Professional Documents
Culture Documents
Introduction 3
VOCs in breath 3
Non-invasive biomarkers 3
Scope of this guide 6
Historical breath analysis 6
Breath analysis in the modern era 6
Established breath tests 8
Breath analysis in clinical trials 8
What are VOCs? 10
Where do breath VOCs originate from? 12
VOCs as biomarkers 16
Metabolomics vs. Genomics 16
Metabolomics for diagnosis and precision medicine 16
Whole body blood sampling via breath 18
Breath Biopsy vs. Liquid Biopsy 21
Applications of VOC biomarkers 23
Wide disease relevance 24
Novel Biomarkers 25
Precision Medicine applications 25
Clinical Trials patient matching 27
Drug metabolites and disease monitoring 27
Early detection and screening of cancer 27
Asthma and COPD 28
Oncology 32
Inflammatory diseases 39
Liver disease 42
Infectious disease 44
How to analyze VOC biomarkers 49
Overcoming challenges in breath sampling and analysis 50
Study design 51
Breath Analysis Workflow 54
Reliable collection of breath samples 54
VOC Biomarker analysis 57
Quality control 59
Data analysis 61
Beyond Breath: VOC Analysis in other Biospecimens 64
Biomarker Services 65
Breath Biopsy Products 70
Further reading 72
References 74
2
Introduction
VOCs in breath
Exhaled breath contains over 1,000 volatile organic compounds (VOCs).
Accessible by a completely non-invasive Breath Biopsy, these VOCs can be
used as disease biomarkers for early detection and screening, diagnosis, and
precision medicine including patient stratification for therapy selection and
treatment monitoring (Figure 1).
VOCs are products of metabolic activity taking place in the body, hence they
directly reflect the current state of cells, tissues and the microbiome - providing
a rich source of valuable information about the health of an individual.
Non-invasive biomarkers
As exhaled breath sampling is also inherently non-invasive, Breath Biopsy is
preferable to patients over uncomfortable and potentially unpleasant
diagnostic procedures, such as stool sampling, blood sampling, and tissue
biopsies.
3
BREATH BIOPSY:
NON-INVASIVE VOC BIOMARKERS FOR DIAGNOSTICS
AND PRECISION MEDICINE
APPLICATIONS
CLINICAL PRECISION
DIAGNOSTICS MEDICINE
Figure 1. Breath Biopsy is a completely non-invasive means to access valuable information about the health of individuals.
VOC biomarkers have numerous applications in clinical diagnostics and precision medicine across a wide range of
diseases.
4
Why breath?
Metabolites relate directly to current disease activity
Breath enables whole body blood sampling
VOC biomarkers in breath are relevant for a wide range of diseases
Breath biomarkers have wide-ranging applications in diagnostics and
precision medicine
Sampling is completely non-invasive and pain-free
Learn more at
owlstonemedical.com/breath-biopsy
www.
5
Scope of this guide
In this guide we introduce VOCs, and discuss the scientific basis for the
application of VOCs in breath as biomarkers for a wide range of diseases. We
provide an overview of current status of VOC biomarkers in the disease areas
of asthma, oncology, inflammatory and infectious disease.
We discuss some of the challenges that have previously limited the potential of
breath analysis, and describe how these challenges have now been overcome
with the introduction of new technology for breath sampling and analysis.
We will outline the workflows for breath collection and VOC analysis that have
introduced Breath Biopsy as a new diagnostic modality. Now, for the first time,
it is possible to perform robust biomarker discovery using breath as a sample
type, on a platform with the potential to transition to point-of-care.
One example familiar to doctors for centuries is the fruity smell in exhaled
breath of patients with diabetic ketoacidosis. This symptom is due to ketones
produced when the body is unable to use glucose due to the lack of insulin.
Instead, it breaks down fat as an alternative source of fuel, producing pear
scented acidic ketones including acetone [2].
During this era, the first discoveries relating to the origin of certain VOCs in
breath were made. This included the identification of small chain hydrocarbons,
which were found to be products of lipid peroxidation, possible biomarkers for
oxidative stress [5]. Another example is the abundant breath VOC isoprene
[68], which was established to be produced in the mevalonate pathway, and
subject to large increases in concentration during physical exertion [9,10].
6
Technologies for breath analysis
Techniques well suited to the analysis of breath VOCs include gas
chromatography-mass spectrometry (GC-MS), proton transfer-reaction mass
spectrometry (PTR-MS) [11], selected ion flow tube-mass spectrometry
(SIFT-MS) [12], arrays of gas sensors (eNoses) and ion mobility spectrometry
(IMS) including field asymmetric ion mobility spectrometry (FAIMS) [1316].
150
100
50
0
1980 1985 1990 1995 2000 2005 2010 2015
YEAR
Figure 2. Increase in annual publications in the area of VOCs and breath. Graph shows the results
of a Web of Knowledge search for publications including the terms volatile organic compound AND
breath.
We have been using FAIMS for almost five years and have found it able
to non-invasively detect a broad range of diseases, including cancer,
with high sensitivity and specificity.
Professor James Covington, University of Warwick
7
Established breath tests
Some breath tests that use volatile biomarkers have already become
established in clinical practice (Table 1 shows approved breath tests). However,
much of the potential shown by VOC tests has remained untapped due to a
lack of reproducibility between studies looking for disease biomarkers. The
variability in the results of breath research has stemmed largely from a lack of
high quality, standardised breath sampling technology [17,18].
Capnography CO2
450
CUMULATIVE NUMBER OF TRIALS
400
350
300
250
200
150
100
50
0
1995 2000 2005 2010 2015
YEAR
Figure 3. Increasing numbers of clinical trials using breath analysis are being registered. This
includes 75 trials analysing exhaled breath condensate. Data from https://clinicaltrials.gov/.
https://www.owlstonemedical.com/media/uploads/files/Breath-analysis-clinical-trials-March-2017.xlsx
Download the list of trials as Excel spreadsheet.
https://www.owlstonemedical.com/media/uploads/files/Breath-analysis-clinical-trials-March-2017.xlsx
8
The identified clinical trials cover a wide range of conditions, demonstrating
the breadth of applications where breath analysis is thought to be useful. A
breakdown of the generalized disease category of the trials is shown in Figure
4.
CANCER
INFLAMMATORY
INFECTIOUS
OTHER (GASTROINTESTINAL)
OTHER (PULMONARY)
LIVER DISEASE
OTHER
Figure 4. Breakdown of disease category of 448 clinical trials using breath analysis in their
workflow. Data from https://clinicaltrials.gov/. https://www.owlstonemedical.com/media/uploads/files/Breath-analysis-clinical-trials-March-2017.xlsx
Download Excel spreadsheet including list of clinical
htps:/w w.owlstonemedical.com/ edia/uploads/files/Breath-an lysi -clincal-trials.xls
trials.
owlstonemedical.com/services
www.
9
What are
VOCs?
Volatile organic compounds (VOCs) are gas phase chemicals that have a high
vapor pressure at room temperature. VOCs form part of the composition of
exhaled breath, and represent the volatile fraction of metabolites. These are
low molecular weight (<1 kDa) compounds produced by the bodys
metabolism, and represent the endpoint of gene, transcription and protein
expression (Figure 5).
Thousands of breath borne metabolite VOCs have already been identified [39],
and they form a library of information about an individuals health. VOCs are
also emitted from other clinical samples types such as urine, feces and saliva
(see section on Beyond Breath on page 64).
Figure 5. Metabolites provide information about actual disease activity, in contrast to genomics
which only provides a starting blueprint.
10
O
O
O
OH
H H
N O
O
OH
OH
O
H N
H
HO 1
O O
OH H
H
HN OH
NH 1 H O
HO
NH
1
HN
H
H
O
H
O
H
H
H
HO
OH
H
O
O
HN
NH1
OH
O
HN
NH
HO
1
H
O
H
Whole body
blood sampling
Breath provides a window into a much larger volume of blood. By
sampling breath over a few minutes, VOC biomarkers originating
from all around the body are captured with high sensitivity.
