Epigenomic analysis of bronchial epithelial cells in smokers at risk of developing lung cancer

A91

Background: DNA methylation abnormalities are both very early events in cancer development and very widespread events in advanced neoplastic disease. Due to the early nature of DNA methylation disturbance, it also has applications as a biomarker in serum, sputum, and biopsies. However, analysis of epigenetic events has been limited by technique throughput and by sample material requirement, thus limiting this approach to the analysis of only a few genes. The ability to analyze many loci from minute starting materials in the small airways of the lung would greatly facilitate the use of DNA methylation as both a discovery tool and as a biological marker.
 >Objective: The objective of the current study is to improve current technologies and adapt them to the investigation of exfoliated bronchial epithelial cells collected by bronchoscopy. With the new techniques, we will monitor the effect of steroid treatment on the epigenome of bronchial cells.
 >Methods: Patients were treated for 4 weeks with inhaled steroid. Exfoliated bronchial cells were collected during bronchoscopy from airways ≤ 2mm in diameter both before and after treatment. Nucleic acids (DNA/RNA) were extracted by standard biochemical means. DNA methylation profiles were generated for the epithelial cells using a whole genome tiling path (WGTP) array by methylation dependent immunoprecipitation (MeDIP) array CGH. In MeDIP, immunoprecipitation with anti-5’-methylcytosine isolates methylated DNA fragments. The methylated fragments and input DNA (without immunoprecipitation) from the same sample are differentially labeled with Cyanine-3 and Cyanine-5 respectively, and competitively co-hybridized to the genomic array. Dye ratios at individual spots on the array are mapped to their genomic location, and are used to infer methylation status. This approach generates a DNA methylation map spanning the entire genome.
 >Results: Our results indicate that whole genome DNA methylation profiles can be produced reliably from airway epithelial cells. We have reduced the material requirement to only 1μg of genomic DNA, without requiring subsequent sample amplification. Analysis of DNA methylation profiles before and after treatment with steroid identified 17 regions of DNA hypermethylation and 16 regions of DNA hypomethylation corresponding to steroid treatment that were present in a minimum of 50% (5/10) of patients analyzed.
 >Conclusion: We have demonstrated that MeDIP aCGH is a valuable tool for the analysis of DNA methylation profiles in the small airways of the lung. This suggests the potential of using this technique for genome-wide assessment of methylation changes in lung diseases.