Abstract 1049: Quantitative immunofluorescence assessment of MET and epithelial to mesenchymal transition (EMT) biomarker modulation by antiangiogenic inhibitors in xenograft tumor tissues
Authors: Navas Tony , Lawrence Scott L. , Butcher Donna , Dutko Lindsay M. , Hollingshead Melinda G. , Kinders Robert J. , Parchment Ralph E. , Bottaro Donald P. , Linehan W. Marston , Tomaszewski Joseph E. , Srivastava Apurva K. , Doroshow James H. 
Published: May 2015

Abstract
We have previously reported the development of ELISA assays for intact MET, pY1235-MET, and pY1356-MET in tumor lysates (Srivastava et al, ASCO 2011). Here we report the development of a multiplex quantitative immunofluorescence assay (qIFA) for simultaneous detection of pY1235-MET and total MET in formalin-fixed paraffin-embedded (FFPE) tissues. We utilized a rabbit monoclonal antibody specific to pY1235-MET (with no detectable crossreactivity to pY1234-MET) to develop a highly specific and sensitive IFA assay with total MET (MET4 Mab; from G. Vande Woude, Van Andel Research Institute). Specificity of the IFA was demonstrated by HGF stimulation of A549 cells showing increased intensity of pY1235-MET which was completely inhibited by crizotinib but not by the non-MET specific multi-kinase inhibitor sorafenib, in vitro. The pY1235-MET epitope is stable in FFPE, and IFA detection was enhanced by EDTA (vs. Citrate) for antigen retrieval. Quantitative assessment of pY1235-MET was determined in xenograft models that demonstrate very high IFA expression of pY1235-MET and total MET via gene amplification (GTL-16 and SNU-5), lower expression in HGF-induced paracrine or autocrine cell lines (A549 and HT29), and no detectable staining for pY1235-MET in negative controls (SNU-1 and MDA-MB-231). We have demonstrated assay fitness for purpose using SNU-5 FFPE xenograft tumor samples from animals treated with increasing doses of crizotinib in vivo. Quantitative measurement by Definiens analysis of pY1235-MET and total MET in tissue regions of interest (ROI) showed 50% and 95% pY1235-MET inhibition with 12.5 mg/kg and 25 mg/kg crizotinib, respectively. There were no significant changes in total MET by IFA. A high correlation (R=0.899) was observed between % pY1235-MET/total MET ratio measured by IFA levels (expressed as the marker area/# nuclei for pY1235-MET and total MET per ROI) and % pY1235-MET/total MET ratio determined by ELISA levels (pM/ug protein). This quantitative MET IFA assay is currently being applied in conjunction with a previously developed EMT IFA assay (Navas et al, AACR 2013) for focal tissue analysis of pY1235-MET and total MET IFA expression as well as changes in epithelial to mesenchymal transition (EMT) in gastric tumor xenograft tissues treated with the VEGF inhibitor pazopanib, the MET inhibitor tivantinib (ARQ197), or the drug combination in vivo. These studies will determine whether pY1235-MET or total MET is induced by anti-VEGF inhibitors via increased EMT transition, and whether this effect can be reversed by combination with tivantinib. Funded by NCI Contract No. HHSN261200800001E
Citation Format: Tony Navas, Scott L. Lawrence, Donna Butcher, Lindsay M. Dutko, Melinda G. Hollingshead, Robert J. Kinders, Ralph E. Parchment, Donald P. Bottaro, W. Marston Linehan, Joseph E. Tomaszewski, Apurva K. Srivastava, James H. Doroshow. Quantitative immunofluorescence assessment of MET and epithelial to mesenchymal transition (EMT) biomarker modulation by antiangiogenic inhibitors in xenograft tumor tissues. [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 1049. doi:10.1158/1538-7445.AM2014-1049