Abstract
The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is a strong predictor for the efficacy of temozolomide chemotherapy and survival periods. However, the correlation between the extent of methylation and the difference in survival times has not been fully clarified. Simple and quantitative evaluations of the methylation status in the promotor region of the MGMT gene are expected to be worldwide standardized diagnostics. We applied real-time semi-quantitative methylation-specific polymerase chain reaction (SQ-MSP) of the MGMT gene promoter region to 84 glioblastoma patients. The SQ-MSP result showed that the ΔCt value, which represents the difference between uCt and mCt (uCt value – mCt value), is inversely correlated with overall survival. With adequate cutoff setting, this assay showed that those patients suffering from a tumor with low ΔCt (methylated) survived significantly longer than those having tumors with high ΔCt (un-methylated). The most significant difference was observed when the cutoff was set at a ΔCt of 2. Using this cutoff point, the result of MGMT immunohistochemical analysis was also significantly correlated with the methylation status examined with real-time SQ-MSP. These results collectively show that MGMT promoter methylation status actually affects patients’ survival and protein expression depending on its methylation level, and the extent of methylated CpGs would be better assessed with real-time SQ-MSP than with the standard gel-based MSP. This method is cost- and labor-saving compared with pyrosequencing, and significantly contributes to the accurate and objective prediction of patient survival.
Highlights
Glioblastoma is one of the most malignant cancers of the central nervous system
We showed that the real-time semi-quantitative methylation-specific polymerase chain reaction (MSP) (SQ-MSP) is an effective method for evaluating the methylation status of the methylguanine-DNA methyltransferase (MGMT) gene promoter for the accurate prediction of patient survival
We clearly showed that the information obtained from real-time semiquantitative methylation-specific polymerase chain reaction (SQ-MSP) concerning MGMT promoter methylation status is highly correlated with the survival period of the GBM patients treated with the standard Stupp’s protocol
Summary
Glioblastoma is one of the most malignant cancers of the central nervous system. Standard treatment includes radiotherapy and temozolomide chemotherapy after surgery. Silencing the MGMT gene by promoter methylation results in decreased MGMT expression and improves the effects of temozolomide [2,3]. It is important to accurately evaluate the methylation status of the MGMT www.oncotarget.com gene promoter in clinical decision making for treatment selection and in the development of novel therapies including MGMT silencing by tumor-targeted siRNA delivery [6,7,8]. It is difficult to interpret PSQ data into clinically-relevant information correlating with MGMT protein expression or patient survival. This method is cost- and labor-intensive, with a necessity for initial investment in the novel sequencer, which prevents this assay from being a world-wide standard essential for the clinical routine and clinical trials. An easier and more quantitative method for analyzing the MGMT promoter methylation status is needed in order to obtain clinically-relevant information in daily medical practice
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