Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most
common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between
mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic
importance. In this thesis, we describe our work developing and optimizing a novel in situ
proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants
prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek
to systematically interrogate the dimerization capacity and activation status amongst EGFR and
EGFR mutants.
Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric
properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with
EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected
using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR
measurements currently undertaken with IHC or IF. These aberrant features are not
overexpression dependent but appear independent of cellular expression levels, suggesting
inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that
various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and
that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments.
Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM
specimens, in keeping with our prior cell line data, and a potential feature of prognostic or
diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this
platform for measuring pEGFR and total EGFR expression on tissue samples, which has not
been efficacious to date with conventional antibody-mediated techniques. Results from this
thesis may therefore provide novel insights into the interaction and activation characteristics of
EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to
target these mutants. In summary, these findings demonstrate the successful application of a
novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic
evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore,
since PLA allows specimen assessment of not only expression and activation, but also
dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to
evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and
activation.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31755 |
| Date | 09 January 2012 |
| Creators | Gajadhar, Aaron |
| Contributors | Guha, Abhijit |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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