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Background  Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients.    Results  We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value << 0.0001). Using the current tissue collection and 5-fold cross validation, the four most significant loci (CDKN2A EX2, CDX2, HOXA1 and OPCML) individually distinguish lung adenocarcinoma from non-cancer lung with a sensitivity of 67–86% and specificity of 74–82%. DNA methylation of these loci did not differ significantly based on gender, race, age or tumor stage, indicating their wide applicability as potential lung adenocarcinoma markers. We applied random forests to determine a good classifier based on a subset of our loci and determined that combined use of the same four top markers allows identification of lung cancer tissue from non-lung cancer tissue with 94% sensitivity and 90% specificity.    Conclusion  The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.

Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma