Non-invasive Testing for the Diagnosis and Assessment of Colorectal Disease
Conditions: Colorectal Cancer, Colorectal Polyps, Colorectal Adenocarcinoma
Sponsor: Imperial College London

 
 
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CLINICAL PROTOCOL 
 
 
Full Title  
Non-invasive testing for the diagnosis and assessment of 
colorectal disease 
 
Short Title 
Colorectal breath analysis (COBRA) 
 
Sponsor 
Imperial College London 
 
Ruth Nicholson 
 
Room 501B, 5th Floor 
                                            Lab Block, 
Charing Cross Hospital 
Fulham Palace Road 
London 
W6 8RF 
 
 
Chief Investigator 
Professor George Hanna 
 
Head of Division of Surgery 
 
Imperial College Healthcare NHS Trust 
 
Principal Investigator 
Professor George Hanna 
 
Management Group 
Dr Georgia Woodfield, Mr Geng-Ping Lin, Mr Piers Boshier, Mr 
Andrea Romano 
 
NCT number  
         NCT03699163 
 
 

 
 
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TABLE OF CONTENTS 
 
1. Signature Page ......................................................................................................... 3 
2. Summary ................................................................................................................... 4 
3. Introduction .............................................................................................................. 5 
    3.1 Background ………     ………………………………………………………………………5 
    3.2   Rationale for study…………………………………………………………………………..7 
4. Study Aims ............................................................................................................... 8 
4.1 
Aims ................................................................................................................................. 8 
5. Methodology ............................................................................................................. 8 
5.1 
Inclusion Criteria ............................................................................................................... 8 
5.2 
Exclusion Criteria ............................................................................................................. 8 
5.3 
Study Design and Plan ..................................................................................................... 8 
6. Study Procedures .................................................................................................. 10 
6.1 
Study Timetable ............................................................................................................. 10 
6.2 
Recruitment and Informed Consent Procedures ............................................................ 10 
7. Statistical Considerations ..................................................................................... 10 
8. Ethical Considerations .......................................................................................... 11 
8.1 
Declaration of Helsinki and Good Clinical Practice ........................................................ 11 
8.2 
Ethical Committee Review ............................................................................................. 11 
   8.3    Consent…………………………………………………………………………………….11 
9. Safety Considerations and Reporting .................................................................. 12 
9.1 
Adverse Event ................................................................................................................ 12 
9.2 
Serious Adverse Event (SAE) ........................................................................................ 12 
9.3 
Unexpected Adverse Event ............................................................................................ 12 
9.4 
Expected Adverse Events .............................................................................................. 12 
9.5 
Reporting Unexpected Adverse Events ......................................................................... 12 
9.5.1 
Assessment of Intensity ........................................................................................... 12 
9.6 
Urgent Safety Measures ................................................................................................. 13 
10. Data Handling and Record Keeping ................................................................... 13 
11. Specimen anonomisation and storage .............................................................. 13 
12. Specimen analysis ............................................................................................... 14 
13. Devices and Techniques ..................................................................................... 15 
13.1 
Devices ......................................................................................................................... 15 
14. Monitoring and Auditing ..................................................................................... 15 
15. Study Committees ............................................................................................... 15 
15.1 
Study Steering Committee ........................................................................................... 15 
16. Finance and Funding ........................................................................................... 15 
17. Indemnity .............................................................................................................. 15 
18. Dissemination of Research Findings ................................................................. 15 
19. References ........................................................................................................... 16 
 
 

 
 
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1. SIGNATURE PAGE 
 
 
Chief Investigator Agreement 
 
 
The clinical study as detailed within this research protocol (), or any subsequent 
amendments will be conducted in accordance with the Research Governance Framework for 
Health & Social Care (2005), the World Medical Association Declaration of Helsinki (1996) 
and the current applicable regulatory requirements and any subsequent amendments of the 
appropriate regulations. 
 
Chief Investigator Name:  
Chief Investigator Site:  
Signature and Date:  
 
 
 
Principal Investigator Agreement  
The clinical study as detailed within this research protocol (), or any subsequent 
amendments will be conducted in accordance with the Research Governance Framework for 
Health & Social Care (2005), the World Medical Association Declaration of Helsinki (1996) 
and the current applicable regulatory requirements and any subsequent amendments of the 
appropriate regulations. 
 
