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 GBM epigenetic biomarker used clinically to guide temozolomide (TMZ) therapy. Although MGMT promoter methylation status as a GBM biomarker has proven to be clinically useful but highly 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 standardized protocols for assessment of MGMT promoter methylation status in GBM.
To study the transcriptional and epigenetic cancer heterogeneity, 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 comprise 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 on reliability of epigenetic cancer biomarkers including GBM have not been rigorously examined. The goal of this U01 application is to define pre-analytical for GBM biospecimen procedures as a way 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 promoter methylation to guide sampling requirements for detection in individual tumors
Aim2. To test effects of ex-vivo warm and cold ischemia on GBM MGMT promoter methylation analysis and histology.
Aim3. To define the effects of tissue freezing/thawing on GBM MGMT promoter methylation analysis and histology.
Aim4. To define the effects of formalin fixation and paraffin embedding (FFPE) tissue preservation on the GBM MGMT promoter methylation analysis and histology. Advances in biospecimen science are critical to facilitate 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.
National Institutes of Health (NIH)