Hjelm B, Brennan DJ, Zendehrokh N, Eberhard J, Nodin B, Gaber A, Pontén F, Johannesson H, Smaragdi K, Frantz C, Hober S, Johnson LB, Påhlman S, Jirström K, Uhlen M
Proteomics Clin Appl 5 (11-12) 624-635 [2011-12-00; online 2011-10-01]
In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.