Individualized Longitudinal ctDNA Monitoring in Melanoma Using Multiplexed USE-PCR

Circulating tumor DNA enables longitudinal monitoring in cancer, but current sequencing-based assays are limited by cost, turnaround time, performance in difficult genomic regions, and infrequent sampling. Universal Signal Encoding PCR (USE-PCR) is a highly multiplexed digital PCR chemistry that enables measurement of up to 32 tumor-informed targets per reaction. Here, we evaluated USE-PCR for dense longitudinal circulating tumor DNA monitoring in patients with metastatic melanoma undergoing immunotherapy.

Tumor and plasma sequencing identified subject-specific variants from nine subjects with melanoma, including variants in GC-rich and historically challenging loci such as the TERT promoter. Subject-specific USE-PCR panels were designed using an automated cloud workflow and applied to plasma cell-free DNA and matched peripheral blood mononuclear cell sample DNA, with up to 21 timepoints per subject, collected over a 2-year window. Variant allele fractions were quantified on a commercial digital PCR platform and interpreted with a peripheral blood mononuclear cell genomic DNA background.

USE-PCR resolved variant-specific circulating tumor DNA trajectories across longitudinal plasma samples. Dense sampling revealed rapid and low-level circulating tumor DNA shifts, including subclonal variants below the 0.2% variant allele fraction that may not be captured through less frequent surveillance intervals. Panels supported the incorporation of new variants without compromising analytical performance.