Abstract 4666: DREAMing as a simple and low cost alternative for the assessment of methylation in ultra rare DNA

Abstract

Abstract Background: Current approaches for the assessment of methylation, such as methylation-specific PCR (MSP) and next-generation bisulfite sequencing (BS-Seq) are fundamentally limited in their ability to detect and assess heterogeneous methylation patterns (epialleles) in ultra-rare (<0.1%) DNA. These limitations critically compromise diagnostic utility and render them ill suited for many emerging applications in cancer diagnostics, such as the analysis of methylation heterogeneity in cell-free DNA (cfDNA) and rare cell populations. We recently addressed the need for a low cost alternative to the assessment of methylation of ultra-rare DNA with the development of DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of DNA at single copy sensitivity and single-CpG-site resolution. Here, we seek to demonstrate the advantages of the DREAMing method over conventional approaches to methylation assessment. Methods: We expand upon the underlying theory of DREAMing and provide guidelines for the development of single-copy sensitive DREAMing assays. We further elucidate methods for tailoring DREAMing assays to samples of interest and compare the performance of these assays to commonly employed techniques including quantitative MSP (qMSP) and BS-Seq. Results: Development of single-copy sensitive DREAMing assays for a number of loci associated with classic tumor-specific methylation such as CHFR and RASSF1A as well as a candidate pan-cancer locus are reported. These assays are then used to analyze methylation in cfDNA derived from the plasma of cancer-positive and healthy patients. DREAM analysis reveals that DREAMing can readily detect over an order of magnitude more epialleles when directly compared to qMSP and BS-Seq assays of the same locus. Some of the challenges associated with distinguishing potential tumor-specific aberrant methylation from background methylation are then discussed and proposed solutions are demonstrated. Lastly, methods for optimizing DREAMing assays for specific sample types are discussed. Conclusions: DREAMing is a recently introduced method for the assessment of locus-specific methylation in samples containing ultra rare target DNA. Its low cost and simplicity coupled with the ability to provide enhanced, single-copy detection of heterogeneous methylation make DREAMing an attractive option over traditional techniques for demanding specimens such as cfDNA and rare cell populations. DREAMing has potential utility in the evaluation of DNA methylation dynamics in cell populations, prenatal testing, as well as clear use in early cancer diagnostic, companion diagnostic and predictive applications. Citation Format: Thomas R. Pisanic, Pornpat Athamanolap, Brendan F. Miller, Vincent Wu, Laura Elnitski, Tza-Huei Wang. DREAMing as a simple and low cost alternative for the assessment of methylation in ultra rare DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2017-4666