Abstract
Early non-invasive detection of lung cancer is a precondition for enabling better prognosis supported by new innovative therapy regimes. The aim of our study was to evaluate angiogenic and inflammatory proteins in exhaled breath condensate (EBC) as markers for lung cancer. Our report presents a diagnostic study of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α) in EBC of 300 individuals, 84 patients with lung cancer, 111 patients with stable chronic obstructive pulmonary disease (COPD), and in 105 healthy controls. Detection of VEGF and bFGF in EBC was applicable to discriminate cancer patients from COPD patients as well as from healthy volunteers. Especially VEGF seems to be suitable to discriminate between non-small cell lung cancer (NSCLC) patients and control groups with highest VEGF values in EBC of patients with progressive NSCLC. The concentration of angiogenic factors correlated with disease progression as well as higher tumor stage. This study supports cytokine analysis in EBC as a suitable noninvasive diagnostic screening method for lung cancer detection and monitoring.
Reviewed Publication:
Sack U. Redaktion Conrad K.
Introduction
Despite intensive research in tumor biology, early diagnosis of lung cancer and subsequent resection therapy remains the only measure to reduce mortality. Treatment efficiency in lung cancer has been improved during the last 10 years but resulted only in marginally prolonged survival time [1]. Lung cancer still has a poor prognosis and is the most common cause of cancer-related death in men [2], [3], [4]. Unfortunately, most screening methods including blood analysis, low dose CT scanning, or cellular analysis of induced sputum has been proven unreliable or elaborate in clinical practice so far [5], [6].
Analysis of exhaled breath condensate requiring little time or effort for collection either from the patient and the examiner, seems to be a promising method as a non-invasive screening test for lung cancer detection [7], [8], [9]. In recent studies authors reported elevated cytokine levels in the exhaled breath condensate (EBC) of non-small cell lung cancer (NSCLC) patients, such as interleukin-6 (IL-6) [10], TNF-α and IL-2 [11], and recently of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin, TNF-α and IL-8 [12].
Bearing in mind those recent advances, we conducted an investigative study in a large cohort of known NSCLC patients to prove the reliability of this new diagnostic approach.
Materials and methods
Study subjects and clinical scores
In this prospective study EBC was collected from 300 individuals (193 men, 107 women; age: 64±12): (a) 34 patients with newly diagnosed NSCLC (b) 50 patients with known and by chemotherapy treated NSCLC with progressive disease (i.e. progress in primary target tumor diameters of at least 20%) following two to four cycles of a platinum based chemotherapy) (c) with 111 patients with stable chronic obstructive pulmonary disease (COPD) (defined by lack of symptoms typical for an acute exacerbation and no need for a change in medication for at least 8 weeks prior to presentation) (d) 105 healthy controls. Patients’ characteristics are shown in Table 1. NSCLC was verified in all patients by histological examination of tumor tissue. All patients and healthy volunteers were recruited in 2007 in an academic hospital specialized in pulmonary oncology and in a private practice of a pulmonary specialist, respectively.
Clinical parameters of patients.
| Patients | Volunteers | Stable COPD | NSCLC_PDc | NSCLC_NDd |
|---|---|---|---|---|
| Total number | 105 | 111 | 50 | 34 |
| Age (mean±SD)a | 56.8±13.5 | 70.5±9.1 | 65.0±8.9 | 62.6±10.5 |
| Sex | ||||
| Male | 50 | 75 | 39 | 29 |
| Female | 55 | 36 | 11 | 5 |
| Smoking statusb | Smokers/non-smokers 38/67 | Smokers/non-smokers 55/56 | Smokers/non-smokers 28/22 | Smokers/non-smokers 27/7 |
| Pack years | 14.6±12.8 | 30.1±18.3 | 29.6±13.9 | 29.0±18.0 |
| Protein in EBC, μg/mL (mean±SD)a | 102.1±12.1 | 99.3±20.1 | 106.1±17.7 | 106.1±18.2 |
All data are shown as mean±SD. ap>0.05 (no significant difference between investigated groups) in ANOVA test. bSmoking was defined as current smokers or ex-smokers that discontinued smoking no longer than 12 month, non-smoking as no smoking longer than 1 year. cPD, progressive disease. dND, newly diagnosed.
