Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model.
Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model.
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DOI：
10.1007/s11060-008-9737-8
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发表时间：
2009-03
影响因子：
3.9
通讯作者：
Sarkaria, Jann N.
中科院分区：
文献类型：
--
作者：
Kitange, Gaspar J.;Carlson, Brett L.;Mladek, Ann C.;Decker, Paul A.;Schroeder, Mark A.;Wu, Wenting;Grogan, Patrick T.;Giannini, Caterina;Ballman, Karla V.;Buckner, Jan C.;James, C. David;Sarkaria, Jann N.
O 6-甲基鸟嘌呤-DNA-甲基转移酶（MGMT）启动子内的CpG甲基化与接受替莫唑胺（TMZ）治疗的多形性胶质母细胞瘤（GBM）患者的生存率提高相关。尽管MGMT启动子在约50%的GBM中甲基化，但几项研究报道了MGMT甲基化和蛋白表达水平之间缺乏相关性，因此TMZ敏感和耐药患者的区分不准确。为了了解目前使用的测定的局限性，将13种GBM异种移植物系的TMZ反应性与MGMT蛋白表达和MGMT启动子甲基化相关，所述MGMT蛋白表达和MGMT启动子甲基化通过1）标准甲基化特异性聚合酶链反应（MS-PCR）、2）定量MS-PCR（qMS-PCR）和3）亚硫酸氢盐测序测定。对于每个异种移植物系，用溶剂对照或TMZ（66 mg/kg × 5天）处理已建立颅内异种移植物的小鼠，TMZ反应定义为TMZ处理小鼠与对照处理小鼠的中位生存期相对延长。TMZ的相对生存获益与MGMT蛋白表达呈负相关（r=-0.75; p=0.003），与qMS-PCR直接相关（r=0.72; p=0.006）。通过qMS-PCR得到的MGMT甲基化信号与通过MS-PCR扩增的区域内甲基化CpG位点的数目之间存在直接相关性（r =0.78，p=0.002）。然而，亚硫酸氢盐测序揭示了具有稳健qMS-PCR信号的那些肿瘤中CpG甲基化程度的异质性。qMS-PCR信号大于10%的4个GBM系中有3个具有至少1个未甲基化的CpG位点，而只有1个系在所有12个CpG位点处完全甲基化。这些数据突出了通过MS-PCR测定评价MGMT甲基化的一个潜在局限性，并表明相对于TMZ反应，更详细地评价单个CpG位点的甲基化可能值得追求。
CpG methylation within the O6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in ~50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by 1) standard methylation-specific polymerase chain reaction (MS-PCR), 2) quantitative MS-PCR (qMS-PCR) and 3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kg × 5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated vs. control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r= −0.75; p=0.003) and directly correlated with qMS-PCR (r=0.72; p=0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r =0.78, p=0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.
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