Functional allelic loss detected at the protein level in archival human tumours using allele-specific E-cadherin monoclonal antibodies

Immunohistochemical analysis has been used to show that expression of the homophilic cell‐to‐cell adhesion molecule, E‐cadherin, is frequently altered in human cancers, including gastric and breast carcinoma. Besides genetic down‐regulation, structural mutations such as in‐frame deletions of exon 8 and exon 9 were frequently found; these may affect the binding of monoclonal antibodies used for immunohistochemical analysis. In this study it was found that antibodies HECD‐1 and E9, two monoclonal antibodies often used in E‐cadherin immunoanalysis, react with epitopes present at least in part in exon 8 and exon 9, respectively. This study generated and characterized a mutation‐specific monoclonal antibody, E‐cad delta 8‐1, reacting with the mutant protein lacking exon 8 but not with the wild‐type molecule. By using E‐cad delta 8‐1 and HECD‐1, it was possible separately to analyse the immunoreactivity of mutant and normal E‐cadherin proteins, respectively, in an allele‐specific manner in archival material. A similar analysis was performed using E9 and the previously characterized mutation‐specific antibody E‐cad delta 9‐1. Typically, in gastric and breast cancer harbouring E‐cadherin splice site gene mutations, the mutant proteins were expressed but the wild‐type protein was not detected in malignant tissues. These results indicate that variant‐specific monoclonal antibodies can be used to identify differentially expressed E‐cadherin proteins. For immunohistochemical analysis of E‐cadherin, at least two different monoclonal antibodies should be used to exclude alterations of the epitopes resulting in failure to detect a mutant protein. Copyright © 2002 John Wiley & Sons, Ltd.