Principal Investigator Name:  
Principal Investigator Site:  
Signature and Date: 
 

 
 
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2. SUMMARY  
Short Title 
Colorectal cancer breath analysis (COBRA) 
 
Methodology 
 
Cohort Observational & Laboratory study 
Research Site 
 
St Mary’s Hospital 
Charing Cross Hospital 
St Mark’s Hospital 
St George’s Hospital 
Homerton University Hospital 
Chelsea and Westminster Hospital 
West Middlesex Hospital 
Royal Marsden Hospital 
 
Aims 
 
Determine the diagnostic accuracy of the exhaled breath and 
urine test for the prediction of colorectal disease. 
To assess the acceptability of using breath and urine testing 
 
Main Inclusion Criteria 
 
Any patient who: 
1. Patients aged 18 - 90 years  
2. Patients seen in secondary care with symptoms of colorectal 
disease or referred for a lower gastrointestinal endoscopy as 
part of their routine clinical investigation programme 
3. Patients able to understand and retain the information 
provided, thereby being able to give informed consent for 
inclusion in this study  
Number of Participants 
Target 2000 
 
Endpoints 
Primary endpoint 
• 
Once samples have been collected from 2000 patients. 
 
Study Duration 
 
April 2017 to April 2022 
 
 

 
 
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3. INTRODUCTION  
 
3.1 Background 
Colorectal cancer (CRC) is the 2nd commonest cause of cancer death in the UK. Survival 
rates are among the lowest in Europe (1) and 26% of patients present as an emergency, 
frequently having visited their GP before diagnosis (2-4). If diagnosed early, CRC survival 
rates exceed 90% (5). Whilst, reducing delay in diagnosis is therefore considered key to 
improving outcomes [6], initiatives to reduce delay have thus far been largely unsuccessful 
(6, 7). In order to improve the diagnosis of CRC, efforts should be made to determine how 
the risk of CRC can be assessed using sensitive primary care tests that are: valid; easy to 
perform, and; well tolerated by patients.  
 
CRC screening programmes rely on detection of early-stage cancer using the faecal occult 
blood test (FOBT) with colonoscopy in those patients with a positive test. Trials have shown 
that this screening method reduced CRC mortality but not incidence rates. Even if UK CRC 
incidence rates remain constant, as they have for the past 30 years, with an aging 
population the number of cases and treatment cost are set to double in the next 50 years. 
The Bowel Scope programme is also being rolled out across the UK currently, aiming to 
perform flexible sigmoidoscopy on every person at the age of 55. Invasive screening 
reduces mortality but is not without risk to patients and pressurises an already over 
burdened healthcare service. 
 
Risk of CRC in symptomatic patients can be assessed by different methods: 
(I) 
Flexible sigmoidoscopy and colonoscopy: The NHS currently has insufficient trained 
endoscopists to perform the number of procedures that would be required if flexible 
sigmoidoscopy were used as a first-line test for symptoms with predictive values below 
3%. 
(II) 
Guaiac FOBT has good sensitivity (87-98% in CRC detection), but highly variable and 
often unsatisfactory specificity (13-79%), requiring the repetition of the test on multiple 
stool samples. To date, the FOBT is neither recommended nor available for use as an 
intermediate test (8-11). 
(III) Faecal immunochemical testing (FIT) requires sampling only a single stool. It has been 
extensively tested; and is now used by many CRC screening programmes with very 
low prevalence rates of CRC (12). Four FIT systems are fully automated, and provide 
a quantitative measure of haemoglobin, allowing selection of a threshold of positivity to 
fit specific circumstances. As a result the research data available on sensitivity and 
specificity for CRC is based on small numbers of cancers. The few studies that have 
included adequate numbers of cancers have used older qualitative tests with a fixed 
threshold for positivity and were not automated. Nevertheless, the data suggest that, 
depending on the selected threshold for positivity, sensitivity for CRC varies between 
35% and 86% with specificity between 85% and 95% (8, 9). However, there are no 
data on the sensitivity of the newer quantitative test for early-stage cancers. 
(IV) Multi-target stool DNA testing: When compared with the FIT, in a large multicentre 
study, the test showed better specificity (92 vs. 73%), but lower sensitivity (90 vs. 96%) 
(13). 
 
At Imperial College London, we have developed a breath test for the detection of 
oesophagogastric adenocarcinoma from biomarker discovery to clinical trials. The 
assessment of volatile organic compound (VOCs) within exhaled by selected ion flow tube 
mass spectrometry (SIFT-MS) (14) was followed by the construction and validation of a 
diagnostic model using a panel of 9 VOCs in 225 patients with an area under the ROC curve 
(±SE) of 0.92±0.01 and 0.87±0.03 for the model and validation subsets respectively (15). We 
also performed cross-platform chemical validation using GC-MS. A NIHR-funded multicentre 
blind validation study on 396 patients (16) showed a sensitivity of 86% and specificity of 

 
 
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80%. In parallel with this work we have also investigated the exhalation kinetics and 
molecular drivers of VOCs in oesophagogastric adenocarcinoma (17-19). 
 
