Laboratory of Oncology, IRCCS "Casa Sollievo della Sofferenza", San Giovanni Rotondo, Italy.
Epigenetics. 2011 Mar;6(3):317-25. doi: 10.4161/epi.6.3.14408. Epub 2011 Mar 1.
In light with the view that KEAP1 loss of function may impact tumour behavior and modify response to chemotherapeutical agents, we sought to determine whether KEAP1 gene is epigenetically regulated in malignant gliomas. We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Characteristics (ROC) curve analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman's Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearman's Rho -0.656 P=0.0001) and in a cell line before and after treatment with 5-azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results further suggest that KEAP1 expression is epigenetically regulated. In addition we demonstrated that KEAP1 is frequently methylated in malignant gliomas and a predictor of patient's outcome.
鉴于 KEAP1 功能丧失可能影响肿瘤行为并改变对化疗药物的反应，我们试图确定 KEAP1 基因是否在恶性神经胶质瘤中受到表观遗传调控。我们开发了一种定量甲基化特异性 PCR（QMSP）测定法来分析 86 例恶性神经胶质瘤和 20 例正常脑组织。通过接收者操作特征（ROC）曲线分析评估测定法的判别能力。曲线的 AUC 值为 0.823（95%CI：0.764-0.883），最佳截断值为 0.133，灵敏度为 74%（95%CI：63%-82%），特异性为 85%（95%CI：64%-95%）。亚硫酸氢盐测序分析证实了 QMSP 结果，并显示 CpG 甲基化百分比与甲基化水平之间存在直接相关性（Spearman's Rho 0.929，P=0.003）。值得注意的是，在肿瘤组织（Spearman's Rho -0.656，P=0.0001）和在用 5-氮杂胞苷处理前后的细胞系中，观察到甲基化水平与 KEAP1 mRNA 转录之间存在强烈的负相关（Spearman's Rho -0.656，P=0.0001）。在接受放疗和替莫唑胺治疗的 70 名患者中，RECPAM 多变量统计分析研究了 MGMT 和 KEAP1 甲基化之间的相互作用，确定了三种不同风险进展的胶质瘤患者的预后类别。同时，MGMT 和 KEAP1 启动子的甲基化与进展风险最低相关，而仅显示 MGMT 甲基化的患者则处于更高风险的亚组（HR 5.54，95%CI 1.35-22.74）。我们的结果进一步表明，KEAP1 表达受到表观遗传调控。此外，我们证明 KEAP1 在恶性神经胶质瘤中经常发生甲基化，是患者预后的预测因子。
