, IL-10, TNFα, MIC-1, IGF) which 
will be conducted within the Benaroya Research Institute for samples collected from 
both study sites. 

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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 of what is involved by taking part in the 
study, including potential risks and benefits.  
 
i. 
Patient demographics and meta-data will be recorded for all patients (please see 
data collection section above). There is a small 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) mouthpiece attached to a hand-held breath 
acetone analyzer. This test is entirely non-invasive and no sample is retained. At 
St Mary’s Hospital, an additional breath sample will be collected for analysis by 
PTR-MS. This sample is stored temporarily (typically <24hrs) on a Thermal 
Desorption (TD) tube and destroyed during the analysis process. 
iii. Blood samples: blood samples (15ml) will be collected at each study time point. 
Where possible blood sampling will be combined with routine blood tests in order 
to avoid the requirement for repeated needle sticks. It is recognized that blood 
sampling can lead to temporary discomfort and a bruise where the needle enters 
the vein. Patients will be counselled about these risks as part of the process of 
giving informed consent. 
iv. Resting energy expenditure measurement by indirect calorimetry: this is an entirely 
non-invasive technique, however patients will be counselled regarding the 
possibility of feeling discomfort when the sample mask is applied over their mouth 
and nose. 
v. 
Quality of life questionnaires, PG-SGA food intake and three day food diary: 
patients will complete these at each study time point. There is a small risk of 
patients feeling uncomfortable with some of the questions; they will be counselled 
they may skip any questions they do not wish to answer. 
 
 
 
 

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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 and the requirement for minimally invasive blood sampling. 
 
Whilst the collection of patient data at collaborative sites will be governed by local 
policies, St Mary’s Hospital’s sharing of linked anonymized date with Virginia Mason will 
be subject to prior regulatory approval in the UK. 
 
All study related documents and patient information for subjects enrolled at Virginia 
Mason will be stored securely within a password protected networked computer within 
the department of Thoracic Surgery at Virginia Mason. Hard copies of consent forms 
and paper questionnaires will be stored in a locked filing cabinet within a locked office 
in the department of Thoracic Surgery. The same standards regarding data storage are 
practiced at St Mary’s Hospital.   
 
Where possible blood sampling will be performed at the time of a routine blood draw 
so as to prevent patients having to undergo a further needle stick. Blood sampling will 
be performed by an appropriately trained individual. Blood sampling has the potential 
to cause temporary discomfort and bruising at the site of needle entry. Risk of serious 
harm from this intervention is considered to be low.  
 
The risk of additional non-invasive interventions including breath sampling and 
measurement of resting energy expenditure, quality of life questionnaires and food 
diaries are considered to be negligible. 
 
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 esophageal 
cancer 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, Professor Hanna St Mary’s Hospital). Members of the research team will meet 
regularly with the multidisciplinary clinical team to ensure suitable identification and 
recruitment of patients.  
 
Patients will be provided with details of the study in an Informed Consent Form. They 
will be allowed adequate time to decide whether they wish to take part in this study. 
Patients will be approached either at the time of a routine outpatient appointment or 
by telephone prior to surgery. At the time when patients are initially approached the 
details and requirement of the study will be explained to them. If initial contact is in-
person patients will be provided with relevant study documents including the consent 
form. If initial contact is via telephone patients will be offered the opportunity to have 
these study documents sent to them either in hard copy or electronically. Written 
consent will be obtained in person prior to enrollment at the time of either routine 
outpatient appointment or on the day of sample collection. 
 
When informed written consent is being sought within the hospital setting it will be 
obtained in a place of privacy by a trained member of the research team. In all cases 
the patient will be helped to understand the purpose of the intended research project 
as well as the potential benefits and risks of involvement in the study.  
 
Where a patient does not adequately understand verbal explanations or written 
information in English then interpreters will be used to translate this information, in 
line with local codes of practice/regulations. 
 
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 or designated members of the research team. 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. 

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All of this information shall be included in the Informed Consent Form. We will not seek 
informed consent from vulnerable groups including: children, prisoners or patients 
lacking mental capacity to provide informed consent for this study.  
 

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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. The principal investigator at each site will have access to the code 
linking patient identities to pseudonymized data. 
 