In colorectal cancer we performed a prospective pilot study to identify discriminative VOCs 
(n=150); a diagnostic validation study (n=110); a clinical prognostic study following surgery 
and after tumour recurrence (n=60); cross platform chemical validation using GC-MS (n=30). 
The diagnostic model had a sensitivity of 85% and specificity of 84% for cancer diagnosis. 
Following surgery the biomarker normalised to control level, but levels significantly increased 
with recurrence (20). The volatile biomarkers for oesophago-gastric and colorectal cancers 
are different with no overlap.  
 
Volatile Organic Compounds (VOCs)  
VOCs emitted from the human body have been of interest to researchers for several 
decades. In 1971, Pauling et al (21), reported that breath and urine contained approximately 
250 and 280 VOCs respectively in normal human subjects. VOCs are traditionally measured 
in food industry and counter-terrorism (22-24). 
 
The analysis of exhaled breath for the non-invasive detection and monitoring of disease is 
an attractive and evolving field of clinical research. Routine clinical applications of breath 
testing include the alcohol breathalyser, 13C urea breath test for H. pylori, nitric oxide in 
asthma, and hydrogen/methane test for bacteria overgrowth (25-29).  
 
Alteration in VOC production in patients with cancer has been postulated to relate to 
oxygenation of cell membrane-based polyunsaturated fatty acids resulting from genetic 
and/or protein mutations within tumour cells and the increased relative prevalence of 
reactive oxygen species (30, 31). These VOCs produced from the cancer site within the 
body, travel within blood to reach pulmonary alveoli where they are exhaled, permitting an 
objective quantitative measurement (32). There have been early reports using VOCs to 
detect lung, breast, bladder, prostate and gastric cancers (33, 34). Reports on colorectal 
cancer had small patient cohorts and lacked validation (32, 35, 36). 
 
Due to the complex composition of human breath a number of sophisticated mass 
spectrometer based technologies have been employed for the detection of exhaled 
VOCs including Gas Chromatography coupled with Mass Spectrometry (GC/MS), Proton 
Transfer Reaction-Mass Spectrometry (PTR-MS), Selected Ion Flow Tube-Mass 
Spectrometry (SIFT-MS) and Ion Mobility Spectrometry (IMS). These instrumental 
methods all have their strengths and limitations for applications in breath analysis. We 
intend to use multiple analytical platforms in our proposed work as these techniques are 
complementary and will allow us to acquire more robust findings.  
 
Thermal-desorption (TD) coupled to Gas Chromatography Mass Spectrometry (GC/MS) is a 
widely adopted method for measuring breath VOCs. Although this method requires the pre-
concentration of breath volatiles prior to analysis, it allows for the comprehensive 
identification and semi-quantification of VOCs in unknown complex mixtures such as breath. 
It can separate and detect hundreds of compounds from a breath sample, allowing for VOC 
profiling and has therefore been used in the study of disease biomarkers for lung cancer (34, 
37), breast cancer (33), colorectal cancer (32) and liver diseases (38, 39).   
 
Selective Ion Flow Tube Mass Spectrometry (SIFT-MS) permits online, real-time VOC 

 
 
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quantification (40). SIFT-MS has been utilised in the study of VOCs in breath and urine from 
patients with conditions including cystic fibrosis and bladder cancer (41, 42). The SIFT-MS 
technique allows real-time detection and quantification of VOCs within biological samples 
such as exhaled breath without any sample preparation minimising diagnostic delay.  
Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) is a relatively 
new instrumental tool that is gaining interest in breath research applications. Similarly to 
SIFT-MS, Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) is a direct injection-
based technology, obviating problems related to pre-concentration and separation steps 
typical of GC-MS. The most important advantage guaranteed by the ToF mass analyzer over 
quadrupole-based instruments (GC-MS and SIFT-MS) is represented by the high time 
resolution. In a ToF analyser a complete mass spectrum is generated at each ionisation 
event and spectra can be recorded at a frequency of 1 Hz or lower. This improves the 
accuracy of measurement during in vivo studies; moreover, full spectral scans can be 
performed without compromising sensitivity, thus enabling simultaneous targeted and 
untargeted analysis.  
 
The use of multi-platform analytical methods will enable both compound identification and 
quantification which are very important in the search and validation of biomarkers.  
 
