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

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.

Grant Number: 5U01CA246503-05
NIH Institute/Center: NIH
Principal Investigator: KAROL BOMSZTYK