11
Where do breath VOCs originate from?
The VOCs in exhaled breath have a number of potential sources. They can be
produced biochemically by an individuals cells and tissues (endogenous
VOCs) or be the result of external influences. These exogenous VOCs in breath
can be the product of metabolism by the bodys own resident microbiome, or
may be introduced from the environment, including downstream drug
metabolites from therapeutic interventions (Figure 6).
>1,000 VOLATILE
BREATH
O
METABOLITES O
O
OH
H H
N O
O
OH
OH
O
H N
H
HO 1
O O
OH H
H
HN OH
NH 1 H O
HO
NH
1
HN
H
H
O
H
O
H
H
H
HO
OH
H
O
O
ENVIRONMENT
HN
NH1
OH
O
HN
NH
HO
1
H
O
H
DRUGS GENOMICS
MICROBIOME
External factors like diet, exercise, A snapshot of the current state Our genes can be markers of
medications, microbiota and even of health that can be used to predisposition or presence of disease,
where we live influence our prevent, diagnose and treat but they do not necessarily give
metabolic state. disease or improve health. information about disease activity.
12
ORIGINS OF VOCs IN BREATH
Oxidative stress (OS) has been implicated in the initiation and development of
many systemic and organ-specific diseases, including cardiovascular,
pulmonary, autoimmunological, neurological, inflammatory diseases and
cancer [1]. OS is also known to play an important role in the production of VOC
biomarkers [40].
At the cellular level, higher than normal ROS levels damage key cellular
structures like phospholipid membranes, proteins and DNA. This damage
causes dysfunction, and can lead to apoptosis or programmed cell death.
Many VOCs are produced by these ROS interactions with cellular structures,
including alkanes ethane and pentane, nitric oxide and aldehyde
2-hydroxy-nonenal (Figure 8) [40].
13
OXIDATIVE STRESS RESULTS IN VOC PRODUCTION
Reactive Oxygen
Species (ROS)
Volatile organic
compounds (VOCs)
Figure 8. Oxidative stress, which is implicated in many diseases, is linked to the production of VOCs
from ROS damage to cell structures and other interactions. Adapted from [40].
Environmental exposure
Compounds present in an individuals environment can alter the presence and
concentration of VOCs observed in breath. VOC biomarkers that typify
exposure have been referred to previously as the exposome [41], and have
been used to measure and assess exposure to certain chemicals in the
environment. For example, breath analysis has been used by the US military to
monitor exposure to volatile compounds in jet fuel - in this case, aromatic and
aliphatic hydrocarbons served as biomarkers of this type of exposure [4143].
14
Metabolic processes of the bodys microbiota also generate VOCs. This can be
from bacterial fermentation of unadsorbed complex carbohydrates and
proteins in the gut. This generates gases including methane, hydrogen,
hydrogen sulphide and ammonia [45]. VOCs from the microbiome include
those produced by pathogenic microorganisms, such as C. difficile [46] or M.
tuberculosis [14], making them valuable for the diagnosis of these infections.
Drug metabolites
Breath analysis has been identified as a useful, non-invasive route to
monitoring certain pharmaceuticals (e.g. Propofol) during or after treatment.
Measuring specific drug related volatiles can provide a non-invasive means of
gaining insight into pharmocokinetics and pharmacodynamics. Breath analysis
has also been suggested for the detection of drugs of abuse, which could be
useful for roadside intoxication tests [47].
15
VOCs
as biomarkers
Metabolomics vs. Genomics
Genes act as the blueprint for an individuals biological functions. Despite years
of research focusing on the human genome, and a number of significant
advances, genomics has not yet delivered on its full potential to help us
understand and cure diseases.
In the last decade, hundreds of genome wide association studies (GWAS) have
been performed to identify regions of the genome associated with a range of
human disease. GWAS have failed to account for all the heritability of any
disease [48]. Only 10% of the variants that lead to common diseases are
located within coding DNA sequences. The remainder are located in
non-coding regions of the genome, or intergenic regions.
16
Metabolites make excellent disease biomarkers because alterations to an
organisms biological systems caused by disease, genetic mutations, the
microbiome or environmental factors change the profile of metabolites
produced. Hence, metabolomics has the closest biological proximity to the
phenotype of the observed individual, and allows rapid observations of
perturbations in this system.
Metabolites can provide early insight into developing disease states because
they reflect and magnify perturbations at lower genomic, transcriptomic and
proteomic levels several thousand fold [50,51]. As metabolic changes are
present at the earliest stages of disease, metabolites offer a route to earlier
diagnoses that can save lives.
Breath Biopsy
A new diagnostic modality
www. owlstonemedical.com/breath-biopsy
17
Whole body blood sampling via breath
Traditional approaches to breath sampling have relied upon collecting a single
exhaled breath, providing a small snapshot of VOCs present in the body. New
advances in breath sampling technology allow breath to be collected over a
longer time period, enabling VOCs originating from the whole body to be
collected.
The ability to capture VOCs originating from throughout the body in a breath
sample is extremely powerful. Firstly, breath enables whole body sampling of
the volatile metabolome. Secondly, this makes VOCs in breath applicable as
biomarkers for a wide range of diseases, not just pulmonary conditions.
18
WHOLE BODY BLOOD SAMPLING VIA BREATH
1 min 10 mins
Sample Capture even
whole low level VOC
body biomarkers
Figure 9. The volatile metabolome of the entire body can be accessed by continuously sampling breath over a few
minutes. Capturing VOCs from exhaled breath onto ReCIVAs integrated sorbent tubes allows the pre-concentration of
VOC biomarkers originating from the entire body via the circulatory system.
19
Breath Biomarkers
for a wide range
of diseases
and applications
From breath, you can non-invasively access the volatile metabolome of the
entire body, making Breath Biopsy relevant for a wide range of conditions.
20
Breath Biopsy vs. Liquid Biopsy
Breath Biopsy has inherent advantages over liquid biopsy techniques, which
look for circulating tumor cells (CTCs) or tumor-derived cell-free DNA/RNA
circulating in the blood. There has been much enthusiasm due to the less
invasive nature of liquid biopsies.
Liquid biopsy shows promise for therapy selection in late stage cancers, but
suffers from three fundamental challenges:
1. Circulating tumour DNA/RNA (ctDNA/RNA) is present only at limited
amounts, especially during the early stages of disease (Figure 10)
2. Only a limited amount of blood can be feasibly sampled and made available
for analysis, which limits the sensitivity of the liquid biopsy approach
3. Detection of a single mutation in ctDNA may not be a reliable indicator of
cancer, hence the liquid biopsy approach may suffer from low specificity for
early diagnosis of cancer.
Bettegowda et al. [52] reported that less than 50% of patients with Stage I
cancer had detectable ctDNA. Notably, the study was conducted using a
relatively large volume of plasma (5 ml). This highlights the limitations of liquid
biopsy, especially for early detection and screening applications.
Fraction of patients with detectable ctDNA in localized Fraction of patients with detectable ctDNA.
(stages I to III) and metastatic (stage IV) colorectal,
gastroesophageal, pancreatic, and breast cancers. 100
Frequency of cases with detecable ctDNA (%)
Frequency of cases with detecable ctDNA (%)
100
80
80
60
60
40
40
20
20
0 0
COLORECTAL GASTROESOPHAGEAL PANCREATIC BREAST STAGE I STAGE II STAGE III
STAGE IV
Localized Disease Metastatic Disease
Figure 10. Liquid biopsy shows promise for therapy selection in late stage cancer, but low
concentrations of ctDNA, and limitations in the amount of blood that can be feasibly sampled, limit
the sensitivity for early stage cancer detection (Adapted from [52]).
Sensitivity
Metabolite VOC biomarkers are altered at the earliest stages of disease. Breath
Biopsy can capture and detect these early warning VOCs that are present at
extremely low concentrations. This is possible because, unlike blood, breath
can be sampled on demand in effectively unlimited volumes.
