Correlation between testing MGMT gene promoter methylation status of glioblastoma tissue using nested gel-based and direct real-time fluorescence-based methylation-specific PCR.

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Introduction: The treatment options for primary malignant brain cancers are limited and available therapies produce modest benefit highlighting the need for more targeted, patient specific treatments. Epigenetic silencing of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation in the tumor tissue is associated with longer survival in patients with glioblastoma who receive alkylating agents, such as carmustine (1) and temozolomide (2, 3). OncoMethylome Sciences (ONCO) has developed a direct real-time fluorescence-based methylation-specific PCR assay (real-time MSP assay) to define the methylation status of the MGMT promoter.
 Materials and Methods: Sample preparation: 125 tissue samples (formalin fixed paraffin embedded (FFPE) sections), predominantly glioblastoma, were used for this study. When processing FFPE samples a deparaffination step (standard reagents) was performed prior to DNA isolation using a standard method and standard equipment. Real-time MSP: DNA was bisulphite modified using a commercially available kit. In addition to MGMT, the independent reference gene β-actin (ACTB) was also measured. The analyte (m_MGMT and ACTB) quantitations were done in real-time PCR assays. These consisted of parallel amplification / quantitation processes. The amplicons created during the amplification process were quantified by real-time measurement of emitted fluorescence. The ratio between m_MGMT and ACTB was calculated. Gel-based MSP: was conducted as published previously (2, 3). Briefly, bisulphite modified DNA was subjected to MSP using a two step approach with nested primers. The first round of PCR amplifies both the methylated as well as the unmethylated version of the MGMT sequence (m_MGMT and u_MGMT). The specific primers for m_MGMT recognize the fully methylated sequence. The products from the second PCR were visualized on 4% agarose gels to determine the MGMT methylation status.
 Results: A cut-off for the MGMT methylation status was suggested by the bimodal distribution of the quantitative results fitting a normal mixture model, supporting the hypothesis that there are two distinct populations within the test samples.
 Conclusions: Comparison between gel-based and real-time MSP showed high concordance of the results. The real-time MSP assay was highly reproducible, and had an excellent evaluability rate with valid test results. Moreover the use of a real-time PCR platform provides the possibility for high throughput analysis and it also opens the possibility of determining in the future a test threshold optimized for clinical applications.
 References: 1) Esteller M et al. N Engl J Med 2000; 343(19):1350-4., 2) Hegi ME et al. Clin Cancer Res 2004; 10(6):1871-4, 3) Hegi ME et al. N Engl J Med 2005; 352(10):1036-8,