MGMT Promoter Methylation in Gliomas. Comparison of Methods: Methylation Specific PCR (MSP) vs Pyrosequencing.
Giovanna DeMaglio, Luca Morandi, Giovanni Falconieri, Giovanni M Guarrera, Miran Skrap, Giovanni Tallini, Stefano Pizzolitto. University Hospital, Udine, Italy; University Hospital Bellaria, Bologna, Italy
Background: Methylation of MGMT gene promoter is predictive of response to alkylating chemotherapy and a prognostic biomarker for improved survival in patients affected by glioma and treated with radiotherapy combined with temozolomide. MGMT promoter is methylated in about 40% of primary glioblastomas and also low levels of methylation predict pharmacological response. Therefore testing for MGMT promoter methylation is increasingly performed as routine test in neuropathology laboratories. MSP was introduced firsty and is still the most popular assay, however other quantitative techniques have been then developed. The aim of our study was to compare a qualitative and a quantitative method for MGMT promoter methylation status, i.e. MSP and Pyrosequencing.
Design: A total number of 119 cases of gliomas have independently been tested by means of MSP and Pyrosequencing at 2 different laboratories. All samples were from formalin-fixed and paraffin embedded tissues. After DNA extraction, conversion of unmethylated cytosines to uracils has been carried out. MSP was performed following the directions published elsewhere. For all cases a quantitative PCR assay (MS-qPCR) was applied to confirm MSP results. Pyrosequencing was done using a commercially available kit (PyroMarkTM MGMT kit, Qiagen) according to manufacturer's instructions on a PyroMarkTM Q96 ID instrument (Qiagen). The test is designed to detect and quantify methylation level in five CpG sites in exon 1 of MGMT gene.
Results: Concordance between MSP and Pyrosequencing has been observed in 114/119 (96%) cases. Four patients evaluated by MSP techniques revealed non-amplifiable DNA, while all but 1 case analysed by Pyrosequencing gave a diagnostic DNA. Pyrosequencing internal control for bisulfite treatment always showed complete conversion. Only 1 case was unmethylated by MSP while Pyrosequencing showed methylation status. Eight cases gave discordant results, but after test repeating Pyrosequencing demonstrated correct results.
Conclusions: Our study indicates that the yield of MSP and Pyrosequencing is highly comparable, however Pyrosequencing has the added value of quantificating the methylation status of single CpG islands. Furthermore it allowed identification of very low levels of methylation even in single CpG sites.
Tuesday, March 1, 2011 1:00 PM
Poster Session IV # 234, Tuesday Afternoon