Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor. Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in GBM. The most common variant is EGFRvIII, an exon 2-7 deletion mutant resulting from amplification and rearrangement of the EGFR locus. We have hypothesized that EGFRvIII could mark a cancer stem cell or tumor initiating cell population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM EGFRvIII expression was focal and sporadic. To understand the relationship between EGFRvIII expression and the underlying genomic alterations, we utilized manual dissection to separate EGFRvIII positive and negative cells and a quantitative PCR assay that detects independently both EGFR amplification and rearrangement. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression. This supports our hypothesis and further suggests that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII expression and corresponding EGFR amplification and rearrangement, confirmed by Western blot, RT-PCR and FISH. By flow cytometry analysis EGFRvIII expression was heterogeneous, with 9-50% EGFRvIII positive cells in each cell line. Both positive and negative populations maintained the genetic alterations, recapitulating what we observed in primary GBM. Importantly, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. However, only cells that had recently lost EGFRvIII expression could re-express EGFRvIII. Epigenetic mechanisms played a role in modulating EGFRvIII expression to maintain a heterogeneous population. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFRvIII does follow a stem cell model for hierarchical expression and sheds light on the existence of a transient population that is able to re-express EGFRvIII, an important buffer for maintaining EGFRvIII-positive cell numbers. Furthermore, we provide the first evidence that EGFRvIII expression can be silenced by epigenetic mechanisms, suggesting that drugs which modulate the epigenome might be used successfully in glioblastoma tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 10. doi:1538-7445.AM2012-10