Abstract 2301: Cross validation of NGS methylated targets using droplet digital PCR (ddPCR)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA methylation is one of the most studied epigenetic modifications. DNA methylation plays an important role in a number of physiological processes as well as common diseases such as cancer and neurodegenerative disorders. In mammals, DNA methylation occurs at the C-5 position of cytosine in CpG dinucle¬otide sequences, which are mainly concentrated in regions known as CpG islands. Methylation in CpG islands within gene promoters usually leads to gene silencing. Recent data have shown the correlation of methylation and disease status. However, there are significant limitations in both biology and technology. Biologically, methylation status is highly heterogeneous in nature. In normal controls there is never a complete lack of methylation marks, and conversely, never a completely saturated methylation signature. In addition methylation sites of interest are often located in CpG islands, regions where multiple methylation sites occur side by side, making assay design and discrimination challenging. Technological hurdles add significant variability as well. The typical method for determining methylation is bisulfite conversion, which uses harsh chemicals to convert cytosines to uracil. When a cytosine is methylated, it is largely, though not completely, protected from the conversion process. However, bisulfite conversion also causes a great deal of DNA damage, causing the strands to separate and fragment. In most cases of research, the DNAs that researchers want to examine are from FFPE or cfDNA samples, samples which are already both limited and damaged. Because of these challenges, there is an unmet need for a sensitive yet robust method to analyze DNA methylation in clinical samples in a rapid, inexpensive, and high throughput manner. Herein we showcase the use of ddPCR for detection of DNA methylation in bisulfite converted gDNA samples. First we demonstrate the superior sensitivity, linearity and robustness of ddPCR methylation detection in the SNRPN promoter, a model system for an imprinted gene involved in neurological disorders. Secondly we show the detection of DNA methylation in the vimentin promoter, which is known to be methylated in cancer (Li et al. Nat. Biotech. 2009). We will demonstrate reproducible detection in replicate bisulfite converted samples and sensitivity. Citation Format: Dawne N. Shelton, Claudia Litterst, John F. Regan, Helen R. Moinova, Sanford D. Markowitz. Cross validation of NGS methylated targets using droplet digital PCR (ddPCR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2014-2301