COPD was defined according to Global Initiative for Chronic Obstructive Lung Disease (GOLD); www.goldcopd.com, 2007). All COPD patients were in GOLD severity classes II–IV. None of the patients in this series were treated with oral steroids prior to admission. Instead all patients were on inhalation therapy (LABA and/or long acting anticholinergic and/or inhaled corticosteroid) according to GOLD recommendations. All patients were regularly seen by a pulmonary physician.
COPD patients with an acute exacerbation were excluded as exacerbation can influence levels of inflammatory parameters in EBC [12].
Healthy control individuals were identified by the absence of pulmonary symptoms, history of pulmonary, malignant or other chronic disease requiring medical therapy and normal lung function. Patients with any acute disease exacerbation, history of any active condition requiring a change of chronic medication or medication possibly interfering the biochemical tests (see below), such as diabetes, immunosuppression, or unstable coronary artery disease, were excluded from the study.
This study was approved by the Ethics Committee of the Leipzig University, Germany (serial no. 090-2007).
EBC collection and markers
The collection of EBC was performed during normal ventilation with an EcoScreen® system (Jaeger/Cardinal Health, Hoechberg, Germany). Average collection time was up to 15 min. After collection, samples were frozen at −20 °C immediately. To exclude contaminating saliva, all EBC samples were examined for amylase activity (100 μL EBC; α-Amylase ESP1491300 kit; Roche, Mannheim, Germany). The total protein concentration was measured by a Micro-BCA-Protein-Assay in 100 μL of unprocessed EBC (Pierce, Rockford, USA; detection limit 0.5 μg/mL).
Lyophilization of EBC fluid and multiplex cytokine detection
Immediately upon collection, condensate samples were frozen at −20°C. The major portion of the samples (2 mL) to be analyzed by the fluorescent bead array was lyophilized using a conventional evaporator (Uniequip, Martinsried, Germany). The resulting pellet was resuspended in 60 μL of double-distilled water (ddH20) for direct use in the assay. This procedure resulted in a 33-fold concentration.
A multiplex fluorescent bead immunoassay [cytometric bead array (CBA) Becton Dickinson, San Jose, CA, USA] was adapted to analysis in breath condensate to detect angiogenic marker/cytokine concentrations according to Sack et al. [13]. A mixture of four bead populations with capture antibodies specific for bFGF, VEGF, IL-8, and TNF-α was incubated with freeze-dried breath condensate (1 mL reconstituted 60 μL water). Angiogenic factors in EBC samples and recombinant standards bound to capture beads were detected by matched phycoerythrine-conjugated detection antibodies using a conventional flow cytometer (FC500™, Beckman Coulter, Krefeld, Germany). All measurements were done in duplicates.
Calibration of immunoassay
Calibration and validation of the immunoassay was performed as recently described [12], [13].
The test system was validated for use in low protein content in EBC by measuring known concentrations of each mediator in 1 mL samples of spiked reconstitution buffer. Three separate series of six concentrations (7.3, 14.7, 29.3, 58.7, 117.3, 234.5 pg/mL) were prepared and frozen at −20°C. Test samples were then lyophilized and reconstituted at days 1, 7 and 30. Accuracy and retrieval of VEGF, bFGF, IL-8 and TNF-α were determined with these test samples.
Effects of vacuum concentration, reduction of reagent volumes, and buffer composition had no effect of test accuracy as previously demonstrated [13]. In addition, four concentrations of the respective recombinant protein standard between 4.9 and 39.2 pg/mL (4.9, 9.8, 19.6, 39.2 pg/mL) were utilized to prove linearity and lower limits of detection for each molecule [12].
