Lung carcinogenesis tracked by DNA methylation mapping in exhaled breath.

PR-14

Early detection strategies for lung cancer all require the non-invasive identification of a population of smokers at particularly high risk. Recently, exhaled breath condensate (EBC) has been proposed as a useful tool to obtain biological information on non-malignant lung diseases. Because silencing of tumor suppressor genes by aberrant promoter hypermethylation is recognized as a crucial component in lung cancer initiation and progression, we developed a method for comprehensive mapping of DNA methylation (tagged bisulfite genomic sequencing, tBGS) across kilobase-scale DNA. This method was then applied to assaying DNA methylation in EBC. Enhanced methods of DNA extraction from EBC increased DNA recovery to ~50-200ng per EBC sample, and bisulfite modification was adapted. Approximately 300bp fragments from the transcription site regions of five tumor suppressor gene promoters (P16, RASSF1A, MGMT, DAPK and CDH1) were simultaneously amplified by multiplex PCR. The detailed methylation map of the promoter of each gene was piloted initially in the EBC of seven subjects by tBGS. The results showed the methylation degree was variable among individuals for RASSF1A, where an association with smoking exposure dose and proximity may be present, and DAPK, where an there was no apparent association with smoking status and dose. P16, MGMT and CDH1 were not methylated in these seven samples. These preliminary results suggest that DNA methylation in exhaled breath condensate can be reliably detected, and it is technically possible to evaluate this whole lung sampling specimen for utility as a risk stratification tool, or disease-screening method.