By sampling for an extended period of time (~10 mins) using the ReCIVA Breath
Sampler, VOCs are concentrated onto sorbent tubes. Pre-concentration of VOC
biomarkers greatly increases the sensitivity of Breath Biopsy compared to
liquid biopsy techniques, enabling detection of VOC biomarkers present at the
onset of disease.
21
THE ADVANTAGE OF BREATH vs. BLOOD
1min
INVASIVE/ NON-INVASIVE/
PAINFUL PAIN-FREE
Figure 11. Breath enables higher sensitivity and whole body blood sampling as it captures VOCs
from the entire blood volume completely non-invasively. In contrast, a blood test samples only a
fraction of the blood volume.
Analytical advantages
Breath Biopsy also offers analytical advantages compared to liquid biopsy
techniques. The analysis of VOCs present in breath or other biospecimens can
be accomplished in a matter of minutes, using technology which has potential
to transition to point-of-care (see page 58). Hence, breath analysis is far more
rapid and cost-effective than the lengthy workflows needed for analysis of
nucleic acid by PCR or sequencing, which also typically require significant
sample preparation.
22
Applications of
VOC biomarkers
Exhaled breath contains a vast untapped resource of chemical information in
the form of over 1,000 volatile organic compounds. As products of underlying
metabolic activity, VOCs directly reflect the state of cells and tissues, and
represent a goldmine of valuable non-invasive biomarkers.
23
Wide disease relevance
VOCs originating from all parts of the body are captured in breath, making
Breath Biopsy applicable to a wide range of diseases including:
t Cancer
t Inflammatory disease
t Infectious disease
t Metabolic disease
t Cardiovascular disease
www. owlstonemedical.com/case-studies
Figure 12. VOC biomarkers in breath have been reported for a large number of diseases in the
literature [53,54], highlighting the wide disease relevance of Breath Biopsy.
24
Novel Biomarkers
Until relatively recently, the identification of robust VOC biomarkers in breath
has been hampered by the lack of standardized sampling and analytical
methods. Progress is now being accelerated by the advent of new technologies
for the reliable collection of patient breath samples (ReCIVA) and advances in
VOC analysis technology (FAIMS); when combined, these new technologies
have the potential to bring Breath Biopsy to use at point-of-care.
Breath and the metabolome are relatively under-explored in a clinical context,
so VOC biomarkers identified in breath are likely to be completely novel, which
has advantages for their utilization as companion or complementary
diagnostics.
The advances in breath sampling technology now enable Breath Biopsy to be
embedded into early stage clinical trials, allowing pharmaceutical companies
to perform biomarker discovery early on in the drug development process. This
enables the development of companion diagnostics which can be critical to
maximize the chance of achieving regulatory approval.
explore this.
Similarly, VOC biomarkers offer a non-invasive means to accurately stratify
inflammatory bowel disease patients to assist with therapy selection (see also
page 40).
VOC biomarkers that predict therapy response can improve treatment efficacy
within the intended population and also lead to development of an easily
deployable breath-based test for identification of responders.
www. owlstonemedical.com/ddw
25
Metabolites relate to
actual disease
activity
DNA provides the blueprint for disease predisposition, but metabolites give
information on the current state of tissues and cells, and the health of the
individual.
Altered metabolism occurs at the earliest stages of disease, and magnifies
changes at the genomic, transcriptomic and proteomic levels several thousand
fold.
Tracking changes in the VOC profile in breath over time could provide an early
warning of changes in the underlying disease state.
www. owlstonemedical.com/services
27
Asthma and COPD
As many as 334 million people globally have asthma and up to 14% of the
worlds children experience asthma symptoms [56]. Asthma management is
focused on achieving control of symptoms to minimize the risk of future
exacerbations.
28
VOC Biomarkers for improved asthma diagnostics
One breath based biomarker, FENO (fractional exhaled nitric oxide), is already
being used to support asthma diagnosis. FENO is useful because it reflects a
protective biochemical pathway of the lungs, but it does lack specificity, as it is
a single biomarker affected by many processes other than asthma.
A study of VOCs in asthma found that they outperform FENO and lung function
tests in discriminating asthmatics and healthy controls. Furthermore,
combining VOCs with FENO gave an even higher diagnostic performance [57].
It has also been shown that VOCs can be used to discriminate with a high
degree of accuracy between asthmatic children and those with transient
wheezing [58]. In both this and two other studies [59,60], increases in
methylated alkanes were associated with asthma prevalence: these VOCs are
potential markers of oxidative stress which have been linked to airway
inflammation.
There are also promising initial results from Owlstone Medicals NHS funded
LuCID clinical trial [34], which aims to recruit 3,000 patients and uses the
ReCIVA Breath Sampler as well as FAIMS technology (Lonestar VOC Analyzer)
to analyze patient breath samples.
Data collected so far indicates that GC-MS combined with FAIMS can
discriminate between asthmatic and non-asthmatic patients (AUC 0.92, Figure
13) in a cohort of patients under clinical suspicion of lung cancer (a
heterogeneous population with a wide variety of medical conditions). Such an
approach allows identification of disease specific volatiles greatly facilitating
translation to clinical practice.
www. owlstonemedical.com/asthma-vocs
29
ASTHMATIC vs. NON-ASTHMATIC PATIENTS
1.0
0.8
True Positive Rate
0.6
0.2
0.0
The existing evidence suggests that VOCs are strongly affected by the airway
inflammation that characterizes obstructive pulmonary diseases such as
asthma, and that they could be useful tools in non-invasive asthma diagnosis.
30
There is already evidence that shows VOCs can be used to predict steroid
responsiveness in mild to moderate asthma sufferers [61]. In this study, VOCs
were considerably more accurate at predicting responsiveness to steroid
treatment (AUC 0.88 0.16) than either FENO or measurements of eosinophil
cells from coughed up sputum samples (Figure 14).
1.0
Sensitivity
0.5
0.0
0.0 0.5 1.0
1-specificity
Figure 14. Predicting steroid responsiveness in steroid naive patients [61].
Blood based biomarker IgE has shown reasonable positive predictive potential
for treatment response to the targeted anti-IgE treatment
XOLAIR/Omalizumab [62]. Therapy failure still occurs in a high percentage of
adult and pediatric patients [63], however, and a significant fraction of patients
lose responsiveness to XOLAIR over time.
Additionally, a large portion of patients that would benefit from XOLAIR are
currently not eligible based on the IgE entry criteria. Similar issues have arisen
with the use of periostin and blood eosinophils to identify potential responders
to other targeted asthma therapies.
31
Promising initial results from the U-BIOPRED (Unbiased BIOmarkers in
PREDiction of respiratory disease outcomes) consortium project show that
VOCs measured using a FAIMS device could be used to stratify asthmatic
patients into treatment sub-groups. For example, VOCs discriminated between
anti-IgE-treated XOLAIR and non-treated severe asthma patients with 83%
accuracy [64,65].
www. owlstonemedical.com/strata
Oncology
Cancer has many complex and interacting causes, including genetic
predisposition, mutations in the genetic code, lifestyle, diet and many other
environmental factors. As metabolites are the conclusion of gene, transcription
and protein expression, they reflect gene-protein-environment interactions.
This unique position means they can provide an accurate representation of the
metabolic phenotypes of different cancers [66].
Tumor metabolomics
Cancer cells display altered metabolism from day one. The mechanisms by
which oncogenes reshape a range of metabolic pathways to meet the
nutritional demands of cancer cells is beginning to be elucidated [67].
One of the best described metabolic changes is the Warburg effect: cancer
cells have glycolytic rates up to 200 times above normal cells, even when
sufficient oxygen is present. This metabolic change is key to cancer survival as
it provides the building blocks a growing tumor requires. The Warburg effect is
a major contributor to a cancers volatile signature [68] and also affects the
profile of VOCs in breath [69].
32
Since altered metabolites are present in the first stages of disease, this enables
earlier detection and diagnosis. As disease prognosis is greatly dependent on
cancer stage and tumor type, these factors make metabolites ideal biomarkers
for cancer.
