3106-LB: Validation and Clinical Application of a ddPCR Unmethylated Insulin cfDNA Methylation Assay: Enhanced Beta-Cell Injury Detection in Immunotherapy-Treated and Islet Infusion Recipients

Introduction and Objective: Circulating unmethylated insulin cell-free DNA, released during beta-cell apoptosis, offers a direct biomarker of active beta-cell destruction. We report validation of a CLIA methylation assay quantifying INS cfDNA and its application in monitoring. Methods: The assay uses HpaII restriction digestion and ddPCR with proprietary INS gene primers. Validation used methylated/unmethylated gDNA controls and EndoC-βH5 across five independent runs assessing accuracy, precision (CV), linearity (5.0×10³-2.5 copies/µL), and specificity (BLAST; exogenous DNA challenge). Results: Treated participants showed lower % unmethylated INS cfDNA vs placebo with treatment-placebo separation of 10.85%, a ~2.2× effect size. Longitudinal decline in treated participants was consistent with resolving beta-cell stress. Islet infusion: % unmethylated cfDNA rose 2-to 5-fold from baseline (~0.19%) within hours post-infusion. Conclusion: This ddPCR methylation assay demonstrates robust analytical performance sensitivity than prior methods for detecting treatment-associated beta-cell changes. It captures acute peri-infusion dynamics with high temporal resolution. These data support unmethylated INS cfDNA as a versatile biomarker across immunotherapy, cell therapy, and early disease detection. Disclosure C. Morris: Employee; Current; Kihealth, Inc.