Introduction

The liver plays a crucial role in modulating the immune system, ensuring its protection via immunotolerance.1 The liver plays a significant role in eliminating microbial compounds and pathogens from the bloodstream, along with detecting and removing infectious organisms.2 The dysregulation of immunological networks is a crucial factor in the development and progression of chronic liver diseases and hepatocellular carcinoma (HCC).345

HCC is considered as one of the most prevalent malignant tumors and the third leading cause of cancer death.67 Hepatocellular carcinogenesis involves numerous genetic and epigenetic changes that are influenced by environmental factors. C-myc, cyclin D1, p53, p16, E-cadherin, and PTEN genes have been linked to hepatocarcinogenesis.8 The experimental research data indicated a correlation between HCC development and exposure to hepatotropic virus infection, aflatoxin, algal hepatotoxins in contaminated water, and alcohol abuse resulting in liver cirrhosis.9 The primary underlying cause of HCC is infection with the hepatitis B virus, especially in individuals with the aforementioned risk factors.10 Historically, patients with HCC have had a poor prognosis primarily due to late diagnosis and high rates of recurrence. The treatment options currently available have limited success rates, with approximately 50% of patients experiencing a recurrence within 3 years after surgery. The long-term survival rate is also quite low, ranging from 30 to 40% at 5 years postsurgery.11

Various factors contribute to the development of tumor antigen tolerance, including reduced recognition of cancerous cells, immune suppression, and chronic inflammation caused by either a viral infection or immune dysregulation, ultimately contributing to the formation of cancer.1213 Recent studies have shown that an imbalanced immune system, characterized by alterations in immune cell count or activity, cytokine levels, and the presence of inhibitory receptors or their ligands, plays a significant role in the progression of HCC.14 The immune response can be shifted toward tumor tolerance due to changes in the function or expression of immune components, leading to the progression of the tumor. Various immune cells, including cytotoxic T cells, CD4+ T cells, regulatory T cells (Tregs), myeloid-derived suppressor cells, natural killer cells, and their interactions, have been found to play a role in the progression of HCC.15 The interactions are sustained by proinflammatory cytokines and anti-inflammatory cytokines.10

Interleukins (ILs), also known as cytokines, regulate inflammatory and immune responses by activating and regulating immune cells and participating in an inflammatory cascade. Moreover, chemokines, the largest member of the cytokine family, may serve pivotal roles in inducing organ-specific metastasis.161718 After the establishment of HCC, various cytokines are released by the tumor itself, nearby nontumor cells, or immune cells, acting on the malignant lesion and supporting tumor survival through multiple mechanisms.1920

The impact of chemokines and their receptors on HCC varies, encompassing both the progression and inhibition of tumor growth. Cytokines control the recruitment of white blood cells and have a vital function in various processes, including angiogenesis, Th1/Th2 development, inflammatory diseases, and tumor growth, having a strong association with HCC and correlating with distant organs and lymph nodes metastasis.1 The dysregulation of proinflammatory cytokines in the tumor microenvironment can impact metastasis by suppressing the immune system's ability to fight against cancer cells and inhibiting the activity of metastasis suppressors.21 Furthermore, these cytokines have the ability to influence the tumor microenvironment, leading to immune evasion and metastasis,22 making it necessary to identify new targets for the early detection and treatment of the disease.2324

This systematic review and meta-analysis aim to comprehensively evaluate existing research studies focusing on intricate connections between immune dysregulation, biomarkers, and cytokine signaling in HCC metastasis, offering valuable insights for advancing clinical strategies and improving outcomes in patients. A deeper understanding of the dysregulation of proinflammatory cytokines and their impact on immune evasion and metastasis may help identify new therapeutic strategies.

Methodology

Data Extraction

Literature screening was performed based on predefined inclusion and exclusion criteria. Data from each study investigating the HCC metastasis was extracted. Notably, the data extracted from each study included first author name and publication year, patient characteristics, sample sizes (n), event (patients reporting occurrence of metastasis), and nonevent (patients that did not report occurrence of metastasis). Moreover, the tumor size was also considered to evaluate the metastasis risk. Each dysregulated gene was further categorized into their respective families. The data also included findings of each gene to understand its potential in immune dysregulation resulting in HCC metastasis. This study aimed to gather a list of biomarkers responsible for immune dysregulation, further leading to HCC metastasis. A comprehensive range of evidence was considered in this meta-analysis.

Statistical Analysis

The “metabin” function of the “meta” library in R was used to conduct the meta-analysis. The function requires the number of patients reporting metastasis as the event group, the patients that did not report any metastasis as the nonevent group, and the sample sizes of the groups. Statistical analysis was conducted to obtain insights into the combined study outcomes. This involved calculating effect sizes, confidence intervals (CIs), weight percentages, and measures of heterogeneity (such as I² and τ²). A statistical significance assessment was performed with a significance threshold set at p < 0.05. Moreover, the publication bias was assessed using Egger's test.