The search for VOC signatures associated with cancer has produced studies
looking at breath VOCs related to many cancers, including lung, breast,
colorectal, gastric, ovarian, liver, head and neck and malignant mesothelioma
[70].
www. owlstonemedical.com/reciva
Lung cancer
Lung cancer is the most common cancer worldwide and also one of the
deadliest, with around 1.8m cases and 1.6m deaths each year. It has one of the
lowest 5-year survival rates of all cancers, at just 17.7% in the US.
However, early diagnosis can greatly improve the clinical outlook. Patients
diagnosed at Stage 1 in the US have a 55% 5-year survival rate, compared to
just 4.3% at stage IV [71].
33
A number of studies have already found evidence that lung cancers can be
detected based on VOCs present in breath [72], but a lack of sampling
standardization and underpowered clinical trials has so far prevented the
introduction of a breath test for lung cancer.
Table 2. Lung cancer VOC biomarkers reported in the literature [44,7380], their approximate
concentrations in lung cancer patient breath, and the concentrations at which they have been
detected using the Lonestar VOC Analyzer (FAIMS).
To make lung cancer breath tests a reality, Owlstone Medicals LuCID (Lung
Cancer Indicator Detection) project is currently underway. LuCID includes
highly powered clinical trials for early detection of lung cancer, and is funded
by an SBRI Healthcare development contract.
www. owlstonemedical.com/lucid
34
Colorectal cancer
Colorectal cancer (also referred to as bowel cancer) is the fourth most common
form of cancer in the US, with around 135,000 new cases and 50,000 deaths
each year. It is the second most common cause of death from cancer in the US,
and has a mortality rate of around 35% within five years of diagnosis.
As with many other types of cancer, the stage at which colorectal cancer is
diagnosed has a huge impact on the patient's prognosis. Patients identified at
Stage I have around a 90% five-year relative survival rate, which drops to 13.5%
if the cancer has metastasized (stage IV) [81].
To increase the the number of early diagnoses, the way in which patients are
screened for colorectal cancer needs to be improved. Existing non-invasive
colorectal cancer screening methods such as gFOB (guaiac based fecal occult
blood) and the now more common FIT (Fecal Immunochemical Testing) show
relatively low sensitivities (FIT sensitivity = 66 - 88%, specificity = 87 - 96%). FIT
also lacks sensitivity to advanced adenoma (27 - 41%, specificity = 91 - 97%).
35
PERCENTAGE OF POPULATION SCREENED
FOR COLORECTAL CANCER USING FECAL TESTING
USA
8% of 89 million population screened with FIT
Age range* 50-74 years old
UK 8%
SCREENED
FRANCE
42% of 19 million population screened with FIT
Age range* 50-74 years old
GERMANY
18% of 28 million population screened with FIT
Age range* >56 years old
ITALY 10M
60%
SCREENED
18%
SCREENED
28M
27% of 16 million population screened with FIT
Age range* 50-69 years old 42%
SCREENED
SPAIN 19M
27%
SCREENED
JAPAN
17% of 75 million population screened with FIT
Age range* >40 years old
CHINA 1%
SCREENED
INDIA 2%
SCREENED
75M
36
There is clear need for a new, non-invasive screening system with the potential
for high patient compliance and low cost. Studies have previously identified at
least 15 discriminating VOCs that can be used to identify between patients with
and without colorectal cancer (e.g. [82]). It has also been shown that breath
VOCs can be used to discriminate patients with colorectal cancer from those
with other cancers, specifically breast, lung and prostate [83].
There is also evidence that VOCs from urine can be used to discriminate
between patients with and without colorectal cancer [84]. Arasaradnam et al.
found that Owlstone Medicals FAIMS technology can be used to differentiate
between patients with colorectal cancer and healthy controls, with a sensitivity
of 88%, by rapid, non-invasive analysis of volatile organic compounds (VOCs)
in urine (Figure 16 and Figure 17).
These results show that detection of colorectal cancers VOC profile with
FAIMS from patient urine samples can prove valuable as part of a non-invasive
screening program for colorectal cancer.
60
40
20
0
-6 -4 -2 0 2 4
COMPENSATION VOLTAGE (V)
Figure 16. Lonestar VOC Analyzer FAIMS spectrum from urine sample of patient with colorectal
cancer.
37
FAIMS DISCRIMINATES BETWEEN PATIENTS
WITH COLORECTAL CANCER AND CONTROLS
0.006
CRC
CONTROL
0.004
DISCRIMINANT ANALYSIS
0.002
-0.000
-0.002
-0.004
-0.006
1 2
GROUP
Figure 17. To determine whether the patients with colorectal cancer could be distinguished from
the other healthy volunteers using the Lonestar VOC Analyzer (FAIMS), Arasaradnam et al. used
Fisher Discriminant Analysis on the FAIMS data collected from the urine samples of patients with
colorectal cancer and controls. They found that it was possible to use the entire VOC profile
measured with FAIMS to detect colorectal cancer cases with 88% sensitivity.
To investigate the performance of both breath and urine VOCs in a larger at risk
population of using FAIMS to screen for colorectal cancer, Owlstone Medical
have launched a 1,400 patient clinical trial in collaboration with Warwick
University and the University Hospital Coventry and Warwickshire NHS Trust.
Known as InTERCEPT, the trial aims to evaluate the accuracy of both breath
and urine VOC biomarkers in the detection of colorectal cancer at an early
stage, when patient survival rates are at their highest.
InTERCEPT
The worlds largest breath-based study for
early detection of colorectal cancer
www. owlstonemedical.com/intercept
38
Inflammatory diseases
The term inflammatory disease covers an array of disorders and conditions
that are characterized by the bodys complex inflammatory reaction to harmful
stimuli, including irritants, pathogens and damaged cells.
The cause of IBD is not entirely understood, but it is widely believed to be the
result of complex interactions between an individuals genetic susceptibility,
environmental triggers (e.g. diet, lifestyle, etc.) and the influence of an
individuals gastrointestinal bacterial colonies.
At present the diagnosis of IBD and the differentiation of disease types takes a
multi-modal approach that includes clinical, histological, serological,
radiological and endoscopic observations [86].
39
Accurate disease stratification is vital for determining the best treatment
strategy, but at present 10 - 15% of patients elude correct categorization.
Accurate phenotyping often requires endoscopy of the intestine, which high
cost, uncomfortable, has high non-compliance rates and can result in
complications such as perforation of the bowel [87,88].
This was used to classify samples and calculate sensitivities and specificities as
part of a 10-fold cross-validation. This box plot (Figure 18) shows the predictive
power of IBD (UC and CD) vs. controls, UC vs. controls, CD vs. controls and
finally UC vs. CD.
0.8
0.6
0.4
0.2
0.0
UC & CD V UC V CD V UC CD
Figure 18. Predictions of classifier to different combinations of diseases and controls (UC
ulcerative colitis, CD Crohns disease, V volunteer (healthy control)). The study used the
Lonestar VOC Analyzer (FAIMS) to analyze VOC biomarkers in breath [16].
40
The analysis showed that patients with IBD could be distinguished from control
patients using FAIMS analysis of VOCs in breath samples with a sensitivity of
74% (95% confidence intervals (CI): 0.650.82) and specificity of 75% (95% CI:
0.530.90), p-value 6.2 107. The AUC (area under curve) was 0.82 (95% CI:
0.74-0.89) (Figure 19).
FAIMS could distinguish those with UC from those with CD with a sensitivity of
67% (95% CI:0.540.79) and specificity of 67% (95% CI: 0.540.79), p-value
9.23 104. The AUC was 0.70 (95% CI: 0.600.80).
0.6
0.4
0.2
0.0
SPECIFICITY
Figure 19. Receiver operator curve (ROC) plot of IBD (UC and CD) vs. healthy controls.The study
used the Lonestar VOC Analyzer (FAIMS) to analyze VOC biomarkers in breath [16].
