Abstract 3566: Effect of afatinib on lung cancer burden induced by an exon 19 EGFR mutation in transgenic mice
Authors: Ninomiya Takashi , Takigawa Nagio , Kubo Toshio , Yasugi Masayuki , Honda Yoshihiro , Minami Daisuke , Murakami Toshi , Ichihara Eiki , Tanimoto Mitsune , Kiura Katsuyuki 
Published: June 2012

Abstract
Afatinib (BIBW2992) is an irreversible dual inhibitor of EGFR and HER2. Afatinib was reported to have anti-tumor effect against L858R/T790M EGFR lung cancer model (Oncogene 27, 2008). Kinase inhibitors had different dissociation constant for binding deletion mutation and point mutation (Nat Biotechnol 26, 2008). Thus, the effect of afatinib on lung cancer harboring deletion or deletion/T790M EGFR double mutations should be clarified because it is possible that afatinib may affect differently tumors with deletion mutations compared to tumors with L858R or L858R/T790M point mutations. Our present study was carried out to investigate the preclinical efficacy of afatinib in lung cancer harboring exon19 EGFR del mutations.
Growth inhibition was measured by MTT assays. RPC-9 cells that acquired T790M mutation of EGFR were established from parental PC-9 cells harboring exon 19 del mutation (delE746-A750) in our laboratory (Cancer Res 67, 2007). The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effect of afatinib and gefitinib. Protein expression was determined by Western blotting. The mean IC50s of afatinib were 0.00032 µM for PC-9 cells and 0.164 µM for RPC-9 cells and were less than those of gefitinib for PC-9 (0.0033 µM) and RPC-9 (11.45 µM), respectively. Phosphorylated EGFR, AKT and ERK of PC-9 were suppressed by treatments with afatinib (1 nM) and with gefitinib (100 nM). In RPC-9 cells, afatinib was 100-fold less active (100 nM) compared to parental cells while gefitinib was ineffective (10000 nM). In addition, 1 nM and 100 nM of afatinib completely inhibited phosphorylation of HER2 in PC-9 and RPC-9, respectively.
We generated transgenic mice expressing the delE748-A752 mutant of mouse EGFR, which is equivalent to the delE746-A750 mutant, driven by the SP-C promoter (Cancer Sci 99, 2008). The mice invariably developed multi-focal pulmonary adenocarcinomas of various sizes at 5 to 6 weeks of age and died of lung cancer about 2 months later, if left untreated. To investigate the effect of afatinib on lung tumors induced by the activating EGFR mutation, the transgenic mice were treated with oral afatinib (5 mg/kg/day), gefitinib (5 mg/kg/day) or vehicle alone from 11 to 15 weeks of age. The number of lung tumors (long axis exceeding 1 mm) in the afatinib-treated group, gefitinib-treated group and vehicle group was 0.12 ± 0.13, 1.25 ± 0.60 and 9.00 ± 1.5 respectively. There were significant differences of the tumor numbers between afatinib-treated group and vehicle group (P &lt; 0.01), while afatinib tended to reduce the tumors compared to gefitinib (P = 0.06). In addition, afatinib was more effective than gefitinib in pathological specimens as the tumors in afatinib-treated group almost disappeared.
In conclusion, our data suggest that afatinib may be more effective for treatment of lung cancer induced by the EGFR mutation in exon 19 than gefitinib.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2011-3566