An exploratory hypoxia signature comprising four genes shows limited external prognostic transferability in esophageal squamous cell carcinoma

Background Hypoxia-associated transcription is an important component of the esophageal squamous cell carcinoma (ESCC) microenvironment. We developed an exploratory four-gene signature and evaluated its prognostic performance, internal stability, cross-cohort transferability, and biological context. Methods TCGA, GTEx, and GEO transcriptomic data were analyzed. A four-gene signature was derived using univariate Cox screening, 10-fold cross-validated LASSO Cox regression, and stepwise multivariable Cox regression. Internal validation used 1,000 bootstrap resamples, optimism correction, and out-of-bag calibration. Fixed-coefficient external survival testing was conducted in GSE53624 and GSE53622. We additionally audited clinical heterogeneity across cohorts, performed exploratory continuous-score subgroup Cox analyses and interaction tests, and synthesized 16 reviewer-directed hypoxia-associated pathways using the original database-wide FDR values. TCGA-only differential-expression sensitivity, paired GSE23400 expression, patient/sample-level GSE160269 single-cell, immune functional scoring, and exploratory in silico drug-sensitivity analyses were also performed. Results The signature comprised ATF3, EGFR, KDELR3, and PNRC1 in 91 ESCC patients. High-risk patients had poorer overall survival (log-rank P = 0.0031), but the complete feature-selection pipeline was unstable under bootstrap resampling. Apparent 1-, 3-, and 5-year AUCs were 0.730, 0.696, and 0.567; optimism-corrected AUCs were 0.691, 0.657, and 0.546, respectively, and the corrected C-index was 0.660. Direct application in GSE53624 ( n = 119) and GSE53622 ( n = 60) did not reproduce derivation-cohort performance. TNM-stage and follow-up distributions differed across cohorts, and TCGA treatment data were unavailable. Exploratory subgroup analyses did not demonstrate consistent external performance. Focused GSEA identified heterogeneous directions across hypoxia-adjacent inflammatory, angiogenic, metabolic, and redox processes. Conclusion The four-gene signature showed moderate short-term discrimination in the derivation cohort but weak long-term performance, selection instability, and limited cross-cohort reproducibility. It should be regarded as a hypothesis-generating research signature rather than a platform-independent or clinically deployable model, and requires prospective multicenter and experimental validation.