SERIES ‘‘LUNG CANCER’’
Edited by C. Brambilla
Number 7 in this Series
Exhaled biomarkers in lung cancer
I. Horva´th*
,#
,Z.La´za´r*, N. Gyulai*, M. Kollai
#
and G. Losonczy*
ABSTRACT: Lung cancer is the leading cause of cancer death. Results of therapeutic
interventions are particularly discouraging when the disease is discovered in an advanced
stage. Early diagnosis is limited by the fact that the disease usually develops asymptomatically
and available screening methods do not fulfil the requirements for reliable discrimination between
patients with lung cancer and subjects not suffering from the disease. Breath sampling is
completely noninvasive and provides a potentially useful approach to screening lung cancer.
Exhaled biomarkers contain both volatile and nonvolatile molecules. The profile of volatile organic
compounds is different in patients with lung cancer than in control subjects. In exhaled breath
condensate, the proteomic profile of breath from cancer patients differs from that of healthy
smokers. We reviewed the scientific evidence demonstrating that a unique chemical signature
can be detected in the breath of patients with lung cancer and that the exhaled breath biomarker
profile could aid clinical decision making.
KEYWORDS: Biomarker, electronic nose, exhaled breath, exhaled breath condensate, lung
cancer, smell
L
ung cancer is the leading cause of global
cancer death in both males and females.
According to the most recent projection of
global mortality, by 2030 it will emerge as the
third and the fifth leading cause of death in high-
and middle-income countries, respectively [1, 2].
Figures on disease outcome measures are very
discouraging as even with the most advanced
treatment strategies ,86% of lung cancer patients
die within 5 yrs of diagnosis. With early detec-
tion and treatment, however, the 5-yr survival
rate improves dramatically from 20% in patients
with stage III lung cancer to 70% in patients with
stage I disease [3]. Researchers, therefore, have
sought out screening tests to detect lung cancer in
the earliest stages and several promising new
approaches have been proposed for this purpose,
such as computer-assisted image analysis of chest
radiographs, spiral computed tomography (CT)
scanning, PCR-based assays of sputum and
fluorescence bronchoscopy [4–7].
Breath chemical tests have a broad spectrum of
applications ranging from the US Food and Drug
Administration-approved exhaled nitric oxide
fraction (FeNO) measurement to monitor the effect
of anti-inflammatory treatment in asthma, to
volatile organic compound (VOC) determination
and nonvolatile biomarker profiling in the cooled
breath sample called exhaled breath condensate
(EBC) [8–11]. Being completely noninvasive, sam-
pling of the breath allows clinicians and research-
ers to assess different body functions in a flexible
manner. Breath collection can be performed even
in very severe patients and also repeated within
short intervals. Therefore, breath testing is con-
sidered to be a potentially ideal candidate for
screening purposes. Besides widely known con-
stituents such as nitrogen, oxygen, carbon dioxide,
inert gases and water vapour, exhaled breath also
consists of thousands of volatile and nonvolatile
components, mainly in trace amounts, making
detection a challenging task. Application of highly
sensitive cutting-edge technologies in sample
analysis provides firm background for proper
evaluation of this type of human sample. The use
of innovative ‘‘-omics’’ technologies, including
proteomics, metabolomics, mass spectromics, gas
AFFILIATIONS
*Dept of Pulmonology, and
#
Institute of Human Physiology and
Clinical Experimental Research,
Semmelweis University, Budapest,
Hungary.
CORRESPONDENCE
I. Horvath
Semmelweis University
Dept of Pulmonology
Dio´sa´rok u. 1/c.
1125 Budapest
Hungary
E-mail: hildiko@elet2.sote.hu
Received:
Sept 16 2008
Accepted after revision:
Feb 10 2009
European Respiratory Journal
Print ISSN 0903-1936
Online ISSN 1399-3003
Previous articles in this series: No. 1: De Wever W, Stroobants S, Coden J, et al. Integrated PET/CT in the staging of nonsmall cell lung cancer: technical
aspects and resection for lung cancer. Eur Respir J 2009; 33: 201–212. No. 2: Rami-Porta R, Tsuboi M. Sublobar resection for lung cancer. Eur Respir J 2009; 33:
426–435. No. 3: McWilliams A, Lam B, Sutedja T. Early proximal lung cancer diagnosis and treatment. Eur Respir J 2009; 33: 656–665. No. 4: Sculier J-P,
Moro-Sibilot D. First- and second-line therapy for advanced nonsmall cell lung cancer. Eur Respir J 2009; 33: 916–930. No. 5: van Tilburg PMB, Stam H,
Hoogsteden HC, et al. Pre-operative pulmonary evaluation of lung cancer patients: a review of the literature. Eur Respir J 2009; 33: 1206–1215. No. 6: Brambilla E,
Gazdar A. Pathogenesis of lung cancer signalling pathways: roadmap for therapies. Eur Respir J 2009; 33: 1485–1497.
