Promoter hypermethylation is a frequent mechanism for the repression of gene transcription in cancer and is regarded as one of the hallmarks of cancer. Analysis of DNA methylation is a promising tool for early cancer detection, risk assessment, and response to therapy. In a recent study (1), quantitative highresolution melting analysis (HRM) with the Roche (SIX: RO, ROG; OTCQX: RHHBY) LightCycler Instrument was used for promoter methylation for analysis of formalinfixed paraffinembedded (FFPE) tissues. Since FFPE tissue samples are the largest source of material from normal controls and diseased tissues, their use is of inestimable value for research. Methodical evaluations are of high importance for demonstrating robustness and sensitivity of the assay, thus facilitating its establishment as a research tool and possibly a future routine test.
The aim of the study was to establish and evaluate HRM assays for detection of promoter methylation on archival FFPE tissues from individuals with colorectal cancer. As proof of the principle, the researchers demonstrated the applicability of HRM for detection of promoter methylation using assays for O6methylguanine-DNA methyltransferase (MGMT), adenomatous polyposis coli (APC), glutathione Stransferase 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. 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. 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.
The most popular approaches for DNA methylation detection rely on the treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil while leaving 5methyl cytosine unmodified. This modification results in a sequence difference allowing for identification of methyl cytosines in a subsequent PCR amplification. The most precise methylation profiling can be achieved by bisulfite sequencing, which allows identification of single methyl cytosines. Several simpler PCRbased methods, which are especially important for small scale research labs, have also been developed. One of these newer and easier methods is highresolution melting analysis (HRM), based on the "melting" properties of DNA in solution. The principle of this method is that bisulfitetreated DNA templates with different contents of methyl cytosine can be distinguished by melting analysis based on differences in melting temperatures. HRM is a relatively simple and costeffective 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.
(1) Roche Applied Science Cancer Research Application Note No. 3: M. Malic, M. Pichler, E. Heitzer, J. Strutz, N. Dandachi (2009) High quality assessment of DNA methylation in archival tissues from patients with colorectal cancer using quantitative highresolution melting analysis, www.cancer-research.roche.com.
The aim of the study was to establish and evaluate HRM assays for detection of promoter methylation on archival FFPE tissues from individuals with colorectal cancer. As proof of the principle, the researchers demonstrated the applicability of HRM for detection of promoter methylation using assays for O6methylguanine-DNA methyltransferase (MGMT), adenomatous polyposis coli (APC), glutathione Stransferase 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. 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. 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.
The most popular approaches for DNA methylation detection rely on the treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil while leaving 5methyl cytosine unmodified. This modification results in a sequence difference allowing for identification of methyl cytosines in a subsequent PCR amplification. The most precise methylation profiling can be achieved by bisulfite sequencing, which allows identification of single methyl cytosines. Several simpler PCRbased methods, which are especially important for small scale research labs, have also been developed. One of these newer and easier methods is highresolution melting analysis (HRM), based on the "melting" properties of DNA in solution. The principle of this method is that bisulfitetreated DNA templates with different contents of methyl cytosine can be distinguished by melting analysis based on differences in melting temperatures. HRM is a relatively simple and costeffective 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.
(1) Roche Applied Science Cancer Research Application Note No. 3: M. Malic, M. Pichler, E. Heitzer, J. Strutz, N. Dandachi (2009) High quality assessment of DNA methylation in archival tissues from patients with colorectal cancer using quantitative highresolution melting analysis, www.cancer-research.roche.com.
