Tumor tissue specimens were fixed in 10% formalin and embedded in paraffin wax using routine processing. For each subject, H&E‐stained sections were examined to classify the tumors according to the WHO International Histological Classification of Tumours. The MGMT promoter methylation status was determined by qMSP. The qMSP process involves the treatment of genomic DNA, denatured by sodium hydroxide with hydroquinone and sodium bisulfite to convert unmethylated C into T. The target gene was amplified by primers designed according to the gene sequence after C‐to‐T conversion. Because the C in the methylated CPG island cannot be converted into T, the target gene will not be amplified with the above primers. Therefore, the purpose of identifying genomic DNA methylation can be achieved by the above methods. To detect the MSP amplification products, if the fragments can be amplified with the primers for the methylated DNA chain, it means that there is methylation at the detected site. If the primers for the unmethylated DNA chain after treatment are used to amplify the fragments, it means that there is no methylation at the detected sites. To reduce the influence of tumor heterogeneity, we selected the solid components of the tumor for amplification, avoiding necrotic or hemorrhagic areas. DNA extracted from unstained tissue slides made from paraffin‐embedded blocks of tumor tissues was subjected to bisulfite modification by a Simlex OUP FFPE DNA extraction kit (TIB), according to the manufacturer’s instructions. Polymerase chain reaction was carried out with a DRR007 kit (Takara) using the Verity 96‐Well Thermal Cycler (Thermo Fisher Scientific), and pyrosequencing was done using the PyroMark Q96 system (Qiagen) in the 5 CpG island region within the MGMT promoter. A glioma was defined as “methylated” if the average methylation rate of the CpG regions was 8% or higher, otherwise, the tumor was classified as “unmethylated” (Figure 1). 11

Sequencing peak map of methylguanine methyltransferase (MGMT) promoter methylation in glioma tumor tissue specimens. A, MGMT promoter methylated. B, MGMT promoter unmethylated

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