Longitudinal ctDNA monitoring in melanoma using highly multiplexed USE-PCR incorporating bespoke targets enables real-time detection of treatment response and recurrence

Purpose: Circulating tumor DNA (ctDNA) is a promising biomarker for real-time monitoring of treatment response and recurrence, but current sequencing-based assays are limited by cost, TAT, performance on difficult regions, and sampling frequency. Universal Signal Encoding PCR (USE-PCR) is a highly multiplexed digital PCR chemistry that enables >30 tumor-informed targets to be measured per reaction. We evaluated its performance in a longitudinal melanoma cohort with dense serial sampling. Methods: Tumor/ctDNA sequencing identified subject-specific variants, including challenging GC-rich loci such as TERT c.-124C>T. Multiplexed, tumor-specific USE-PCR panels were designed using Apollo, an automated cloud-based workflow. Panels flexibly incorporated additional targets and were compatible with low-shedding ctDNA. Plasma cfDNA and matching PBMC samples were collected from 8 melanoma subjects at high density (up to 20 timepoints per patient over ~2 years). Plasma ctDNA VAF trajectories, corrected for PBMC, were quantified on a commercial dPCR platform and compared with clinical course and treatment events. Analytical validation included sensitivity, precision, linearity, specificity, cross-platform performance, and robustness with low DNA input (<10 ng). Results: USE-PCR detected distinct molecular phenotypes including clear treatment response (rapid VAF decline), molecular recurrence (0→5%→15% VAF within ~80 days), stable disease (VAF ~0), and mixed responses with target-specific fluctuations often preceding clinical progression or therapy change. An average of four variants were measurable per subject. Sampling at ~20-day intervals uncovered rapid, low-level shifts in ctDNA, including subclonal SNVs at <0.2% VAF, not achievable with quarterly surveillance. Detection was reliable down to 80 ppm with ≤14 SNV targets, strong precision (CV <15%), and linearity over 4 logs. The platform was robust on GC-rich and historically difficult NGS targets, including the TERT promoter, maintained performance with low cfDNA input (<10 ng), and allowed seamless addition of new variants (e.g. BRAF V600E/K, NRAS Q61) for longitudinal monitoring. Conclusions: USE-PCR is a rapid (median TAT < 6 hrs), low-cost, and highly scalable platform for tumor-informed ctDNA monitoring, enabling high multiplexing, compatibility with difficult genomic loci, and frequent sampling suitable for real-time clinical management. High-density longitudinal data reveal molecular patterns aligned with disease activity supporting USE-PCR for dynamic treatment response assessment and recurrence monitoring. Processing of a larger cohort is in progress to enable prospective clinical validation.