Bettstetter Marcus, Dechant Stefan, Ruemmele Petra, Vogel Corinna, Kurz Katrin, Morak Monika, Keller Gisela, Holinski-Feder Elke, Hofstaedter Ferdinand, Dietmaier Wolfgang
Institute of Pathology and Molecular Diagnostics, University of Regensburg, Regensburg, Germany.
Lab Invest. 2008 Dec;88(12):1367-75. doi: 10.1038/labinvest.2008.100. Epub 2008 Oct 20.
Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.
启动子高甲基化发生在多种肿瘤中，并导致肿瘤相关基因沉默。因此，启动子甲基化分析（MA）已成为癌症研究和诊断中的一项重要工具。在此，我们介绍了MethyQESD（抗核酸内切酶DNA的甲基化定量），这是一种快速、简便、精确且可靠的定量MA方法，无需亚硫酸氢盐处理或荧光探针。尽管MethyQESD原则上可用于任何基因启动子，但我们针对错配修复基因MLH1建立了MethyQESD，并通过定量MLH1 MA测试了其区分散发性微卫星不稳定（MSI-H）结直肠癌和遗传性非息肉病性结直肠癌（HNPCC）的效用。我们研究了来自先前发表的、特征明确的肿瘤群体的福尔马林固定石蜡包埋组织样本，该群体包括25例HNPCC、14例散发性MSI-H CRC和16例散发性微卫星稳定（MSS）CRC。通过用甲基化（SW48癌细胞系）和未甲基化（血液）DNA的稀释系列进行加标实验，我们发现MethyQESD具有很高的准确性（Pearson相关系数r = 0.9997（MLH1启动子近端区域），r = 0.9976（MLH1启动子远端区域））。使用96例福尔马林固定石蜡包埋的CRC进行的MethyQESD和传统定量MA显示两种方法具有高度一致性（Pearson相关系数r = 0.885）。HNPCC肿瘤要么MLH1甲基化为零，要么MLH1甲基化程度明显低于散发性MSI-H CRC（P < 0.001）。所有MSS肿瘤的MLH1甲基化均为阴性。受试者工作特征（ROC）曲线分析确定了16.5%的MLH1甲基化临界值，用于特异性和敏感地识别散发性MSI-H CRC（ROC曲线下面积：1.000；渐近显著性：P < 0.001）。因此，通过MethyQESD进行的定量MLH1 MA提供了一种简单、快速且有价值的工具来识别HNPCC候选者。此外，MethyQESD对福尔马林固定石蜡包埋组织可靠有效，并且简化了用于研究和诊断目的的DNA MA。
