Extrachromosomal DNA amplification defines a high-risk subgroup and unique molecular features in gastric cancer

Gastric cancer (GC) is the fifth most prevalent cancer and the fourth leading cause of cancer mortality. However, clinical strategies targeting these amplifications in GC have been unsuccessful, often leading to treatment resistance and poor prognosis. Extrachromosomal DNA (ecDNA) has emerged as a major mechanism associated with oncogene focal amplification and adverse outcomes. In this study, we performed whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) on paired tumor-normal samples from 76 Korean GC patients collected through Seoul National University Bundang Hospital to understand the prevalence of ecDNAs and their clinical relevance in GC patients. Focal amplification regions amplicons were identified and classified using AmpliconArchitect (AA) and Amplicon Classifier into ecDNA (circular amplification) and non-ecDNA (Chromosomal Amplicons, ChAmps). Focal amplifications were highly frequent and showed a strong association with the chromosomal instability (CIN) subtype (P = 2.37e-09). Of the 76 patients, 17 (22.4%) were classified as "ecDNA positive patients". Notably, 75% of CIN subtype patients carried one or more ecDNA amplicons. Genomic analysis revealed that ecDNA amplicons were significantly larger (P = 0.00056) and more structurally complex than ChAmps, exhibiting a higher frequency of structural variants. ecDNA amplicons also harbored significantly more canonical cancer genes (P = 3.90e-03) and displayed significantly higher copy numbers of these genes compared to ChAmps (P = 6.50e-04). Furthermore, ecDNA regions were significantly enriched with putative transcriptional regulatory elements (P = 1.20e-04) and GC-specific accessible chromatin regions. In WTS data, genes within ecDNA exhibited significantly higher expression compared to in ChAmp (P = 3.59e-05). Gene Set Enrichment Analysis (GSEA) revealed that ecDNA cohorts displayed a significantly more pronounced immunosuppressive phenotype-characterized by downregulation of immune response gene sets-compared to ChAmp patients in both SNUBH and TCGA cohorts. Clinically, the presence of ecDNA conferred a significantly worse Overall Survival (OS) rate compared to ChAmp cohorts (Log-rank test, P = 0.012). Multivariate Cox proportional hazards analysis confirmed that ecDNA status acts as an independent risk factor for OS (HR = 14.4, P = 0.001)Our findings demonstrate that ecDNA amplification is frequent in GC, particularly within the CIN subtype, and is associated with distinct genomic complexity, higher oncogene burden, unique transcriptional consequences (immune suppression), and poor patient prognosis. The presence of ecDNA amplification may serve as a critical prognostic factor in GC, highlighting the need for personalized treatment, including the development of ecDNA-targeted therapies to improve treatment outcomes.