DIGITAL DETECTION OF TUMOR-DERIVED CIRCULATING METHYLATED DNA


Year of Award:
2014
Award Type:
R21
Project Number:
CA186809
RFA Number:
RFA-CA-13-001
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
WANG, TZA-HUEI
Other PI or Project Leader:
N/A
Institution:
JOHNS HOPKINS UNIVERSITY
Aberrantly methylated DNA is found abundantly in all forms of cancer and the tumors that produce them. In fact, it has been estimated that within the cells of every tumor are several hundreds of aberrantly methylated CpG islands, many of which are promoters of tumor suppressor genes. The ability to detect and quantify promoter methylation will allow much more refined diagnostic and prognostic stratification. Recently, several groups including ours have reported the detection of tumor-associated methylated DNA circulating in serum/plasma. The use of circulating methylated DNA is a particularly attractive for screening and companion diagnostics for cancer, as serum/plasma is obtained through a simple, relatively noninvasive procedure. Currently, detection of circulating methylated DNA is mainly performed using bisulfite-based methods such as methylation specific PCR (MSP) due to their high sensitivity and specificity. However, clinical applications of these tests have been encumbered by a number of hurdles, resulting in highly variable success. For any bisulfite based methods, the process of DNA extraction and bisulfite conversion involves several disconnected steps on different platforms, resulting in substantial sample loss. Furthermore, MSP is designed to detect a specific methylation pattern; however, the promoter methylation patterns may be highly variable in tumors, compromising its clinical sensitivity. While sequence-based methods including bisulfite sequencing and pyrosequencing can be used to analyze methylation heterogeneity, these methods do not have the requisite sensitivity to detect the extremely low ratios (