Background/Objectives: The clinically validated multiplex Oncuria bladder cancer (BC) assay quickly and noninvasively identifies disease risk and tracks treatment success by simultaneously profiling 10 protein biomarkers in voided urine samples. Oncuria uses paramagnetic bead-based fluorescence multiplex technology (xMAP^®; Luminex, Austin, TX, USA) to simultaneously measure 10 protein analytes in urine [angiogenin, apolipoprotein E, carbonic anhydrase IX (CA9), interleukin-8, matrix metalloproteinase-9 and -10, alpha-1 anti-trypsin, plasminogen activator inhibitor-1, syndecan-1, and vascular endothelial growth factor]. Methods: In a pilot study (N = 36 subjects; 18 with BC), Oncuria performed essentially identically across three different common analyzers (the laser/flow-based FlexMap 3D and 200 systems, and the LED/image-based MagPix system; Luminex). The current study compared Oncuria performance across instrumentation platforms using a larger study population (N = 181 subjects; 51 with BC). Results: All three analyzers assessed all 10 analytes in identical samples with excellent concordance. The percent coefficient of variation (%CV) in protein concentrations across systems was ≤2.3% for 9/10 analytes, with only CA9 having %CVs > 2.3%. In pairwise correlation plot comparisons between instruments for all 10 biomarkers, R² values were 0.999 for 15/30 comparisons and R² ≥ 0.995 for 27/30 comparisons; CA9 showed the greatest variability (R² = 0.948–0.970). Standard curve slopes were statistically indistinguishable for all 10 biomarkers across analyzers. Conclusions: The Oncuria BC assay generates comprehensive urinary protein signatures useful for assisting BC diagnosis, predicting treatment response, and tracking disease progression and recurrence. The equivalent performance of the multiplex BC assay using three popular analyzers rationalizes test adoption by CLIA (Clinical Laboratory Improvement Amendments) clinical and research laboratories.

Background: Bladder cancer (BC) is a prevalent malignancy with muscle-invasive bladder cancer (MIBC) accounting for 20% of cases and a poor prognosis with a 5-year survival of ~50%. Although neoadjuvant chemotherapy (NAC) followed by radical cystectomy is recognized as the optimal approach for treating locally advanced bladder cancer, it is important to acknowledge the limitations of NAC, which include toxicity and a lack of response in certain individuals. There is an unmet need for predictive assays to stratify patients who are likely to benefit from NAC. We identified 10 mRNA bladder cancer associated biomarkers that can predict response to NAC. This study aimed to develop and analytically validate a multiplex qPCR array-based assay to evaluate the mRNA expression of these 10 biomarkers in MIBC. Methods: We analyzed RNA extracted from 8 formalin-fixed paraffin-embedded (FFPE) and 4 fresh-frozen (FF) specimens of MIBC. The effect of procedures and tissue quality on RNA yield and quality were assessed under various pre-analytical and analytical conditions. Gene expression was evaluated using a custom qPCR array targeting 10 biomarkers and 3 reference genes. The robustness, reproducibility, and effect of necrosis on RNA yield and quality were also examined. Results: The assay demonstrated high concordance between FFPE and FF specimens with no significant difference in gene expression patterns. FFPE curls stored at or below 4°C maintained assay accuracy for up to 2 weeks. The assay was robust to variations in RNA input (5–100 ng) with a quality cut-off (DV200 of >15%) ensuring reliable analysis. Reproducibility studies revealed consistent biomarker profiles across independent technicians and dates, confirming the assay’s reliability in clinical settings. Necrotic tissue proportions up to a certain level had minimal impact on gene expression results. Conclusions: The optimized qPCR array-based assay provides a reliable platform for evaluating 10 mRNA bladder cancer associated biomarkers in FFPE tissues, offering potential as a diagnostic tool to predict clinical response to NAC. This assay represents a step forward in precision medicine for managing advanced BC.