Evaluation of flow-cytometric three-parameter analysis for EGFR quantification and DNA assessment in human bladder...

Flow-cytometric multi-parameter staining is an excellent method for defining tumour subpopulations. This provides further understanding of tumour heterogeneity and defines the biological relevance of tumour subpopulations. A method of quantifying the epidermal growth factor receptor (EGFR) in parallel with DNA staining, which was previously established in bladder carcinoma cell lines, was applied to twenty-five biopsies of urothelium and urothelial neoplasms. Uro5, a surface glycoprotein, was used to identify urothelial cells. Objective quantification of receptor content via flow cytometry was achieved with beads of defined numbers of antigen-binding sites, and receptor numbers obtained from urothelial and nonurothelial cells were compared with staining intensity in a three-step immunoperoxidase detection of the EGFR. The data obtained matched the immunohistochemical findings and were more sensitive in the low range (ca. 5x103) of receptors. Parallel definition of the proliferative fraction and DNA-ploidy of tumour cells means that this method satisfies the requirements of objective quantification for oncological diagnosis.