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 1 261
Eur Respir J 2009; 34: 261–275
DOI: 10.1183/09031936.00142508
Copyright
ERS Journals Ltd 2009
c

chromatography/mass spectrometry (GC-MS) and ion mobility
spectrometries, offers great potential for the field of exhaled
biomarker profiling [12]. Exhaled breath biomarkers have been
assessed to understand disease pathomechanism and also to aid
clinical decision making. For each purpose, completely different
strategies have been implemented, including the determination
of individual biomarkers and the recognition of signal patterns
created by undefined compounds. Although pattern recognition
is a challenging task from the statistical point of view, it is a
powerful tool to analyse samples that comprise of a large
number of different constituents.
BREATH TESTS TO DETECT LUNG CANCER
Exhaled VOCs and ‘‘smellprints’’
Exhaled VOCs and their origin
VOCs in human breath were first described by PAULING et al.
[13] in 1971. Now, it is known that exhaled breath contains
thousands of different VOCs, most of them in picomolar
(10
-12
mol?L
-1
or particles per trillion) concentrations. In
normal subjects, more than 3,000 different VOCs can be
detected; however, only 20–30 of these VOCs are present in
all subjects. These are principally isoprene, alkanes, methylalk-
anes and benzene derivatives [14].
Exhaled organic compounds can originate from two main
sources: exogenous volatiles that are inhaled (or absorbed
through the skin) and then exhaled and those endogenously
produced by different biochemical processes. Basic cellular
functions including maintenance of cell membrane integrity,
energy metabolism and especially oxidative stress are all
known to be linked with VOC formation. Alkanes are
generated during the lipid peroxidation of polyunsaturated
fatty acids by reactive oxygen species and, although debated,
the so-called breath alkane profile has been postulated as a
new biomarker of oxidative stress [15, 16]. Aldehydes, ethane
and penthane are all produced during lipid peroxidation and
can be detected in exhaled breath [17]. Additionally, acetone
formed via the decarboxylation of acetoacetate also arises from
lipid peroxidation. Furthermore, the production of isoprene
from acetyl-coenzyme A is associated with cholesterol bio-
synthesis [18]. These examples highlight that widely different
biochemical pathways result in VOC formation and also imply
that endogenously produced VOCs originate from several cell
types.
Catabolism of many VOCs, including camphor, occurs through
the cytochrome P450 (CYP) mixed oxidase enzymes [19].
Regardless of the distance of the organ where produced, VOCs
can be transported by the blood to the lungs and exhaled
during breathing. Therefore, the origin of exhaled VOCs is
assumed to be mainly alveolar; however, direct comparison of
VOC profiles from different parts of the lung and the airways
is lacking. Changes either in the production or clearance of
VOCs may result in alteration in their exhaled concentration,
which can also be influenced by the gas-exchange properties of
the lung. Several studies have assessed the changes in exhaled
VOC profile in diseases of different organs. No detailed
information is available, however, on the question of whether
organ specific VOC profile exists and if functional changes in
different organs are linked with altered exhaled VOC profile
specific to a disease.
Collection of exhaled VOCs
Collection of exhaled breath for VOC detection is a simple
procedure; however, there are several important methodolo-
gical issues to consider (fig. 1). First, most of the components
known to be present in exhaled breath can also be found in the
environment. It is therefore necessary to distinguish the breath
signal from an artefact of contamination with room air. One
approach is to simultaneously collect VOCs in the breath and
also in the air in order to determine the alveolar gradient
(concentration in breath minus concentration in air) of each
VOC as proposed by PHILLIPS et al. [9]. Although this method is
easy to perform, it does not fully take into account the
complexity of pulmonary absorption and exhalation of volatile
substances. This approach has failed in FeNO studies and those
studies may serve as examples for addressing the problem in
another way. Another approach might be the use of a VOC
filter at the inhalation port of the breath collecting apparatus
which ensures the capture of environmental VOCs before the
exhaled sample is taken. Nonetheless, no approach considers
that exposure to environmental VOCs may have a sustained
effect on exhaled VOC pattern since volatiles can readily be
absorbed from the ambient air entering the blood, from where,
along with endogenously produced molecules, they can be
continuously released. This issue can be addressed by follow-
up studies performing serial measurements with subjects
inhaling VOC-free gases.
Secondly, due to the very low concentrations of volatiles in
exhaled breath, optimised sample collection and very sensitive
instruments are required for exhaled VOCs detection. Different
research groups applied widely different approaches for breath
collection (table 1). In the study of POLI et al. [20] a bag
nonpermeable for VOCs was used for collection and VOCs were
captured with a solid phase microextraction technique from the
obtained sample. A portable breath collection apparatus has
been developed by PHILLIPS and colleagues [9, 21], which collects
breath VOCs onto sorbent traps for subsequent analysis by GC-
MS. The potential influence of breathing pattern and the
contribution of volatiles, from the upper airways and the
mouth, to exhaled biomarkers might be of significance.
Therefore, these aspects of sampling procedures have been
FIGURE 1. Sampling of exhaled breath for smellprint analysis by electronic
nose. The subject inhales though a volatile organic compound filter and exhales
with stable flow rate against resistance.
SERIES: LUNG CANCER I. HORVA
´
TH ET AL.
262 VOLUME 34 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL