Abstract 4624: Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents.
Authors: Yang Xiaojing , Han Han , Treppendahl Marianne B. , Tsai Yvonne C. , O'Connell Casey , Weisenberger Dan , Lay Fides , Grønbæk Kirsten , Liang Gangning , Jones Peter A. 
Published: August 2014

The successful use of DNA methyltransferase inhibitors (DNMTis) in Myelodysplastic Syndromes (MDS) therapy has brought epigenetic drugs to the forefront of cancer management. However it has been difficult to find a consistent association between patients’ outcome and DNA methylation changes. This could be attributed to a variety of factors including the choice of DNA methylation markers to track the response to treatment.

LINE-1 methylation changes are widely studied as a marker predictive of genome-wide DNA methylation changes; however recent reports have shown that its methylation levels are variable across tissues. In addition, we have found that LINE-1 remethylation after DNMTis withdrawal occurs faster than that of other regions, suggesting that LINE-1 methylation changes might not reflect the overall demethylation effects of DNMTis. Thus, it is imperative to find more sensitive DNA methylation markers to accurately track the methylation change that occurs after DNMTi treatment. Ideally, such markers could be applied to various tumor types and to samples collected by invasive and non-invasive methods. To find improved DNA methylation markers, we took advantage of the well-established Infinium DNA methylation platform and found 1429 probes, which were consistently methylated in both normal/tumor bladder tissue samples as well as white blood cells from healthy donors. The remethylation pattern of those 1439 probes was investigated in the T24 bladder cancer and HL60 leukemia cell lines treated with 5-Aza-CdR for 24 hours. Probe consensus clustering yielded a group of 79 probes that significantly responded to the demethylation treatment and remained demethylated beyond 30 days. In silico analysis of the DNA methylation patterns of the top two probes show consistent hypermethylation in both normal and tumor samples. As a proof of principle, we tested the DNA demethylation levels of these two markers in urine sediments from 7 MDS patients treated with Azacitidine using pyrosequencing. Our results showed that the two markers were significantly demethylated; in contrast, LINE-1 methylation showed no clear decreasing trend. Demethylation of these two markers was also observed in peripheral blood samples from MDS patients treated with Azacitidine. We are in the process of testing more samples to find the association between marker demethylation and patient's outcome. In summary, we have identified and validated two DNA methylation markers, which unlike LINE-1, show consistent demethylation in response to the DNMTi treatment irrespective of the type of sample tested. The wider range of demethylation provided by these markers may offer a more accurate representation of the patient's response to treatment.

Citation Format: Xiaojing Yang, Han Han, Marianne B. Treppendahl, Yvonne C. Tsai, Casey O'Connell, Dan Weisenberger, Fides Lay, Kirsten Grønbæk, Gangning Liang, Peter A. Jones. Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2013-4624