Hepatocellular carcinoma (HCC) is a classic example of inflammation-associated malignancy, predominantly arising in patients with pre-existing chronic liver disease (1). The pathogenesis of HCC involves a complex, multistep process influenced by multiple etiological factors. Early-stage diagnosis remains particularly challenging because initial disease progression is asymptomatic. Consequently, there is a pressing clinical need for reliable diagnostic approaches capable of differentiating among distinct stages of HCC progression, which could significantly improve patient outcomes and survival rates.

Chronic hepatitis B virus (HBV) infection is a well-established risk factor for HCC development. As a non-cellular pathogenic agent, HBV induces chronic liver injury through aberrant immune-mediated mechanisms. Growing evidence supports the pivotal role of immune pathogenesis in HBV-related HCC progression. Recent studies indicate that immune escape mechanisms, mediated by the suppression of various immune cell subpopulations, significantly contribute to HCC initiation and progression (2). A comprehensive understanding of immune landscape alterations during HCC progression may provide valuable insights into developing early diagnostic strategies.

Immune checkpoints represent critical regulatory mechanisms that maintain immune homeostasis through both positive and negative regulation of immune cell activity. During tumorigenesis and progression, dysregulation or functional impairment of immune checkpoint proteins may facilitate tumor immune evasion and promote malignant progression (3). Programmed cell death protein 1 (PD-1), predominantly expressed on activated T cells, B cells, and myeloid cells, exerts immunosuppressive effects by binding to its ligands programmed death-ligand 1 (PD-L1) and PD-L2, thereby inhibiting T-cell activation and cytokine production. In HCC, tumor cells frequently exhibit upregulated PD-L1 expression, which engages PD-1 expressed on tumor-infiltrating T cells and suppresses their anti-tumor activity. Activation of the PD-1/PD-L1 axis significantly impairs T cell-mediated immune responses by inhibiting the secretion of key cytokines, including IFN-γ and TNF-α (3). Furthermore, cytokines such as IL-6 and TGF-β can modulate tumor immune evasion through regulation of immune checkpoint expression and function. Nevertheless, the precise interplay between PD-1 signaling and cytokine networks during HCC progression remains poorly understood. Recent research has expanded beyond PD-1 to investigate the role of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) as a novel immune checkpoint molecule in various pathological conditions (4). However, the expression dynamics and functional significance of Tim-3 in HCC pathogenesis remain elusive. Given these knowledge gaps, systematic analyses of immune checkpoint proteins and cytokine expression profiles are necessary to identify reliable biomarkers for early HCC detection.

To identify potential diagnostic biomarkers for different stages of HCC progression, the expression profiles of immune checkpoint proteins and cytokines were systematically analyzed in serum samples from healthy donors and patients with chronic hepatitis B (CHB), liver cirrhosis (LC), and HCC. Through statistical analysis using one-way analysis of variance (ANOVA) and multivariate logistic regression, interferon (IFN)-γ and interleukin (IL)-22 emerged as significantly differentially expressed cytokines. Receiver operating characteristic (ROC) curve analysis demonstrated that the combination of IFN-γ and IL-22 exhibited superior diagnostic performance compared with conventional α-fetoprotein (AFP) in predicting HCC. These findings provide a robust theoretical foundation for developing early clinical diagnostic strategies for HCC.

Current clinical detection of HCC primarily relies on imaging modalities, including ultrasound, MRI, CT, and AFP measurement (8). Although AFP is widely regarded as the most reliable serum biomarker for HCC diagnosis (9), its diagnostic accuracy remains limited, especially in early-stage HCC with tumors smaller than 2 cm, where AFP levels often remain below clinical detection thresholds (9). This limitation was apparent in the cohort of the present study, where only 4 out of 20 patients with HCC exhibited AFP levels exceeding 100 ng/ml. These findings underscore the critical need to develop more sensitive, non-invasive serological diagnostic approaches for early HCC detection.

