Abstract 694: Immunohistochemical discrimination of wild type EGFR from EGFRvIII in fixed tumor specimens using anti-EGFR mAbs ICR9 and ICR10

Abnormal expression of the epidermal growth factor receptor (EGFR) has been reported in a wide range of human epithelial malignancies and in some studies has been associated with poor prognosis and treatment resistance. Currently, three different types of EGFR inhibitors have been approved by the FDA for the treatment of patients with head and neck, metastatic colorectal, pancreatic or breast cancers: anti-EGFR monoclonal antibodies [(mAbs), cetuximab and panitumumab], small molecule EGFR tyrosine kinase inhibitors [(TKIs) gefitinib, erlotinib] or a dual EGFR and HER-2 tyrosine kinase inhibitor (lapatinib) However, there has been no clear association between the expression levels of EGFR in the tumours determined by the FDA approved EGFR PharmDx™ kit (DakoCytomation) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors. One reason could be that the antibody used in the diagnosis of EGFR is not specific to the wild type EGFR and can also bind to the type-III mutant form of EGFR (EGFRvIII). We have extensively profiled our unique panel of high affinity rat anti-EGFR mAbs for use in the diagnosis and therapy of human cancers. The aim of the present investigation was to evaluate the potential of several of these antibodies (ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild type EGFR and/or EGFRvIII in formalin fixed paraffin embedded tumour specimens. We prepared 10% (v/v) neutral buffered formalin paraffin embedded sections of HN5 and HC2 20d2/c pelleted cells, which overexpress wild type EGFR and EGFRvIII respectively. Tumour sections were incubated with primary anti-EGFR mAbs followed by HRP linked secondary antibody (ABD Serotec Ltd., UK). The sections were then treated with DAB+ substrate-chromogen solution (Dako), counterstained with haematoxylin and mounted. The EGFR PharmDx™ kit, containing positive and negative control cell lines was purchased from Dako (UK) and the immunostaining of sections was conducted according to the manufacturer9s protocol. We found that the mouse anti-EGFR mAb in the EGFR pharmDx™ kit stained both wild type and EGFRvIII expressing cells in the formalin fixed paraffin embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR9 and ICR10, which are directed against two distinct epitopes on the external domain of the EGFR, were specific for wild-type EGFR in formalin-fixed paraffin embedded tumour specimens. We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild type EGFR (membranous, nuclear, and cytoplasmic) in tumour specimens using immunohistochemistry and to determine their prognostic significance as well as predictive value for response to therapy with EGFR antibodies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 694.