一种使用限制性酶切偶联定量聚合酶链反应在RCL2固定石蜡包埋肿瘤上进行MGMT DNA甲基化检测的简单定量诊断替代方法。

A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

作者信息

Pulverer Walter, Hofner Manuela, Preusser Matthias, Dirnberger Elisabeth, Hainfellner Johannes A, Weinhaeusel Andreas

出版信息

Clin Neuropathol. 2014 Jan-Feb;33(1):50-60. doi: 10.5414/NP300633.

DOI:10.5414/NP300633

PMID:23993306

Abstract

BACKGROUND

MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation.

METHODS

DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF.

RESULTS

qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR.

CONCLUSIONS

The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

摘要

背景

O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）启动子甲基化与多形性胶质母细胞瘤（GBM）的良好预后和化疗敏感性相关，尤其是在老年患者中。我们旨在开发一种基于甲基化敏感限制酶（MSRE）的简单定量PCR（qPCR）检测方法，以实现MGMT启动子甲基化的定量分析。

方法

在三种不同的样本保存条件下（-80°C、福尔马林固定石蜡包埋（FFPE）；RCL2固定），从非肿瘤性脑（n = 24）和GBM样本（n = 20）中提取DNA。我们评估了每种固定方法对与MSRE偶联的qPCR甲基化分析的适用性。甲基化数据通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）进行验证。

结果

qPCR用于评估替代组织保存程序。来自FFPE组织的DNA未能通过可靠检测；来自RCL2固定组织和新鲜冷冻组织的DNA表现同样良好，并进一步用于定量MGMT甲基化检测的验证（检测限（LOD）：19.58 pg），使用个体未消化的样本DNA进行校准。非肿瘤性脑中的MGMT甲基化分析确定背景甲基化为0.10±11%，我们将其用于定义患者分层的临界值为0.32%。在GBM患者中，9例MGMT甲基化呈阳性（范围：0.56 - 91.95%），11例检测为阴性。与qPCR相比，MALDI-TOF测量结果使94%的GBM样本分类一致。