HCC typically develops in the context of chronic liver disease, contributing to an immunosuppressive hepatic microenvironment and T-cell dysfunction (10,11). During hepatocarcinogenesis, the anti-tumor immune surveillance mechanisms of the liver become impaired, with immune checkpoint pathways, particularly the PD-1/PD-L1 axis, playing a pivotal role in this process (12). In the present study, serum PD-L1 and PD-1 levels were quantified using ELISA in samples from healthy donors and patients with CHB, LC, and HCC. Results revealed a progressive increase in PD-1 and PD-L1 expression, with the lowest levels observed in healthy donors and the highest expression in patients with HCC, reflecting disease progession. PD-L1 and PD-1 expression were significantly elevated in patients with HCC compared with healthy controls (P<0.05). These results align with previous research demonstrating that HCC is characterized by high PD-L1 expression (13). Furthermore, observations of PD-1 upregulation in patients with HCC are consistent with reports of PD-1 induction in circulating monocytes from patients with HBV-associated HCC (13).

Although Tim-3 expression was assessed across the four study groups, no significant differences were observed among the cohorts. This suggests Tim-3 may have less influence on liver disease progression compared with the PD-1/PD-L1 pathway. However, the precise role of Tim-3 in liver disease pathogenesis requires further investigation. Unlike previous research utilizing cell lines (14), the present research used patient serum samples, providing more clinically relevant data to support the development of non-invasive diagnostic strategies for HCC detection.

HBV infection represents a global health challenge, affecting over 2 billion individuals worldwide and is one of the primary etiological factors for liver disease (15,16). A total of ~350 million infected individuals progress to CHB, with one-third developing LC, and >75% eventually progressing to HCC (17). This disease continuum of CHB, LC, and HCC is widely recognized as the natural progression of chronic HBV infection (18). While immune-mediated chronic inflammation is established as a critical driver of HCC pathogenesis, the precise molecular mechanisms underlying this progression remain poorly understood. In this investigation, expression profiles of eight cytokines across different stages of chronic HBV infection were systematically analyzed. Initial screening using one-way ANOVA identified statistically significant cytokines, which were subsequently incorporated into multivariate logistic regression analysis. Two particularly noteworthy cytokines emerged: IFN-γ and IL-22. One-way ANOVA demonstrated significant upregulation of IL-22 in patients with HCC compared with healthy donors, CHB, and LC groups, whereas IFN-γ exhibited significant downregulation in patients with HCC relative to these groups. Given that most HCC cases develop from HBV-related CHB, the analysis focused on disease progression by excluding healthy donors and stratifying the remaining cohorts into HCC and non-HCC groups. Multivariate logistic regression analysis of differentially expressed proteins between these groups identified IFN-γ as a protective factor against HCC development (OR=0.991; 95% CI, 0.984-0.997), while IL-22 emerged as a significant risk factor (OR=1.020; 95% CI, 1.004-1.036).

IFN-γ, a pleiotropic cytokine, plays crucial roles in immune regulation, antiviral defense, and anti-proliferative processes (19). Previous research demonstrated that sustained IFN-γ exposure could suppress hepatocarcinogenesis despite concurrent liver injury (20). The findings of the present study revealed a progressive decrease in IFN-γ expression associated with HCC development. This downregulation might be mediated by Tim-3, which has been shown to inhibit IFN-γ production in T cells (21). However, the precise molecular mechanisms require further investigation. Notably, Li et al (20) reported that patients with HCC with low serum IFN-γ levels or negative IFN-γ receptor expression in tumor tissue exhibited larger tumor volumes, increased metastatic potential, and higher recurrence rates following treatment.

IL-22, a member of the IL-10 cytokine family, plays a significant role in antimicrobial defense, tissue homeostasis, and repair mechanisms (22). As a hepatocyte survival factor, IL-22 has substantial effects in various liver pathologies, including hepatitis, cirrhosis, and HCC, through interactions with IL-22R1 and IL-10R2 receptors (22). Although IL-22 demonstrates hepatoprotective properties during hepatic inflammation (23), emerging evidence suggests its dual role in liver pathophysiology. Previous research established an association between elevated levels of IL-22 and alterations in the hepatic microenvironment, with progressive increases observed from normal tissue to cirrhotic liver and subsequently to tumor tissue (24), a trend consistent with the findings of the present study in the sera of patients with LC and HCC. Furthermore, excessive IL-22 levels may promote hepatocarcinogenesis in chronic hepatitis patients, potentially facilitating tumor development through regulatory mechanisms in viral hepatitis (25).

