Jeuken Judith W M, Cornelissen Sandra J B, Vriezen Martine, Dekkers Marieke M G, Errami Abdellatif, Sijben Angelique, Boots-Sprenger Sandra H E, Wesseling Pieter
Department of Pathology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Lab Invest. 2007 Oct;87(10):1055-65. doi: 10.1038/labinvest.3700664. Epub 2007 Aug 13.
Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.
由O6 - 甲基鸟嘌呤（O6 - mG）-DNA - 甲基转移酶（MGMT）DNA修复基因编码的DNA修复蛋白O6 - 烷基鸟嘌呤 - DNA - 烷基转移酶（AGT）的表达导致对烷化剂产生抗性，而MGMT启动子的高甲基化与化学敏感性相关，因为它会阻止AGT的表达。由于评估AGT表达的免疫组化结果的解读被证明很困难，我们当前研究的目的是建立一种可行、可靠且稳健的MGMT启动子高甲基化检测方法，该方法可在诊断环境中轻松实施且适用于常规处理的组织。对55个个体肿瘤（包括12个细胞系）的62个胶质瘤样本进行了使用甲基化特异性（MS - ）多重连接依赖探针扩增（MLPA）的MGMT高甲基化分析，并与更常用但经过改进的MS - 聚合酶链反应（PCR）进行比较。与MS - PCR不同，MS - MLPA：（i）不基于未甲基化胞嘧啶的亚硫酸氢盐转化（MS - PCR中有点麻烦的步骤）；（ii）提供所有样本的甲基化状态；（iii）被证明是半定量的；（iv）可用于同时评估多个序列（CpG二核苷酸）的甲基化状态；（v）允许联合拷贝数检测和甲基化特异性分析。在一组接受替莫唑胺化疗的20例胶质母细胞瘤患者中显示了使用MS - MLPA评估MGMT高甲基化的潜在治疗价值。我们得出结论，MS - MLPA是一种稳健且可靠的方法，可轻松应用于不同处理的组织，包括那些用福尔马林固定并石蜡包埋的组织。MS - MLPA的半定量方面可能被证明具有很大价值，特别是在预测对烷化剂的反应方面，不仅对于本研究中评估的胶质瘤，而且对于一般肿瘤也是如此。
