HRM is based on the melting properties of DNA in solution. The principle of this method is that bisulfite-treated DNA templates with different contents of methyl cytosine can be distinguished by melting analysis based on differences in melting temperatures. Analysis of DNA methylation is a promising tool for early cancer detection, risk assessment, and response to therapy.

A recent study evaluated HRM assays for detection of promoter methylation on archival FFPE tissues from individuals with colorectal cancer. This sensitive assay can be adapted and used to detect low amounts of methylated cells within a tumor, or even to detect low numbers of tumor cells in the background of nontumor cells in lymph nodes and other organs. Neoplasia is characterized by "methylation imbalance" where genome-wide hypomethylation is accompanied by localized hypermethylation and an increase in expression of DNA methyltransferase.

The applicability of HRM for detection of promoter methylation was demonstrated using assays for O6-methylguanine-DNA methyltransferase (MGMT), adenomatous polyposis coli (APC), glutathione S-transferase P1 (GSTP1), and phosphatase and tension homolog deleted on chromosome 10 (PTEN) promoters in methylated DNA dilution matrix. In a second step, HRM assays for MGMT and APC were tested on DNA isolated from fresh and FFPE human cancer cell lines. These established MGMT and APC HRM assays were analyzed using archival FFPE colorectal tumor specimens. HRM assays were performed with the Roche LightCycler Instrument (Roche Diagnostics; Basel, Switzerland). Methylated DNA levels as low as 1% were reproducibly detected in a background of unmethylated DNA. For certain applications, such as detection of rare events or risk stratification of individuals based on methylation status of specific markers, high sensitivity of the assay is important. The results of the study were issued in 2009 in Roche Applied Science Cancer Research Application Note, No. 3.

HRM is a relatively simple and cost-effective method, since it does not require expensive probes and reference gene assays for normalization. With HRM, all CpGs within the amplicon are analyzed, enabling the assay to distinguish heterogeneous from homogeneous methylation by the shape of the melting curve. This ability can be of importance, because methylation patterns at promoter CpG islands are typically not homogeneous.

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