Dataset Information

MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR.

ABSTRACT:

Background

Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method.

Method

We examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP) and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR.

Results

When examined by qMSP, 26% of low-grade and 37% of high-grade gliomas were found to be methylated, whereas 97% of low-grade and 55% of high-grade gliomas were found methylated by pyrosequencing. The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection. Primary glioblastoma patients with a methylated MGMT promoter (as evaluated by both methylation detection methods) had approximately 5 months longer median survival compared to patients with an unmethylated promoter (log-rank test; pyrosequencing P = .02, qMSP P = .06). One third of the analyzed samples had conflicting methylation results when comparing the data from the qMSP and pyrosequencing. The overall survival analysis shows that these patients have an intermediate prognosis between the groups with concordant MGMT promoter methylation results when comparing the two methods.

Conclusion

In our opinion, MGMT promoter methylation analysis gives sufficient prognostic information to merit its inclusion in the standard management of patients with high-grade gliomas, and in this study pyrosequencing came across as the better analytical method.

SUBMITTER: Havik AB

PROVIDER: S-EPMC3311573 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Publications

MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR.

Håvik Annette Bentsen AB Brandal Petter P Honne Hilde H Dahlback Hanne-Sofie Spenning HS Scheie David D Hektoen Merete M Meling Torstein Ragnar TR Helseth Eirik E Heim Sverre S Lothe Ragnhild A RA Lind Guro Elisabeth GE

Journal of translational medicine 20120306

<h4>Background</h4>Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method.<h4>Method</h4>We examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP) and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR.<h4>Results</h4>When examine ...[more]

PMID: 22390413

Similar Datasets

Project description:BACKGROUND: O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is reported to be a prognostic and predictive factor of alkylating chemotherapy for glioblastoma patients. Methylation specific PCR (MSP) has been most commonly used when the methylation status of MGMT is assessed. However, technical obstacles have hampered the implementation of MSP-based diagnostic tests. We quantitatively analyzed the methylation status of the entire MGMT promoter region and applied this information for prognostic prediction using sequencing technology. METHODS: Between 1998 and 2012, the genomic DNA of 85 tumor samples from newly diagnosed glioblastoma patients was subjected to bisulfite treatment and subdivided into a training set, consisting of fifty-three samples, and a test set, consisting of thirty-two samples. The training set was analyzed by deep Sanger sequencing with a sequencing coverage of up to 96 clones per sample. This analysis quantitatively revealed the degree of methylation of each cytidine phosphate guanosine (CpG) site. Based on these data, we constructed a prognostic prediction system for glioblastoma patients using a supervised learning method. We then validated this prediction system by deep sequencing with a next-generation sequencer using a test set of 32 samples. RESULTS: The methylation status of the MGMT promoter was correlated with progression-free survival (PFS) in our patient population in the training set. The degree of correlation differed among the CpG sites. Using the data from the top twenty CpG sites, we constructed a prediction system for overall survival (OS) and PFS. The system successfully classified patients into good and poor prognosis groups in both the training set (OS, p?=?0.0381; PFS, p?=?0.00122) and the test set (OS, p?=?0.0476; PFS, p?=?0.0376). Conventional MSP could not predict the prognosis in either of our sets. (training set: OS; p?=?0.993 PFS; p?=?0.113, test set: OS; p?=?0.326 PFS; p?=?0.342). CONCLUSIONS: The prognostic ability of our prediction system using sequencing data was better than that of methylation-specific PCR (MSP). Advances in sequencing technologies will make this approach a plausible option for diagnoses based on MGMT promotor methylation.

Dataset Information

MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR.

Background

Method

Results

Conclusion

Publications

MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR.

Similar Datasets

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