脑胶质瘤 O6-甲基鸟嘌呤-DNA 甲基转移酶启动子甲基化分析：基于锁核酸的定量 PCR 检测法，以印迹基因（SNURF）作为内参
Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene (SNURF) as a reference.
机构信息
Department of Haemathology and Oncological Sciences Section of Pathology, Bellaria Hospital, University of Bologna, Italy.
出版信息
BMC Cancer. 2010 Feb 18;10:48. doi: 10.1186/1471-2407-10-48.
BACKGROUND
Epigenetic silencing of the MGMT gene by promoter methylation is associated with loss of MGMT expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference.
METHODS
An analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The SNURF promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization.
RESULTS
Concordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). MGMT promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with MGMT unmethylated and methylated, respectively. Considering MGMT methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring MGMT promoter unmethylated and other patients with any percentage of MGMT methylation (p = 0.003). This difference was retained using other cut off values for MGMT methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of MGMT methylated allele was used as cut-off.
CONCLUSIONS
We report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for the detection and quantification of methylated MGMT alleles in GBM samples. Using MS-qLNAPCR we demonstrate that even low levels of MGMT promoter methylation have to be taken into account to predict response to temozolomide-chemotherapy.
背景
MGMT 基因启动子甲基化导致的表观遗传沉默与 MGMT 表达缺失、DNA 修复活性降低以及胶质母细胞瘤患者总生存期延长有关，这些患者在接受卡莫司汀或替莫唑胺烷化化疗联合放疗的同时。我们描述并验证了一种快速甲基化敏感定量 PCR 检测（MS-qLNAPCR），该方法使用锁核酸（LNA）修饰引物和印迹基因作为参考。
方法
对 2004 年 4 月至 2008 年 10 月期间随访的 159 例 GBM 患者的数据库进行了分析。经亚硫酸氢盐处理后，LNA 引物和分子信标探针识别甲基化和未甲基化的 CpG。印迹基因 SNURF 启动子位于 15q12 上，用作参考。之所以选择这种方法，是因为印迹基因的甲基化和未甲基化等位基因具有平衡的拷贝数，这种特性允许进行简单而精确的归一化。
结果
在 159 个样本中的 158 个样本（99.4%）中发现已经描述的巢式 MS-PCR 与 MS-qLNAPCR 之间存在一致性。MS-qLNAPCR 检测法显示 LNA 修饰引物的 PCR 效率为 102%，灵敏度为 0.01%，而未修饰引物的效率（69%）和灵敏度（0.1%）较低。使用 MS-qLNAPCR 在 70 例患者（44.02%）中发现 MGMT 启动子甲基化，在 89 例样本（55.97%）中完全未甲基化。MGMT 未甲基化和甲基化患者的中位总生存期分别为 20 个月和 36 个月。考虑到 MS-qLNAPCR 提供的 MGMT 甲基化数据作为二进制变量，MGMT 启动子未甲基化的 GBM 样本患者的总生存期与其他 MGMT 甲基化率存在差异（p=0.003）。当使用 MGMT 甲基化等位基因的其他截断值（即 10%和 20%）时，仍保留这种差异，但当使用 50%的 MGMT 甲基化等位基因作为截断值时，这种差异则消失。
结论
我们报告并临床验证了一种准确、稳健且具有成本效益的 MS-qLNAPCR 协议，用于检测和定量 GBM 样本中的甲基化 MGMT 等位基因。使用 MS-qLNAPCR，我们证明即使是低水平的 MGMT 启动子甲基化也必须考虑在内，以预测替莫唑胺化疗的反应。
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