Journal of Cancer Therapy, 2012, 3, 460-466
http://dx.doi.org/10.4236/jct.2012.324059 Published Online September 2012 (http://www.SciRP.org/journal/jct)
Tumour Necrosis Factor Alpha and Oxidative Stress in the
Breath Condensate of Those with Non-Small Cell Lung
Cancer
Enoch Chan1, Thevaki Sivagnanam2, Qi Zhang1, Craig R. Lewis1,3, Paul S. Thomas1,2*
1Inflammation and Infection Research Centre and Prince of Wales Clinical School, Faculty of Medicine, University of New South
Wales, Sydney, Australia; 2Department of Respiratory Medicine, Prince of Wales Hospital, Randwick, Australia; 3Department of
Medical Oncology, Prince of Wales Hospital, Randwick, Australia.
Received July 31st, 2012; revised August 30th, 2012; accepted September 15th, 2012
ABSTRACT
Background and Aims: Lung cancer is a leading cause of cancer mortality worldwide and is associated with the re-
lease of tumour necrosis factor-α (TNF-α), subsequent cellular apoptosis and the generation of oxidative stress. Exhaled
breath condensate (EBC) analysis is a non-invasive method for sampling biofluids from the lower respiratory tract. This
study aimed to evaluate possible biomarkers of lung cancer by measuring the levels of TNF-α and the oxidation of
ascorbic acid in EBC. Patients with lung cancer were enrolled into the study prior to treatment, during treatment and
post-treatment, and results compared with an age-matched control population. Material and Methods: Patients with
Stages II-IV non small cell lung cancer (NSCLC) were recruited prior to and at stages of their treatment. EBC levels of
TNF-α, and rate of ascorbic acid oxidation were measured. Results: A total of 19 patients with NSCLC (mean age
71.37 ± 7.77 yr) and 25 age-matched control subjects were enrolled. Levels of EBC TNF-α were elevated in the EBC of
patients with lung cancer compared with control subjects (1.02 ± 0.07 pg/ml vs 0.51 ± 0.06 pg/ml, p < 0.0001). More-
over, the rate of ascorbic acid oxidation was significantly greater in the EBC of patients with lung cancer compared
with control subjects (2.20% [0.4 - 11.0] vs 1.00% [0.1 - 8.5], p = 0.0244). Conclusion: TNF-α and the rate of ascorbic
acid oxidation was elevated in the EBC of patients with lung cancer regardless of treatment. Longitudinal studies in a
larger population are required to evaluate these markers for the effect of treatment and prognosis.
Keywords: Exhaled Breath Condensate; Lung Cancer; Ascorbic Acid Oxidation; Tumour Necrosis Factor Alpha
1. Introduction
Lung cancer is a leading cause of cancer mortality
worldwide, with over 1.6 million new cases and over 1.3
million deaths each year [1-3]. It contributes to 157,000
deaths and 28% of cancer deaths annually in the USA,
and 19% of cancer deaths in Australia [4-5]. Despite ad-
vances in treatment strategies, long-term mortality has
not improved significantly in the last 20 years, with the
overall 5-year survival being 16% in the USA. This is
mainly due to the late onset of symptoms resulting in
patients who present with advanced and incurable disease.
Such symptoms often relate to disseminated disease such
as bony metastases or to locally advanced disease with
breathlessness, lung infection or haemoptysis. However,
when lung cancer is discovered earlier, radical treatment
and potentially curative surgery may improve 5-year sur-
vival rates (from 20% in those with Stage III lung cancer
to 70% in patients with Stage I disease [6-8]. Current
methods of diagnosing lung disease such as CT guided
tissue biopsy or bronchoscopic biopsy and bronchial-
veolar lavage are associated with invasive risks [9,10].
Therefore a simple, non-invasive and relatively inexpen-
sive method of screening for lung cancer in at-risk popu-
lations may potentially detect early disease and reduce
lung cancer mortality.
