A multiplex immunoassay for the non-invasive detection of bladder cancer.

Hideki Furuya,Mark D Thornquist,Peter Bryant Greenwood,Owen Chan,Yoshiko Shimizu,Yunfeng Dai,Charles J Rosser,Steve Goodison

doi:10.1186/s12967-016-0783-2

Abstract

BackgroundUrine based assays that can non-invasively detect bladder cancer (BCa) have the potential to reduce unnecessary and invasive procedures. The purpose of this study was to develop a multiplex immunoassay that can accurately and simultaneously monitor ten diagnostic urinary protein biomarkers for application as a non-invasive test for BCa detection.MethodsA custom electrochemiluminescent multiplex assay was constructed (Meso Scale Diagnostics, LLC, Rockville, MD, USA) to detect the following urinary proteins; IL8, MMP9, MMP10, ANG, APOE, SDC1, A1AT, PAI1, CA9 and VEGFA. Voided urine samples from two cohorts were collected prior to cystoscopy and samples were analyzed blinded to the clinical status of the participants. Means (±SD) and receiver operating characteristic (ROC) curve analysis were used to compare assay performance and to assess the diagnostic accuracy of the diagnostic signature.ResultsComparative diagnostic performance analyses revealed an AUROC value of 0.9258 for the multiplex assay and 0.9467 for the combination of the single-target ELISA assays (p = 0.625), so there was no loss of diagnostic utility for the MSD multiplex assay. Analysis of the independent 200-sample cohort using the multiplex assay achieved an overall diagnostic sensitivity of 0.85, specificity of 0.81, positive predictive value 0.82 and negative predictive value 0.84.ConclusionsIt is technically feasible to simultaneously monitor complex urinary diagnostic signatures in a single assay without loss of performance. The described protein-based assay has the potential to be developed for the non-invasive detection of BCa.

Highlights

Urine based assays that can non-invasively detect bladder cancer (BCa) have the potential to reduce unnecessary and invasive procedures
These tests include the measurement of soluble proteins such as bladder tumor antigen (BTA) [1], and nuclear matrix protein 22 (NMP22) [2, 3], proteins detected on fixed urothelial cells (ImmunoCyt) [4], and chromosomal aberrations detected by
Integration of data and selection based on p value, fold change and availability of antibodies resulted in a 14-protein biomarker panel for subsequent testing and refinement in independent cohorts

Summary

Introduction

Urine based assays that can non-invasively detect bladder cancer (BCa) have the potential to reduce unnecessary and invasive procedures. The purpose of this study was to develop a multiplex immunoassay that can accurately and simultaneously monitor ten diagnostic urinary protein biomarkers for application as a non-invasive test for BCa detection. Urine based assays that can non-invasively detect bladder cancer (BCa) have the potential to improve the diagnosis of BCa and help to avoid unnecessary and invasive diagnostic procedures. Several urine-based commercial molecular tests have been FDA-approved for BCa detection and surveillance. We have previously coupled high throughput, discovery-based technology (i.e., genomics and proteomics) with bioinformatics in order to derive diagnostic signatures that show promise for the accurate detection of BCa in voided urine samples [9,10,11,12]. A custom multiplex assay, using MULTI-ARRAY® technology (Meso Scale Diagnostics, LLC), was constructed and the analytical performance was compared with data obtained from individual ELISA assays directed at each of the same ten urinary proteins

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