Results

Study Characteristics

Four sequential steps were followed for literature screening: identification, screening, eligibility, and final inclusion, illustrated in Fig. 1. After a thorough screening of 64 papers and the removal of duplicate studies, a total of 10 which met the criteria were selected. The study aimed to investigate the biomarkers that lead to immune dysregulation, eventually resulting in HCC metastasis. Further analyses were performed on these studies.

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Fig. 1: Systematic flow diagram of literature screening for meta-analysis.

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The final studies ranged from 2009 onwards. This meta-analysis included a total of 1,008 patients (850 males and 158 females), comprising 357 patients in the event group and 651 patients in the nonevent group. Moreover, each study classified patients based on an average age of 58 years, creating two distinct groups: one comprising individuals with an average age greater than 58 years and the other with an average age less than 58 years. However, the studies conducted by Yang et al and Zhang et al lacked a clear division of patients into age groups, reporting an average age of 52 and 56 years for both groups, respectively. The clinical data of the patients is represented in Table 1. Furthermore, the biomarkers were considered in each study. These genes were then divided into their respective family categories. Notably, six genes were from the chemokine family, two were from the cytokines family, and the remaining two were from the deubiquitinating enzyme (DUB) and antigen family, respectively. The baseline patient characteristics, such as age and gender, are depicted in Fig. 2.

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Fig. 2: Baseline patient characteristics across studies. (A) Age distribution ratio across studies. (B) Gender distribution ratio across studies.

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Table 1

Clinical patient characteristics across studies

Study Id	Sample size (n)		Sex		Age (y)		References
	Event	Nonevent	Male	Female	≤ 58	> 58
Li et al (2012)25	28	53	64	17	46	35	(25)
Li et al (2015)26	32	70	87	15	49	53	(26)
Xue et al (2013)27	24	38	56	6	47	15	(27)
Li et al (2017)28	40	38	67	11	54	24	(28)
Lan et al (2014)29	22	23	31	14	30	15	(29)
Yang et al (2012)30	69	219	254	34	52		(30)
Li et al (2023)31	15	70	70	15	54	31	(31)
Kang et al (2018)32	20	25	39	6	20	25	(32)
Gao et al (2020)33	52	28	63	17	35	45	(33)
Zhang et al (2009)34	55	87	119	23	56		(34)

Immune Biomarkers across Studies

A comprehensive literature search was performed to enlist the biomarkers responsible for immune dysregulation causing HCC metastasis. This meta-analysis encompasses a total of 10 genes from various families, including two cytokine genes (IL23 and IL17A), six chemokine genes (CXCL8, CXCR7, CXCL13, CXCL9, CCL22, and IP10), one DUB gene (USP13), and one antigen gene (MAGE1). The distribution ratio of these genes into their respective families across the studies is listed in Table 2 and illustrated in Fig. 3.

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Fig. 3: Distribution ratio of genes into their respective families.

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Table 2

Distribution of genes and their respective families across studies

Study Id	Genes	Family
Li et al (2012)	IL23	Cytokine
Yunzhi Dang (2020)	CXCL8	Chemokine
Xue et al (2013)	CXCR7	Chemokine
Li et al (2017)	CXCL13	Chemokine
Lan et al (2014)	CXCL9	Chemokine
Yang et al (2012)	CCL22	Chemokine
Li et al (2023)	IP10	Chemokine
Kang et al (2018)	IL17A	Cytokine
Gao et al (2020)	USP13	Deubiquitinating enzyme (DUB)
Zhang et al (2009)	MAGE1	Antigen

Tumor Size across Studies

The distribution of tumor sizes categorized as 5 cm or less and greater than 5 cm was reported across studies. In this meta-analysis, a total of 617 cases of tumor size data was extracted, where 330 were reported to be less or equal to 5 cm and 287 greater than 5 cm. Four studies by Yunzhi Dang, Li et al, Kang et al, and Zhang et al reported cases with tumor sizes less than 5 cm.57 Conversely, five studies by Li et al, Xue et al, Li et al, Lan et al, and Gao et al reported higher cases of tumor sizes greater than 5 cm. Notably, the study conducted by Yang et al did not provide any specific information regarding tumor sizes. A comprehensive overview of the studies, accompanied by the corresponding patient cases and reported tumor sizes, is represented in Table 3. A quantitative analysis was conducted to compare the reported tumor sizes among patient cases. The study by Zhang et al was excluded from the analysis due to its aberrant data, leading to a significantly higher number of cases with tumor sizes greater than 5 cm. The comparative analysis of the remaining studies is illustrated in Fig. 4.