3.2 Rationale 
Colonoscopy remains the gold-standard investigation for the assessment of patients with 
lower gastrointestinal symptoms and considered at risk of colorectal cancer. Colonoscopy is 
an expensive investigation that is uncomfortable for the patient and not without important 
risks including visceral perforation and bleeding.  
A breath test is a non-invasive investigation. The use of a breath test as a triage 
investigation in symptomatic patients could identify high-risk patients who should be referred 
for colonoscopy at an earlier stage, increase the proportion of appropriate referrals from 
primary care, and improve NICE guidelines which may currently miss particularly younger 
patients (43). If a GP is presented with a patient with gastrointestinal symptoms that do not 
prompt referral under NICE guidelines, he/she would not need to watch-and-wait to see if 
symptoms worsen but could offer the test immediately. The GP would order a breath test in 
much the same way as routine blood tests, with a single breath collection assessing for 
individual or multiple cancers. A nurse can perform the test and send breath samples to a 
regional laboratory for analysis. A positive result would warrant immediate referral. A 
negative test would permit the GP to reassure the patient and offer retesting if symptoms 
persist. Furthermore if we could develop a breath test that can discriminate colonic 
adenomas, the test would have further a potential application within the screening 
programme.  
 
A breath test could also be used to detect recurrence in patients post-resection for their 
colorectal cancer, allowing earlier detection and limiting invasive testing in those who are 
free of recurrence.  
 
Previous pilot study data for colorectal cancer detection by our group produced a diagnostic 
model with a sensitivity of 85% and specificity of 84% for colorectal cancer. The next stage 
of this research and the subject of this current proposal is to assess the influence of lower 
gastrointestinal diseases on VOC exhaled breath and urine. The external validation of this 
VOC breath model in a large multi-centre study will provide this risk-stratification tool for 
future clinical practice.  

 
 
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4. STUDY AIMS 
 
4.1 Aims 
 
The primary aim of this study is to determine the diagnostic accuracy of the exhaled breath 
and urine test for the prediction of colorectal disease. 
 
  
Secondary aims are: 
(i) Refine the current diagnostic model using technologies that allow identification of 
additional biomarkers at a lower detection level for CRC. 
(ii) Confirm the diagnostic accuracy of the test in a new and independent group of patients 
who have CRC compared to subjects with benign diseases or normal colon. 
(iii) Confirm the prognostic accuracy of the test in detecting tumour recurrence after surgical 
removal of CRC. 
(iv) Profile VOCs in patients with colonic adenomas and construct a diagnostic model. 
(v) Profile VOCs from colonic air in a subset of patients, for use as a comparison against 
breath testing in order to improve understanding of underlying pathological mechanisms.  
(vi) To assess the acceptability of using breath and urine testing 
 
5.   METHODOLOGY 
 
5.1 Inclusion Criteria 
 
Any patient who is:  
• 
≥ 18 years old and below 90 years of age, AND: 
• 
undergoing a lower gastrointestinal endoscopy as part of their routine clinical care 
OR: 
• 
seen in another hospital clinical setting with symptoms of lower gastrointestinal 
disease 
• 
able to provide informed written consent 
 
 
5.2 Exclusion Criteria  
 
Any patient who: 
• 
Lacks capacity or is unable to provide informed consent. 
• 
Any patient below 18 years of age or over 90 years of age. 
 
 
5.3 Study Design and Plan 
 
Patients recruited from the endoscopy unit will have been referred by their responsible 
clinician to undergo a lower gastrointestinal endoscopy (which involves a telescope test to 
visualise the colon). Others may be attending for colonoscopy as part of the bowel cancer 
screening programme or as part of routine surveillance. When patients attend the endoscopy 
unit, a member of the research team will discuss the research project with the patient, 
explain that the study involves sampling of their breath and urine while they are waiting for 
their endoscopy. Patients may also be asked to consent to the use of the colonic air 
collected during colonoscopy for analysis. Patients will then be provided with a patient 

 
 
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information leaflet, (see Participant Information Leaflet version 3.0 24/04/2017). Should the 
patient agree to participate they will be asked to sign a written consent form (see Consent 
Form Version 2.0 15/02/2017).  
 
Patients recruited from secondary care will be approached either before or after a routine 
appointment that they will be attending as part of their clinical care. This could include 
general colorectal clinics, pre-operative colorectal clinics, oncology clinics, theatres or on 
hospital wards.  
 
The project will be explained to them and they will be provided with an information leaflet. If 
they agree to participate they will be asked to sign a written consent form in order to provide 
breath and urine samples for the research project. Consent for colonic air sampling is not 
applicable to the majority of secondary care patients, only to those having a colonoscopy. No 
additional invasive procedures will be undertaken for the purposes of this study. A subset of 
patients with known cancer who are due to have a cancer resection may be asked to provide 
additional breath and urine samples 6–8 weeks following surgical resection of their tumour 
and/or if their disease should recur.   
 
