精准肿瘤学是由生物标志物驱动的。对于多形性胶质母细胞瘤（GBM）这种最常见的恶性成人原发性脑肿瘤，O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化是重要的预后和治疗临床生物标志物。生物样本储存、固定、取样和处理等耗时的预分析步骤是数据不可重复性的根源，并且所有这些预分析变量都被 MGMT 启动子甲基化的肿瘤内异质性所混淆。为了评估分析前变量对 GBM DNA 甲基化的影响，使用了组织储存/采样 (CryoGrid)、样品制备多超声仪 (PIXUL) 和 5-甲基胞嘧啶 (5mC) DNA 免疫沉淀 (Matrix MeDIP-qPCR/seq) 平台用过的。通过 MeDIP-qPCR 检测的 MGMT 启动子甲基化状态通过甲基化特异性 PCR (MS-PCR) 进行了验证。冷冻和福尔马林固定石蜡包埋 (FFPE) 样品对中的 MGMT 启动子甲基化水平没有统计学差异，证实了 FFPE 用于 MGMT 启动子甲基化分析的可靠性。温离体缺血（37°C 下长达 4 小时）和 3 个重复样品解冻和冷冻循环对 MGMT 启动子、外显子和增强子区域的 5mC 没有统计学影响，表明 DNA 甲基化对样品处理条件中常见变化的抵抗力在研究和临床环境中可能会遇到。 26-34% 的样本在 MGMT DNA 启动子甲基化方面表现出肿瘤内异质性。这些数据表明，样品固定、缺血持续时间和温度以及 DNA 甲基化测定技术的变化对 MGMT 启动子甲基化评估没有统计学上的显着影响。然而，肿瘤内甲基化异质性强调了不同 GBM 地理肿瘤位点的多次活检在评估 MGMT 启动子甲基化状态中的价值。 Matrix-MeDIP-seq 分析显示，MGMT 启动子甲基化状态与其他差异甲基化基因组位点（例如 HOXA 和 lncRNA）聚集在一起，这些位点对上述预分析条件的变化具有弹性。这些观察结果为开发更精细的基于数据的表观遗传 GBM 生物标志物提供了新的机会。在这方面，高通量 CryoGrid-PIXUL-Matrix 工具箱可能会很有用。版权所有 © 2024。由 Elsevier Inc. 出版。

Precision oncology is driven by biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is an important prognostic and treatment clinical biomarker. Time consuming pre-analytical steps such as biospecimen storage, fixation, sampling, and processing are sources of data irreproducibility, and all these pre-analytical variables are confounded by intratumor heterogeneity of MGMT promoter methylation. To assess the effect of pre-analytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multi-sonicator (PIXUL), and 5-methylcytosine (5mC) DNA immunoprecipitation (Matrix MeDIP-qPCR/seq) platforms were used. MGMT promoter methylation status assayed by MeDIP-qPCR was validated with methylation specific PCR (MS-PCR). MGMT promoter methylation levels in frozen and formalin fixed paraffin embedded (FFPE) sample pairs were not statistically different, confirming reliability of FFPEs for MGMT promoter methylation analysis. Warm ex-vivo ischemia (up to 4hrs at 37oC) and 3 cycles of repeated sample thawing and freezing did not statistically impact 5mC at MGMT promoter, exon, and enhancer regions, indicating the resistance of DNA methylation to common variations in sample processing conditions that might be encountered in research and clinical settings. 26-34% of specimens exhibited intratumor heterogeneity in the MGMT DNA promoter methylation. These data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have a statistically significant impact on MGMT promoter methylation assessment. However, intratumor methylation heterogeneity underscores the value of multiple biopsies at different GBM geographic tumor sites in the evaluation of MGMT promoter methylation status. Matrix-MeDIP-seq analysis revealed that MGMT promoter methylation status clustered with other differentially methylated genomic loci (e.g. HOXA and lncRNAs) that are resilient to variation in the above pre-analytical conditions. These observations offer new opportunities to develop more granular data-based epigenetic GBM biomarkers. In this regard, the high throughput CryoGrid-PIXUL-Matrix toolbox could be useful.Copyright © 2024. Published by Elsevier Inc.