Expression of epidermal growth factor variant III (EGFRvIII) in pediatric diffuse intrinsic pontine gliomas

Abstract

Despite numerous clinical trials over the past 2 decades, the overall survival for children diagnosed with diffuse intrinsic pontine glioma (DIPG) remains 9-10 months. Radiation therapy is the only treatment with proven effect and novel therapies are needed. Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the epidermal growth factor receptor and is expressed in many tumor types but is rarely found in normal tissue. A peptide vaccine targeting EGFRvIII is currently undergoing investigation in phase 3 clinical trials for the treatment of newly diagnosed glioblastoma (GBM), the tumor in which this variant receptor was first discovered. In this study, we evaluated EGFRvIII expression in pediatric DIPG samples using immunohistochemistry with a double affinity purified antibody raised against the EGFRvIII peptide. Staining of pediatric DIPG histological samples revealed expression in 4 of 9 cases and the pattern of staining was consistent with what has been seen in EGFRvIII transfected cells as well as GBMs from adult trials. In addition, analysis of tumor samples collected immediately post mortem and of DIPG cells in culture by RT-PCR, western blot analysis, and flow cytometry confirmed EGFRvIII expression. We were therefore able to detect EGFRvIII expression in 6 of 11 DIPG cases. These data suggest that EGFRvIII warrants investigation as a target for these deadly pediatric tumors.

Fig. 1

MRI Sagittal T1-post contrast (left) and Axial T2 (right) demonstrating a diffusely infiltrative brainstem lesion

Fig. 2

Immunohistochemistry of pediatric diffuse intrinsic pontine glioma samples reveal EGFRvIII expression. a Example of a case revealing staining in a perinuclear pattern by the EGFRvIII antibody. b Example of a case revealing a diffuse staining pattern by the EGFRvIII antibody. c A negative control example of a case being stained only by secondary antibody. d A negative control example of a case being stained by the EGFRvIII antibody after peptide competition. e Example of a tumor that did not express EGFRvIII by IHC

Fig. 3

FISH reveals EGFR gene amplification. EGFR (red) and centromeric D7Z1 (green) control signals shows innumerable EGFR signal (>10 signals per cell)

Fig. 4

Flow cytometric analysis of DIPG neurospheres reveal EGFRvIII surface expression. Live unfixed cells were analyzed for EGFRvIII surface expression using a 2-step staining protocol. Positive cells were scored as those which showed fluorescence above that of rabbit IgG alone. Flow cytometric analysis of neurospheres grown from DIPG-I showed expression of both EGFRvIII and CD133. EGFRvIII was surface expressed in about 9% of the cells

Fig. 5

Western blot and RT-PCR analysis reveals EGFRvIII expression in the diffuse pontine glioma neurospheres and tumor samples: a Western blot analysis showed the presence of EGFRvIII in DIPG neurospheres, tumor lysate and U87MG-vIII (positive control) but not in the U87MG cell lysate. b RT-PCR was performed on total RNA from DIPG neurospheres and tumor samples. Water and U87MG cells were used as a negative control and total RNA from U87MG-EGFRvIII cells were used as a positive control. EGFRvIII was detected in the tumor and neurosphere samples as well as the positive control but not the negative control