Influence of Pre-Analytical Factors in Globlastoma MGMT Promoter Methylation Biomarker Assay

生物标志物的发现及其临床验证对于个性化医学至关重要。为 胶质母细胞瘤(GBM)是一种统一致命的脑癌，标准治疗的中位生存期仅为12-18个月。 在GBM中，DNA修复酶MGMT基因启动子的甲基化是一个既定的预后表观遗传学 生物标志物，虽然对指导标准护理替莫唑胺(TMZ)治疗具有潜在的关键作用，但目前 没有得到充分利用。此外，MGMT启动子甲基化状态与治疗之间缺乏相关性 一些患者的反应可能与分析前处理的技术方面有关。因此，有一个 对分析前变量的循证知识的需求未得到满足，以便建立标准化 GBM中MGMT启动子甲基化状态的评估方案。 为了研究疾病中的转录和表观遗传变化，我们开发了PIXUL芯片，用于高表达 组织的生产量样品制备和分析。为了方便冰冻组织和FFPE组织的抽样，我们 开发了用于提取多个小组织核心的CryoCore枪。这些工具提供了强大的集成 用于同时处理和分析来自单个肿瘤的多个小样本的平台。 生物样品的分析前处理对数据输出有着深远的影响。然而，相对的 在组织收集、保存、运输、储存、取样和 表观遗传癌症生物标记物(包括GBM)评估可靠性的分析处理尚未 经过了严格的审查。该U01应用程序的目标是定义分析前程序变量 为了最大限度地减少体外MGMT启动子甲基化的变化 保护组织的完整性。提出了以下目标。 目的：1.确定GBM MGMT甲基化及其在肿瘤内异质性的范围 与组织学的关系，以指导个别肿瘤的采样需求。 AIM2.目的：检测体外热缺血对GBM-MGMT启动子甲基化状态的影响。 组织学。 Aim3.确定组织冷冻/冷冻/解冻对GBM-MGMT启动子的影响 甲基化分析和组织学。 目的：4.确定福尔马林固定石蜡包埋(FFPE)组织保存的效果 关于GBM MGMT启动子甲基化分析。 生物谱系科学的进展对于促进表观遗传生物标记物的发现和使用至关重要。 通过询问与组织收集、保存、存储和采样相关的标准变量 临床相关的GBM表观遗传学分析，并通过应用一种新的设备--CryoCore枪--来取样 肿瘤的异质性，这一建议与NCI生物科学U01 FOA的意图高度一致。

Discovery of biomarkers and their clinical validation is critically important for personalized medicine. For glioblastoma (GBM), a uniformly lethal brain cancer, median survival is only 12-18 months with standard therapy. In GBM, methylation of the DNA-repair enzyme MGMT gene promoter is an established prognostic epigenetic biomarker which, while potentially critical to guide standard-of-care temozolomide (TMZ) therapy, is currently underutilized. Further, the lack of correlation between MGMT promoter methylation status and treatment response in some patients may be related to technical aspects of pre-analytical processing. Thus, there is an unmet need for evidence-based knowledge of pre-analytical variables in order to establish standardized protocols for the assessment of MGMT promoter methylation status in GBM. To study transcriptional and epigenetic alterations in disease, we developed PIXUL-ChIP for high- throughput sample preparation and analysis of tissues. To facilitate sampling of frozen and FFPE tissues, we developed the CryoCore Gun for extracting multiple small tissue cores. These tools provide a powerful integrated platform for simultaneous processing and analysis of multiple small samples from individual tumors. Pre-analytical processing of biological samples profoundly impacts data output. However, the relative importance of variables encountered during tissue collection, preservation, transport, storage, sampling and analytic processing for the reliability of assessment of epigenetic cancer biomarkers (including GBM) has not been rigorously examined. The goal of this U01 application is to define pre-analytical procedure variables for GBM biospecimens in order to minimize ex-vivo MGMT promoter methylation changes while preserving tissue integrity. The following aims are proposed. Aim1. To define the scope of intratumoral heterogeneity of GBM MGMT methylation and its relation to histology to guide sampling needs in individual tumors. Aim2. To test effects of ex-vivo warm ischemia on GBM MGMT promoter methylation analysis and histology. Aim3. To define the effects of tissue freezing/cryostorage/thawing on GBM MGMT promoter methylation analysis and histology. Aim4. To define the effects of formalin fixation and paraffin embedding (FFPE) tissue preservation on GBM MGMT promoter methylation analysis. Advances in biospecimen science are critical to facilitate the discovery and use of epigenetic biomarkers. By interrogating standard variables associated with tissue collection, preservation, storage and sampling in a clinically relevant GBM epigenetic assay, and through application of a novel device – CryoCore Gun – to sample tumor heterogeneity, this proposal is highly aligned with the intent of the NCI Biospecimen Science U01 FOA.