The study confirms the utility of FAIMS exhaled VOC analysis to distinguish IBD
from healthy controls, and UC from CD. It conforms to other studies using
different technology, whilst affirming exhaled VOCs as biomarkers for
diagnosing IBD.
www. owlstonemedical.com/lonestar
41
Liver disease
The link between liver diseases and characteristic breath aromas has been
known to clinicians for thousands of years. Hippocrates (460370 BC)
famously described fetor hepaticus in his treatise on breath aroma and disease
[1].
Liver diseases including non-alcoholic fatty liver disease and viral hepatitis can
progress to cirrhosis and eventually hepatocellular carcinoma. Early diagnosis
and treatment of liver diseases is important because progression is common
and has a significant risk of mortality.
Hepatic encephalopathy
Owlstone Medicals FAIMS technology has already been utilised to analyse the
breath of patients with hepatic encephalopathy (HE). HE is a neuropsychiatric
condition which occurs when the liver cannot adequately remove toxins from
the blood. It can occur suddenly in people with acute liver failure but is more
often seen in those with chronic liver disease.
42
In the pilot study [15], the chemical fingerprint of breath samples from patients
with HE and controls was analyzed using a Lonestar VOC Analyzer fitted with
an ultra violet ionisation source. 42 patients were recruited; 22 patients with HE
(13 covert and 9 with overt HE) and 20 healthy controls.
HE vs. CONTROLS
0.6
0.4
0.2
0.0
SPECIFICITY
Figure 20. Receiver operator curve (ROC) plot for distinguishing HE from controls. The study used
the Lonestar VOC Analyzer (FAIMS) to analyze VOC biomarkers in breath [15].
FAIMS analysis of exhaled VOCs was able to classify HE patients from controls
with a sensitivity and specificity of 88% (0.730.95) and 68% (0.510.81)
respectively with an AUC of 0.84 (0.750.93).
This pilot study provides evidence that breath VOC biomarker analysis has
potential as a diagnostic aid in distinguishing HE of all grades from healthy
control subjects, and further suggests potential for VOC biomarkers as an aid
to distinguish between the covert and overt forms of HE.
www. owlstonemedical.com/reciva
43
Infectious disease
Infectious disease mortality can be lowered by correctly targeting
antimicrobial drugs early on in the progress of an infection [91]. At present,
most treatment is untargeted due to a lack of adequate diagnostics.
Tuberculosis
There are around 8.6 million new cases, and 1.3 million deaths globally from
tuberculosis (TB) every year, a disease caused by the bacterium
Mycobacterium tuberculosis. Diagnosis is still mostly performed by trained
technicians examining sputum samples under a microscope [97].
A recently published pilot study by Sahota et al. [14] found that Owlstone
Medical's Lonestar VOC Analyzer (FAIMS) was able to distinguish between
healthy controls and patients with either pulmonary or extra-pulmonary
tuberculosis (the latter type of the disease traditionally more difficult to
diagnose). They analyzed the FAIMS data from patient breath samples using a
wavelet feature extraction process followed by a random forest classifier, to
identify features in FAIMS spectra. This allowed discrimination of cases from
controls with a sensitivity of 81% and a specificity of 79%.
44
CLASSIFICATION OF CONTROL AND TB GROUPS
0.8
CONTROL
0.7
TB
0.6
PREDICTIVE PROBABILITY
0.5
0.4
0.6
0.2
0.1
1 2
GROUPS
Figure 21. Classification probabilities for the control and TB groups in pilot study using a Lonestar
VOC Analyzer by Sahota et al. Applying a Wilcoxon rank-sum test to the sets of classification
probabilities from the two groups, we get a p-value of 2.89E6, showing that there is a highly statis-
tically significant difference between the control and TB groups. The study used the Lonestar VOC
Analyzer (FAIMS) to analyze VOC biomarkers in breath [14].
TB vs. CONTROLS
1.0
0.8
SENSITIVITY
0.6
0.4
0.2
0.0
SPECIFICITY
Figure 22. Receiver Operator Curve (ROC) plot from Sahota et al. (AUC = 0.92; 95% CI: 084, 1) for
breath analysis of patients with TB. The study used the Lonestar VOC Analyzer (FAIMS) to analyze
VOC biomarkers in breath [14].
45
Detection of TB in patients with negative ZN-smear tests
A team from the Pulmonology Department at the Academic Medical Centre,
University of Amsterdam were interested to test whether breath biomarkers
could detect TB in patients suspected to have TB, but who displayed negative
ZN-smear tests.
The results of these pioneering studies indicate that FAIMS, in particular, has
potential as a technology that can be applied to breath testing for infectious
diseases including TB.
www. owlstonemedical.com/lonestar
Clostridium difficile
C. difficile is a bacterium that can infect the bowel, causing diarrhea. It
commonly affects the elderly and patients who have recently received
antibiotics. In recent years, the infection has become increasingly prevalent,
and more severe forms of the disease have emerged. More serious symptoms
include severe pseudomembranous colitis and toxic megacolon. Large hospital
outbreaks have required ward closures and extensive infection control
measures.
While the application of breath VOC biomarkers has not yet been investigated
for C. difficile diagnosis, there is evidence for the utility of VOCs emitted from
stool samples. VOC biomarkers emitted from stool samples could accurately
distinguish C. difficile-positive stool samples from healthy samples.
46
FAIMS SPECTRUM OF A C. DIFFICILE-POSITIVE
STOOL SAMPLE
100
0.001
0.01
0.1
1
80
Ion Current
(arbitary units)
DISPERSION FIELD (%)
60
40
20
0
-6 -4 -2 0 2 4
COMPENSATION VOLTAGE (V)
Figure 23. FAIMS spectrum of a C. difficile-positive stool sample. Stool samples were analyzed
using a Lonestar VOC Analyzer (FAIMS) fitted with an ATLAS headspace sampler [46].
Training and test samples (n=135) were used to identify which characteristics
discriminate between positive and negative samples, and to build machine
learning algorithms interpreting these characteristics.
The best performing algorithm was then validated on new, blinded validation
samples (n=78). The predicted probability of C. difficile (as calculated by the
algorithm) was compared with the microbiological test results (direct toxin test
and culture).
47
C. DIFFICILE POSITIVE vs. NEGATIVE SAMPLES
ROC curve
1.0
0.8
SENSITIVITY
0.6
0.4
0.2
0.0
Figure 24. The Receiver Operating Characteristic (ROC) curve for the (toxin and culture) negative
and (toxin and culture) positive samples. Samples analyzed within 7 days are included. Stool
samples were analyzed using a Lonestar VOC Analyzer (FAIMS) fitted with an ATLAS headspace
sampler [46]
This pilot study shows that FAIMS analysis of unprocessed stool samples can
differentiate between C. difficile-positive and negative samples with high
diagnostic accuracy. FAIMS analysis is also quick, easy to use, relatively cheap,
and can be used on the ward. FAIMS may be the ideal point-of-care test for C.
difficile in stool samples, reducing delays in diagnosis and treatment.
www. owlstonemedical.com/atlas
48
How to analyze
VOC biomarkers
The era of modern breath testing started in 1971, with the work of Nobel Prize
winner Linus Pauling [3]. Since then, hundreds of scientific papers have been
published suggesting the presence of VOC biomarkers across a range of
diseases (Figure 12). There is limited agreement, however, in identified
biomarkers within a disease across these published studies.
49
HISTORICAL CHALLENGES
Figure 25. Historical challenges that have hampered breath analysis, leading to the development of
new technologies that enable the identification of robust VOC biomarkers in breath.
50
CHALLENGES AND SOLUTIONS IN BREATH SAMPLING AND VOC ANALYSIS
Breath High-end analytical instruments e.g MS: Microchip chemical sensor (FAIMS):
analysis Expensive and bulky Easier to operate than MS approaches
Inter-instrument variability Expertise required to Superior robustness and reproducibility
operate correctly Improved stability, sensitivity and selectivity
Limited throughput compared to eNose systems
Challenging to deploy at point-of-care Small size facilitates deployment at
point-of-care
Electronic Nose (eNose):
Limited temporal stability
Poor chemical selectivity
Table 3. Overview of historical challenges to the adoption of breath sampling, and solutions offered
by Owlstone Medical's Breath Biopsy technology.
