Objective To investigate the relationships of the expressions of tumor necrosis factor receptor superfamily 4 (TNFRSF4), neurogenic differentiation factor 1 (NEUROD1), and POU domain, class 2, transcription factor 3 (POU2F3) in peripheral blood mononuclear cells (PBMCs) with the tumor microenvironment (TME) in hepatocellular carcinoma (HCC) patients.

Methods A retrospective case-control study design was employed. Ninety HCC patients who underwent radical surgery in the general surgery department of our hospital from January 2022 to January 2025 were selected as the study subjects. Based on postoperative follow-up results, they were divided into recurrence group (32 cases) and non-recurrence group (58 cases). Additionally, 50 healthy individuals from the health examination center of our hospital were selected as control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of TNFRSF4, NEUROD1, and POU2F3 in preoperative PBMCs from each group. Multiplex immunohistochemistry (mIHC) was employed to assess the infiltration of immune cell subsets in tumor tissues. Spearman correlation analysis was conducted to explore the relationships between the expression of TNFRSF4, NEUROD1, and POU2F3 and the infiltration of immune cells such as CD8⁺ T cells, FoxP3⁺ Tregs, and CD68⁺ M2 macrophages. Receiver operating characteristic (ROC) curves were plotted to analyze the predictive value of the mRNA expression of TNFRSF4, NEUROD1, and POU2F3 for recurrence and metastasis in HCC patients.

Results Statistically significant differences were observed in the China liver cancer staging (CNLC) and microvascular invasion (MVI) grading between the recurrence and non-recurrence groups (P < 0.05). The mRNA expression levels of TNFRSF4, NEUROD1, and POU2F3 were higher in both the recurrence and non-recurrence groups compared to the control group, and they were significantly higher in the recurrence group than those in the non-recurrence group (P < 0.05). The density of CD8⁺ T cells was significantly higher in the non-recurrence group than that in the recurrence group (t=11.751, P < 0.001), while the proportion of FoxP3⁺ Tregs was significantly higher in the recurrence group (t=16.797, P < 0.001). The density of CD68⁺ macrophages was also significantly increased in the recurrence group (t=7.104, P < 0.001), and the CD8/FoxP3 was significantly lower in the recurrence group than that in the non-recurrence group (t=15.632, P < 0.001). Spearman correlation analysis revealed negative correlations between the expression of TNFRSF4, NEUROD1, and POU2F3 and CD8⁺ T cell infiltration, while positive correlations were observed with FoxP3⁺ Tregs and CD68⁺ M2 macrophages. To exclude the influence of CNLC staging and MVI grading on the results, partial correlation analysis was further conducted, which showed that the correlations between gene expression and immune cell infiltration remained significant after adjusting for confounding factors. ROC curve analysis demonstrated that the predictive performance of the combined model of TNFRSF4, NEUROD1, and POU2F3 was significantly improved (AUC=0.900), with a sensitivity of 88.51% and a specificity of 77.87%, which was superior to the predictive value of single indicators (TNFRSF4 vs. combined prediction: Z=3.751, P < 0.001; NEUROD1 vs. combined prediction: Z=4.980, P < 0.001; POU2F3 vs. combined prediction: Z=4.984, P < 0.001).

Conclusion High mRNA levels of TNFRSF4, NEUROD1, and POU2F3 are observed in PBMCs of HCC patients and are significantly correlated with TME characteristics (decreased CD8⁺ T cells and increased FoxP3⁺ Tregs). The combined model of TNFRSF4, NEUROD1, and POU2F3 exhibits high predictive potential for postoperative recurrence in preliminary analysis, providing a new direction for the development of non-invasive monitoring biomarkers.