Recent advances in tumour biology and angiogenesis
has promoted studies into potential tumour biomarkers
[11-13]. Traditionally, blood or tumour samples are used
to identify biomarkers but analysis of exhaled breath
condensate (EBC) provides a promising, non-invasive
approach that could allow screening of smokers for the
diagnosis of lung cancer. EBC analysis is the collection
of epithelial lining fluid from the lower respiratory tract
via condensation of exhaled breath. EBC consists pri-
marily of water vapour as well as various aerosolised
particles including non-volatile organic compounds and
*Corresponding author.

Tumour Necrosis Factor Alpha and Oxidative Stress in the Breath Condensate of Those with
Non-Small Cell Lung Cancer
461
volatile organic compounds (VOCs) that may be used to
identify lung pathology [11-13].
Lung cancer biomarkers and those associated with tu-
mour lysis may be able to aid in screening of lung cancer
and possibly predict a patient’s response to cancer treat-
ment [14]. TNF-α is highly expressed in tumours and is
elevated in the serum of patients with non-small cell lung
cancer (NSCLC) when compared with control subjects
[15,16]. TNF-α is elevated in the EBC of patients with
lung cancer when compared with control subjects [16].
TNF-α is also a mediator for initiating cellular apoptosis
but its apoptotic pathways may be circumvented by cer-
tain tumour cell characteristics [17]. Therefore levels of
TNF-α may be raised in the EBC of patients with lung
cancer and further increased in treatment-responsive tu-
mours. Cigarette smoking and environmental pollutants
can release TNF-α from alveolar macrophages which in
turn generate oxidative stress in the lung. In addition,
there is an increase in the levels of known markers of
oxidation in the EBC derived from smokers, such as hy-
drogen peroxide, 8-isoprostane, as well as those of the
nitrosative pathway e.g. nitrate, and S-nitrosothiols [18-
21]. Oxidative stress is also associated with the induction
of lung cancer, possibly by indirect and direct mutagene-
sis (e.g. DNA adduct formation, DNA double strand
breaks, DNA mutation) [22]. Furthermore, there is in-
creased oxidative stress in cancer cells compared with
normal cells [23,24]. This may be due to oncogenic sig-
nals that increase production of reactive oxygen species
(ROS) [25,26] or the increased metabolic demand of
rapidly growing and differentiating cancer cells [27].
In the presence of oxygen, ROS including hydroxyl
radicals, superoxides and organic radicals generate other
downstream ROS including hydrogen peroxide and or-
ganic hydroperoxides. These highly reactive radicals oxi-
dise anti-oxidants including ascorbic acid. Both radio-
therapy and chemotherapy induce ROS at the site of the
tumour to damage cell lipid membranes and the DNA of
malignant cells, thus capturing this phenomenon for the-
rapeutic gain. The reduced ability of tumour cells to re-
pair damaged DNA increases their vulnerability to chemo-
and radiotherapy when compared with normal, healthy
cells [28,29].
It was postulated that increased ROS as an indication
of elevated oxidative stress could be measured in the
EBC of patients with NSCLC and undergoing treatment.
ROS activity in EBC may be assessed by the ex-vivo
oxidation of ascorbic acid. The ascorbate reacts with the
oxidative species and is degraded, thus its rate of degra-
dation can be analysed and compared between patients
pre- and post-therapy and with control subjects.
2. Materials and Methods
2.1. Subject Recruitment
This study was approved by the South Eastern Sydney
Area Health Service Research Ethics Committee. Par-
ticipants with NSCLC were recruited from the Prince of
Wales Hospital, NSW. Studied subjects were divided into
a pre-treatment group, a group undergoing treatment and
a post-treatment group. The inclusion/criterion for the
pre-treatment group was that the subject had to be eligi-
ble for chemotherapy, radiotherapy or surgery. The ex-
clusion criterion was that they must not have had any
chemotherapy, radiotherapy or surgery for their lung
cancer before EBC sample collection. Treatment define-
tion for EBC collection in subjects during treatment and
post-treatment was defined as chemotherapy and/or ra-
diotherapy, ± surgery. EBC for the post-treatment group
was collected at least three weeks after the final treat-
ment. Control subjects without lung cancer were recruited
from the orthopaedic ward and were age and gender
matched. The exclusion criterion for control subjects was
a past or current history of lung cancer.