Patient demographics, meta-data, food diary and questionnaire responses will be 
collected either in paper or electronic (e.g. Microsoft Office Excel or REDCap platform) 
form. Where data are collected in paper form they will be subsequently transcribed to 
electronic form. 
 
Blood samples will be labelled using a unique study identifier composed of patient’s 
study ID and date sample was collected. 
 
Any data transferred from St Mary’s Hospital to Virginia Mason Medical Center will done 
so in a linked anonymized from, such that patients cannot be identified by outside 
parties. No data from Virginia Mason Medical Center will be passed to St Mary’s 
Hospital. 
 
Study data will be securely stored for a period of 10 years following study conclusion. 
 
Risk of physical harm: 
It is considered that the risk of physical harm associated with this study is low. With 
the exception of blood testing, all other interventions are non-invasive. To minimize 
the risks of blood sampling, which include discomfort and bruising, where possible 
blood samples will be collected at the time of routine venipuncture.  

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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 emailed to the local Principal 
Investigator (Virginia Mason Medical Center, Dr Low; St Mary’s Hospital, Professor 
Hanna) 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 (Mon to Fri 09.00 – 17.00) 
 
UK 
Professor George Hanna 
g.hanna@imperial.ac.uk 
Tel: 07980323539 (Mon to Fri 09.00 – 17.00) 
 
 

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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 esophageal cancer. 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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G.  REFERENCES 
 
1. Bozzetti F, Group SW. Screening the nutritional status in oncology: a preliminary 
report on 1,000 outpatients. Support Care Cancer. 2009;17(3):279-84. 
2. Daly JM, Fry WA, Little AG, Winchester DP, McKee RF, Stewart AK, et al. Esophageal 
cancer: results of an American College of Surgeons Patient Care Evaluation Study. 
J Am Coll Surg. 2000;190(5):562-72; discussion 72-3. 
3. Laviano A, Meguid MM, Inui A, Muscaritoli M, Rossi-Fanelli F. Therapy insight: 
Cancer anorexia-cachexia syndrome--when all you can eat is yourself. Nat Clin 
Pract Oncol. 2005;2(3):158-65. 
4. Cespedes Feliciano EM, Kroenke CH, Meyerhardt JA, Prado CM, Bradshaw PT, Kwan 
ML, et al. Association of Systemic Inflammation and Sarcopenia With Survival in 
Nonmetastatic Colorectal Cancer: Results From the C SCANS Study. JAMA Oncol. 
2017:e172319. 
5. Ebadi M, Martin L, Ghosh S, Field CJ, Lehner R, Baracos VE, et al. Subcutaneous 
adiposity is an independent predictor of mortality in cancer patients. Br J Cancer. 
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6. Palmela C, Velho S, Agostinho L, Branco F, Santos M, Santos MP, et al. Body 
Composition as a Prognostic Factor of Neoadjuvant Chemotherapy Toxicity and 
Outcome in Patients with Locally Advanced Gastric Cancer. J Gastric Cancer.  
2017;17(1):74-87. 
7. Rangel EL, Rios-Diaz AJ, Uyeda JW, Castillo-Angeles M, Cooper Z, Olufajo OA, et 
al. Sarcopenia Increases Risk of Long-Term Mortality in Elderly Patients Undergoing 
Emergency Abdominal Surgery. J Trauma Acute Care Surg. 2017. 
8. Anderson JC. Measuring breath acetone for monitoring fat loss: Review. Obesity 
(Silver Spring). 2015;23(12):2327-34. 
9. Evans WJ, Morley JE, Argiles J, Bales C, Baracos V, Guttridge D, et al. Cachexia: a 
new definition. Clin Nutr. 2008;27(6):793-9. 
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Academy of Nutrition and Dietetics and American Society for Parenteral and Enteral 
Nutrition: characteristics recommended for the identification and documentation of 
adult malnutrition (undernutrition). JPEN J Parenter Enteral Nutr. 2012;36(3):275-
83. 
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decade of experience in patients with cancer. Cancer TreatRev. 2013;39(5):534-
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12. Simmons CP, Koinis F, Fallon MT, Fearon KC, Bowden J, Solheim TS, et al. 
Prognosis in advanced lung cancer--A prospective study examining key 
clinicopathological factors. Lung Cancer. 2015;88(3):304-9. 
13. Tsai VW, Husaini Y, Manandhar R, Lee-Ng KK, Zhang HP, Harriott K, et al. 
Anorexia/cachexia of chronic diseases: a role for the TGF-beta family cytokine MIC-
1/GDF15. J Cachexia Sarcopenia Muscle. 2012;3(4):239-43. 

