Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas
Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas
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DOI：
10.3892/ijo_00000029
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发表时间：
2008-09-01
影响因子：
5.2
通讯作者：
Nagase, Hiroki
中科院分区：
文献类型：
--
作者：
Yachi, Kazunari;Watanabe, Takao;Nagase, Hiroki
O-6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT) 启动子高甲基化最近已成为胶质母细胞瘤化疗敏感性的强大决定因素。为了使如此重要的表观遗传生物标志物适应临床环境中的常规应用，我们验证了常规使用的甲基化特异性聚合酶链反应（MSP）测定法在确定 MGMT 甲基化状态中的相关性。对 25 个原代胶质母细胞瘤样本和 7 个细胞系进行了采用 MSP 的 MGMT 启动子高甲基化分析，并与更可靠的直接启动子测序进行了比较，后者描绘了 MGMT 启动子内 27 个 CpG 位点的甲基化状态。此外，使用免疫组织化学在原发性肿瘤样品中评估蛋白质水平的 MGMT 表达，并使用蛋白质印迹分析评估细胞系中的 MGMT 表达。我们的 MSP 分析产生了可重复的结果，除了一个原发肿瘤在 MSP 上呈阴性之外，所有结果都与亚硫酸氢盐测序数据相同。原发性胶质母细胞瘤样本中免疫组化染色结果与 MGMT 启动子甲基化状态之间存在较差的相关性。 MSP-MGMT 甲基化和免疫组织化学 MGMT 表达在这一小规模且不均匀的患者组中均没有预后意义。在所有 MGMT 表达缺失的细胞系中，MSP 检测到甲基化 DNA 信号。我们的数据支持 MSP 分析的可行性和可靠性，可以在诊断环境中常规实施。
O-6-Methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation has recently emerged as a powerful determinant of chemotherapy sensitivity in glioblastomas. To adapt such an important epigenetic biomarker to routine application in the clinical setting, we validated the conventionally used methylation-specific polymerase chain reaction (MSP) assay for its relevance in the determination of MGMT methylation status. MGMT promoter hypermethylation analysis employing MSP was performed on 25 primary glioblastoma samples and 7 cell lines, and compared with the more robust direct promoter sequencing that profiled the methylation status of 27 CpG sites within the MGMT promoter. In addition, the MGMT expression at the protein level was evaluated in the primary tumor samples using immunohistochemistry and in the cell lines using Western blotting analysis. Our MSP analyses yielded reproducible results, which were identical to the bisulfite sequencing data in all except one primary tumor that was negative on MSP. A poor correlation existed between the immunohistochemical staining results and the methylation status of the MGMT promoter in primary glioblastoma samples. Neither MSP-MGMT methylation nor immunohistochemical MGMT expression had prognostic implications in this small and non-uniform group of patients. In all of the cell lines with loss of MGMT expression, signals of methylated DNA were detected by MSP. Our data support the feasibility and reliability of MSP analysis, which could be routinely implemented in the diagnostic setting.