Zwarthoff, Ellen C.

Abstract Background: Because of the faltering sensitivity and/or specificity, urine-based assays currently have a limited role in the management of patients with bladder cancer. The aim of this study was to externally validate our previously reported protein biomarker panel from multiple sites in the United States and Europe. Methods: This multicenter external validation study included a total of 320 subjects (bladder cancer = 183). The 10 biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) were measured using commercial ELISA assays in an external laboratory. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values. Results: Utilizing the combination of all 10 biomarkers, the area under the ROC for the diagnostic panel was noted to be 0.847 (95% confidence interval, 0.796–0.899), outperforming any single biomarker. The multiplex assay at optimal cutoff value achieved an overall sensitivity of 0.79, specificity of 0.79, positive prediction value of 0.73, and negative prediction value of 0.84 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, muscle invasive bladder cancer, and non-muscle invasive bladder cancer were 0.81, 0.90, 0.95, and 0.77, respectively. Conclusions: Urinary levels of the biomarker panel enabled discrimination of patients with bladder cancer and controls, and the levels of biomarker subsets were associated with advancing tumor grade and stage. Impact: If proven to be reliable, urinary diagnostic biomarker assays can detect bladder cancer in a timely manner such that the patient can expect improvements in overall survival and quality of life. Cancer Epidemiol Biomarkers Prev; 23(9); 1804–12. ©2014 AACR.

Abstract Introduction The presence of blood in the urine (hematuria) is one of the hallmark symptoms of bladder cancer. Thus, the current best practice policy suggests that patients with hematuria undergo cystoscopic examination. However, hematuria can be present in healthy individuals as well as in individuals with various non-life threatening conditions such that a majority of patients with blood in the urine (∼95%) who are screened by cystoscopy do not have bladder cancer. In addition, recent studies have found that a majority of patients with hematuria are not referred on to a specialist for further invasive tests, despite the guidelines. This suggests that the prevalence of cancer in this population may be lower than 5%, making screening by cystoscopy inefficient and, in a large number of cases, unnecessary. We are developing a non-invasive diagnostic assay utilizing urinary protein and DNA biomarkers to triage those patients who do not have cancer from those who should go on to be existing invasive screening modalities. Methods Urine samples were obtained from 48 cancer patients and 256 patients who were evaluated for hematuria but who did not have cancer upon cystoscopic evaluation (Hem+/Cysto−). Twist1 and Nid2 methylation status was assessed using methylation-specific PCR primers and FGFR3 mutational status was determined by quantitative PCR. Matrix Metalloproteinases (MMPs) levels were determined by ELISA. Results Using a novel combination of these DNA and protein markers we identified with high confidence (99% negative predictive value) those patients who do not have cancer and who could be excluded from further testing. Cutoffs could be adjusted such that at 94% sensitivity, 65% of patients who do not have cancer could be excluded. Conclusions The Multi-Analyte Diagnostic Readout (MADR) assay described here combines the best performance characteristics of protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of hematuria patients undergoing screening for bladder cancer effectively increases the sensitivity and negative predictive value at an established MMP protein cutoff. This non-invasive urinary diagnostic assay could lead to the more efficient triage of hematuria patients by identifying those patients who do not have cancer and could be excluded from receiving invasive procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-313. doi:10.1158/1538-7445.AM2011-LB-313