Abstract
Abstract Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase with elevated expression in most human cancers, most notably in invasive metastasis. FAK is involved in cell adhesion, motility, and apoptosis, and it is a target for oncology therapeutics. FAK activation occurs by phosphorylation at Tyr397 in response to integrin clustering caused by cell adhesion. The development of a sensitive and specific immunohistochemical assay for phosphorylated FAK (pFAK) is important for pharmacodynamic biomarker studies. The purpose of this study was to investigate 4 commercially available anti-pFAK antibodies in human tissues and cell lines. Antibodies included 3 rabbit monoclonals (EP2160Y, 141-9, and 31H5L17) and 1 rabbit polyclonal. Antibody specificity was evaluated in a variety of cell lines (BxPC3, HCT-116, LOVO, MDA-MB-231, and MiaPaca2) treated with 2 FAK inhibitors: TAE226 (NVP-TAE226) and PF-562271. Decreased immunohistochemical staining for pFAK was observed in post-treatment samples of cell lines with baseline pFAK expression. TAE226 was more potent at inhibiting pFAK staining. Membrane staining was observed with the rabbit polyclonal and rabbit clone EP2160Y only. Each of the 4 antibodies was tested in human breast cancer tissues using tissue microarrays created by Mosaic Laboratories. The percentage of breast cancer samples that were pFAK positive was 96% with clone EP2160Y, 83% with clone 141-9, 92% with clone 31H5L17 and 96% with the polyclonal. The percentage of cells that were positive within each breast cancer sample ranged from 63% to 83%, depending on the antibody used (standard deviations between 30% and 44%). Immunohistochemistry with the polyclonal and clone EP2160Y antibodies demonstrated membrane and cytoplasmic staining, while clones 141-9 and 31H5L17 demonstrated primarily cytoplasmic staining. Based on reduction of pFAK staining in treated cells and acceptable performance in human cancer tissues, pFAK immunohistochemistry is “fit for purpose” for evaluation of FAK inhibitor activity in pre- and post-treatment clinical trial tissue biopsies. Citation Format: Lisa M. Dauffenbach, Gela C. Sia, Patricia A. Cash, Sherif K. Girees, Ryan S. Lim, Jianping Zheng, Eric P. Olsen, Rana Richeh, Christopher A. Kerfoot. Optimization of an assay for the detection of phosphorylated FAK by immunohistochemistry in formalin-fixed, paraffin-embedded human tissue and cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2014-3745
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