Patients attending for colonoscopy will be fasted for a minimum of six hours prior to their 
breath sample collection. This is normal routine practice for patients undergoing endoscopy 
and therefore will not alter their treatment pathway. All breath samples will be retrieved prior 
to endoscopy. In other secondary care settings patients will not be specifically asked to fast 
prior to giving breath or urine samples but note will be made of how long they have fasted 
for. Patients will be asked to give a number of exhalations into a breath sampling device until 
a defined volume of breath has been sampled. This involves performing normal tidal 
breathing whilst wearing a face mask, and takes around 2 minutes on average. Patients will 
also be asked to perform a single deep nasal inhalation followed by complete exhalation via 
their mouth into a secure GastroCHECK steel breath bag (500 mL) via a 1 mL Luer-Lok 
syringe (Terumo Europe, Leuven, Belgium). Patients at St Mary’s Hospital will also be asked 
to breathe directly into a mass spectrometer machine. The mass spectrometer is a self-
contained mobile device which can be easily moved between clinical areas within St Mary’s 
Hospital. Sampling by this method will involve a single deep nasal inhalation followed by 
complete exhalation via their mouth into a plastic cylinder connected to a tube leading to the 
mass spectrometer itself. We expect the breath sampling process to take no longer than 5-
10 minutes in total. Patients will be requested to pass urine into a standardised 60ml urine 
specimen vial, which will be immediately sealed. 20ml of urine will be aliquoted into a 
standard 60ml specimen vial.  
 
Of patients attending for colonoscopy, a subset will be asked to consent to have air from 
their colon collected. This air comes up from the colon through the colonoscope suction 
channels. Suctioning of air is a routine part of the examination, but for this study we will be 
collecting it for analysis. Collection is done via a small suction pump connected to a fine bore 
tube that is passed through the suction channel of the colonoscope. Sampling in this way will 
add no more than a few minutes (<4 minutes) on to the procedure time. 
 
The VOCs from the breath/colonic air will be trapped onto adsorbent tubes inserted into the 
breath taking device. In the case of colonic air sampling, an adsorbent tube is attached to 
the pump end of the fine bore disposable suction tube. After sampling the adsorbent tube is 
removed from the device, capped and transported back to the laboratory at St Mary’s 
Hospital London for analysis. Samples will be transported by courier or by a member of the 
research team. The sample(s) shall undergo analysis via our mass spectrometry 
instruments, at St Mary’s Hospital London.  
Breath samples collected in steel bags will be immediately disposed of following analysis 
within 6 hours of the sample being taken. Breath VOCs sampled on adsorbent tubes will be 

 
 
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analysed within 48 hours of collection. The sample constituents are destroyed within the 
mass spectrometer for detection meaning that no clinical material will remain after analysis 
by GC-MS. Urine samples will be stored within a registered tissue bank (REC Ref no. 
04/Q0403/119, custodian Prof G Hanna) in accordance with the Human Tissue Act and 
disposed of following analysis.  
 
This study has been specifically designed to ensure that any disruption to patient’s routine 
care and management is kept to an absolute minimum. 
 
 
6. PROCEDURES 
 
6.1 Study Timetable 
 
January 2017 – March 2017 
Research & Development and ethical approval. 
 
April 2017 – March 2022 
Participant recruitment and sample collection. Laboratory analysis of samples, write up and 
dissemination of results. 
 
 
6.2 Recruitment and Informed Consent Procedures 
 
A member of the research team will identify and approach patients when they attend for a 
lower gastrointestinal endoscopy. All patients attending for colonoscopy within the specified 
age range are potentially eligible for this study. If the patient is in agreement, a member of 
the research team will explain the research project and provide a written information leaflet 
(see Participant Information Leaflet version 3.0 24/04/2017). Patients will be given adequate 
time to decide if they wish to participate and should they agree they will complete a written 
consent form (see Consent Form Version 2.0 15/02/2017). 
 
A member of the research team will identify patients when they attend for a clinic 
appointment by discussion with the Consultant clinician responsible for the clinic. A member 
of the research team will approach patients when they attend for their clinic or pre-
assessment appointment prior to planned surgery. Patients will have the research project 
explained to them and will be provided with a written information leaflet (see Participant 
Information Leaflet version 3.0 24/04/2017). Patients will be provided with adequate time to 
decide if they wish to participate and should they agree they would complete a written 
consent form (see Consent Form Version 2.0 15/02/2017).  
 
All of the above would occur at one of the patient’s routine clinical attendances and would 
not require any additional patient attendances.  
 
7. STATISTICAL CONSIDERATIONS 
 
Both univariate and multivariate data analysis techniques will be applied to the results to 1) 
identify VOC components with the best discriminating ability between the groups and 2) to 
develop a multivariate discriminant analysis model.  
 
