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

• DOI: 10.20944/preprints202504.2055.v1
• Publication Date: 2025-04-28T01:23:40Z
• Authors: Toru Sakatani; Sunao Tanaka; Kaoru Murakami; Francisco Aguilar; Owen T.M. Chan; Catherine Bresee; Daniel J. Luthringer; Charles J. Rosser; Wayne Hogrefe; Hideki Furuya
• Keywords: Multiplex; Neoadjuvant Therapy

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