Informed consent was obtained and a questionnaire
completed for demographic details and relevant medical
details including information on smoking history, (ex-
smokers defined as not having smoked for at least one
year), medical history, medication, and staging and his-
tological typing of lung cancer.
2.2. EBC Collection
EBC was collected as previously described [11]. Briefly,
prior to collecting EBC, the subjects were instructed to
rinse their mouths with water. EBC was then collected
for 15 min with tidal breathing into a unidirectional
mouthpiece connected to a custom unsiliconised glass
collection device and condensed using wet ice at appro-
ximately 4˚C. Excess saliva was swallowed.
The EBC was aliquoted into 120 μl samples in 1.5 ml
Lobind Eppendorf tubes (POCD Scientific, Artarmon,
NSW) and degassed at 0.5 L/min with high purity argon
gas (BOC, Sydney) for one minute. All samples were
immediately stored at –80˚C for subsequent analysis.
2.3. Measurements of Tumour Necrosis Factor-α
TNF-α levels were measured using a commercial sand-
wich enzyme-linked immunosorbent assay (ELISA) (In-
vitrogen, Mulgrave, Victoria), with a sensitivity of <0.09
pg/ml. EBC samples were assayed in duplicates. The
range for this assay was 0 - 32 pg/ml and the limit of de-
tection was 0.2 pg/ml.

Tumour Necrosis Factor Alpha and Oxidative Stress in the Breath Condensate of Those with
Non-Small Cell Lung Cancer
462
2.4. Ascorbic Acid Oxidation
Ascorbic acid oxidation was measured by analysing the
rate of oxidation of ascorbic acid by ROS in EBC using
the reaction: Ascorbic Acid + RO• → Ascorbyl radical +
ROH. [30] Briefly, an aliquot of 20 μl of 1.65 M ascorbic
acid solution (Sigma Aldrich, Australia) was added to
200 μl of de-ionised water and 100 μl of EBC in a black
quartz cuvette. The rate of ascorbic acid oxidation as de-
termined by change in absorbance at 265 nm was moni-
tored at 30 second intervals for 210 seconds. At the end
of this period and to determine the presence of any tran-
sition metals, 10 μl of 660 μM ethylene diamine tetra
acetic acid solution was added as a chelator and the rate
of oxidation was monitored at 30 second intervals for a
further 150 seconds.
2.5. Statistical Analysis
Statistical analyses were performed using Graph Pad
Prism 5 (Graph Pad, La Jolla CA). TNF-α data were
normally distributed and analysed by parametric statistics
(one-way ANOVA, Bonferroni post-hoc test and inde-
pendent unpaired 2-tailed t-test where appropriate). For
follow-up patients Repeated Measures ANOVA was used.
Data for parametric statistics are presented as mean ±
S.E.M. Ascorbic acid oxidation did not follow a Gaus-
sian distribution and non-parametric tests were used;
Kruskal-Wallis test for data with multiple groups and
Mann-Whitney U test for data with two groups. Data for
non-parametric statistics are presented as median [range].
3. Results
Of the 44 individuals that were enrolled into the study, 19
had been diagnosed with NSCLC (11 male, 8 female).
Control subjects comprised 11 healthy smokers/ex-smokers
(6 males, 5 females), and 14 healthy non-smokers (5 males,
9 females). Subjects gave variable volumes of EBC and
as a result, some patients had inadequate samples col-
lected for the analysis of every marker. Subject demo-
graphics are summarised in Table 1. All patients with
lung cancer undergoing treatment or who were post-
treatment had a positive response to treatment with re-
duction in tumour size. Demographic characteristics are
summarised in Table 2.
3.1. Tumour Necrosis Factor-α
Patients with lung cancer had significantly higher con-
centrations of TNF-α than control subjects (–0.08 ± 0.03
pg/ml vs –0.42 ± 0.05 pg/ml, p < 0.0001; Figure 1).
There was no significant difference in TNF-α concentra-
tion between pre-treatment, treatment and post treatment
groups. However, each treatment group was significantly
Table 1. Subject demographics.