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14. Anandavadivelan P, Lagergren P. Cachexia in patients with oesophageal cancer. 
Nat Rev Clin Oncol. 2016;13(3):185-98. 
15. Prado CM, Lieffers JR, McCargar LJ, Reiman T, Sawyer MB, Martin L, et al. 
Prevalence and clinical implications of sarcopenic obesity in patients with solid 
tumours of the respiratory and gastrointestinal tracts: a population-based study. 
Lancet Oncol. 2008;9(7):629-35. 
16. Popuri K, Cobzas D, Esfandiari N, Baracos V, Jagersand M. Body Composition 
Assessment in Axial CT Images Using FEM-Based Automatic Segmentation of 
Skeletal Muscle. IEEE Trans Med Imaging. 2016;35(2):512-20. 
17. U.S.Department Of H, Human S, National Institutes of H, National Cancer I. 
Common Terminology Criteria for Adverse Events (CTCAE) v.4.03. 2010. 
18. Low DE, Alderson D, Cecconello I, Chang AC, Darling GE, D'Journo XB, et al. 
International Consensus on Standardization of Data Collection for Complications 
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(ECCG). Ann Surg. 2015;262(2):286-94. 
 
 
 

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H. CONSULTANTS 
 
Professor G. B. Hanna 
Head of Division of Surgery 
Imperial College London, St Mary’s Hospital Campus, London, UK 
g.hanna@ic.ac.uk 
Area of expertise: surgical management of esophagogastric cancer surgery and 
breath analysis. 
Intended role: local principal investigator St Mary’s Hospital (UK) 
 
Professor V. E. Baracos 
Professor, Department of Oncology 
Division of Palliative Care Medicine 
University of Alberta 
vbaracos@ualberta.ca 
Area of expertise: expert in the field of cancer-associated wasting and assessment 
of body composition 
Intended role: assistance with body composition assessment and interpretation of 
study findings in a consulting and instructional capacity 
 
Mr P. R. Boshier 
Honorary Clinical research Fellow, 
Imperial College London, St Mary’s Hospital, London, UK 
p.boshier@imperial.ac.uk 
Area of expertise: surgical outcomes of esophagogastric surgery 
Intended role: link person between Virginia Mason Medical Center and St Mary’s 
Hospital with responsibilities for daily running of the study 
 
Dr A. Long PhD 
Research Associate Member; Manager, Human Immunophenotyping Core Lab 
Benaroya Research Institute at Virginia Mason, Seattle, USA 
along@benaroyaresearch.org 
Area of expertise: translational immunology 
Intended role: ELISA analysis of selected inflammatory markers in blood samples 
collected as part of this research study 
 
 

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I. APPENDIX 
APPENDIX I 
 
 
Results of meta-analysis: 
 
Figure IA. Summary meta-analysis of studies reporting the effect of sarcopenia on 
the survival of patients undergoing esophagectomy for esophageal cancer (HR 1.70, 
95% CI 1.33 to 2.17, P<0.0001) 
 
 
 
 

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Figure IB. Summary meta-analysis of studies reporting the effect of sarcopenia on 
the occurrence of postoperative respiratory complications in patients undergoing 
esophagectomy for esophageal cancer (OR 2.03, 95% CI 1.32 to 3.11, P=0.001) 
 
 
Figure IC. Reported incidence of sarcopenia in esophageal cancer patients 
determined prior to esophagectomy 
 
A draft of the manuscript of this meta-analysis is available from Dr Donald 
Low upon request 
Donald.Low@viginiamason.org 
 

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APPENDIX II 
 
Synthetic pathway if endogenous acetone synthesis: 
 
 
Figure IIA. Synthetic pathway of endogenous acetone synthesis 
 
 

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APPENDIX III 
 
Influence of pre-operative low carbohydrate diet and bariatric surgery on 
exhaled acetone levels:  
 
Methodology: Patients at different stages of treatment for obesity were recruited to 
this single center cross-sectional study. Sample time points were: (i) at the time of 
initial attendance prior to dietary or surgical interventions; (ii) on the day of surgery 
following low carbohydrate diet, and; (iii) >3 months after either Roux-en Y gastric 
bypass or sleeve gastrectomy. The concentration of acetone within breath samples, 
collected into steel bags, was analyzed by selected ion flow tube mass spectrometry 
(SIFT-MS). Exhaled acetone was correlated with measures of weight loss and 
biochemical markers of nutrition. 
 
