Abstract 4429: Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells.

Abstract

Erlotinib is a first-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor and have well-established efficacy in non-small cell lung cancer patients with activating EGFR mutations. Afatinib, on the other hand, is a second-generation EGFR tyrosine kinase inhibitor with anti-HER2 activity and was found to be of benefit to patients with advanced lung adenocarcinoma who failed previous gefetinib or erlotinib. In previous study we have demonstrated the in vitro and in vivo radiosensitising activity of afatinib in a murine bladder cancer model. However, the radiosensitizing effect of afatinib and erlotinib, has never been compared in cancer cells including bladder cancer. We performed RTK (Receptor Tyrosine Kinase) antibody arrays to investigate the relative levels of phosphorylation in T24 human bladder cancer cell line. We found that EGFR and HER2 signals were activated after radiation10 Gy. Afatinib 100 nM suppressed both irradiation-induced EGFR and HER2 signals but erlotinib 100 nM only suppressed irradiation-induced EGFR signal. Clonogenic assay of T24 and NTUB1 human bladder cancer cell lines was then examined. Afatinib (100-500 nM) showed better radiosensitizing effect (radiation dose: 2-10 Gy) than erlotinib (300-1500 nM) in both T24 and NTUB1 cells by significantly decreasing the numbers of colonies 7 days after treatment. The effect is more prominent in high doses of afatinib. Flow cytometry was used to determine the distribution of cells among various cell cycle phases. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 200 nM, erlotinib 200 nM or the combination of radiation and afatinib or erlotinib. When compared with radiation or drug alone, treatment combining radiation and afatinib in T24 cells resulted in a significant increase of cells in sub-G1 phase but the phenomenon was not observed in the combination of erlotinib and radiation. A similar trend was found in NTUB1 cells. The results implicated that afatinib might enhance radiation effect by increasing apoptosis in bladder cancer cells. To study the DNA damage status after treatment, we checked intra-nuclear γH2AX foci by mmunofluorescence microscopy. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 100 nM, erlotinib 100 nM or the combination of radiation and afatinib or erlotinib. Radiation alone significantly increased γH2AX foci and combining radiation with afatinib further increased the foci in both T24 and NTUB1 cells. The enhancement was absent in the combination of radiation and erlotinib. Our data clearly show that afatinib, a new generation EGFR tyrosine kinase inhibitor with anti-HER2 activity, is superior to erlotinib as a radiosensitizer in the treatment of human bladder cancer cells. Citation Format: Yu-Chieh Tsai, Tsung-Fan Tuan, Pei-Yin Ho, Wei-Lin Liu, Liang-Yu Chang, Yeong-Shiau Pu, Ann-Lii Cheng, Jason Chia-Hsien Chen. Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4429. doi:10.1158/1538-7445.AM2013-4429