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疗至关重要。为 胶质母细胞瘤（GBM）是一种均匀致死的脑癌，采用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种确定的预后表观遗传学因素， 生物标志物，虽然对指导标准护理替莫唑胺（TMZ）治疗可能至关重要，但目前 利用不足。此外，MGMT启动子甲基化状态与治疗之间缺乏相关性， 某些患者的反应可能与分析前处理的技术方面有关。因此， 对分析前变量的循证知识的需求未得到满足， 用于评估GBM中MGMT启动子甲基化状态的方案。 为了研究疾病中的转录和表观遗传改变，我们开发了PIXUL-ChIP，用于高水平的 通过量样品制备和组织分析。为了便于冷冻和FFPE组织的取样，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 该平台可同时处理和分析来自单个肿瘤的多个小样本。 生物样本的分析前处理对数据输出产生深远影响。然而，相对 组织采集、保存、运输、储存、取样和 表观遗传癌症生物标志物（包括GBM）评估可靠性的分析处理尚未 被严格审查。此U 01应用程序的目标是定义分析前程序变量 用于GBM生物标本，以最大限度地减少离体MGMT启动子甲基化变化， 保持组织完整性。提出了以下目标。 目标1.为了确定GBM MGMT甲基化的肿瘤内异质性的范围及其在肿瘤内的表达， 与组织学的关系，以指导个体肿瘤的采样需求。 目标2。为了测试离体热缺血对GBM MGMT启动子甲基化分析的影响， 组织学 目标3。确定组织冷冻/冻存/解冻对GBM MGMT启动子的影响 甲基化分析和组织学。 目标4。确定福尔马林固定和石蜡包埋（FFPE）组织保存的效果 GBM MGMT启动子甲基化分析。 生物标本科学的进步对于促进表观遗传生物标志物的发现和使用至关重要。 通过询问与组织收集、保存、储存和取样相关的标准变量， 临床相关的GBM表观遗传测定，并通过应用一种新的设备- CryoCore枪-样品 肿瘤的异质性，这个建议是高度一致的NCI生物标本科学U 01 FOA的意图。

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.