Aminopeptidase activities as prospective urinary biomarkers for bladder cancer

Purpose: Proteases have been implicated in cancer progression and invasiveness. We have investigated the activities, as opposed to simple protein levels, of selected aminopeptidases in urine specimens to serve as potential novel biomarkers for urothelial cancer. Experimental design: The unique urinary proteomes of males and females were profiled to establish the presence of a gender-independent set of aminopeptidases. Samples were also collected from patients with urothelial cancer and matched controls. A SOP for urine processing was developed taking into account hydration variation. Five specific aminopeptidase activity assays, using fluorophoric substrates, were optimized for evaluation of marker potential. Results: Nineteen exopeptidases and 21 other proteases were identified in urine and the top-five most abundant aminopeptidases, identical in both genders, selected for functional studies. Depending on the enzyme, activities were consistently lower (p <= 0.05), higher or unchanged in the cancer samples as compared to controls. Two selected aminopeptidase activities used as a binary classifier resulted in a ROC curve with an AUC = 0.898. Conclusion and clinical relevance: We have developed functional assays that characterize aminopeptidase activities in urine specimens with adequate technical and intraindividual reproducibility. With further testing, it could yield a reliable biomarker test for bladder cancer detection or prognostication.