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

Section for Cancer Cytogenetics, Institute for Medical Informatics, Oslo University Hospital-The Norwegian Radium Hospital, P,O, Box 4950 Nydalen, N-0424 Oslo, Norway.
Journal of Translational Medicine (Impact Factor: 3.93). 03/2012; 10(1):36. DOI: 10.1186/1479-5876-10-36
Source: PubMed


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.
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.
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.
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.

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    • "Internal control for completion of the bisulfite treatment was included in the sequence. The cutoff value to distinguish between methylated and unmethylated samples was established at 11 %, based on the results obtained on control non-cancerous brain tissue, according to the previously described formula (Majchrzak-Celin´ska et al. 2015; Håvik et al. 2012). "
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    • "970032) provided by Qiagen. These kits are adapted for the PyroMark Q96 ID System and PyroMark Q24 MDx System, respectively and have already been used in several studies [10, 14–17]. CpGs 76–79 are tested in another MGMT PSQ kit (PyroMark Q24 CpG MGMT) developed by Qiagen for the PyroMark Q24 MDx System (ref. "
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    ABSTRACT: The methylation of O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a key biological marker in clinical neuro-oncology. Nevertheless, there is no consensus concerning the best technique for its assessment. In a recent study comparing five methods to analyze MGMT status, we found that the best prediction of survival was obtained with a pyrosequencing (PSQ) test assessing methylation of 5 CpGs (CpGs 74–78). In the present study we extended our PSQ analysis to 16 CpGs (CpGs 74–89) identified as critical for transcriptional control of the gene. The predictive value of the methylation levels at each CpG, as well as the mean methylation levels of selected sets of consecutive CpGs was tested in a cohort of 89 de novo glioblastoma patients who had received standard of care treatment (Stupp protocol). Using an optimal risk cut-off, each CpG or combination of CpGs, was associated with overall survival (OS) and progression free survival. The best predictive models for OS after stratification on performance score and age were obtained with CpG 89, CpG 84 and mean methylation of CpG 84–88 (Hazard ratio (HR), 0.31; p < 0.0001). The improvement compared to the predictive value of the test analyzing average methylation of CpG 74–78 (HR, 0.32; p < 0.0001) was however marginal. We recommend to test CpGs 74–78 when analyzing MGMT methylation status by PSQ because a commercial kit that has successfully been used in several studies is available, allowing reproducible and comparable results from one laboratory to another. Electronic supplementary material The online version of this article (doi:10.1007/s11060-013-1332-y) contains supplementary material, which is available to authorized users.
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    • "The widely used semi-quantitative methods, such as MSP, bear a significant risk of false-positive or false-negative results, especially when the DNA quality and/or quantity is low, which is often the case in clinical settings. In comparison to the other quantitative methods as COBRA (combined bisulfite restriction analysis), SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography) or Q-MSP (quantitative methylation specific PCR), pyrosequencing has been shown to be the most reliable method that fulfils also the basic requirements of clinical settings, such as robustness, cost efficiency and easy use [43] [44]. As a general observation from DNA methylation studies, the RASSF1A gene appeared to be a promising biomarker of BC in plasma/serum. "
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    ABSTRACT: Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated. Keywords: breast cancer, protein expression, DNA methylation, invasivity and metastasising associated genes, pyrosequencing analysis.
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