Abstract A18: CpG island microarray analysis of minute bronchial epithelial cell specimens
Authors: Wilson Ian , Lam Stephen , Lam Wan 
Published: May 2012

Abstract
A18

Background
DNA methylation abnormalities are both very early events in cancer development and very widespread events in advanced neoplastic disease. Given their early occurrence in neoplasia, DNA methylation changes have many potential applications as biomarkers in serum, sputum, and exfoliated bronchial cells. To date, analysis of epigenetic events has been limited by technique throughput and by DNA requirement, limiting this approach to larger specimens, or only a few genes at a time. The ability to determine methylation status simultaneously at many loci using the limited amounts of starting tissue material recoverable from the small airways of the lung or sputum would greatly facilitate the use of DNA methylation as both a discovery tool and as a biological marker.

Objective
To improve on current methylation profiling technologies and adapt them to the investigation of minute amounts of DNA from exfoliated bronchial epithelial cells collected by bronchial brushing.

Materials and Methods
Exfoliated bronchial cells collected during bronchoscopy from peripheral airways from patients with and without non-small cell lung cancer (NSCLC). DNA was extracted by standard biochemical means. Using only 250 ng of genomic DNA, which is available from a single bronchial brush, methylation profiles were generated for the epithelial cells using a 244k oligonucleotide microarray from Agilent that covers 27,800 CpG islands across the entire genome. Methylated segments of the genome were differentiated from unmethylated segments by adapting the methylated DNA immunoprecipitation (MeDIP) protocol which relies on the specificity of anti-5'-methylcytosine for immunoprecipitation. The methylated fragments and input DNA (without immunoprecipitation) from the same sample were differentially labeled with Cyanine-3 and Cyanine-5 respectively and competitively co-hybridized to the CpG island array. Dye ratios at individual spots on the array were mapped to their genomic location and are used to infer methylation status at that locus. This approach generates a DNA methylation map spanning nearly every CpG island in the genome.

Results
Importantly, we have reduced the material requirement for oligonucleotide microarray profiling of DNA methylation levels to only 250 ng of genomic DNA, without requiring subsequent sample amplification (thus eliminating this source of potential bias). Using this new technique, we showed that CpG island DNA methylation profiles can be produced reliably from airway epithelial cells using very limited amounts of DNA with high correlations between replicate experiments. We also demonstrated DNA methylation alterations that are specific to the airways of patients with NSCLC, highlighting the potential role of this technique as a new tool in screening and early detection of lung cancers.

Conclusion
We have demonstrated that CpG island profiling of airway epithelial cells using a modified MeDIP procedure is a powerful tool for the analysis of DNA methylation profiles. This technique can be used for large-scale genome-wide assessment of methylation changes occurring in the peripheral airways and provide a method to study lung cancer development in former smokers as well as for developing early detection biomarkers and targets for chemoprevention.
Citation Information: Cancer Prev Res 2008;1(7 Suppl):A18.