Statistical analysis
Statistical analysis was performed using the SPSS software program (SPSS Inc., Chicago, IL, USA). Linear regression analysis was applied to investigate the correlation of cytokine levels in EBC. Comparison of patient groups (three or more) was performed using the Kruskal-Wallis or the Mann-Whitney rank sum test. Statistical significance was accepted at the 5% level.
Results
Exhaled breath condensate characteristics
None of the condensate samples exhibited amylase concentrations measurable with the assay used. So a relevant saliva contamination is excluded as amylase concentration in saliva is 10,000 times higher than that in EBC.
Total EBC protein concentration was measured from 100 μL aliquots in all unprocessed samples. Results are shown in Table 1. There was no significant difference in any of the subgroups analyzed (p=0.68).
Angiogenetic and inflammatory factors in EBC
VEGF, bFGF and TNF-α were clearly elevated to varying levels in patients with newly diagnosed NSCLC (VEGF: 21.1±3.3 pg/mL; bFGF: 19.0±3.2 pg/mL; TNF-α: 2.9±0.8 pg/mL) and in patients with progressive disease of NSCLC following chemotherapy (VEGF: 26.1±5.6 pg/mL; bFGF: 72.9±16.5 pg/mL; TNF-α: 4.1±1.4 pg/mL) compared to stable COPD patients (VEGF: 12.8±2.0 pg/mL; bFGF: 14.7±2.4 pg/mL; TNF-α: 1.81±0.3 pg/mL) and volunteers (VEGF: 3.9±0.8 pg/mL; bFGF: 14.0±3.0 pg/mL; TNF-α: 1.3±0.3 pg/mL) (Figure 1A–C).
VEGF (A), bFGF (B), and TNF-α (C) in patients with newly diagnosed NSCLC, in patients with progressive disease of a NSCLC following chemotherapy, in stable COPD patients and in volunteers.
Means are indicated by horizontal lines.
We observed no influence of age, gender, tumor histology and tumor size (T) or lymph node status (N) on the levels of VEGF, bFGF and TNF-α in this limited number of patients in investigated subgroups (Table 2). However, in patients with distant metastasis status and consecutively in total tumor stage there were found increased marker levels (Table 2, Figure 2).
Tumor parameters and correlation with VEGF, bFGF and TNF-α (ANOVA Rank Test for three or more groups, Mann-Whitney rank sum test for two groups).
| VEGF | bFGF | TNF-α | |||
|---|---|---|---|---|---|
| T-stage of primary tumor | |||||
| T1 | 7 (8%) | p=0.53 | p=0.67 | p=0.66 | |
| T2 | 40 (48%) | n.s. | n.s. | n.s. | |
| T3 | 10 (12%) | ||||
| T4 | 27 (32%) | ||||
| Lymph node metastases | |||||
| N0 | 8 (9%) | p=0.36 | p=0.33 | p=0.50 | |
| N1 | 14 (17%) | n.s. | n.s. | n.s. | |
| N2 | 33 (39%) | ||||
| N3 | 29 (35%) | ||||
| Distant metastasis | |||||
| M0 | 19 (23%) | p=0.041 | p=0.015 | p=0.021 | |
| M1 | 65 (77%) | ||||
| Tumour stage groups | |||||
| I | 0 (0%) | p=0.041 | p=0.015 | p=0.021 | |
| II | 0 (0%) | ||||
| III | 19 (23%) | ||||
| IV | 65 (77%) | ||||
| Histological classification | |||||
| Adenocarcinoma | 49 (58%) | p=0.81 | p=0.60 | p=0.48 | |
| Squamous cell carcinoma | 27 (32%) | n.s. | n.s. | n.s. | |
| Large cell carcinoma | 8 (10%) | ||||
Significant changes are shown in bold.
VEGF (A), bFGF (B), and TNF-α (C) in NSCLC patients compared by tumor stage III and tumor stage IV.
Means are indicated by horizontal lines.
