Evaluation of HHLA2 expression predicts poor survival in patients with esophageal squamous cell carcinoma

Background/Aims Esophageal squamous cell carcinoma (ESCC) remains a major health burden in China, and the clinical benefit of immunotherapy is limited by the lack of reliable predictive biomarkers. Human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2), a recently identified immune checkpoint molecule of the B7 family, has been implicated in tumor immune evasion and disease progression across multiple malignancies. Therefore, this study investigated the expression pattern and prognostic significance of HHLA2 in ESCC. Methods Multiplex immunofluorescence staining for HHLA2, CD68, and CD8 was performed on an ESCC tissue microarray to characterize HHLA2 expression within the tumor microenvironment (TME). Quantitative analyses were subsequently conducted to evaluate its associations with clinicopathological features and patient survival. Results HHLA2 expression was significantly elevated in ESCC tissues compared with normal esophageal tissues. Patients with high HHLA2 expression exhibited significantly poorer overall survival (OS) than those with low expression in CK + tumor area and CK - stroma area. The combined analyses of HHLA2 expression and infiltration levels of CD8 + , CD68 + provided novel exploratory risk stratification biomarkers for ESCC patients. Increased HHLA2 expression on both CD68⁺tumor-associated macrophages (TAMs) and CD8⁺T-cells were likewise associated with unfavorable prognosis. Moreover, univariate and multivariate analyses both revealed that HHLA2 could served as an independent prognostic biomarker in esophageal cancer patients. Conclusion HHLA2 was frequently overexpressed in ESCC and was associated with adverse clinical outcomes. The combination of HHLA2 expression and immune cell CD8 + , CD68 + infiltration levels could offer enhanced exploratory risk stratification biomarkers. These findings identified HHLA2 as a promising prognostic biomarker and a potential target for immunotherapeutic intervention in ESCC.