Bettstetter Marcus, Dechant Stephan, Ruemmele Petra, Grabowski Monika, Keller Gisela, Holinski-Feder Elke, Hartmann Arndt, Hofstaedter Ferdinand, Dietmaier Wolfgang
Institute of Pathology and Molecular Diagnostics, University of Regensburg, Regensburg, Germany.
Clin Cancer Res. 2007 Jun 1;13(11):3221-8. doi: 10.1158/1078-0432.CCR-06-3064.
Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics.
We developed a new real-time PCR-based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50).
The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases).
According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.
启动子高甲基化在肿瘤中频繁发生，并导致肿瘤相关基因（如肿瘤抑制基因）沉默。在一部分散发性结直肠癌（CRC）中，由于启动子甲基化导致错配修复基因MLH1失活，引起高水平的微卫星不稳定性（MSI-H）。MSI-H也是遗传性非息肉病性结直肠癌（HNPCC）的一个标志，其中错配修复失活是由种系突变引起的。为了区分散发性和遗传性MSI-H肿瘤患者，MLH1启动子甲基化分析是一种有前景的工具，但由于目前只有未经标准化的定性技术，尚未用于日常诊断。本研究的目的是建立一种可靠的定量MLH1甲基化分析技术，并确定用于HNPCC诊断的有效MLH1甲基化临界值。
我们开发了一种基于实时PCR的新技术，用于检测和定量近端和远端hMLH1启动子区域的甲基化。我们在一组108例CRC（94例MSI-H和l6例微卫星稳定（MSS）病例）中建立并验证了该技术，该组包括一个参照（n = 58）和一个测试肿瘤组（n = 50）。
参照肿瘤组包括28例经证实有种系突变或符合阿姆斯特丹I标准阳性（中位年龄37岁）且MLH1表达缺失的HNPCC，14例MLH1表达缺失且BRAF V600E突变的散发性MSI-H CRC肿瘤（中位年龄80.5岁），以及16例散发性MSS CRC（中位年龄76.5岁）。在任何MSS肿瘤中均未发现MLH1启动子甲基化。HNPCC患者MLH1启动子甲基化水平无或较低。本研究可确定18%甲基化程度的临界值，以定义散发性MSI-H病例特有的MLH1高甲基化。甲基化也可通过熔点分析进行定性验证。在任何HNPCC患者（n = 22例信息性病例）中均未检测到BRAF V600E突变。
根据目前的数据，MSI-H CRC中的定量MLH1甲基化分析是区分HNPCC和散发性MSI-H CRC的一种有价值的分子工具。BRAF V600E突变的检测进一步支持排除HNPCC。
