Abstract 4960: In situ proximity ligation-based analysis reveals aberrant dimerization and activation of epidermal growth factor receptors prevalent in glioblastoma multiforme
Authors: Gajadhar Aaron , Guha Abhijit 
Published: February 2011

Gliomas are the most common form of primary brain tumor, with Glioblastoma multiforme (GBM) representing the most malignant grade and an average life expectancy of ∼12-16 months. Aberrations in epidermal growth factor receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human GBMs. Several growth promoting mutant forms of EGFR have been identified in GBMs, the most common of which are EGFRvIII and EGFRc958. Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. Utilizing our novel in situ platform for EGFR dimerization analysis, we seek to directly ascertain the dimerization capacity and activation status amongst EGFR mutants and ErbB members in GBM operative specimens and whether this feature has important prognostic or diagnostic value in GBMs harboring them.

We have recently described our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization analysis of EGFR mutants and ErbB members prevalent in GBMs (Biotechniques, in submission). The ability to directly analyze cell lines in situ and tissue sections from clinical specimens, allows detection and resolution of individual EGFR dimers in intact cells and tissue samples. This approach also affords the ability to measure the phospho-activation status of detected homo- and hetero-dimers.

In vitro results demonstrate the homo- and hetero-dimeric abilities of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. Furthermore, in situ dimer activation analysis has revealed aberrant receptor activation of mutant homo- and hetero-dimers. Preliminary GBM operative specimen analysis has demonstrated the in situ detection of EGFRwt and EGFRvIII mutant heterodimers which corroborates our in vitro cell culture observations. We propose to use this innovative PLA platform to directly assay GBM operative specimens in a tissue microarray format (TMA). We will initially assess the EGFRwt and EGFRvIII heterodimer, by PLA screening the GBM TMA from Dr. Paul Mischel (UCLA) composed of 90 total GBMs (Primary, n=74; Secondary, n=16) to investigate the presence of this heterodimer. Interaction and activation status of this heterodimer will be clinically correlated with regards to molecular alterations (eg. ErbB2, p53, PTEN, Ink4A/Arf, etc.), treatment regimen, and overall survival data.

The aforementioned studies will be instructive for understanding the dimerization behaviour of oncogenic EGFRs using a methodology in intact cells where relevant physiological and compartmentalization aspects are preserved. This in situ PLA EGFR dimerization platform will provide unique insight into the clinical relevance of mutant EGFR and ErbB member dimerization and hopefully guide development of rational targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4960.