Abstract LB-84: Multiplex detection of urinary biomarkers for rapid bladder cancer diagnosis using an automated cartridge-based platform.
Authors: Wallace Ellen , Lai Edwin W. , Mach Kathleen E. , Haque Neda , McCann Leena , Hsiao Shelly , Bui Daniel , Mohan Ruchika , Satya Malini , Wong Edith , Bridge Julia A. , Persing David , Higuchi Russell , Liao Joseph C. 
Published: August 2014

Abstract
Introduction and Objectives
Bladder cancer, a common cancer with high recurrence rate, currently depends on invasive endoscopy for diagnosis and surveillance. A non-invasive, rapid, and accurate urine test will significantly improve management. We have developed a near-patient, fully automated bladder cancer test using the GeneXpert® platform (GX, Cepheid, Sunnyvale CA) for multiplex detection of urinary biomarkers. This test, requires simply mixing voided urine with a liquid preservative followed by loading into the GX cartridge.
Materials and Methods
Thirty candidate mRNA markers from published studies were selected for evaluation. These markers were screened in urine using RT-qPCR targeting intron spanning amplicons for each candidate mRNA. Urinary expression was measured relative to ABL1 reference gene. Four markers, IGF2, KRT20, ANXA10 and CRH, were chosen based on sensitivity and specificity with analysis of &gt;200 patient cases and control urines. A urine preservative was developed to preserve urothelial cells and their RNA and to lyse red blood cells and white blood cells. Four mL preserved urine was added to an integrated cartridge and inserted into a GX module where the on-board sample preparation steps and RT-qPCR took place. The test results, cancer detected or not, are returned within 90 minutes.
With local IRB approval, clinical urine samples were collected from research subjects prior to undergoing cystoscopy for cancer screening or resection. The Xpert® Bladder assay results were compared to urine cytology, tissue histopathology and in selected samples, UroVysion, a commercially available bladder cancer test based on fluorescent in-situ hybridization.
Results
In our pilot validation study, the Xpert® Bladder assay demonstrated 89.7% sensitivity (61/68) for the detection of high grade bladder cancer; 54.1% sensitivity (33/61) for the detection of low grade bladder cancer; 77.5% specificity (248/320) for cystoscopy negative patients; and 92.7% specificity (51/55) for the healthy control group. In the split sample study the Xpert® Bladder assay demonstrated equal performance compared to UroVysion for patients with high grade bladder cancer and performed better than UroVysion for patients with low grade bladder cancer.
Analysis of tumor biopsy tissue from patients whose urine samples were falsely negative showed that all tumors tested had up-regulation of at least one of the four markers, suggesting the failure to detect these markers in the urine may be due to insufficient quantity of urinary tumor cells.
Conclusion
We have developed a rapid, on-demand urine test for bladder cancer diagnosis based on multiplex detection of a 4-marker panel using a well-established molecular diagnostic platform. Pending further validation, the Xpert® Bladder assay may be integrated as an important part of bladder cancer management.
Citation Format: Ellen Wallace, Edwin W. Lai, Kathleen E. Mach, Neda Haque, Leena McCann, Shelly Hsiao, Daniel Bui, Ruchika Mohan, Malini Satya, Edith Wong, Julia A. Bridge, David Persing, Russell Higuchi, Joseph C. Liao. Multiplex detection of urinary biomarkers for rapid bladder cancer diagnosis using an automated cartridge-based platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-84. doi:10.1158/1538-7445.AM2013-LB-84