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Cancer Stem Cells |
1 Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom
2 Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom; Department of Pathology, Royal Group of Hospitals Trust, Belfast, United Kingdom
3 Northern Ireland Regional Department of Thoracic Surgery, Royal Group of Hospitals Trust, Belfast, United Kingdom
* To whom correspondence should be addressed. E-mail: p.maxwell{at}qub.ac.uk.
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Abstract |
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Immunohistochemical studies on formalin-fixed paraffin embedded (FFPE) tissue utilising polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently Elliott and colleagues (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 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) utilising tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with non-absorbed antibody. Four tumors which showed a membranous pattern of staining initially, retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in six of 30 tumors which 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. Pre-incubation 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 emphasise the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue.
Key Words. Non-Small Cell Lung Carcinoma, Erythropoietin Receptor, C20 antibody, Heat Shock Proteins
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