SHORT COMMUNICATIONDetection of p53 gene mutations in exhaled breath condensate of non-small cell lung cancer patients
Introduction
Despite extensive research lung cancer still has a poor prognosis and remains the most frequent cause of cancer-related death [1], [2], [3]. Early diagnosis of lung cancer is a major problem, as symptoms occur predominantly in late stages [4], [5]. Screening methods, which allow examination of greater numbers of individuals at risk will have to be developed [6]. Most techniques so far are invasive (BALF, fluorescence bronchoscopy) [7], [8] or associated with considerable radiation such as low dose or regular dose CT scans. Other sensitive detection techniques require a great deal of technical equipment and are costly like MR tomography and PET [9]. Automatic sputum cytology may be an appropriate technique, but acquisition of sputum is not always easy and algorithms for automatic evaluation will have to be validated further [10].
The detection of molecular markers specific for lung cancer is a relatively new strategy for early cancer screening and detection [11]. Molecular markers such as p53 or K-ras mutations are viewed as more general indicators of the progress of the multistep process of cancerisation in high risk patients. The detection of p53 and other somatic gene mutations may therefore be an important diagnostic tool in determining the degree of DNA damage and cancer risk and possibly in early cancer detection. Of cause, mutations of these genes of interest have to be analyzed in tissues participating in the process of field cancerisation. Biopsies of the airway mucosa would be one way to obtain relevant tissue. However, bronchial biopsies involve bronchoscopy and are not suited for broader screening. Exhaled breath condensate collection is a non-invasive technique that involves quiet breathing, e.g. 10 min. The exhalate which is collected in a cooled condenser contains mostly water but also many different molecules most likely derived from the lining fluid of airways and alveoli [12], [13] albeit in low concentrations. We set out to investigate the question whether human DNA can be detected in exhaled breath condensate (EBC) and whether this material can be assayed for specific gene mutations that are relevant in the process of cancerisation.
In this report, we demonstrate detection of human DNA in EBC of healthy volunteers and patients with NSCLC and in addition we demonstrate the detection of p53 gene mutations in patients but not in volunteers.
Section snippets
EBC collection
EBC was collected using the EcoScreen breath condensate collection device (Jaeger/Toennies, Hoechberg, Germany). Sampling time was 20 min as previously described [14]. EBC was collected from (a) 18 patients with a histologically proven NSCLC and (b) 18 healthy non-smoking volunteers. Main clinicopathologic parameters of investigated NSCLC patients and healthy volunteers are given in Table 1.
From patients with NSCLC tumor tissue was obtained bronchoscopically. Bronchoscopy was performed in NSCLC
Methodological aspects of detecting human DNA in EBC
We first investigated the effectiveness of PCR for the human β-actin gene fragment in extracted and native samples of EBC-DNA. The detection limit of the PCR product was 0.05 pg DNA as determined using DNA from NSCLC cells (Fig. 1). In a separate set of experiments we compared PCR for the human β-actin gene fragment in extracted and native samples of EBC-DNA. For this propose in 10 samples DNA extracted from EBC was added to the PCR starting material. The same was done for native EBC fluid. Both
Discussion
A major finding of this report is the demonstration of the amplification of human DNA in EBC of healthy individuals and patients with NSCLC. The human β-actin gene a ‘house keeping’ gene has been chosen to examine methodological aspects of detecting human genes in DNA of EBC. Because the amount of DNA in EBC is very low, we investigated the reproducibility of detection. A second PCR increased the fraction of samples with positive human β-actin gene fragment detection by 10% while further
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