Ultra-specific high-multiplex PCR liquid biopsy assays to enable high-frequency treatment and disease monitoring in urothelial malignancies.

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Background: In urothelial cancer, several high-impact decisions are made with limited real-time molecular evidence of residual disease or treatment response. In non–muscle-invasive bladder cancer (NMIBC), early repeat TURBT is often performed due to concern for residual tumor, and response to intravesical BCG is typically assessed only after completion of induction—potentially delaying escalation to salvage therapy or consideration of radical cystectomy when indicated. Tumor-informed NGS MRD can be useful, but cost and turnaround time may limit sampling frequency. A rapid, quantitative, highly sensitive and specific PCR-based liquid biopsy assay platform could support earlier escalation/de-escalation decisions. Deep multiplexing may further reduce per-test reaction burden by compacting panels into a single/few PCR reaction chambers. Methods: An emerging platform (Infiniplex) combines: (1) ultra-specific allele-selective primer design for single-nucleotide variants (SNVs), with extensions to additional mutation classes and methylation targets, compatible with qPCR and d/ddPCR; This, in turn, facilitates (2) deep-combinatorial multiplexing schemes that enable deconvolution of co-expressed mutations from a single/few wells. Analytical performance was assessed using contrived variant mixtures and relevant liquid biopsy matrices (urine and plasma), with a focus on sensitivity, specificity, and multiplex decoding robustness. Outputs are designed for serial quantification to generate molecular response trajectories (decay/plateau/rise). Results: Ultra-specific SNV assays achieved detection down to 0.1–0.01% VAF in liquid biopsy workflows, supporting applications where very low-frequency variants are clinically relevant. Combinatorial multiplexing enabled practical high-multiplex panel designs while maintaining rapid turnaround suitable for frequent longitudinal testing. In NMIBC, proposed clinical readouts include: (i) post-TURBT urine monitoring to help identify patients most likely to benefit from repeat TURBT; (ii) early BCG response/failure trend assessment after ≥3 instillations to support earlier therapy adaptation; and (iii) urine-based molecular trajectories to inform discussions around escalation to definitive management in BCG unresponsive NMIBC. Conclusions: A combinatorial, ultra-specific qPCR/dPCR platform may enable increased frequency of bladder cancer liquid biopsy monitoring with low cost and fast turnaround, without compromising analytical sensitivity or specificity. Prospective clinical studies are planned to establish actionable thresholds and outcome-linked performance at key NMIBC decision points.