Abstract P4-06-09: Synergistic effect of EGFR1 inhibitor and paclitaxel in newly patient derived metaplastic carcinoma cell line which harbored EGFR gene amplification

Abstract

Abstract Background Metaplastic breast cancer (MBC) is a rare but aggressive subtype of invasive breast cancer, which is generally resistance to conventional chemotherapy. The majority of MBCs do not express the estrogen receptor, progesterone receptor and negative for HER-2/neu over expression. Thus, targeted treatment options to treat MBCs are also limited. Cancer cell lines are important tools used for us to test the therapeutic efficacy of anticancer agents. However, few in vitro systems for studying MBC are developed. Here we report a newly established novel MBC cell line using the recently developed conditionally reprogrammed cell method and search for effective therapeutic regiments to treat MBCs. Methodology The tumor tissue used for cell line establishment was obtained from a 40 year-old Taiwanese female, who was diagnosed with T2N2aM1 metaplastic carcinoma of breast cancer (with squamous metaplasia). The MBC tumor tissue was first digested by collagenase/ dispase mixture. After digestion, the MBC tumor cells were cultured in F medium with irradiated 3T3-J2 fibroblasts supplemented by 5 uM ROCK inhibitor (Liu et al., Am. J. Pathol. 2012). Subsequently, the MBC tumor tissue cells were passaged and propagated in these conditions until the majority of culture is tumor cells. The MBC tumor cell line was further passaged in DMEM/F12 medium without feeder cells and ROCK inhibitor. Finally, the MBC cell line (MBC-1) was characterized using DNA sequencing, immunofluorescence, Western blotting and viability assays. Results The genomic DNA of MBC1 was examined using DNA sequencing, and the MBC-1 maintained known patient's tumor mutation at the TP53 locus (p.V73fs*76). The MBC cell line also harbored EGFR gene amplification as the patient's tumor, according to quantitative PCR assays. To evaluate tumorigenicity, the MBC cell line was injected subcutaneously into immunocompromised mice, and the MBC tumors formed four weeks after injection. The MBC-1 stained positively using pan-cytokeratin antibody and negative of vimentin, confirming the epithelial origin of the cell line. To explore the possible effective treatment options, the cell line was incubated with different targeted therapy and chemotherapy agents, and the MBC cell line was relatively resistant to most of targeted therapy and chemotherapeutic agents. However, EGFR inhibitors (gefitinib and afatinib) could partially inhibit growth of the MBC cell line. We hypothesis that the inhibitory effect could act through inhibiting the amplified EGFR receptors. To further enhance the effect of EGFR inhibitors in the MBC-1, we combine the EGFR inhibitors with chemotherapy agents, and discovered that EGFR inhibitor and paclitaxel were synergistic across the range of concentration tested. The study on downstream mechanism of the synergism is ongoing. Conclusions We have successfully established a novel metaplastic carcinoma cell line with EGFR amplification from a patient using the conditionally reprogrammed cell method. Based on the preclinical study in the cell line, we found that combining EGFR inhibitor and paclitaxel could be a promising strategy to treat MBC with EGFR amplification. Citation Format: Chung P-H, Chen TW-W, Chang D-Y, Lin C-H, Huang S-M, Lu Y-S. Synergistic effect of EGFR1 inhibitor and paclitaxel in newly patient derived metaplastic carcinoma cell line which harbored EGFR gene amplification [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-09.