Mann-Whitney U test will be used to compare the measured metabolite concentrations 
between the study groups. A p value <0.05 will be taken as the level to indicate statistical 
significance.  
 

 
 
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Non-parametric (Kruskal-Wallis) ANOVA test will be used to compare the measured 
metabolite levels between the study groups. Statistically significant metabolites will be 
selected using False Discovery Rate (FDR) adjusted p value threshold such that less than 
5% of discovered candidate markers are false positives (i.e. q-value <0.05). 
The FDR strategy will be based on Benjamini–Hochberg procedure. 
 
Receiver operating characteristic (ROC) curves will be used to determine the accuracy of a 
diagnostic test in classifying those with and without colorectal disease. The metabolite 
concentrations that exhibit significant differences (q<0.05) between the cancer and positive 
controls (including adenomatous polyps) will be included as variables for the proposed 
diagnostic test. The linear discriminant analysis based on multivariate logistic regression will 
be subsequently used to derive a combination of the metabolite concentrations for sample 
classification. The optimum threshold for group discrimination will be calculated to achieve 
the highest possible classification accuracy. The predictive capacity of the model will be 
assessed on independent sample set, as described below. 
 
Validation 
The Breath and Urine metabolite model for the prediction of colorectal disease developed in 
the previous studies will be used to assign risk of disease in a prospective cohort. 
Investigators undertaking metabolite sampling and analysis will be blinded to the results of 
the endoscopy. Comparison of predicted disease risk and actual endoscopy findings will 
then be made, and the overall diagnostic accuracy (sensitivity, specificity, positive and 
negative predictive value, ROC curve analysis) for this non-invasive diagnostic investigation 
will be determined. Colonic air VOC profiles will be used for validation of paired breath 
results, and to gain further understanding of the mechanism of VOC production in humans. 
 
8. ETHICAL CONSIDERATIONS 
 
8.1 Declaration of Helsinki and Good Clinical Practice 
 
The study will conform to the spirit and letter of the declaration of Helsinki and in accordance 
with the Good Clinical Practice Guidelines 
 
8.2 Ethical Committee Review 
 
The study protocol will be used when approval has been obtained from a UK Research 
Ethics Committee. The study must be submitted for Site Specific Assessment (SSA) at each 
participating NHS Trust.  The Chief Investigator will require a copy of the Trust R&D 
approval letter before accepting participants into the study.  The study will be conducted in 
accordance with the recommendations for physicians involved in research on human 
subjects adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions. 
Additionally, in order to sample breath from any patient who is having a colonoscopy via the 
UK Bowel Cancer Screening Programme (BCSP) pathway, there also has to be specific 
approval from the UK BCSP Research Advisory committee. Formal approval has been 
gained for an initial 100 BCSP patients to be approached for this study, as a pilot, with a 
view to further approval after this period (BCSP ID189, approval given on 18/1/17). Further 
approval will therefore be sought for the recruitment of additional BCSP patients, after 100 
patients have been approached, as per BCSP panel request. 
 
 
8.3 Consent 
 
Consent to enter the study must be sought from each participant only after a full explanation 
has been given, an information leaflet offered and time allowed for consideration.  Signed 

 
 
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participant consent should be obtained. The right of the participant to refuse to participate 
without giving reasons must be respected. All participants are free to withdraw at any time 
from the protocol without giving reasons and without prejudicing further treatment. 
 
 
9. SAFETY CONSIDERATIONS AND REPORTING 
 
This is not a study of an investigational medicinal product and therefore all untoward 
occurrences will be adverse events rather than adverse reactions. 
 
9.1 Adverse Event 
 
An adverse event is defined as any untoward medical occurrence affecting a participant. The 
terms ‘mild, moderate or severe’ are used to describe the intensity of a specific event or 
reaction. An event would be considered ‘serious’ based upon participant/event outcome or 
action criteria as defined below. 
 
9.2 Serious Adverse Event (SAE) 
 
A serious adverse event (SAE) will be considered any untoward medical occurrence/effect 
that: 
• 
Results in death 
• 
Is life-threatening 
• 
Requires hospitalisation or prolongation of existing inpatient’s hospitalisation. 
• 
Results in persistent or significant disability or incapacity. 
• 
Is otherwise considered medically significant by the investigator 
‘Life threatening’ refers to an event in which the subject was at risk of death at the time of the 
event; it does not refer to an event which hypothetically may have caused death if it had 
been more severe. 
 
9.3 Unexpected Adverse Event 
 
An unexpected adverse event is considered an adverse event of which the nature and 
severity is not consistent with the expected consequences of sample collection.  
 