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Fig. 4: Comparative analysis of reported patient cases based on tumor sizes (5 cm).

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Table 3

Reported patient cases based on tumor sizes (5 cm) across studies

Study Id	Tumor data
	Tumor size (cm)
	≤ 5	> 5
Li et al (2012)	34	46
Yunzhi Dang (2020)	27	11
Xue et al (2013)	6	18
Li et al (2017)	34	44
Lan et al (2014)	13	32
Yang et al (2012)	NA	NA
Li et al (2023)	50	35
Kang et al (2018)	27	18
Gao et al (2020)	26	54
Zhang et al (2009)	113	29

Abbreviation: NA, not available.

Quantitative Synthesis (Meta-Analysis)

Role of Dysregulated Immune Biomarkers in Hepatocellular Carcinoma Metastasis

All 10 studies reported the biomarkers responsible for immune dysregulation causing the occurrence of HCC metastasis in the event group. The data set representing 357 number of events from 1,008 total samples, along with 651 nonevents and their respective outcome ratios, are represented in Table 4. The patient cases reporting metastasis were considered as events within the total sample size. Only studies by Li et al and Gao et al reported a higher number of events while the remaining reported higher nonevents. The meta-analysis results showed significant heterogeneity in the role of biomarkers in immune dysregulation causing HCC metastasis (risk ratio = 0.54, 95% CI: 0.4972, 0.6048, I² = 92.4%, p < 0.01). The publication bias analysis reported insignificant results (test result: t = 0.90, degrees of freedom = 8, p-value = 0.39). The meta-analysis results are depicted in Fig. 5.

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Fig. 5: Meta-analysis results. (A) Forest plot analysis. (B) Publication bias via funnel plot.

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Table 4

Event outcome analysis across studies

Study Id	Event	Nonevent	Total	Outcome
Li et al (2012)	28	53	81	Yes
Yunzhi Dang (2020)	32	70	102	Yes
Xue et al (2013)	24	38	62	Yes
Li et al (2017)	40	38	78	Yes
Lan et al (2014)	22	23	45	Yes
Yang et al (2012)	69	219	288	Yes
Li et al (2023)	15	70	85	Yes
Kang et al (2018)	20	25	45	Yes
Gao et al (2020)	52	28	80	Yes
Zhang et al (2009)	55	87	142	Yes

Discussion

HCC is a common lethal cancer globally, particularly in areas with a high prevalence of chronic viral hepatitis infection, notably hepatitis B infection, causing both intrahepatic and extrahepatic metastases. HCC often metastases to the lungs, lymph nodes, adrenal gland, and bones, including the skull.3536 Factors such as immune dysregulation lead to carcinogenesis.13 The functions of diverse immune cells undergo dysregulation as the disease progresses from liver cirrhosis to HCC. This dysregulation manifests as a decreased secretion of Th1 cytokines, leading to a reduction in the antigen presentation capabilities of dendritic cells, playing a crucial role in the development of HCC.15 Alterations in immune function or expression can cause the immune response to shifting toward tumor tolerance, leading to the progression of the tumor.1437

Chemokines, a family of small cytokines, are crucial in coordinating immune cell trafficking and shaping the immune profile of tumors. They influence the migration patterns of immune cells into tumors, often favoring a protumorigenic state, directly impacting the nonimmune cells within the tumor microenvironment, including cancer cells, stromal cells, and vascular endothelial cells. Given their integral role in the tumor immune response and tumor biology, the chemokine network has emerged as a potential target for immunotherapy. Hence, in this study, 10 genes, particularly those encoding cytokines (IL23 and IL17A) and chemokines (CXCL8, CXCR7, CXCL13, CXCL9, and CCL22), as well as DUB (USP13) and antigen (MAGE1), have been identified as implicated in dysregulation, influencing immune cell behavior within the tumor microenvironment.

The aforementioned genes are intimately linked to both immune dysregulation and the metastatic progression of HCC. Notably, the dysregulation of IL23 and IL17A cytokines has been observed to actively promote tumorigenesis.3839 Additionally, in a murine HCC model generated using a specific HCC cell line, the overexpression of IL23 resulted in enhanced growth of HCC, demonstrating a dependence on IL17 for this effect,40 underscoring the intricate relationship between these cytokines and their consequential impact on HCC progression, as evident in the experimental mouse model.41