In a subgroup analysis of patients with NSCLC following two up to four courses of chemotherapy resulting in progressive tumor disease (>20% increase in primary tumor diameters) significantly higher EBC levels of all angiogenic factors were observed compared to newly diagnosed NSCLC. Lowest levels were seen in volunteers followed by stable COPD, which was also true for TNF-α (Table 3, Figure 1).
Tumor parameters and correlation with VEGF, bFGF and TNF-α (ANOVA Rank Test for three or more groups).
| VEGF | bFGF | TNF-α | ||
|---|---|---|---|---|
| Patient subgroups | ||||
| Newly diagnosed NSCLC | 34 (11%) | p=0.0001 | p=0.0001 | p=0.0001 |
| NSCLC with progressive disease | 50 (17%) | . | ||
| Stable COPD | 111 (37%) | |||
| Volunteers | 105 (35%) | |||
Significant changes are shown in bold.
Discussion
A previous comparative analysis of EBC from patients with lung cancer and healthy volunteers prompted us to confirm the release of specific angiogenic factors as predictive biomarkers, which are evident for the diagnostic determination in lung cancer [14]. Therefore, to confirm these observations and moreover to prove the discrimination of those factors between lung cancer and chronic-inflammatory lung diseases (i.e. COPD), differential display for the release of angiogenic factors into EBC form patients suffering from NSCLC in comparison to patients with COPD or healthy volunteers was performed by specific CBA. Thereby, three cytokines, two related to angiogenesis and one related to inflammation, were considered as potential predictive biomarkers for NSCLC.
In the present study we evaluated the concentrations of VEGF, bFGF and TNF-α in EBC from patients with NSCLC. We were able to show that all three cytokines are detectable in the EBC samples. However, in discordance to former studies also TNF-α was found to be increased in NSCLC patients. However, increase of TNF-α was most prominent in newly diagnosed or progressive NSCLC probably indicating a tumor-induced inflammatory response in these cases. Highest levels of angiogenic markers were observed in NSCLC under progress.
Diagnosis of lung cancer in an early stage of disease is strongly requested for a curative treatment as well as successful therapy. In this context, the continuous debate regarding a more effective screening of malignant lung diseases and its pro vs. contra is independent from the previous consideration [15], [16]. The proposed clinical application of diagnosing lung cancer has to be easily performed in a non-invasive manner and with very high specificity and sensitivity. However, all patients included in the present study had already advanced tumor stages – a situation which is very often found in clinical practice. The proposed analysis of angiogenic markers is expected to be also suitable in earlier stages of disease as shown in a very small cohort of patients in a previous study of our group and is intended to be verified by means of a larger screening study.
Furthermore, the outcome of differential secretion for angiogenic and inflammatory factors was independent from any kind of clinical features such as age, sex, smoking pack years, T and N classification and histology. Only in M classification and tumor stage increased levels of angiogenic markers and TNF-α were observed, which could be explained by the assumption that a larger total tumor mass is capable of releasing higher concentrations of angiogenic markers. Earlier studies revealed biologic activities of cytokines in the pathogenesis and progression of solid neoplasms. In the present study we have validated the concentration of VEGF in the EBC from lung cancer patients. VEGF is a very important angiogenic mediator of particular effects on cancer development [17]. Additionally, it has been shown that VEGF is responsible for recruitment of a higher blood vessel density within malignant tissues, for metastasis, and for cancer prognosis [18], [19]. VEGF was detected in serum, tissue, bronchoalveolar lavage, and pleural fluid of lung cancer patients [20], [21], [22], [23]. The first measurement of VEGF in EBC was published in 2009 by Dalaveris et al. that proposed this molecule as a trend marker for prescreening of lung cancer [14]. The VEGF molecule has been proofed in a number of studies as a detectable and suitable biomarker in EBC of patients with lung cancer.
Highest protein concentrations in EBC were found for VEGF. Here, NSCLC patients with progressive disease following chemotherapy had highest values followed by newly diagnosed NSCLC. For VEGF the overlap in the range of values with stable COPD or healthy volunteers was very low suggesting that VEGF is the most suitable marker for further studies.
