Expression of the immune checkpoint receptors PD-1, LAG3, and TIM3 in the immune context of stage II and III gastric cancer by using single and chromogenic multiplex immunohistochemistry

We sought to determine the clinicopathological significance of PD-1, LAG3, and TIM3 in gastric cancer (GC) by examining their expression and immune context. Immunohistochemistry (IHC) for PD-1, TIM3, LAG3, and tumor-infiltrating immune cell (TIIC) markers was performed in 385 stage II/III GCs. Epstein-Barr virus (EBV) and microsatellite stability (MSI) testing were performed for molecular classification. Chromogenic multiplex IHC (mIHC) for PD1, TIM3, LAG3, CD3, CD8, FOXP3, CD68, and cytokeratin was performed in 58 of the total samples. PD-1, LAG3, and TIM3 expression in TIICs was observed in 91 (23.6%), 193 (50.1%), and 257 (66.8%) GCs by single IHC, respectively. The expression was associated with EBV⁺ and MSI-H molecular subtypes (p ≤ 0.001). A positive expression of LAG3 in the invasive margin of the tumor was associated with better prognosis in univariate (p =.020) and multivariate (p =.026) survival analyses. The expression of different immune checkpoint receptors (ICRs) was significantly positively correlated. Dual or triple ICR expression was more frequent in high PD-1 and TIM3 density groups than in low-density groups by mIHC (all p ≤ 0.05). ICRs were mainly expressed in CD3⁺CD8⁺ and CD3⁺CD8⁻ T cells. Fifty-eight GCs were classified into three groups by clustering analysis based on mIHC, and the group with the highest ICR expression in TIICs showed significantly better outcomes in progression-free survival (p =.020). In GC, PD-1, LAG3, and TIM3 expression is positively correlated and associated with better prognosis. Our study provides information for the application of effective immune checkpoint inhibitors against GC.