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.
Becker
Karl-Friedrich
KF
Technische Universität, Klinikum rechts der Isar, Institut für Pathologie, Trogerstrasse 18, D-81675 München, Germany. kf.becker@lrz.tum.de
Kremmer
Elisabeth
E
Eulitz
Manfred
M
Schulz
Stephan
S
Mages
Jörg
J
Handschuh
Gabriele
G
Wheelock
Margaret J
MJ
Cleton-Jansen
Anne-Marie
AM
Höfler
Heinz
H
Becker
Ingrid
I
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D015820:Cadherins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D019656:Loss of Heterozygosity; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015820:Cadherins; D009363:Neoplasm Proteins