Chun-Hua Wang,1,2,* Shi-Lu Chen,1,2,* Xia Yang,1,2,* Ting Wu,1,3 Li-Li Liu,1,2 Jing-Ping Yun1,2 

1State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-Sen University Cancer Center, Guangzhou, People’s Republic of China; 2Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, People’s Republic of China; 3Department of Gastric Surgery, Sun Yat-Sen University Cancer Center, Guangzhou, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jing-Ping Yun, Department of Pathology, Sun Yat-sen University Cancer Center, 651# Dong Feng Road East, Guangzhou, 510060, People’s Republic of China, Tel/Fax +86-20-8734-3693, Email yunjp@sysucc.org.cn Chun-Hua Wang, Department of Pathology, Sun Yat-sen University Cancer Center, 651# Dong Feng Road East, Guangzhou, 510060, People’s Republic of China, Tel/Fax +86-20-8734-2272, Email wangchunh@sysucc.org.cn

Background: HHLA2, a member of the B7 family, is extensively expressed in various cancers and plays a pivotal role in modulating the immune microenvironment. However, its prognostic significance in hepatocellular carcinoma (HCC) remains poorly understood. This study aims to elucidate the expression patterns of HHLA2 and PD-L1 in HCC, their associations with tumor-infiltrating lymphocytes (TILs), and their impact on clinical outcomes.
Methods: Immunohistochemistry (IHC) was employed to evaluate HHLA2 and PD-L1 expression in 547 HCC tissue samples. PD-L1 positivity was defined as ≥ 1% membranous or cytoplasmic staining. Hematoxylin and eosin (H&E) staining was utilized to quantify TILs (percentage/area), while IHC was used to measure the densities of CD3+, CD4+, and CD8+ TILs (cells/mm²).
Results: HHLA2 and PD-L1 exhibited similar positivity rates. HHLA2 positivity was associated with older age, lower alpha-fetoprotein (AFP) levels, well-differentiated tumors, and improved overall survival (OS). HHLA2 expression was inversely correlated with stromal TIL density. In contrast, tumor cell (TC)-PD-L1 and inflammatory cell (IC)-PD-L1 positivity were positively correlated with higher stromal TIL density and increased levels of CD3+, CD4+, and CD8+ TILs. Patients with HHLA2(+)/PD-L1(-) status demonstrated the longest OS. A novel classification system based on HHLA2/PD-L1 expression identified distinct immune profiles and prognostic subgroups.
Conclusion: HHLA2 significantly influences the immune microenvironment of HCC and serves as an independent prognostic marker. The combined assessment of HHLA2 and PD-L1 expression facilitates risk stratification, providing a framework to optimize immunotherapy strategies. These findings contribute to the advancement of precision medicine in the management of HCC.

Keywords: HHLA2, PD-L1, TILs, prognosis, HCC