Circulating Tumor DNA Monitoring in Patients with Uveal Melanoma Using Mutation-Agnostic Multiplex Drop-Off ddPCR...

To address the limitations of existing approaches─targeted next-generation sequencing (NGS) and simplex droplet digital PCR (ddPCR) assays─for circulating tumor DNA (ctDNA) detection in uveal melanoma (UM), we developed and validated mutation-agnostic multiplex drop-off ddPCR assays at the targeted loci as a novel strategy. Two multiplex drop-off ddPCR assays covering hotspot mutations in GNAQ p.Q209, GNA11 p.Q209, p.R183, SF3B1 p.R625, PLCB4 p.D630, and CYSLTR2 p.L129Q were developed. Analytical sensitivity and specificity were determined. Clinical validation was performed using DNA from tumor tissue and plasma samples from a prospective cohort of Metastatic UM (MUM) (ALCINA, NCT02866149) or archived clinical samples. The multiplex assays demonstrated high sensitivity, with detection limits ranging from 0.06% to 0.13%, comparable to those of simplex ddPCR assays. Somatic mutations in tumor DNA (N = 12) were successfully identified using the multiplex assays, and all were confirmed by NGS. ctDNA was detected in 62.8% of 43 plasma samples, with a median mutant allele frequency (MAF) of 2.9% (interquartile range [IQR] 0.6-7.4%). Strong concordance with simplex ddPCR results was observed (r = 0.98, p < 0.0001 for multiplex 1, N = 32; r = 0.97, p < 0.0001 for multiplex 2, N = 11), further supporting the accuracy of the multiplex assays. Our mutation-agnostic multiplex drop-off ddPCR assays provide a sensitive, specific, and cost-effective alternative to targeted NGS and simplex ddPCR for ctDNA monitoring in UM. By minimizing reliance on prior knowledge of tumor genotype with NGS, these assays enable broader clinical applicability for real-time treatment monitoring in UM.