Control Subjects
Non-smokersSmokers/Former
smokers Total
Subjects
with Lung
Cancer
N 14 11 25 19
Age
(mean ± SD)73.6 ± 11.575.4 ± 8.7 74.4 ± 10.2 71.4 ± 7.8
Male/Female5/9 6/5 11/14 11/8
Table 2. Demographic characteristics for subjects with lung
cancer.
N 19
Age (mean ± SD) 71.4 ± 7.8
Male/Female 11/8
Smoking (current/former/never) 3/12/4
Adenocarcinoma 8
Large cell carcinoma 3
Squamous cell carcinoma 5
NSCLC (NOS) 3
Stage I/II/III/IV 1/3/6/9
Treatment pre/during/post 8/7/10
Follow-up during/post 3/3
Figure 1. EBC TNF-α levels in control subjects (n = 25)
versus subjects with lung cancer (n = 23) in EBC (1.02 ±
0.07 pg/ml vs 0.51 ± 0.06 pg/ml, p < 0.0001). Note: Two sub-
jects with lung cancer had pretreatment, during treatment
and post treatment follow up results included.
different when compared with both non-smoker control
subjects and control smokers/ex-smokers (p < 0.05, Fig-
ure 2). Treatment did not significantly affect the level of
EBC TNF-α nor were there significant differences ob-
served between the stages of lung cancer (Stage II (n = 6)
0.86 ± 0.12, Stage III (n = 6) 1.09 ± 0.19, Stage IV (n =
10) 1.09 ± 0.08).

Tumour Necrosis Factor Alpha and Oxidative Stress in the Breath Condensate of Those with
Non-Small Cell Lung Cancer
463
Figure 2. Comparison of EBC TNF-α levels during treat-
ment in comparison with control groups. Significant diffe-
rences were found betwee n any of the treatment sub-groups
to both control subgroups groups (p < 0.05). (Means: pre-
treatment (n = 7) 1.02 ± 0.11, treatment (n = 7) 1.00 ± 0.13,
post-treatment (n = 9) 1.04 ± 0.13, smokers/ex-smokers (n =
10) 0.54 ± 0.08, non-smokers (n = 15) 0.49 ± 0.08). Note:
Two subjects with lung cancer had pretreatment, during
treatment and post treatment follow up r e sults inc l ude d.
3.2. Ascorbic Acid Oxidation
Patients with lung cancer had significantly greater per-
centage degradation of ascorbic acid from baseline than
control subjects (2.20% [0.4 - 11.0] vs 1.00% [0.1 - 8.5],
p = 0.024; Figure 3). No significant difference between
the rates of ascorbic acid degradation was found between
treatment and control subgroups (Figure 4). No signifi-
cant difference was observed between lung cancer stages.
4. Discussion
This study shows a significant increase in the levels of
EBC TNF-α and in the oxidation rate of ascorbic acid in
the EBC of patients with primary lung cancer when
compared with control subjects.
TNF-α can activate the cell-mediated response against
neoplastic cells as well as mediate tumourigenesis in lung
cancer, such as promoting neoplastic cell growth and
inhibiting the anti-tumour immune response [31]. Pa-
tients with lung cancer had greater levels of EBC TNF-α,
independent of whether they were yet to commence
treatment, undergoing treatment at the time of collection
or had completed their treatment, when compared with
healthy controls. This finding is consistent with a previ-
ous study that reported statistically elevated levels of
TNF-α in the EBC of patients with lung cancer prior to
treatment when compared with controls [16].
This study also observed sequential elevation of TNF-
α levels from samples of EBC from two patients as they
Figure 3. The rate of decline of ascorbic acid in the EBC of
patients with lung cancer (n = 19) versus control subjects (n
= 18). (2.20% [0.4 - 11.0] vs 1.00% [0.1 - 8.5], p = 0.024).
