Detection of Hepatocyte Growth Factor (HGF) Ligand-c- MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay Rajiv Dua*, Jianhuan Zhang, Gordon Parry, Elicia Penuel¤ Research and Development, Oncology, Monogram Biosciences Inc., South San Francisco, California, United States of America Abstract Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels of c-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens. Citation: Dua R, Zhang J, Parry G, Penuel E (2011) Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay. PLoS ONE 6(1): e15932. doi:10.1371/journal.pone.0015932 Editor: Syed Aziz, Health Canada, Canada Received November 10, 2010; Accepted November 30, 2010; Published January 21, 2011 Copyright: 2011 Dua et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded by Monogram Biosciences, Inc. Monogram Biosciences played role in study design, data collection and analysis, preparation of manuscript, and decision to publish this work. No outside funding was received for performing this research work. Competing Interests: All authors are employees of Monogram Biosciences, Inc. The authors also declare that they have read Plos ONE policies on sharing data and materials and will adhere to these as mentioned. * E-mail: rdua@monogrambio.com ¤ Current address: Department of Molecular Diagnostics, Genentech Inc., South San Francisco, California, United States of America Introduction Arguably, the most significant opportunity to improve response rates for targeted therapeutics in solid tumors resides in the ability to accurately match patient specific molecular alterations to drugs that antagonize those alterations. This is the goal of personalized medicine for oncology. In that context, measuring receptor activation signatures constitutes an integral part of an overall diagnostic strategy aimed at identifying potentially responsive patients, stratifying patients for clinical trials and monitoring therapeutic responses to a given drug. Post-translational modifi- cations such as phosphorylation either at the level of the receptor or downstream proteins, are likely to be better indicators of signal pathway activation and thus drug susceptibility, than mere quantification of receptor levels. In a clinical setting, formalin- fixation followed by paraffin embedding (FFPE) is the most common format of tissue preservation used by pathologists. This format maintains antigenicity and ensures excellent cellular morphology for diagnosis and immunohistochemistry (IHC) applications. However, detection of phosphoproteins in FFPE specimens is not robust in clinical settings, especially in clinical surgical tissue samples. One reason may be due to rapid dephosphorylation during ischemic stress after surgery [1,2]. During ischemia, opportunistic phosphatases in the cell are activated and dephosphorylate proteins. It has been shown that when tissues are not fixed immediately the ability to detect phosphoproteins is lost within 60 minutes after biopsy [1]. Consequently, there is an urgent need to develop alternative methods to measure receptor activation. Although not all of the intricate details are known, there are a series of key steps leading to receptor activation and subsequent signaling of cell growth and proliferation. These key steps, ligand-receptor binding, receptor dimerization, and receptor transphosphorylation resulting in the production of substrate and adaptor protein binding sites, can be measured as potential indicators of receptor activation. One can also measure total receptor expression by IHC or FISH, or total ligand levels by either IHC or ELISA, however, these are not direct indicators of receptor activation. Receptor tyrosine kinases (RTK) play essential functions in regulating important cellular processes [3]. Aberrant activation of RTK’s resulting from either by mutation, gene amplification, or overexpression is significantly associated with many human PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e15932