VEGF, TNF-α and 8-isoprostane levels in exhaled breath condensate and serum of patients with lung cancer

Abstract

The aim of the present study was to evaluate the levels of VEGF, 8-isoprostane and TNF-α in EBC and serum of patients with primary lung cancer prior to the initiation of any treatment, in order to evaluate their possible diagnostic role. Furthermore, associations between VEGF, 8-isoprostane and TNF-α levels in EBC and serum with clinicopathologic factors were investigated.

We enrolled 30 patients with lung cancer (mean age 65.2 ± 10.5 years) and 15 age and gender-matched healthy smokers as controls. Serum and EBC were collected before any treatment. TNF-α, VEGF and 8-isoprostane levels in EBC and serum were analyzed by an immunoenzymatic method (ELISA).

A statistically significant difference was observed between lung cancer patients and the control group regarding the values of TNF-α, both in EBC (52.9 ± 5.0 pg/ml vs. 19.4 ± 3.9 pg/ml, p < 0.0001) and serum (44.5 ± 6.3 pg/ml vs. 22.2 ± 4.3 pg/ml, p = 0.035). Moreover, EBC VEGF levels were higher in patients with T3–T4 tumor stage compared to T1–T2 (9.3 ± 2.8 pg/ml vs. 2.3 ± 0.7 pg/ml, p = 0.047). A statistically significant correlation was also observed between serum and EBC values of VEGF (r = 0.52, p = 0.019). In addition, serum levels of VEGF were higher in lung cancer patients than in controls (369.3 ± 55.1 pg/ml vs. 180.5 ± 14.7 pg/ml, p = 0.046). VEGF serum levels were also found higher in patients with advanced stage of disease (IIIB–IV) and distant nodal metastasis (N2–N3). No differences were observed in 8-isoprostane in EBC between lung cancer patients and controls. In contrast, serum 8-isoprostane levels were higher in lung cancer patients compared to controls (24.9 ± 3.6 pg/ml vs. 12.9 ± 1.6 pg/ml, p = 0.027) and were higher in patients with advanced disease. All three biomarkers presented acceptable reproducibility in the EBC on two consecutive days.

In conclusion, we have shown that TNF-α, VEGF and 8-isoprostane are elevated in the serum of lung cancer patients and increased serum VEGF and 8-isoprostane levels are related to advanced disease. In EBC, increased TNF-α levels were observed in lung cancer patients, whereas increased VEGF levels were observed in advanced T-stage. Further longitudinal studies are warranted for the evaluation of the prognostic role of these biomarkers in lung cancer.

Introduction

Lung cancer was the leading cause of cancer death in Europe for 2006 and the third most common form of malignancy, gathering 12.1% of the entire number of cancer incidence [1]. Despite the efforts to find new, more sensitive diagnostic tools, able to anticipate the diagnosis of lung cancer, a significant percentage of patients still have poor prognosis because of the wide extent of disease during diagnosis. There is currently renewed interest in biological factors that are not only involved in the development of malignant diseases but may also serve as prognostic markers for survival and as predictive signals for the efficacy of a specific therapy [2].

Among these markers, TNF-α has emerged from recent studies, as one of the mediators interferes with both antiproliferative and tumorigenic effects [3]. However, the association of this factor with tumor prognosis is not yet clearly defined. Additionally, an interesting line of research is presently directed towards the study of angiogenetic factors, such as vascular endothelial growth factor (VEGF) [4]. Angiogenesis plays an important role in tumor biology, especially in tumor progress and growth of metastasis [5]. Tumor cells themselves, are able to release many microangiogenetic stimulating substances [6]. Furthermore, various markers of oxidative stress have been reported in the past to interfere with carcinogenesis [7], [8]. We have recently shown that serum vascular endothelial growth factor is related to systemic oxidative stress in patients with lung cancer [9]. 8-Isoprostane is a stable prostaglandin-like product that is formed from arachidonic acid by the non enzymatic action of reactive oxygen species and it is considered a marker of oxidative stress, though its exact role in lung cancer biology is still not clear [10].

Exhaled breath condensate (EBC) represents a totally non-invasive method for the evaluation of airways pathology during lung diseases [11], and recently considerable burden of information has accumulated and efforts for the standardization [12] and the assessment of normal values of biomarkers in EBC [13] have been presented in the literature. Despite its advantages and its widespread use in other lung disorders [12], the role of EBC in the diagnosis and the evaluation of patients with lung cancer has not been widely evaluated, as the major part of biomarkers in lung cancer have been determined until nowadays in samples obtained with more invasive techniques, such as induced sputum, bronchial biopsies and bronchoalveolar lavage (BAL). However, EBC represents an attractive means of airway sampling since it represents a totally non-invasive and easily repeatable method. Studies have shown increased cytokine levels in the EBC of non-small cell lung cancer patients, such as IL-6 [14] and TNF-α, IL-2 and leptin [15]. Additional studies in EBC have shown increased levels of endothelin-1, a mitogenic factor [16], as well as microsatellite alterations in the DNA of lung cancer patients [17], providing a rationale for further evaluation of EBC in lung cancer.

The aim of the present study was to evaluate the levels of VEGF, 8-isoprostane and TNF-α in the EBC and serum of patients with primary lung cancer prior to the initiation of any treatment, in order to evaluate their possible diagnostic role. Furthermore, associations between VEGF, 8-isoprostane and TNF-α levels in the EBC and serum with clinicopathologic factors were investigated.