Moreover, the chemokine family genes, including CXCL8, CXCR7, CXCL13, CXCL9, and CCL22, activate the signaling pathways for the development of tumor diseases.42 The CXCL family not only regulates leukocyte migration but also controls tumor growth, immune cell activity, tumor cell proliferation, invasion, neoplastic microvascular formation, and tumor cell transformation, altering the angiogenic environment and promoting local tumor cell growth and metastasizes to distant organs. The role of the CXCL family is closely related to HCC.43 The upregulation of CXCL8 and CXCL9 promoted tumor cell proliferation, migration, and metastasis through the interaction between HCC cells and macrophages. Moreover, these genes are also considered potential biomarkers of chronic hepatitis B.4445

The CXCL13, identified as a B-cell chemoattractant, plays fundamental roles in inflammatory, infectious, and immune responses.46 While the CXCL13 gene has been previously associated with prostate cancer cell proliferation and abnormal signaling activation, recent findings suggest its potential involvement in the development and progression of HCC due to higher serum levels.284748 Furthermore, the study by Zheng et al reported that inhibition of CXCR7, a chemokine receptor, decreases angiogenesis and tumor growth in the murine HCC model.49 It is noteworthy that CCL22 plays a role in recruiting Treg cells, and heightened levels of this chemokine correlate with poorer survival outcomes in individuals with HCC.50

Lastly, the USP13, a deubiquitination enzyme, is involved in various cellular processes like mitochondrial energy metabolism, autophagy, deoxyribonucleic acid damage response, and endoplasmic reticulum-associated degradation,51 whereas MAGE1, with strong tumor antigen properties, holds significance in immune responses against cancer cells. Thus, the dysregulation of USP13 and MAGE1 is reported in many malignant tumors, including HCC.5253 The reported dysregulation of USP13 and MAGE1, encompassing their involvement in diverse cellular processes and immune responses, underlines their potential significance in the metastasis of HCC. Hence, in this meta-analysis, 10 studies consisting of the aforementioned genes played a crucial role as biomarkers in immune dysregulation within the tumor microenvironment, causing HCC metastasis. All studies, except Li et al and Gao et al, reported higher cases of metastasis from the sample size. A significant heterogeneity was observed among studies with insignificant publication bias.

Additionally, a quantitative analysis of tumor parameter (tumor size 5 and > 5 cm) data was also extracted across studies. Tumor size is an independent predictor of HCC progression, metastases formation, and overall survival. Tumor size exhibits a direct correlation with the likelihood of metastasis. As the size of the tumor increases, the probability of metastatic spread also increases.545556 Four studies reported a higher incidence of cases for tumors measuring 5 cm or less, whereas five studies reported larger tumor sizes, indicating a difference of 41 more cases in the category of larger tumor sizes. Only one study by Yang et al did not provide information on tumor size. Despite some studies focusing on tumor sizes of 5 cm or less, it is noteworthy that all studies across both categories consistently reported cases of metastasis.

This pooled analysis of biomarkers in immune dysregulation within the tumor microenvironment has highlighted the intricate interplay between various cytokines, chemokines, and regulatory elements, emphasizing their crucial roles in HCC metastasis. Additionally, the quantitative analysis of tumor size reaffirms it as an independent predictor of HCC progression and metastasis. The correlation between larger tumor sizes and an increased likelihood of metastatic spread, as evidenced by the observed difference in cases across studies, highlights the clinical relevance of tumor size as a prognostic factor.

However, it is important to acknowledge certain limitations within the meta-analysis. A notable constraint lies in the variability among studies regarding the clear division of patient cases into HCC metastasis events or nonevents. Furthermore, the absence of a comprehensive understanding of the specific pathways and interactions through which these dysregulated genes directly contribute to metastasis underscores a current limitation in the studies. Future studies with a more focused exploration of the functional implications of these genes in the metastatic process will be crucial for unraveling the underlying biology and advancing targeted therapeutic strategies for HCC. Despite these limitations, this meta-analysis provides valuable insights into the complex relationship between biomarkers, tumor size, and HCC metastasis, emphasizing the need for standardized reporting practices and comprehensive data in future studies.

Conclusion

In conclusion, the comprehensive meta-analysis of the 10 selected studies sheds light on the intricate relationship underlying immune dysregulation and metastatic progression in HCC. The identified genes, encompassing cytokines (IL23 and IL17A), chemokines (CXCL8, CXCR7, CXCL13, CXCL9, and CCL22), as well as DUB (USP13) and antigen (MAGE1), have emerged as crucial players in influencing immune cell behavior within the tumor microenvironment. These biomarkers exhibit significant associations with both immune dysregulation and HCC metastasis. The heterogeneity observed among the studies, along with the absence of publication bias, underscores the robustness and reliability of the meta-analysis results. Moreover, the quantitative analysis of tumor parameters, specifically tumor size, reaffirms its role as an independent predictor of HCC progression and metastasis. Although this meta-analysis findings underscore the importance of these biomarkers as potential targets for therapeutic interventions, the limitations should be considered for comprehensive evaluation and validation of the results.