Study design
As with any omics technique, the identification of robust VOC biomarkers in
breath involves broadly three stages:
1. Discovery
2. Verification
3. Validation
The above stages encompass the studies involved in discovering the optimal
classifier, verifying the classifier in a larger patient cohort, and performing an
external blinded validation in a larger patient cohort representative of the
intended use population, to validate both the VOC biomarker algorithm and
the analytical instrumentation that together comprise a diagnostic test ready
for regulatory approval.
51
Tips for a successful breath biomarker study
Use a robust, reproducible, standardized breath sampling protocol.
Select a patient cohort representative of the intended use population.
Record potential confounding factors in a Case Report Form (CRF)
accompanying the study, and assess systematic bias or confounders that
might impact the biomarker signal.
It is vital that all breath samples in the study should be collected using a robust,
reproducible, standardized breath sampling protocol (see section on Reliable
collection of breath samples on page 54).
There are three complementary approaches that can be taken to minimize the
impact of potential variables on your study outcome, and maximize the ability
to detect a biological signal:
1. Selection criteria for subjects should reflect the actual population in which
the test is to be used as closely as is practically feasible. This means potential
confounders are less likely to impact biomarker discovery.
2. It is crucial to assess systematic bias or confounders that are unevenly
distributed between case and control groups that might impact the
biomarker signal, hence potential confounding factors should be detailed in
a Case Report Form (CRF) accompanying the study. Clinical background
data can be used to assess the accuracy with which the breath biomarkers
can identify the presence of certain confounders.
3. Signal-to-noise ratio should be maximized by reducing background noise
(e.g. using a clean air supply), and using a well standardized and validated
breath collection method and analytical pipeline.
52
FACTORS THAT CAN CONTRIBUTE TO THE VOC PROFILE
GENDER
AGE BMI
SMOKING DRUGS
Figure 26. Factors that can contribute to the VOC profile [100]. It is crucial to assess systematic
bias or confounders that are unevenly distributed between case and control groups that might
impact the biomarker signal, hence potential confounding factors should be detailed in a Case
Report Form (CRF) accompanying the study.
Cohort size
Many of the breath analysis studies reported in the literature involve small
patient cohorts, which limits the statistical significance of the findings, and
makes it challenging to investigate the impact of possible confounding factors.
The sample size needed to reach statistical significance depends both on the
inter-sample variation, and on the expected amplitude of differences the study
is intended to show. In a cohort including a diverse set of human individuals,
the population is likely to be fairly heterogeneous with a high degree of
biological variation.
Patient population
Care should be taken to select not only the optimal cohort size, but also the
appropriate patient population. This patient population is likely to vary
according to the stage of the study.
53
Initial discovery or pilot studies may involve more clearly defined and
homogeneous groups. Whilst this increases chances of finding a signal, there is
a trade off with the external validity of these results which should be carefully
considered. Trying to focus on a population representative of the intended use
population is key to minimize this risk.
In some cases, e.g. screening, the target condition only occurs infrequently in
the population in which the breath test is to be used. In these cases, recruiting
an enriched population is often an effective way of running the discovery phase
of the study. However, in order to obtain the evidence required for regulatory
approval, the validation study will always need to be in the intended
population.
Breath is collected continuously over a ~10 minute time period using the
ReCIVA Breath Sampler, and VOCs are pre-concentrated onto sorbent tubes
for analysis using the Lonestar VOC Analyzer which incorporates the proven
FAIMS microchip chemical sensor technology.
Figure 27. Workflow for VOC biomarker collection and analysis in breath. *Sorbent tubes must be
thermally desorbed prior to VOC analysis. Abbreviations: GC (Gas Chromatography), LC (Liquid
Chromatography), MS (Mass Spectrometry).
54
The ReCIVA Breath Sampler provides a much-needed solution to the
challenges of breath using containers such as Tedlar bags which have been
shown to result in the loss of sampled VOCs [101]. Instead, the ReCIVA Breath
Sampler collects breath samples onto stable sorbent tubes, which can be
analyzed immediately or stored for later transport under ambient conditions to
an off-site laboratory for analysis. Sorbent tubes are used by National Institute
for Occupational Safety and Health (NIOSH), and are the accepted industry
standard.
4000
3000
2000
1000
0
22 23 24 25 26 27
RETENTION TIME (MINS)
ii)
BREATH COLLECT B
7000
SORBENT TUBE 1
6000 SORBENT TUBE 2
5000
INTENSITY
4000
3000
2000
1000
0
22 23 24 25 26 27
RETENTION TIME (MINS)
iii)
7000 BREATH COLLECT A
BREATH COLLECT B
6000
5000
INTENSITY
4000
3000
2000
1000
0
22 23 24 25 26 27
RETENTION TIME (MINS)
Figure 28. Chromatogram of VOCs in breath samples collected on independent sorbent tubes
using ReCIVA and analyzed using GC-FAIMS (Lonestar VOC Analyzer). High reproducibility is
observed within a single breath collect (i and ii). Small changes in the chromatogram are observed
between different breath collects performed on the same individual (iii), which is to be expected
because some VOCs in breath do vary over time.
55
REPRODUCIBLE MEASUREMENT OF VOCs
IN BREATH WITH ReCIVA AND FAIMS
12 12
10 10
6 6
4 4
2 2
0 0
1 2 SORBENT 1 2 SORBENT
TUBE TUBE
BREATH COLLECT A BREATH COLLECT B
Figure 29. D-limonene in breath samples collected on independent sorbent tubes (1 and 2) using
ReCIVA and analyzed using GC-FAIMS (Lonestar VOC Analyzer) shows high reproducibility within
a single breath collect. Small changes in D-limonene peak area are observed between different
breath collects (A and B, performed on the same individual), which is to be expected because some
VOCs in breath do vary over time.
Pressure and CO2 sensors in the ReCIVA Breath Sampler provide real-time
monitoring of patient breathing, allowing specific breath fractions to be
selected (e.g. enriched bronchial vs. end-tidal). ReCIVA is designed to be used
with the CASPER clean air supply, in order to ensure breath samples are not
contaminated by external VOCs.
Named Invention of the Year in the 2017 Top 50 in Digital Health Awards,
ReCIVA is the first breath sampler able to provide an out-of-the-box means to
reproducibly capture breath samples for analysis.
56
ReCIVA is CE marked and is already in use in over 60 academic research
groups and clinical labs around the world. In addition, a paediatric ReCIVA
breath sampler enables breath collection from children as young as 5.
ReCIVA is currently being deployed as part of the NHS funded lung cancer
detection project (LuCID) - the worlds largest breath-based study ever
https://www.owlstonemedical.com/clinical-pipeline/lucid/
https://www.owlstonemedical.com/clinical-pipeline/lucid/
www. owlstonemedical.com/reciva
Owlstone Medical has incorporated its FAIMS technology into the Lonestar
VOC Analyzer for medical applications, enabling improved stability and
sensitivity compared to eNose systems. In addition, the Lonestar VOC Analyzer
is easier to use than mass spectrometry approaches, and offers superior
robustness and reproducibility, due to its excellent intra-device stability (Figure
30) and ability to selectively detect VOCs in complex biological samples
containing thousands of chemical species.
57
FAIMS spectra are very information rich. This richness is due to the fact that the
technique directly measures the presence and concentration of distinct ionized
molecular species, as opposed to eNose chemical sensors which perform
pattern recognition based on chemical interactions between the sample and
sensor. Multivariate data analysis techniques allow for simple interpretation of
complex FAIMS data to classify different patient groups through highly
sensitive, highly specific detection of relative biomarker concentrations.
Clinical applications
The FAIMS chip is also programmable using powerful software which can
incorporate a range of clinical information. This makes the FAIMS platform
suitable for a wide range of medical applications, some of which are detailed in
a recent review [13].
