Expression and clinical significance of LAG-3, FGL1, PD-L1 and CD8⁺T cells in hepatocellular carcinoma using multiplex quantitative analysis

Background: Fibrinogen-like protein 1 (FGL1)-Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown.

Methods: The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8⁺T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker's expression and clinical significances were studied.

Results: We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3⁺cells but not PD-L1. CD8⁺ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3⁺cells and low levels of CD8⁺ T cells were correlated with poor disease outcome. Moreover, LAG-3⁺cells deteriorated patient stratification based on the abundance of CD8⁺ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC⁺) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC^-). Furthermore, PD-L1 TC^- in combination with high densities of LAG-3⁺cells showed the worst prognosis, and PD-L1 TC⁺ patients with low densities of LAG-3⁺cells had the best prognosis.

Conclusions: LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3⁺cells and CD8⁺ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.