title: Detection of hepatocyte growth factor (HGF) ligand-c-MET receptor activation in formalin-fixed paraffin embedded specimens by a novel proximity assay.
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 ofc-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.
authors.affiliation: Research and Development, Oncology, Monogram Biosciences Inc, South San Francisco, California, United States of America. rdua@monogrambio.com
authors.forename: Rajiv
authors.initials: R
authors.lastname: Dua
authors.forename: Jianhuan
authors.initials: J
authors.lastname: Zhang
authors.forename: Gordon
authors.initials: G
authors.lastname: Parry
authors.forename: Elicia
authors.initials: E
authors.lastname: Penuel
mesh_terms: D001706:Biopsy; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005557:Formaldehyde; D017228:Hepatocyte Growth Factor / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D009363:Neoplasm Proteins / Q000032:analysis*; D009369:Neoplasms / Q000473:pathology*; D016612:Paraffin Embedding; D011485:Protein Binding; D019859:Proto-Oncogene Proteins c-met / Q000032:analysis / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000032:analysis / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D009363:Neoplasm Proteins; D017978:Receptors, Growth Factor; D005557:Formaldehyde; D017228:Hepatocyte Growth Factor; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met