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

预分析因素对球母细胞瘤 MGMT 启动子甲基化生物标志物测定的影响

• 批准号: 10415839
• 负责人: KAROL BOMSZTYK
• 金额: $ 38.91万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10415839
• 关键词: Address; Affect; Biological; Biological Assay; Biological Markers; Brain Neoplasms; Chromatin; Clinical; Clinical Management; CryoCore Gun; Cryopreservation; DNA; DNA Methylation; DNA Repair Enzymes; Data; Deamination; Devices; Disease; Eosine Yellowish; Epigenetic Process; Excision; Formalin; Freezing; Genetic Transcription; Glioblastoma; Goals; Histologic; Histology; Histopathology; Ice; Immunoprecipitation; Individual; Ischemia; Knowledge; Laboratories; MGMT gene; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of brain; Methods; Methylation; Modification; Output; Paraffin Embedding; Patients; Preparation; Procedures; Process; Protocols documentation; Research; Sampling; Science; Specimen; Stains; Standardization; Technology; Temperature; Testing; Thymine; Time; Tissue Banks; Tissue Embedding; Tissue Preservation; Tissue Sample; Tissues; Transportation; Validation; Variant; Warm Ischemia; biomarker discovery; cancer biomarkers; chemical fixation; clinically relevant; epigenetic marker; evidence base; methylation biomarker; multidisciplinary; neuropathology; novel; personalized medicine; preservation; prognostic; promoter; pyrosequencing; sample fixation; standard of care; statistics; temozolomide; tool; treatment response; tumor; tumor DNA; tumor heterogeneity; tumorigenesis

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.

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。为了 胶质母细胞瘤 (GBM) 是一种均致死性的脑癌，采用标准治疗后中位生存期仅为 12-18 个月。 在 GBM 中，DNA 修复酶 MGMT 基因启动子的甲基化是一种既定的预后表观遗传因素 生物标志物虽然对于指导标准护理替莫唑胺 (TMZ) 治疗可能至关重要，但目前 未得到充分利用。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预分析处理的技术方面有关。因此，有一个 为了建立标准化，对分析前变量的循证知识的需求未得到满足 GBM 中 MGMT 启动子甲基化状态评估方案。 为了研究疾病中的转录和表观遗传改变，我们开发了 PIXUL-ChIP 组织的通量样品制备和分析。为了便于对冷冻和 FFPE 组织进行取样，我们 开发了 CryoCore Gun 用于提取多个小组织核心。这些工具提供了强大的集成 用于同时处理和分析单个肿瘤的多个小样本的平台。 生物样品的预分析处理深刻影响数据输出。然而，相对的 组织采集、保存、运输、储存、取样和处理过程中遇到的变量的重要性 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实现 都经过严格审查。此 U01 应用程序的目标是定义预分析过程变量 对于 GBM 生物样本，以尽量减少离体 MGMT 启动子甲基化变化，同时 保持组织完整性。提出以下目标。 目标1。定义 GBM MGMT 甲基化的瘤内异质性范围及其影响 与组织学的关系，以指导个体肿瘤的采样需求。 目标2。测试离体热缺血对 GBM MGMT 启动子甲基化分析和 组织学。 目标3。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。 目标4。确定福尔马林固定石蜡包埋 (FFPE) 组织保存的效果 GBM MGMT 启动子甲基化分析。 生物样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。 通过询问与组织采集、保存、储存和采样相关的标准变量 临床相关的 GBM 表观遗传测定，并通过应用新型设备 – CryoCore Gun – 进行采样 肿瘤异质性，该提案与 NCI 生物样本科学 U01 FOA 的意图高度一致。