However, we observed no association between bronchiolar VEGF and distant nodal metastasis (N2-N3), not supporting previous findings that VEGF correlates with upregulated lymph node angiogenesis and regional nodal metastasis [24]. The assumption that a higher tumor stage is associated with stronger VEGF release will be supported by our finding of increased levels of VEGF in situation with distant metastasis.
A further important mediator of an angiogenic environment is displayed by bFGF in malignant lung cells. In detail, the function of bFGF and its angiogenic potential is given by descriptions of Takanami and colleagues [25], [26]. However, the role of bFGF in tumor growth has not yet been clarified. In several tumors such as melanoma or bladder cancer the suppression of bFGF led to inhibition of tumor growth [27], [28]. The data of our study suggested an elevation of bFGF secretion in NSCLC patients vs. stable COPD and healthy volunteers. The higher levels of bFGF observed in our study, support previous findings showing that angiogenic factors may trigger proliferation of lung cancer cells [29].
Earlier in-vitro and in-vivo studies revealed more terms of cytological pathways by many cytokines in course of the pathogenesis and the progression of malignancies. Thus, informations regarding detection of inflammatory cytokines in EBC are considered helpful for assessment of correlation between an inflammation and a neoplastic situation, or another inflammatory disease without malignant features such as COPD. For example, TNF-α, a potent mediator of antiproliferative effects is capable of triggering neoplastic modulation in malignant tumors. In this context, Boldrini and colleagues have been shown that expression of bcl-2 and neoangiogenesis were significant correlated in lung cancer [30]. Moreover, these authors found a direct association between TNF-α and bcl-2 expression but an inverse association between TNF-α expression and counts of microvessels within the mutated tissue [30]. In the present study, we were able to show increased concentration of TNF-α in the EBC of NSCLC patients compared to the stable COPD cohort and healthy controls. Here, we observed a local inflammatory condition that is very likely mediated by mononuclear cells which infiltrate the tumor [31]. Thus, TNF-α concentrations detected in in EBC in the present study are similar to them ascertained in previous studies in lung cancer patients [10], [32].
Finally, our data support the previously proposed great diagnostic opportunity of early predictive detection of lung cancer by protein analysis in EBC [7]. Nevertheless, the application of EBC protein analysis in clinical routine requires further validation and standardization of specific biomarkers and their assign to each pathogenic issue of the lung [33]. The major achievement of the application of EBC protein analysis in clinical practice could be summarized as a noninvasive method affiliated with high specific and sensitive read-out for detection of various diseases. Moreover, our present study demonstrates the VEGF molecule as prominently detectable in all randomized EBC samples of lung cancer patients. Dalaveris and colleagues considered limitations by using EBC and claimed a proper evaluation, which we proposed here and placed our results for discussion [14]. In previous studies of our group, the proof of principle of angiogenic markers for non-invasive detection of NSCLC was shown [12]. In the present study we could confirm the proof of principle in a large cohort of NSCLC patients compared with more than 100 healthy volunteers or COPD patients.
In summary, this is an experimental diagnostic study for evaluation of VEGF, bFGF, and TNF-α in EBC as anticipated predictors (biomarkers) for diagnosis of NSCLC. As a main result, we suggested VEGF as a suitable biomarker in EBC and thus as a possible sensor for detecting lung cancer especially in advanced tumor stage within unclassified cohorts of patients with higher and also lower risk. However, a limitation of this study is that only patients with high tumor stage were included. Aim of further studies should be to show that investigated biomarkers are also capable to discriminate patients with tumor stage I and II showing that they are also suitable in very early NSCLC.
Acknowledgments
The authors thank Prof. Dirk Roggenbuck (Generic Assays, Dahlenberg) for generous contributions, Dr. Thomas Keller for statistic support, and Ulrike Scholz as well as Heike Knaack for technical assistance.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This study was supported by a grant from Stiftung Industrieforschung e.V., Germany, and by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0313909).
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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