9.4 Expected Adverse Events 
 
We do not believe any risks or adverse events are associated with patients providing 
samples of urine and breath or in the consent process for these procedures. All samples will 
be disposed of following analysis. All hospital records will be handled with strict 
confidentiality in accordance with the Data Protection Act 1998. All other risks will be in 
accordance with usual clinical practice. These include risk of endoscopic perforation, patient 
discomfort during endoscopy, and local bruising following intravenous cannulation. There are 
no other serious adverse events caused by sample collection and tissue biopsy. 
 
9.5 Reporting Unexpected Adverse Events 
 
Given the nature of this investigation, it is unlikely that any will occur. Nonetheless, should 
an unexpected adverse events occur, the investigators will make an assessment of severity 
and report the event to the Research Ethics Committee and the Sponsor as appropriate. 
Usually, this will mean within 24 hours in the case of severe or moderate events, and within 
14 days for mild events. 
 

 
 
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9.5.1 Assessment of Intensity 
 
Mild: The subject is aware of the event or symptom, but the event or symptom is easily 
tolerated. 
Moderate: The subject experiences sufficient discomfort to interfere with or reduce their 
usual level of activity. 
Severe: Significant impairment of functioning; the subject is unable to carry out usual 
activities and/or the subject’s life is at risk from the event. 
 
9.6 Urgent Safety Measures 
 
Should it be necessary to undertake urgent safety measures to ensure the safety and 
protection of the participants the CI will take these measures immediately. The CI would 
inform the sponsor and Main research Ethics Committee of this event immediately via 
telephone and in writing within 3 days in the form of a substantial amendment.  
 
10. DATA HANDLING AND RECORD KEEPING 
 
Information related to study participants will remain confidential and be managed in 
accordance with the Data Protection Act, NHS Caldecott Principles, The Research 
Governance Framework for Health and Social Care, and the conditions of Research Ethics 
Committee Approval. 
 
All case record forms will be held in the site file, which will be kept in a locked drawer in a 
locked office at the study site. They will be destroyed after 10 years. All digital data will be 
stored on a password-protected NHS computer on site at St Mary’s Hospital, under the 
control of the Chief Investigator. Data will be erased securely after 10 years. 
 
11. SPECIMEN ANONOMISATION AND STORAGE 
 
Samples will be anonymised with all patients being assigned a study number that will be 
added to the specimen during storage. Patients would not be identifiable from this study 
number. These study numbers will be linked to the digital database stored on a password-
protected NHS computer on site at St Mary’s Hospital, under the control of the Chief 
Investigator. 
 
Breath samples collected in steel bags will be disposed of immediately following analysis.  
Breath/colonic air VOCs sampled on to adsorbent tubes will be released by thermal 
desorption into the analytical instrument where they will be destroyed during mass 
spectrometric detection. Urine samples will be stored within -80C freezers used solely for the 
storage of human samples within a secure laboratory within the research department at St 
Mary's hospital. The only people who will have access to this tissue bank are key members 
of the research team. These samples will be added to Professor Hanna’s current tissue bank 
for which local ethical approval is already in place for sample collection at St Mary’s Hospital 
(REC Ref no. 04/Q0403/119). Urine samples will be stored until study completion. Analysed 
samples will be destroyed by incineration following analysis and surplus samples will be 
stored in the current tissue bank until study completion in case a repeat analysis is required.  
 
 
 
 
 
 
 

 
 
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12. SPECIMEN ANALYSIS  
 
Selective Ion Flow Tube Mass Spectrometry (SIFT–MS)  
SIFT-MS permits online, real-time Volatile Organic Compound (VOC) quantification. SIFT-
MS has been utilised in the study of VOCs in breath and urine from patients with conditions 
including cystic fibrosis and bladder cancer. The principle of SIFT-MS is selected precursor 
ions are formed in a microwave discharge source and are selected according to their mass-
to-charge ratio, m/z, by a mass filter and injected into a helium carrier gas where they are 
convected as a thermalised swarm along a flow tube. H3O+, NO+, O2+ precursor ions are 
used to ionize the trace gases in an air sample that is introduced into the helium at a known 
flow rate, these ions selectively ionise VOCs present within the sample resulting in 
characteristic product ions. By measuring the count rate of both precursor ions and the 
characteristic product ions at the downstream detection system, a real-time quantification is 
achieved, realising the absolute concentration of trace and volatile compounds at the parts-
per-billion by volume or parts-per-million by volume. The SIFT-MS technique allows real-time 
detection and quantification of Volatile Organic Compounds within biological samples such 
as exhaled breath without any sample preparation minimising diagnostic delay. This is 
particularly advantageous within the clinical environment where samples can be retrieved 
and real-time VOC measurements made with negligible concern for sample degradation. 
 
