Abstract 4146: Quantitative cell signaling analysis for cetuximab sensitivity prediction in colorectal cancer

Abstract

[Background] Anti-human EGFR monoclonal antibody (cetuximab) has been widely used in the treatment of colorectal cancer (CRC). However it is thought that a reliable predictor for therapeutic efficacy should be established because the benefit of cetuximab treatment is often still uncertain. Our preliminary study showed that the prediction of therapeutic efficacy using a conventional chemosensitivity test using growth suppression assay is generally not informative because cetuximab does not seem to have as strong a cytotoxicity as other anticancer drugs. KRAS mutation status has been used as a biomarker to predict therapeutic efficiency but it is not sensitive enough to determine the absolute indication. [Purpose] We hypothesized that we could predict therapeutic efficacy by evaluating expected molecular reaction when cetuximab is administrated in vitro. In the present study, we build up an in vitro EGFR signaling model for a new chemosensitivity test based on protein monitoring in response to cetuximab administration. [Materials and Methods] Three growth factors (EGF, TGF-α, and IGF) and the inhibitor (cetuximab) were added to HT29 (KRAS wild type, EGFR positive) in six combinations. Cell pellets were harvested at 5 time points over a 15min time course, each of which was processed for cell lysate. The lysates were analyzed by Western blotting to see if the proteins involved in the EGFR signaling pathway responded over time. [Results] We confirmed the activation of signaling stimulated by EGF, TGF-α, and IGF; and the effect on signaling by cetuximab in HT29 with Western blotting. Intriguingly, the phosphorylation level of EGFR was rapidly elevated and declined by EGF stimulating in the absence of cetuximab whereas dephosphorylation was prolonged in the presence of cetuximab. In the administration of either TGF-α or IGF, the phosphorylation pattern of EGFR, c-RAF, MEK, MAPK, ERK, PI3K, AKT, PTEN, and STAT was different from that of EGF alone. [Conclusions] These results shows that:(i) EGF is not EGFR's only ligand; (ii) cetuximab can't inhibit every signaling pathway starting from EGFR; and (iii) TGF-α and IGF activate signaling pathway starting from EGFR partially but not thoroughly. An additional reliable predictor of cetuximab efficacy should be identified based on functional protein characteristics. 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 4146.