Development and Analytical Validation of a Multiplex Diagnostic qPCR-Array for Predicting the Response to Neoadjuvant Chemotherapy in Muscle Invasive Bladder Cancer

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.