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.