Multi-Center Clinical Analysis and Single-Cell Sequencing Insights Into the Developmental Mechanisms of Synchronous Multiple Primary Esophageal Squamous Cell Carcinoma

Background Synchronous multiple primary esophageal squamous cell carcinoma (M-ESCC) is a rare condition defined by two or more pathologically confirmed primary tumors in distinct esophageal regions within a 6-month period. However, existing studies are limited to small-sample analyses and no exploration of its genetic background or mechanisms of tumorigenesis. This study compares M-ESCC with S-ESCC using a multi-center, large-sample approach to assess clinical features, prognostic factors, and mechanistic insights at the single-cell level. Methods From January 2010 to December 2022, patients with M-ESCC who underwent esophagectomy at four high-volume centers were propensity score-matched (PSM) to S-ESCC patients in a 1:2 ratio. Overall survival (OS) was analyzed using Kaplan–Meier curves, with log-rank tests for univariate analysis and Cox proportional hazards regression for multivariate analysis. Single-cell sequencing mapped the multi-origin carcinogenic landscape, which was further validated by in vitro experiments. Results Among 7126 esophagectomy cases, 102 M-ESCC patients met inclusion criteria and were matched with 204 S-ESCC patients (1:2 PSM). M-ESCC had higher smoking index grades (P = 0.021), SII values (P = 0.001), and worse prognosis (P < 0.001). Cox analysis identified M-ESCC, pT stage, pN stage, and differentiation as independent prognostic factors. Single-cell RNA sequencing revealed unique immune microenvironments and epithelial subpopulations (C7 C1S+, C6 WNT10A+). IFNγ treatment enhanced organoid formation (P = 0.0164) and upregulated SOX2/KRT13 expression, particularly centrally. Conclusion This study underscores the increased malignancy and poorer prognosis associated with M-ESCC. Single-cell profiling identifies IFNγ-responsive epithelial subpopulations as potential contributors to the tumorigenesis of M-ESCC. These findings offer novel insights into the molecular mechanisms driving synchronous multi-primary carcinogenesis in esophageal squamous cell carcinoma, highlighting key factors that may inform future therapeutic strategies.