Abstract 3274: Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties
Authors: James Keller, Kristen A. VanHeyst, Anjaruwee S. Nimnual, Mathew Varghese, Michael J. Hayman, Edward L. Chan
Journal: Cancer Research (2015)

Abstract Background: Epidermal Growth Factor Receptor (EGFR) is a biologic target for cancer therapy. Clinical response to EGFR inhibitors is variable. Recently, EGFR mutations were found to predict response to anti-EGFR therapy. Since a few children with Neuroblastoma (NB) benefited from EGFR inhibitors in clinical trials, we screened for EGFR mutations in primary NB. Interestingly, we identified the expression of both EGFRvIII and a novel mutation, EGFRΔ768, in these tumors. In this study, we characterized the EGFRΔ768 mutation by comparing its biological and biochemical effect with that of wild type (WT)-EGFR and EGFRvIII. Hypothesis: EGFRΔ768 expression confers an aggressive cancer phenotype in NB cells Materials and Methods: We obtained 62 Snap Frozen primary neuroblastic tumors from the Columbus Bipathology Repository Center and 80 NB RNA from the Children Oncology Group (COG). Primary NB were prepared for RT-PCR and sequencing analyses using EGFR exon 1 &amp; 8 primers. Five NB cell lines were obtained and confirmed by STR profiling. The EGFRΔ768 and EGFRvIII deletions were subcloned into a WT-EGFR expression vector with a C-terminal GFP tag. Next, stable 3T3 and NB clones expressing WT and mutant EGFR were generated. The clones were visualized under fluorescence microscopy. Invasion and XTT assays were performed with the stable clones. Responses to Erlotinib from 0.01 - 1 ng/ul and Etoposide from 0.5 - 2 uM were compared among the clones by XTT and western analyses. EGFR phosphorylation and downstream signaling pathways were examined by Western Blot and phosphokinase array. Results: We previously reported that 32% (20/62) of the Columbus neuroblastic tumors expressed EGFR extracellular deletion mutants, EGFRΔ768 (an in-frame deletion from nucleotides 102 of exon 2 to 869 of exon 7) and EGFRvIII (Δ801). In the 80 COG NB RNA, 7 expressed EGFRΔ768, 7 expressed EGFRvIII and 1 expressed both. EGFRΔ768 expression was also detected in the NB cell line, BE2M17. This novel mutant localized to the cell surface and enhanced the invasion and proliferation of NB cells in vitro when compared to control GFP+ cells. Its expression also confers sensitivity to Erlotinib, but the biochemical inhibition of Erlotinib on EGFRΔ768 is significantly less effective than that on activated WT-EGFR from 0.01 - 0.1 ng/ul. EGFRΔ768+ cells were also significantly more resistant to Etoposide than the GFP and WT-EGFR+ cells. Although both EGFRΔ768 and EGFRvIII autophosphorylated without ligand, the cellular proliferation rate and baseline autophosphorylation of EGFRΔ768+ cells were significantly higher than those of EGFRvIII+ cells. Finally, we found that EGFRΔ768 did not activate downstream signaling pathways of WT-EGFR such as MAPK or STAT. It has a different tyrosine phosphorylation profile than WT-EGFR. Conclusion: EGFRΔ768 confers aggressive cancer cell behaviors and has distinct biological as well as biochemical properties. Citation Format: James Keller, Kristen A. VanHeyst, Anjaruwee S. Nimnual, Mathew Varghese, Michael J. Hayman, Edward L. Chan. Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3274. doi:10.1158/1538-7445.AM2015-3274