Abstract 3844: Longitudinal ctDNA monitoring in melanoma using highly multiplexed USE-PCR incorporating bespoke targets enables real-time detection of treatment response and recurrence

Abstract 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. Citation Format: Hunter Miller, Annalara Fischer, Melissa Hall, Michael E. Egger, Kavitha Yaddanapudi, Lucien Jacky, John Alvarado, Aaron Aguiar, Nathaniel Clark, Paul Belitz, Jeremy Myslinski, Jerrod Schwartz, Paul Flook, Mark Linder. Longitudinal ctDNA monitoring in melanoma using highly multiplexed USE-PCR incorporating bespoke targets enables real-time detection of treatment response and recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3844.