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256 Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate composed of trastuzumab and DM1 (an anti-microtubule agent derived from maytansine) that is approved for the treatment of HER2-positive metastatic breast cancer. HER2-overexpression rate in biliary tract cancer (BTC) is reported as approximately 10%, and HER2-targeted therapy may enhance therapeutic efficacy in HER2-positive BTC. In this study, we investigated the antitumor activity of T-DM1 in several BTC cell lines and xenograft models. Methods: HER2 expression was detected by flow cytometry and Western blotting in 17 BTC cell lines. The cell growth inhibition activity of T-DM1 and DM1 was examined by quantifying the DNA with Hoechst 33258 nucleic acid stain. In vivo antitumor activity of T-DM1 was evaluated in three xenograft mouse models using cell lines with different levels of HER2 expression. HER2 expression in xenografts was assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), according to gastric cancer criteria. Cell cycle was assessed by flow cytometry after staining with propidium iodide. Apoptosis was assessed by caspase 3/7 activity measurement. Results: In the 17 BTC cell lines, HER2 expression was high in KMCH-1, Mz-ChA-1, and TGBC-18-TKB. Although cell growth inhibition of DM1 was similar in all cell lines (50% inhibitory concentration (IC50): 0.79–7.2 nM), that of T-DM1 was HER2-expression-dependent, with IC50 values in KMCH-1, Mz-ChA-1, and KKU-100 (which has low HER2 expression) of 0.031, 1.3, and 4.3 μg/mL, respectively. In KMCH-1 and Mz-ChA-1 xenograft models, T-DM1 treatment once every 3 weeks for 6 weeks showed significant dose-dependent antitumor activity, and tumor growth inhibition 21 days after first dose at 20 mg/kg T-DM1 was 108% (KMCH-1) and 75% (Mz-ChA-1), whereas there was no significant efficacy in KKU-100 xenograft model. Values for HER2 status (IHC score/FISH ratio of HER2 to CEP17) in KMCH-1, Mz-ChA-1, and KKU-100 tumor tissues were 3+/3.5, 2+/4.7, and 0/1.1, respectively. In KMCH-1, T-DM1 induced M phase arrest and apoptosis, as is reported in HER2-positive breast cancer cells. Conclusions: T-DM1 could be an effective therapy for HER2-overexpressed BTC.

e18091 Background: The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects for non-small cell lung cancers (NSCLC) harboring EGFR activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs within several years. The major mechanisms of acquired resistance are acquisition of a secondary EGFR mutation T790M and amplification of MET. However, the mechanisms of other resistance remain unclear. In this study, we established novel erlotinib-resistant NSCLC cells and examined their resistant mechanism. Methods: Resistant cells were established by continuously exposing EGFR mutated NSCLC cells HCC827 to 0.1, 1 or 10 μM of erlotinib in 96 well plates for three months. The resistant mechanisms were determined by direct sequence analysis and EGFR FISH analysis. Results: Resistant cells were emerged from 14/96 and 3/96 wells by 0.1 μM and 1 μM of erlotinib exposure, respectively. No resistant cells appeared in the wells of 10 μM of erlotinib. The IC50 values of these resistant cells were more than 100-fold higher than that of parental cells. No secondary mutation of T790M was detected in any of the resistant cells. Instead, we found that 13/17 resistant cells were dominated by EGFR not amplified cells and one of resistant cells B10 consisted of more than 99.9% of them, while the parental cells consisted of 2.5% of EGFR not amplified cells. Then, we isolated EGFR not amplified clone 4D8 from parental cells and found that this clone also had a resistance to erlotinib comparable to the resistant cells B10. Metaphase FISH analysis showed that EGFR amplified cells in parental cells had a chromosome 7 containing multiple copies of EGFR, while EGFR not amplified cells in B10 did not have it. Furthermore, we found that EGFR not amplified cells were constantly emerged from EGFR amplified clone isolated from parental cells under normal cell culture condition. Conclusions: Loss of a chromosome 7 containing multiple copies of EGFR causes acquired resistance in HCC827 cells when exposed to relatively low concentration of erlotinib, while high concentration of erlotinib deprives HCC827 cells of the chance of emergence of resistant cells.