Abstract B10: Detection of DNA methylation in the serum of lung cancer cases and controls using Digital MethyLight
Authors: Johnson Candace J.S , Galler Janice S. , Anglim Paul P. , Tsou Jeffery A. , Selamt Suhaida A. , Rom William N. , Laird-Offringa Ite A. 
Published: March 2013

Background: Lung cancer is the leading cause of cancer death worldwide. Over 1.3 million people succumbed to lung cancer in 2008 according to the World Health Organization (WHO). The predicted five year survival rate for non-small cell lung cancer (NSCLC) for the year 2010 is 16%. An early diagnosis would significantly increase survival of lung cancer patients. Low dose computed tomography (LDCT) detects tumors as small as T0. However, in a recent study by The National Lung Screening Trial Research Team, the false positive rate was 96%. DNA methylation, which has been shown to be associated with gene inactivation in various cancers, exhibits cancer-specific profiles. We hypothesize that DNA methylation in plasma/serum could be a powerful biomarker to complement LDCT when a lesion is detected.

Results: Using candidate gene analysis in combination with epigenetic profiling we have identified 12 DNA methylation markers based on comparisons between archival lung cancer samples and matched adjacent non-tumor lung: 2C35, GDNF, HOXA1, HOXB4, MT1G, NEUROD1, NID2, OPCML/HNT, SFRP1, TNFRSF25, TRIM58, TWIST1 are all highly significantly and frequently hypermethylated in lung cancer tissues. Because detection of shed DNA in bodily fluids requires a very sensitive method, we have adapted Digital MethyLight to interrogate the 12 markers simultaneously on 384-well plates. Digital MethyLight is a sensitive real time-based limiting dilution method in which free floating DNA extracted from bodily fluids is diluted over a large number of wells (in our case 90) so that the number of detectable methylated DNA molecules can be counted. We used three different colors of probes to generate four sets of multiplexed probes, covering our 12-marker panel. Using this method, we examined the serum of 90 patients with lung cancer and 90 high-risk controls from New York City. We also examined serum from an additional 30 low risk, non-smoker controls. Many of our markers showed a statistically significant difference in methylation when comparing cases to non-smoker controls, but the difference with the high risk smokers was much less pronounced. Several subjects from the high-risk controls showed DNA methylation levels comparable to the cases, and several of these controls were ultimately diagnosed with cancer. Interestingly, we found no correlation between tumor stage and amount of DNA shed, and were able to detect methylated DNA in the serum of several stage 1A patients.

Conclusion: Elevated DNA methylation is observed in lung cancer cases, as well as in a number of high risk control subjects. Serum DNA methylation levels are markedly lower in non-smoking controls. While sensitivity is low, methylated DNA can be detected in stage 1A patients, suggesting that even small tumors can shed sufficient DNA to allow detection. It is not clear why some patients do not show detectable DNA, even with progressed disease. This merits further investigation.

Funding: R01 CA 119029, R01 CA120869, Canary Foundation and Thomas G. Labrecque Foundation.