Published online Dec 15, 2026. doi: 10.4251/wjgo.126494
Revised: September 8, 2026
Accepted: October 29, 2026
Published online: December 15, 2026
Processing time: 96 Days and 9 Hours
Neoadjuvant immunotherapy has emerged as a promising strategy for gastric cancer (GC), yet reliable biomarkers for patient stratification remain an unmet need, and the systemic epigenetic reprogramming associated with therapeutic response is largely unexplored.
To characterize the circulating cell-free DNA (cfDNA) methylation landscapes associated with major pathological response (MPR) to neoadjuvant anti-programmed cell death protein 1 therapy.
In this proof-of-concept study, CpG island enriched cfDNA methylation profiling was performed on pre-treatment plasma from 19 GC patients (8 MPR, 11 non-MPR) receiving neoadjuvant immunotherapy. Differential methylation analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, and Boruta-based feature selection were applied.
In this small cohort, MPR was associated with significantly superior 3-year overall survival (87.5% vs 36.4%, P = 0.046) and progression-free survival (87.5% vs 27.3%, P = 0.017). Unbiased methylome-wide analysis identified 37185 differentially methylated sites, of which 8904 remained significant after false discovery rate correction. The top 50 sites clearly segregated responders from non-responders, and the majority of the differentially methylated sites (61.45%) were located in promoter regions and enriched at CpG islands. Strikingly, functional annotation revealed that MPR-associated regions were predominantly enriched in neuroactive ligand-receptor interaction, G protein-coupled receptor signaling, and synaptic membrane components, pointing to a profound systemic neuro-immune crosstalk. Three top-ranked CpG sites (chr11_111298596, hypermethylated; chr9_136342657, hypermethylated; and chr12_132283098, hypomethylated in MPR) were identified as prioritized CpG sites meriting further validation.
Our findings demonstrate that cfDNA methylation-derived epigenetic reprogramming of neuro-immune axes is associated with MPR following neoadjuvant immunotherapy in GC. While preliminary, these results provide a prioritized set of CpG sites for future validation in larger cohorts.
Core Tip: This proof-of-concept study explored cell-free DNA methylation profiles associated with major pathological response to neoadjuvant immunotherapy in gastric cancer. CpG-island-enriched profiling identified 37185 nominally significant differentially methylated sites, of which 8904 remained significant after false discovery rate correction; functional enrichment of these false discovery rate-confirmed differentially methylated sites implicated neuroactive ligand-receptor interaction and G protein-coupled receptor signaling rather than conventional T-cell pathways. Three CpG sites were prioritized as exploratory candidates. These findings generate a testable hypothesis that baseline neuro-immune signaling may be associated with immunotherapy response through epigenetic mechanisms, offering a new biological perspective and candidate markers for future validation.