Introduction

The association between DNA methylation and diseases such as cancer opens the door to the development of new clinical testing technology, applicable in both diagnostic and prognostic settings. Methylated DNA possesses all of the desired hallmarks of an optimal biomarker amenable to testing in a clinical setting. Compared with protein and mRNA, methylated DNA is chemically stable and less subject to transient alterations due to biological variability. In many cancers, cell-free, tumor-derived DNA is shed and can be detected in a patient's serum (1,2). Therefore, detection of occult malignant cells may be achievable without direct tumor sampling (see Reference 3, for a review). Some of the better studied examples of genes whose transcription is silenced by methylation in cancer include p16^INK4a, hMLH1, and GSTP1, the most frequently hypermethylated gene in prostate cancer (4).

Although a small set of genes have been characterized that are affected by methylation of specific residues within their upstream regions (5,6), for most genes the overall density of methylation surrounding the transcriptional start sites correlates best with gene expression (7). While methylation of specific sites may have an important impact upon expression of the gene, cells rarely modify only those sites (5), suggesting that regional methylation may be the basis of the mechanism of DNA methylation-associated transcriptional inactivation rather than methylation of specific residues. Therefore, a regional perspective seems warranted when determining target DNA methylation status. It would seem that most biologically (and potentially clinically) relevant information should ideally reveal not only whether a DNA target contains methylation, but also the extent of methylation occupancy within the target region and the abundance or load of those aberrantly methylated molecules.

Previously developed methods for methylation detection rely on digestion of target DNA with methylation-sensitive restriction enzymes (MSREs) for which restriction is blocked by methylation. Digestion is followed by qualitative measurements such as Southern blot analysis, or semiquantitative measurements of restriction-refractory template in end point PCR (8), or by quantitative PCR (9). Alternative methods employ bisulfite conversion of unmethylated cytosine residues, followed by direct DNA sequencing (10) or amplification selecting for specific methylated base configurations [i.e., methylation-specific PCR (MSP)] (11) alone or in combination with quantitative PCR [(qMSP) (12) or MethyLight (13)]. Each of these approaches has associated limitations. MSRE-based methods are prone to false-positive results due to incomplete restriction, while bisulfite conversion techniques are laborious in terms of time and reagent consumption (14). In addition, amplification primers and detection probes used in all MSP-based methods by necessity encompass several CpG dinucleotides in the sequences, and only DNA that has the complementary methylation state for all cytosines covered by the primers/probes will be recognized in the assay. Given the large number of differing methylation configurations that may be associated with gene silencing at a given locus, these assays can only detect a tiny fraction of the total number of erroneously methylated molecules present in a sample.

To overcome many of the limitations associated with existing methylation detection technologies and to develop an assay that is particularly suited for the clinical laboratory, we have developed a novel method, MethylScreen, which when coupled with quantitative PCR permits sensitive detection and quantification of cytosine methylation in genomic loci (15). The MethylScreen technology is based upon the combination of methylation-sensitive and methylation-dependent restriction enzymes in single-and double-digests. The inclusion of a methylation-dependent restriction enzyme (MDRE) in the assay design not only eliminates false-positive reporting but also provides additional valuable information about the methylation density present in the region studied. The method is fast and efficient, and amenable to high-throughput clinical formats. Bisulfite conversion of DNA is not required, allowing the assay to be performed on nanogram quantities of starting template. In this report, we present an analysis of the performance characteristics of the MethylScreen assay, along with the methods necessary to interpret results from the assay. Importantly, in this study the MethylScreen measurements obtained were validated using bisulfite genomic sequencing. Our results suggest there may be an inherent benefit of application of the MethylScreen approach to biomarker-based cancer detection in tissue samples.