Summary of key findings relevant to current application: Exhaled acetone was 
observed to vary significantly in patients receiving treatment for obesity.  Higher levels 
of acetone were observed in patients following pre-operative diet and bariatric surgery: 
pre-diet 410 ppb (IQR 161-796); post-diet 1396 ppb (357-2990), and; postoperative 
1693 ppb (1149-2547)(P < 0.0001)(Figure IIIA). 
 
 
 
Figure IIIA. Variation in exhaled acetone concentration 
(Kruskal Wallis test, P<0.001) 
 
Exhaled acetone levels were inversely correlated to patient BMI ( -0.262, P=0.005), 
excess body weight (EBW,  -0.264, P=0.005) and percentage loss of EBW (for post-
diet and postoperative patients;  -0.375, P<0.0001) determined at the time of 
sampling. Exhaled acetone did not however correlate with rate of weight change after 
either dietary modification or surgical intervention (P>0.05). 
 

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For EBW, the results of multivariate linear regression demonstrated that neutrophil 
count, glucose, acetone, and low-density lipoprotein concentration explained a 
significant amount of variation in patients BMI (F(df 3, 43) = 7.4, P = 0.0001, R2 = 
0.41, R2 adjusted = 0.36). With the exception of glucose (Beta = 0.24 , t(46) = 2.00, 
P = 0.053) each of these variables significantly predicted EBW within the model: 
neutrophil count (Beta = 0.41 , t(46) = 3.39, P = 0.002), acetone (Beta = -0.34 , t(46) 
= -2.78, P = 0.008), and low-density lipoprotein (Beta = -0.30 , t(46) = 2.42, P = 
0.020). 
 

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APPENDIX IV 
 
Methodology for calculating specific parameters of body composition: 
 
CT or positron emission tomography with computed tomography scan acquired as part 
of patient’s routine care will be acquired. Images at the anatomical level of L3 or the 
L3-L4 intervertebral disc space will be obtained and exported to a Digital Imaging and 
Communications in Medicine (DICOM) format. Images will be subject to automatic 
segmentation to determine cross sectional muscle and adipose area. Definitions of 
skeletal muscle index, sarcopenia and visceral obesity are provided below (15, 16). 
 
 
 
 
Figure IVA. Example of automatic skeletal muscle (central panel) and adipose tissue 
(right panel) segmentation 
 
 
 
Skeletal muscle index (SMI) will be calculated as the ratio of lean tissue area to height: 
 
𝑆𝑀𝐼 (𝑐𝑚2/𝑚2) = 𝐿𝑒𝑎𝑛 𝑡𝑖𝑠𝑠𝑢𝑒 𝑎𝑟𝑒𝑎 (𝑐𝑚2)
𝐻𝑒𝑖𝑔ℎ𝑡 (𝑐𝑚2)
 
 
Sarcopenia will initially be defined as SMI <52.4 cm2/m2 for men and <38.5 cm2/m2 for 
women (15). The following formulae will be used to determine lean body mass and fat 
mass: 
 
𝐿𝑒𝑎𝑛 𝑏𝑜𝑑𝑦 𝑚𝑎𝑠𝑠 (𝐾𝑔) = 0.30 × [𝐿𝑒𝑎𝑛 𝑡𝑖𝑠𝑠𝑢𝑒 𝑎𝑟𝑒𝑎 (𝑐𝑚2)] + 6.06 
 
𝐹𝑎𝑡 𝑚𝑎𝑠𝑠 (𝐾𝑔) = 0.042 × [𝑇𝑜𝑡𝑎𝑙 𝑓𝑎𝑡 𝑎𝑟𝑒𝑎 (𝑐𝑚2)] + 11.2 
 
Visceral obesity is defined as a fat area >163.8 cm2 for men and >80.1 cm2 for women.