A New Assay for Measurement of the Liberated Domain I of the Urokinase Receptor in Plasma Improves the Prediction of Survival in Colorectal Cancer

BACKGROUND: The liberated domain I of the urokinase plasminogen activator receptor [uPAR(I)] is a significant prognostic marker in lung and ovarian cancer, although the uPAR(I) concentration is below the limit of quantification (LOQ) in a substantial proportion of patient samples (Lung Cancer 2005; 48: 349-55; Clin Cancer Res 2008;14:5785-93; APMIS 2009; 117: 755-61). This study was undertaken to design an immunoassay with improved functional sensitivity for measuring uPAR(I) and to evaluate the prognostic value of uPAR(I) for colorectal cancer (CRC) patients. METHODS: Surface plasmon resonance analysis identified 2 monoclonal antibodies, R3 and R20, that simultaneously bind to the liberated uPAR(I) but not to intact uPAR. We used R3 for capture and Eu-labeled R20 for detection in designing a 2-site sandwich time-resolved fluorescence immunoassay (TR-FIA 4) for measuring liberated uPAR(I). TR-FIA 4 was validated for use with citrated plasma. The prognostic value of the uPAR(I) concentration was evaluated in 298 CRC patients. The Cox proportional hazards model was used for the uni- and multivariate survival analyses. RESULTS: The LOQ was 0.65 pmol/L. Liberated uPAR(I) was measurable in all patient samples with TR-FIA 4. In the multivariate analysis that included sex, age, tumor stage, tumor localization, and adjuvant treatment, the uPAR(I) concentration measured with TR-FIA 4 (hazard ratio, 1.72; 95% CI, 1.15-2.57; P = 0.009), as well as the concentration of intact soluble uPAR plus the cleaved uPAR fragment containing domains II and III, tumor stage, and age were independent predictors of prognosis. CONCLUSIONS: TR-FIA 4 has a functional sensitivity improved 4-fold over that of the previous uPAR(I) assay. The uPAR(I) concentration measured with TR-FIA 4 is an independent predictor of prognosis in CRC patients.