To evaluate the diagnostic potential of these cytokines, comparative analyses using ROC curves for IFN-γ, IL-22, and AFP were performed in patients with HCC. ROC curve analysis demonstrated superior diagnostic performance of both IFN-γ and IL-22 compared with conventional AFP testing. Notably, the combination of IFN-γ and IL-22 exhibited enhanced diagnostic accuracy compared with AFP alone. These findings suggest that the combination of IFN-γ and IL-22 holds promise for early HCC detection, providing a theoretical foundation for clinical application. However, further research is warranted to elucidate the underlying mechanisms and validate these findings in larger clinical cohorts.

Furthermore, additional cytokines, including IL-4 and IL-9, showed significant differential expression during the progression from CHB and LC to HCC. IL-4 is a pleiotropic cytokine predominantly secreted by activated T helper 2 cells. As a key immunomodulatory molecule, IL-4 serves crucial functions in both mediating and regulating immune and inflammatory responses. This cytokine not only orchestrates humoral and cellular immune responses but also acts as a critical regulator of B cell, T cell, and macrophage functions in combating infections and malignant cellular processes (26). Current research indicates that IL-4, along with IL-13, functions as a prototype inducer of M2 macrophages, an alternatively activated macrophage phenotype that has been demonstrated to facilitate HCC progression (26). IL-9 is a pleiotropic cytokine capable of modulating diverse cellular functions. Emerging evidence highlights the dual role of IL-9 in tumorigenesis, establishing it as a molecular double-edged sword in cancer biology. This cytokine exhibits context-dependent immunomodulatory effects in tumor immunity: It functions as a lymphocyte growth factor that promotes progression in hematological malignancies, while in certain solid tumors, it exhibits tumor-suppressive activity by activating both innate and adaptive immune responses. Notably, these antitumor effects are not universal, as IL-9 has been shown to facilitate tumor progression in some solid malignancies (27). Despite these advances, the diagnostic potential of IL-9 in early-stage HCC remains largely unexplored. Consequently, further investigation is required to fully elucidate the mechanistic role of IL-9 in HCC pathogenesis and its broader implications in tumor immunology. The potential utility of these cytokines as predictive biomarkers for HCC development warrants further comprehensive investigation.

While the findings of the present study highlight the diagnostic potential of the IFN-γ/IL-22 cytokine panel, several limitations should be acknowledged. On the one hand, the study cohort was derived from a single center, potentially limiting the generalizability of the results across diverse ethnic populations and etiologies of chronic liver disease. On the other hand, the mechanistic insights into the protective role of IFN-γ and the tumor-promoting effects of IL-22 remain observational. Further in vitro and in vivo functional studies are needed to elucidate the underlying signaling pathways. To address these limitations, multi-center validation in prospective, longitudinal cohorts is needed to confirm the reproducibility of the panel across different clinical settings. Mechanistic investigations using knockout models or cytokine-neutralizing antibodies are required to establish causality between IFN-γ/IL-22 and HCC pathogenesis. Future studies should also explore dynamic changes in cytokine levels during therapeutic interventions (such as immunotherapy or resection), which may further refine their utility as predictive biomarkers. Furthermore, immune checkpoint inhibitors show stage-specific variations in therapeutic efficacy across different liver disease phases. Through systematic analysis of immune checkpoint protein and cytokine expression profiles at various stages of HCC development, a distinct predictive molecular signature could be established. This approach holds significant promise for achieving early and accurate HCC diagnosis, potentially revolutionizing current diagnostic paradigms.

In conclusion, the findings of the present study demonstrated that IFN-γ acts as a protective factor against HCC progression, while IL-22 serves as a significant risk factor for HCC development. Notably, the combination of IFN-γ and IL-22 exhibited superior diagnostic accuracy compared with conventional AFP testing for detecting HCC. These results suggest that the combination of IFN-γ and IL-22 holds substantial promise as a novel diagnostic tool for HCC, potentially improving early detection and clinical outcomes.