Figure 4. Comparison of the rate of degradation of ascorbic
acid in the EBC between different treatment sub-groups
and control sub-groups. No differences were found between
these subgroups. (Medians: pre-treatment (n = 5) 2.04 [1.1 -
3.1], treatment (n = 5) 4.18 [1.2 - 10.1], post-treatment (n = 9)
3.8 [0.4 - 11.0], smokers/ex-smokers (n = 9) 2.6 [0.8 - 8.5],
non-smoker (n = 9) 1.2 [0.1 - 2.6]).
progressed from pre-treatment to treatment and then fi-
nally follow up post-treatment, but this trend was not
observed over the group (Figure 3). Both patients had
positive responses to treatment, which supports the anti-
proliferative effects of TNF-α in higher concentrations,
but this observation would need to be confirmed in larger
studies [17]. This is the first study, to our knowledge, that
has analysed TNF-α in the EBC of patients before, during
and post treatment. The group data did not suggest that
there was a clear response in EBC TNF-α levels to treat-
ment.
Compared to normal cells, neoplastic cells are in a
pro-oxidative state that leads to intrinsic oxidative stress.

Tumour Necrosis Factor Alpha and Oxidative Stress in the Breath Condensate of Those with
Non-Small Cell Lung Cancer
464
Cancer cells have higher levels of ROS than normal cells,
and ROS are, in turn, responsible for the maintenance of
the cancer phenotype [25-27]. Ascorbic acid is oxidised
by ROS produced by these neoplastic cells [32]. In this
study there was an increased rate of ascorbic acid degra-
dation in the EBC of patients with lung cancer compared
with healthy control subjects. This is a novel finding
from our study, as there are no previous publications in
the literature regarding the increased rate of ascorbic acid
oxidation with lung cancer, but it is supported by previ-
ous work which showed elevated H2O2, decreased total
anti-oxidants as measured by a total anti-oxidant assay,
and decreased pH [11]. Interestingly, the current data did
not suggest a significant difference between the smoking
control group and those with NSCLC, suggesting that the
induction of ROS by smoking remains a major contribu-
tor to EBC ROS. Furthermore, ROS did not significantly
differ after the initiation of treatment, which may reflect
the small sample size, but indicates that there is not a
major change in the ROS. It may be that a different and
more sensitive marker may be required to be used for
assessing clinical response to treatment [33].
This study conformed to the recommendations pro-
vided by the ERS/ATS Task Force however, there were
some limitations. [34] It was a single centre, cross-sec-
tional observational study, with a modest sample size,
which undoubtedly affects the power of this study.
ROS are unstable in EBC thus rapid freezing of the
samples at –80˚C is required until they were analysed.
Furthermore, samples were thawed and kept on ice dur-
ing analysis. The ascorbic acid oxidation reaction is
catalysed by transitional metals such as copper (II) ions
[35,36] and thus the presence of different levels of transi-
tional metals may also affect the rate at which ascorbic
acid is oxidised. There was no significant increase in the
rate of ascorbic acid oxidation during treatment for lung
cancer, or between the treatment subgroups and control
subgroups. Despite these limitations, we believe that our
study provides further convincing evidence that analysis
of exhaled breath is promising, as it shows that the tech-
nique is feasible and can be performed in those with sig-
nificant lung disease.
As previously mentioned, further longitudinal investi-
gations are needed, in large multicentre studies, evaluat-
ing levels of biomarkers in patients with lung cancer at
various stages through their treatment to further support
our findings.
5. Conclusion
TNF-α and the rate of ascorbic acid oxidation are in-
creased in the EBC of patients with lung cancer regard-
less of treatment status. Longitudinal studies would assist
in the evaluation of the clinical use of these markers in
diagnosing and assessing treatment efficacy in lung can-
cer.
6. Acknowledgements
The authors thank the participants for their time and Beth
Ivimey for her kind assistance with both the patients and
recruitment.
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Tumour Necrosis Factor Alpha and Oxidative Stress in the Breath Condensate of Those with
Non-Small Cell Lung Cancer
466
Abbreviations
EBC: Exhaled breath condensate
ELISA: Enzyme-linked immunosorbent assay
NSCLC: Non small cell lung cancer
ROS: Reactive oxygen species
TNF-α: Tumour Necrosis Factor-alpha
VOCs: Volatile organic compounds