title: Erythropoietin receptor expression in non-small cell lung carcinoma: a question of antibody specificity.
abstract: Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892-1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue.
authors.affiliation: Department of Pathology, Institute of Pathology, Royal Group of Hospitals Trust, Grosvenor Road, Belfast BT12 6BA, United Kingdom.
authors.forename: W Mark
authors.initials: WM
authors.lastname: Brown
authors.forename: Perry
authors.initials: P
authors.lastname: Maxwell
authors.forename: Alastair N J
authors.initials: AN
authors.lastname: Graham
authors.forename: Anita
authors.initials: A
authors.lastname: Yakkundi
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Dunlop
authors.forename: Zhanzhong
authors.initials: Z
authors.lastname: Shi
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Johnston
authors.forename: Terence R J
authors.initials: TR
authors.lastname: Lappin
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000276:immunology; D045744:Cell Line, Tumor; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017467:Receptors, Erythropoietin / Q000235:genetics* / Q000276:immunology
chemical_list: D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D017467:Receptors, Erythropoietin