The small size of the FAIMS microchip enables analysis of samples with
increased throughput, as required for applications in clinical diagnostics, and
will also facilitate development of a device for deployment at point-of-care.
FAIMS, along with the ReCIVA Breath Sampler, is being used in large clinical
trials relating to lung cancer (LuCID), colorectal cancer (InTERCEPT) and
https://www.owlstonemedical.com/clinical-pipeline/lucid/ https://www.owlstonemedical.com/clinical-pipeline/intercept/
https://www.owlstonemedical.com/clinical-pipeline/lucid/ https://www.owlstonemedical.com/clinical-pipeline/intercept/
asthma (STRATA).
https://www.owlstonemedical.com/clinical-pipeline/strata/
https://www.owlstonemedical.com/clinical-pipeline/strata/
140
120
100
METHYL SALICYLATE
INTENSITY
D-LIMONENE
80
60
40
20
0
JANUARY FEBRUARY MARCH
TIME
Figure 30. FAIMS technology shows excellent reproducibility. Standard compounds were loaded
onto sorbent tubes at a defined amount and analyzed using GC-FAIMS (Lonestar VOC Analyzer).
The experiment was repeated regularly to assess the variation over time. Data for two compounds
over a three month period is shown. The relative standard deviation is 6.1 % for methyl salicylate and
3.4 % for D-limonene.
www. owlstonemedical.com/lonestar
58
Quality Control
Consistency in the breath collection and VOC analysis workflow is key to
obtaining reliable data for biomarker discovery. In this section we provide an
overview of how we monitor the steps in the workflow to identify any outlier
samples and sources of technical variation.
The pressure and CO2 levels within the ReCIVA are monitored throughout
collection to warn the operator of leaks from the mask, hyperventilation,
hypoventilation, shallow or otherwise irregular breath profiles that negatively
impact VOC collection. Feedback is given in real-time to correct poor
collection technique. Breath profiles and sampling time are also stored along
with other patient metadata to inform subsequent data analysis.
Occasionally, it may not be possible collect the required volume of breath in the
collection time specified for the project. In this case, the breath sample can still
be analyzed, but this sample is flagged in case the reduced breath volume may
affect the results.
Sorbent tubes are checked prior to breath collection, and blank samples are
regularly tested, to ensure that any background is below a threshold level
(Figure 32). We use a set of standard compounds with a range of
concentrations to check the linearity (Figure 31), limit of detection and
signal-to-noise ratio.
The VOC profile of each patient sample is also inspected for the presence of
compounds that we expect to be present above certain thresholds in a patient
breath sample. This QC serves as an initial check that the analysis has been
successful, before initiating data analysis.
59
FAIMS SHOWS EXCELLENT REPRODUCIBILITY
AND LINEARITY DOWN TO 70 PPT
3.5
3.0
2.5
1.5
1.0
0.5
0.0
0 1 2 3 4 5 6
D-LIMONENE STANDARD ON-COLUMN MASS (ng)
Figure 31. Calibration plot of D-limonene standard applied to sorbent tubes at a range of amounts
and measured using GC-FAIMS (Lonestar VOC Analyzer). Excellent linearity is observed, enabling
accurate quantification down to 0.2 ng on-column mass which equates to around 70 parts per
trillion (ppt) in breath, with the potential to detect D-limonene down to 35 ppt in breath. Good
precision is demonstrated; data shown is the mean of measurements from 4 - 6 different sorbent
tubes analyzed on 4 different days (error bars represent 1 standard deviation).
7.28E+07
5.46E+07
3.64E+07
BLANK COLLECT
1.82E+07
0
21.6539 21.7 21.8 21.9 22 22.1 22.2 22.277
RETENTION TIME (MINS)
Figure 32. Comparison of the abundance of D-limonene measured in a breath sample, an ambient
air sample or a blank collect sample. All samples were collected using the ReCIVA Breath Sampler
and analyzed using GC-MS. The breath and blank collect samples were collected using the CASPER
clean air supply (in the case of the blank collect sample, breath collect is mimicked). The ambient
air sample was collected by sampling ambient air without the CASPER clean air supply. This
demonstrates the importance of using a clean air supply to avoid contamination by VOCs in the
environment, and measuring the background VOC levels using a blank collect to ensure low level
VOCs are distinguishable from any system-related background.
60
Data analysis
In order to identify VOCs that are potential biomarkers for disease, the raw
spectrometry data must be processed and analyzed. To facilitate this process,
we have developed a data analysis pipeline that takes raw spectrometry data,
patient clinical and outcome data, develops classification algorithms, and
ultimately outputs a classification algorithm that uses multiple biomarkers to
accurately identify patients by disease presence, therapy response or
outcome (Figure 33).
Raw data
Build statistically comparable test and training data sets
Classification
algorithm development
using machine
learning approach
Apply classifiers to
Test Set
Classifier evaluation
Identify confounders
and any sources
of technical variation
Classifier refinement
Iterative validation and
refinement of features
and classifiers
Classifier evaluation
Sensitivity, specificity
and statistical significance
In silico verification
Classifiers are verified using
our proprietary database
of 1000s of patient
VOC profiles
Verification
Verification of the successful classifier in a larger cohort
61
Training and test data sets
The first step is to build training and test data sets that are statistically
comparable, where any confounding factors are represented equally. Both
training and test data sets then undergo unsupervised data pre-processing to
generate calibrated retention times and identify features (peaks in the FAIMS
or mass spectrum data, or absence thereof) that can be used for classifier
development. Any features that are system-related are excluded.
Acetone DCM
Isoprene
Acetonitrile
2-methyl
E-2-pentene
butane
Figure 34.
Classifier development
Using the training data set, a machine learning approach is taken to train a
learning algorithm and develop classifiers using 10-fold stratified cross
validation [103]. The classifiers are applied to the test data set, and a
systematic study of different sources of bias is performed, in order to identify
any sources of technical variation and to verify that the classifier is robust to
confounding factors. The features and classifiers are then further refined
through an iterative process.
Classifier evaluation
In order to select the best performing classifier, classifier performance is
evaluated in terms of sensitivity, specificity and area under the ROC curve
(Figure 35). In the case of on-going studies, we also assess whether the
number of patients is sufficient for statistical significance, or whether the study
would benefit from recruitment of additional patients.
62
Did you know?
Our Biomarker Services include extensive data analysis including classifier
development and in silico verification using Owlstone Medicals database
containing thousands of patient VOC profiles.
In silico verification
We use a proprietary database containing thousands of anonymized patient
VOC profiles to verify classification algorithms in silico. We assemble virtual
patient populations defined by demographics, comorbidity, and outcome, and
apply the classifier to gain early evidence of likely clinical performance, without
the need for additional samples or studies. This step can also identify
potentially confounding variables or relevant sub-populations that may not
have been adequately represented in the original trial population.
THRESHOLD
300 1.0
CONTROL DISEASE
250
True Positive Rate
0.8
g
Frequency
200 sin
es
0.6 gu
om
150 nd
ra
of
0.4 line
100 ce
en
fer
re
0.2
50
0 0.0
0 2 4 6 8 10 12 0 0.2 0.4 0.6 0.8 1.0
Signal False Positive Rate
Figure 35. Adjusting the threshold for discrimination between control and disease states enables
the positive and negative predictive values of the test to be optimized.
Verification
Analysis of candidate VOC biomarkers is performed in an independent patient
cohort to verify the performance of the classifier. In this situation, the same
data analysis pipeline will be applied to analyze the the list of candidate
biomarkers identified in the biomarker discovery phase, and further refine the
classifier.
Validation
Following successful verification of the classifier, the classifier is locked down
and an external blinded validation is performed in a larger patient cohort
representative of the intended use population, to validate both the VOC
biomarker algorithm and the analytical instrumentation that together comprise
a diagnostic test ready for regulatory approval.
63
Beyond Breath: VOC Analysis
in other Biospecimens
In addition to breath, Owlstone Medical have also developed robust workflows
for collection and analysis of VOCs in other biospecimens (Figure 36).
