Elsevier

Lung Cancer

Volume 43, Issue 2, February 2004, Pages 215-222
Lung Cancer

SHORT COMMUNICATION
Detection of p53 gene mutations in exhaled breath condensate of non-small cell lung cancer patients

https://doi.org/10.1016/j.lungcan.2003.08.034Get rights and content

Abstract

Early diagnosis of lung carcinoma is greatly desired. A potential source of early information regarding the process of cancerisation in the airways is exhaled breath condensate (EBC). The direct approach to detecting cancerisation is examining DNA from the area of chronic damage, i.e. airways and lung parenchyma. We therefore investigated DNA in EBC of patients with NSCLC and healthy volunteers. Human DNA was amplified by PCR in exhaled breath condensate and used to detect p53 mutations. A PCR of the β-actin gene fragment was used to detect human DNA in each of the EBC samples. In 65.7% of the samples, the β-actin gene was found. Extracted DNA as well as native EBC were equally suited as starting material for amplification. Mutations of the p53 gene were investigated in all EBC samples of NSCLC patients. p53 exons 5–8 were amplified using nested PCR and subsequently sequenced. Mutations were found in four of the patients (n=11; 36.4%) while no mutation was found in volunteers (n=10). Mutations detected in EBC were also compared with those of corresponding tumor tissue. Different point mutations in EBC and tumor tissue were revealed in all cases. Our findings demonstrate that exhaled breath condensate may be used for analysis of somatic gene mutations in an area of direct tobacco-related DNA damage.

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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