Comprehensive molecular characterization of cfDNA as predictive and monitoring biomarkers in advanced gastric cancer receiving immunotherapy

BACKGROUND: Advanced gastric cancer (aGC) exhibits substantial heterogeneity in response to combination immunotherapy. Circulating cell-free DNA (cfDNA) enables non-invasive profiling of tumor dynamics and may provide biomarkers for response prediction. METHODS: We enrolled 94 patients with aGC undergoing combination immunotherapy and assigned them to a discovery set (n=49) and an internal validation set (n=45). Plasma cfDNA was collected pre-treatment and post-treatment and profiled by low-pass whole-genome sequencing and whole-genome bisulfite sequencing in the discovery set, with targeted bisulfite sequencing used in the validation set. RESULTS: The discovery set included 34 responders and 15 non-responders, and the validation set included 30 responders and 15 non-responders. We found that responders showed longer cfDNA, lower cfDNA tumor fraction (median: 0.06 vs 0.01, p<0.001), reduced chromosomal instability (median genomic instability index: 0.026 vs 0.007, p<0.001), and higher global methylation (median: 0.715 vs 0.724, p=0.007). Additionally, the differentially methylated region (DMR) at chr20:25849353-25849490 consistently showed higher methylation in responders in both the discovery (adjusted p=0.006) and validation sets (adjusted p=0.049). A predictor based on this DMR outperformed programmed death-ligand 1 (PD-L1) combined positive score (CPS) with area under the curve (AUCs) of 0.79 (discovery: 95% CI 0.65 to 0.93) and 0.72 (validation: 95% CI 0.54 to 0.91). When integrating PD-L1 CPS with this DMR, the AUCs were 0.81 (discovery: 95% CI 0.67 to 0.95) and 0.75 (validation: 95% CI 0.56 to 0.94), respectively. For on-treatment monitoring, three DMRs increased specifically in responders; among them, increased methylation of chr8:110479193-110480324 and chr8:50891437-50892120 was associated with improved progression-free survival (median: 4.90 vs 11.57 months, p<0.001; median: 5.20 vs 10.20 months, p=0.007). CONCLUSION: Integrated cfDNA profiling captures immunotherapy-associated molecular dynamics in aGC. A single pretreatment cfDNA methylation marker (chr20:25849353-25849490) improves response prediction beyond PD-L1 CPS and represents a potential predictive biomarker for combination immunotherapy.