Figure 36. Workflow for VOC biomarker collection and analysis for biofluids and other
biospecimens. Abbreviations: GC (Gas Chromatography), LC (Liquid Chromatography), MS (Mass
Spectrometry).
www. owlstonemedical.com/atlas
64
Biomarker
Services
Bring Breath Biopsy to your own research.
www. owlstonemedical.com/services
65
Outsource your Biomarker Discovery
Owlstone Medicals Biomarker Services enable academics, clinicians and
pharmaceutical companies to identify novel breath biomarkers for diagnostics
as well as precision medicine applications including therapeutic response,
patient stratification and outcome prediction.
We offer CRO capabilities to set up and run clinical trials, drawing on our
experience of running VOC biomarker clinical trials as part of our own clinical
pipeline.
Broadly, VOC biomarker discovery studies follow the stages outlined in Figure 37.
VOC BIOMARKERS:
FROM DISCOVERY TO VALIDATION
Discovery Verification Validation
Analyze full spectrum of Analyze candidate VOC Validation of defined VOC
VOCs in a small cohort biomarkers to verify biomarker algorithm and
and develop classifier. classifier performance analytical instrumentation
in a larger cohort. in large patient cohort
representative of the
intended use population.
Figure 37. Stages of a typical VOC biomarker discovery and validation project. During discovery
and verification, the classifier is further verified in silico using a population assembled from
Owlstones proprietary database of patient VOC profiles. Following successful verification of the
classifier, the classifier is locked down and an external blinded validation is performed in a larger
patient cohort representative of the intended use population, to validate both the VOC biomarker
algorithm and the analytical instrumentation that together comprise a diagnostic test ready for
regulatory approval.
Collect
We provide full guidance on optimal collection of breath and other biospecimens. We
install ReCIVA Breath Sampler in clinic and provide training and on-going support.
Analyze
Samples are analyzed using our proven, highly sensitive, FAIMS technology in
state-of-the-art laboratories, generating rich chemical spectral data.
Discover
Our experts identify discriminating features from spectral data and develop robust
classification algorithms using sophisticated machine learning approaches.
Report
Our in-depth report provides the results of our analysis as well as complete raw FAIMS
data. Our analysis includes in silico verification of the classifier using our proprietary
database containing thousands of VOC profiles.
Figure 38.
66
Study design
Prior to initiating a Biomarker Services project, we recommend arranging a
consultation with our team of clinical and technical experts to discuss the study
design. Taking advantage of the experience and knowledge we have developed
through our own clinical programs, we can provide advice on the optimal study
design to ensure that your project has the maximum chance of being
successful (read more on page 51).
Breath collection
We ensure that breath samples are collected reliably and reproducibly by
installing the ReCIVA Breath Sampler at clinical sites, and providing training for
trial nurses to ensure optimal breath collection. Breath samples are
pre-concentrated on stable sorbent tubes and shipped to Owlstone Medicals
service laboratory for analysis.
67
Extensive QC checks are part of our Quality Management System, which covers
the entire analysis workflow starting with preparation of the sorbent tubes for
breath collection. We perform a number of daily and weekly QC checks to
monitor a range of instrument parameters, and use blanks and standard
compounds to monitor background, sensitivity, limit of detection and
signal-to-noise ratio.
Biomarker discovery
Our analysis experts develop robust classification algorithms from rich spectral
FAIMS data, using a sophisticated machine learning approach. Our clinical
experts are on hand to provide biological interpretation of the results in
relation to clinical outcomes. Our data analysis pipeline is designed to take raw
spectrometry data and output a classification algorithm that uses multiple
biomarkers to accurately identify patients by disease presence, therapy
response or outcome.
Classifiers are developed using a training set, and then applied to a test set.
Classifier development includes a systematic study of different sources of bias,
to identify any sources of technical variation and ensure that the classifier is
robust to confounding factors.
Using patient history, comorbidity and outcome information along with VOC
biomarker data we create tests to make actionable patient classifications that
can be deployed in clinic.
In silico verification
Classification algorithms are verified in silico using our proprietary database
containing thousands of patient VOC profiles, to provide early evidence of
likely clinical performance without the need for additional samples or studies.
Our proprietary database contains anonymized chemical spectra records from
all samples collected from trials in the Owlstone Medical clinical pipeline, as
well as Biomarker Service projects (subject to authorization).
68
Report
Our results are delivered in a format which is easily interpretable by a clinician.
We provide in-depth reports on the results of our statistical analysis including
principal component analysis, box plots (Figure 39) and receiver operating
characteristics curves (ROC). In addition, we provide the complete set of raw
FAIMS data for your research.
Our analysts, developers and clinical research staff will walk you through the
analysis and the results either in person or by phone. Our experts are on hand
to answer any questions that you may have about the current project or
possible next steps, including verification of the classifier, and validation of the
diagnostic test.
CLASSIFICATION PROBABILITIES
0.9
CONTROL
GROUP A
GROUP B
0.8
PREDICTIVE PROBABILITY
0.7
0.6
0.5
0.4
0.3
0.2
0.1
PC1 PC2
Figure 39.
Our experts are on hand to provide advice and guidance on the research
protocol, securing ethics, sample collection and storage and managing the
project to meet your individual requirements.
All data is blinded to our statisticians, and the blinding key is provided by an
independent clinician provides only after the test classification is reported,
allowing full confidence in the validity of the results.
69
Breath Biopsy
Products
Bring VOC Biomarker research to your own lab with our
complete range of products for breath collection and analysis.
www. owlstonemedical.com/reciva
70
CASPER Clean Air Supply
CASPER provides a consistent, portable supply of clean air, ideal for use with
the ReCIVA Breath Sampler.
Ensure breath samples are not contaminated by external VOCs
Remove the need for a clean air supply line
Portable to wherever the patient is in clinic
www. owlstonemedical.com/casper
Lonestar VOC Analyzer
The Lonestar VOC Analyzer is an easy to use instrument for the detection of
VOC biomarkers in clinical samples such as breath and other biospecimens.
Rapid, sensitive, and selective VOC analyzer
Based on proven FAIMS technology
Biomarker discovery and targeted analysis
www. owlstonemedical.com/lonestar
Use the ATLAS Headspace Sampler to analyze VOCs emitted from liquid or
solid clinical samples on the Lonestar VOC Analyzer.
71
Further reading
Search for publications, posters etc. on the resources page:
https://www.owlstonemedical.com/resources/
Ebook: Simulating human breath: The complete guide to generating VOC and
humidity calibration standards:
https://www.owlstonemedical.com/download/simulating-human-breath-complete-gui
de
72
LuCID Infographic:
https://www.owlstonemedical.com/media/uploads/files/LuCID_Infographic.pdf
STRATA Infographic:
https://www.owlstonemedical.com/media/uploads/files/STRATA-infographic.pdf
InTERCEPT Infographic:
https://www.owlstonemedical.com/media/uploads/files/INTERCEPT-infographic.pdf
73
References
https://www.owlstonemedical.com/media/uploads/files/Complete-guide-to-Breath-Biopsy.bib
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https://www.owlstonemedical.com/media/uploads/files/Complete-guide-to-Breath-Biopsy.bib
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government regulatory body for sale or use with different indications and restrictions in different countries. Readers are advised that
alternative products and services may be available from other manufacturers and service providers. Breath Biopsy and ReCIVA are
registered trademarks of Owlstone Medical Ltd. Tedlar is a registered trademark of E. I. du Pont de Nemours and Company.
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OWLSTONE MEDICAL LTD
Cambridge, UK
162 Cambridge Science Park
Milton Road
Cambridge CB4 0GH
London, UK
Unit 19
205 Richmond Road
London E8 3FF
OWLSTONE INC
Connecticut, USA
Owlstone Inc.
761 Main Avenue
Norwalk, CT 06851
OW-012042-MC - v1.0 - 04/2017
T: +1 203-908-4848
F: +1 203-908-4849
www.owlstonemedical.com