Multiplex screening of 422 candidate serum biomarkers in bladder cancer patients identifies syndecan-1 and macrophage colony-stimulating factor 1 as prognostic indicators

Background: Serum protein biomarkers that correlate with urothelial bladder cancer (UBC) stage and outcome could accelerate and improve clinical management, but this requires thoroughly validated high-performance biomarker(s). Unbiased discovery of serum biomarkers by mass spectrometry is challenging due to their low abundance in a complex sample, and candidate-based discovery is slow and expensive due to the number of immunoassays required. We have utilised a novel multiplex platform to assay disease-associated proteins in the sera of bladder cancer patients with the aim of identifying novel staging and prognostic biomarkers. Methods: All sera were collected as part of the Bladder Cancer Prognosis Programme. We randomly selected 10 non-UBC, 10 G1pTa, 10 G3pTa, 30 G3T1 and 30 G3T2+ UBC cases. Serum levels of proteins were determined with Proseek multiplex immunoassays (http://www.olink.com/). Multivariate linear regression analysis was used to identify significant associations between protein levels and bladder cancer stage and grade. Kaplan-Meier analyses and log-rank tests were used to identify associations between protein levels and disease-specific survival. Results: There were no significant differences in age and gender between the groups. 422 proteins were successfully measured in the sera of the 10 non-cancer controls and 80 UBC patients. Linear regression identified 5 proteins significantly associated with UBC. In order of statistical significance (lowest P value first) these were nectin-4, syndecan-1, T-cell immunoglobulin mucin receptor 1, macrophage colony-stimulating factor 1 and matrilysin. Although none of these showed clear discrimination between stages of disease, high levels of syndecan-1 and macrophage colony-stimulating factor 1 were significantly associated with worse UBC-specific survival. Conclusions: We have studied the relationship between UBC and the serum concentrations of over 400 proteins. Those which reach statistical significance include known biomarkers and new candidates that may warrant further investigation. Although bladder cancer does cause many biologically plausible changes in the serum proteome none of the proteins studied appears to be suitable for accurate non-invasive staging of bladder cancer. However, syndecan-1 and/or macrophage colony stimulating factor-1 (CSF-1) might prove useful as prognostic indicators.