Transcriptomic and Single-Nucleus Characterization of a Tumor Microenvironment-Associated Four-Gene Score in Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EAC) is molecularly heterogeneous, and prognostic models with clear biological context remain limited. We analyzed RNA-sequencing and clinical data from 46 EAC tumors and 13 adjacent normal tissues in The Cancer Genome Atlas. Differential expression, ESTIMATE, weighted gene co-expression network analysis, and LASSO–Cox regression were used to derive a tumor microenvironment-associated score, followed by bootstrap and nested cross-validation, assessment in independent cohorts, immune deconvolution, and single-nucleus RNA-sequencing analysis. We identified 1571 differentially expressed genes and an 879-gene module associated with stromal, immune, and tumor-purity traits. A four-gene score comprising ACSM1, AGT, IGF2BP3, and ESM1 stratified overall survival in the development cohort. The optimism-corrected C-index was 0.769, whereas the median nested cross-validation C-index was 0.589. In GSE13898, the locked score identified Cluster B with a rank AUC of 0.923; however, survival associations were not reproduced in GSE72873 or GSE254942. Immune-cell comparisons showed nominal differences that did not remain significant after BH correction. Single-nucleus analysis localized IGF2BP3 mainly to malignant cells and ESM1 to endothelial cells. The score provides molecular-subtype and cellular context for hypothesis generation, but its prognostic generalizability remains unestablished.