10557 Background: Early detection of non–small cell lung cancer (NSCLC) within liquid biopies remains limited by the extremely low abundance and heterogeneous nature of circulating tumor DNA (ctDNA) in plasma, particularly in patients with indeterminate pulmonary nodules. DNA methylation is an attractive early biomarker; however, conventional PCR- and sequencing-based assays have limited ability to sensitively and cost-effectively assess heterogeneous epiallelic methylation patterns at low variant fractions. We developed a digital microfluidic platform that enables multiplex, single-molecule profiling of DNA methylation heterogeneity to improve early NSCLC detection. Methods: We applied REM-DREAMing (Ratiometric-Encoded Multiplex Discrimination of Rare EpiAlleles by Melt), a digital high-resolution melt (dHRM) platform combining nanowell-based digital PCR with ratiometrically encoded, methylation-agnostic probes. Following bisulfite conversion, cfDNA was digitized into >40,000 nanoliter wells, enabling copy-by-copy assessment of methylation density across a five-gene biomarker panel previously associated with NSCLC. Analytical sensitivity and multiplex performance were compared against quantitative methylation-specific PCR (qMSP) and digital MSP (dMSP). Clinical feasibility was evaluated in plasma from patients with CT-detected indeterminate pulmonary nodules. Results: REM-DREAMing resolved heterogeneous methylation patterns at single-molecule resolution and detected methylated epialleles at fractions as low as 0.00005% in a high background of unmethylated DNA. In a cohort of 48 low-volume plasma samples, incorporating intermolecular methylation density distributions significantly improved classifier performance relative to binary methylation calls. The five-gene panel achieved 93% sensitivity at 90% specificity for early-stage NSCLC, with an AUC of 0.96. Compared with qMSP and dMSP, REM-DREAMing demonstrated superior sensitivity, multiplex scalability, and discrimination of partially methylated epialleles. Conclusions: Multiplex digital profiling of DNA methylation heterogeneity enables sensitive, low-cost detection of early-stage NSCLC from limited plasma input. By capturing epigenetic heterogeneity at the single-molecule level, this platform improves diagnostic performance in clinically challenging early detection settings and provides a scalable foundation for expanded biomarker panels. Analytical and clinical performance comparison. Method Heterogeneous Methylation Detection Detectable Copy Number Analytical Specificity NSCLC AUC qMSP No 10 ~0.01% 0.78–0.82 dMSP No 1 ~0.005% 0.85 Bulk MS-HRM Yes 10 ~0.1% 0.80 REM-DREAMing Yes (single-molecule) 1 0.00005% 0.96
O'Hersey et al. (Wed,) studied this question.