Abstract
Exhaled breath condensate (EBC) is a non-invasive source that can be used for studying different genetic alterations occurring in lung tissue. However, the low yield of DNA available from EBC has hampered the more detailed mutation analysis by conventional methods. We applied the more sensitive amplicon-based next generation sequencing (NGS) to identify cancer related mutations in DNA isolated from EBC. In order to apply any method for the purpose of mutation screening in cancer patients, it is important to clarify the incidence of these mutations in healthy individuals. Therefore, we studied mutations in hotspot regions of 22 cancer genes of 20 healthy, mainly non-smoker individuals, using AmpliSeq colon and lung cancer panel and sequenced on Ion PGM.In 15 individuals, we detected 35 missense mutations in TP53, KRAS, NRAS, SMAD4, MET, CTNNB1, PTEN, BRAF, DDR2, EGFR, PIK3CA, NOTCH1, FBXW7, FGFR3, and ERBB2: these have been earlier reported in different tumor tissues. Additionally, 106 novel mutations not reported previously were also detected. One healthy non-smoker subject had a KRAS G12D mutation in EBC DNA.Our results demonstrate that DNA from EBC of healthy subjects can reveal mutations that could represent very early neoplastic changes or alternatively a normal process of apoptosis eliminating damaged cells with mutations or altered genetic material. Further assessment is needed to determine if NGS analysis of EBC could be a screening method for high risk individuals such as smokers, where it could be applied in the early diagnosis of lung cancer and monitoring treatment efficacy.
Highlights
In spite of recent improvements in the treatment of many cancers, the prognosis of lung cancer has remained unchanged for 20 years and lung cancer is still the leading global cause of cancer related deaths [1]
Exhaled breath condensate (EBC) is a non-invasive source that can be used for studying different genetic alterations occurring in lung tissue
11 different TP53 mutations were seen, of which only three (Q104*, Y163*, and M169I) have been reported previously in lung tissues and upper aerodigestive tract according to COSMIC database
Summary
In spite of recent improvements in the treatment of many cancers, the prognosis of lung cancer has remained unchanged for 20 years and lung cancer is still the leading global cause of cancer related deaths [1]. Developments in microarray based techniques, generation sequencing (NGS) and bioinformatics tools have made it possible to identify genome-wide gene alterations from an extremely small amount (1-10 ng) www.impactjournals.com/oncotarget of DNA or RNA [3,4,5]. This has made it possible to use exhaled breath condensate (EBC) as a source of testing material since this is a patient-friendly, noninvasive approach. Genetic changes in EBC DNA are thought to reflect alterations present in lung tissue and the sampling process is convenient for the patient and the specimens can be collected repeatedly throughout the follow-up [6]
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