Previously we have initiated a large cDNA microarray effort in collaboration with the Human Genome Project and the Cancer Genome Anatomy Project (CGAP) to develop a comprehensive and novel molecular classification schema for human gliomas based on a gene expression profile using cDNA microarray technology. Most recently, in collaboration with the NCI Molecular Pathology Laboratory, we have developed a state of the art molecular panel that was specifically designed to evaluate central nervous system tumors. An exhaustive search of the scientific literature uncovered 55 specific mutations or copy number variations as well as 25 gene fusions that may be germane to these cancers. All of these molecular findings have been incorporated into a panel that can be readily used to screen patient tumor material for discovery. This will provide a unique opportunity to uncover potential therapeutic targets (after confirmation in a CLIA certified laboratory) or reconcile disparities or inconsistences in diagnosed performed using only traditional histopathologic examination. This project will include hundreds of tumor specimens and offer an unprecedented opportunity for dissecting signal transduction pathways, and learning this exciting new technology. Investigation of glioma stem cells (GSCs) are an integral component of NOB research. GSCs are a tumor subpopulation that can self-renew in culture, perpetuate a tumor in orthotopic transplant in vivo, and generate diversified neuron-like and glia-like postmitotic progeny in vivo and in vitro. In addition to conventional and array-based CGH (aCGH) profiling of the laboratories GSC lines, we have also performed whole exome sequencing and most recently have begun to explore RNA sequencing to evaluate gene expression and look for gene fusions. With these evaluations, we hope to determine if the candidate genes or gene fusions change the biology of these cells in such a way that may be consistent with a role in tumorigenesis (i.e. clonogenecity, proliferation, apoptosis, tumorigenic potential in immunosuppressed animals). In addition, the NOB Laboratory recently began collaborating with Dr. Gordon Hager and the Laboratory of Receptor Biology and Gene Expression. Dr. Hager's work has focused on the reorganization of the nuclear chromatin and the impact of these changes on gene regulation. In the context of brain tumor biology, there are a variety of primary central nervous system tumors that despite a malignant phenotype have few mutations. Therefore, it is possible that alterations in the transcriptional profile may help explain this apparent disparity. The NOB laboratory is using the DHS-seq method to profile genome-wide transcriptional changes in glioma patient samples. As described above, the DHS-seq will reveal dynamic changes in the chromatin, which are important in the development and progression of brain tumors and allow us to identify novel molecular targets to treat this disease. We have tested the DHS-seq protocol on two glioma stem cell lines (827P12 and 923P9) and corresponding xenograft tissues. Preliminary analyses of these data suggest that in combination with gene expression and copy number data, we will obtain novel insights into the genomics underlying brain tumor biology. To this end, the NOB laboratory has begun testing this method on patient samples, using tumor tissues and adjacent normal brain directly from surgical specimens. The plan is to continue processing additional patient samples as they become available with the ultimate goal of incorporating the transcriptome analysis into the comprehensive genomic analysis that is being planned as a component of the molecular tumor board, described in the Clinical Project.