Thermal Desorption (TD) and Gas Chromatography Mass Spectrometry (GC/MS)  
Thermal desorption coupled to GC/MS is one of the most widely used techniques for breath 
biomarker discovery work for a number of reasons. Firstly, VOCs in exhaled breath are 
present at very low concentrations of parts-per-million (ppm) to parts-per-trillion (ppt) and 
pre-concentration is often necessary to allow for detection. This can be achieved by 
adsorption onto sorbent beds (tubes packed with tenax, carbon molecular sieve, or 
graphitized carbon) followed by release through thermal desorption. The coupling of a 
thermal desorber to a GC/MS system allows for the trapped VOCs to be released and 
rapidly injected on to the GC column for analysis. GC/MS is a very well established 
analytical technique that allows for the successful separation, identification, and semi-
quantification of trace VOCs in unknown complex mixtures such as breath. This technique 
has allowed for the identification of over 800 compounds in breath (44). Although this 
technique does not allow for real-time analysis it provides a much more comprehensive 
analysis of the VOC profile. It will complement results obtained with SIFT-MS and can allow 
for cross-platform validation.  
 
Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) 
Similarly to SIFT-MS, Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) is a direct 
injection-based technology, obviating problems related to pre-concentration and separation 
steps typical of GC-MS. In PTR-MS the ion beam generated at the source does not undergo 
selection by means of a mass filter as in SIFT-MS so the detection limits are on average two 
orders of magnitude lower than those obtained with SIFT-MS. However, equipped with a 
Time-of-Flight (ToF) mass analyzer, a higher mass resolution is achieved, signals 
represented by a single mass peak in a quadrupole-generated spectrum, can be resolved in 
two or more distinct mass peaks, generally resulting in an improved discrimination 
performance. In addition to this, accurate masses can be determined with at least two 
decimal digits; this means that the data generated by PTR-ToF-MS, especially when 
integrated with GC-MS, can be used for compound identification and thus permit the 
discovery of new biomarkers. The most important advantage guaranteed by the ToF mass 
analyzer over quadrupole-based instruments (as those in GC-MS and SIFT-MS) is 
represented by the high time resolution. In a ToF analyzer a complete mass spectrum is 
generated at each ionisation event and spectra can be recorded at a frequency of 1 Hz or 
lower. This improves the accuracy of measurement during in vivo studies; moreover, full 

 
 
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spectral scans can be performed without compromising sensitivity, thus enabling 
simultaneous targeted and untargeted analysis. 
 
 
13. DEVICES AND TECHNIQUES 
 
13.1 
Devices 
 
A device that collects and concentrates breath VOCs on adsorbent tubes in one easy step 
will be used to collect breath (Owlstone’s CE-marked hand-held breath sampler, ReCIVA, 
Owlstone Ltd, Cambridge, UK). This device has been designed for easy patient use, it 
contains a pump of filtered air to ensure clean air sampling, it allows for the concentration of 
a defined volume, and is more economical than other sampling methodologies available. A 
steel bag will also be used for the collection of breath. For direct sampling into the mass 
spectrometer, the patient blows into a disposable cardboard tube attached to the inlet to the 
machine, discarded between each patient. All other devices are those used within the 
patient’s routine clinical practice including endoscopic equipment and universal sampling 
containers. These will all be used in accordance with routine clinical practice.  
 
14. MONITORING AND AUDITING 
 
The study will be monitored and audited by the Sponsor, in accordance with usual protocols. 
 
15. STUDY COMMITTEES 
 
a. Study Steering Committee 
 
This will comprise of Professor George Hanna, Professor Wendy Atkins, Professor Brian 
Saunders, Dr Jonathan Hoare 
 
 
16. FINANCE AND FUNDING 
 
Equipment utilised for sample collection will be funded as part of Professor Hanna’s 
research funding. Laboratory analysis of the biological specimens (breath, colonic air and 
urine samples) obtained will also be funded through Professor Hanna’s research group with 
external funding from HCA Hospitals. A grant from the Rosetrees Trust (ref: A1361) has 
already been approved to fund an analytical chemist to work on this study. 
 
17. INDEMNITY 
 
 
The nominated sponsor of our research study is Imperial College London, Research and 
Development Department, Room 501B, Laboratory Block, Charing Cross Hospital, Fulham 
Palace Road, London, W6 8RF. Delegated responsibilities will be assigned to the NHS trusts 
taking part in this study.   
Imperial College London holds negligent harm and non-negligent harm insurance policies 
which apply to this study. Medical co-investigators will also be covered by their own medical 
defence insurance for non-negligent harm.  
 
 
18. DISSEMINATION OF RESEARCH FINDINGS 
 
Results from this study will be presented in a peer-reviewed journal. Authorship will follow 
international guidelines. 
 

 
 
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