Clinical significance of citrullinated glial fibrillary acidic protein in predicting outcomes in hepatocellular carcinoma

Abstract

BACKGROUND

Citrullination is a post-translational modification mediated by calcium-dependent peptidylarginine deiminases that results in notable changes in protein structure and function. Glial fibrillary acidic protein (GFAP), which is highly vulnerable to peptidylarginine deiminases-mediated modification, has been found to be elevated in activated hepatic stellate cells, with GFAP-positive hepatic stellate cells and myofibroblasts accumulating within and around areas of hepatic fibrosis. Although recent studies have shown that the expression of citrullinated GFAP (cit-GFAP) increases during hepatic fibrosis, its expression pattern and functional roles in hepatocellular carcinoma (HCC) remain unclear.

AIM

To determine whether cit-GFAP expression influences the recurrence and survival of patients undergoing hepatic resection for HCC.

METHODS

We retrospectively analyzed 169 patients with HCC who underwent hepatic resection. Based on the immunohistochemical staining of resected specimens, the enrolled patients were stratified into two groups according to cit-GFAP expression: Low (-/1 +) or high (2 +/3 +) levels of expression. Kaplan-Meier survival curves were constructed to assess overall survival and recurrence-free survival, and comparisons between groups were performed using the log-rank test.

RESULTS

The median follow-up duration was 33 months (range, 1-183). High cit-GFAP expression, identified in 81 patients (48.2%), was significantly associated with male sex, hepatitis B virus positivity, and higher Edmonson-Steiner grade. No associations were found between age, diabetes, hypertension, cirrhosis, Child-Pugh classification, major portal vein invasion, hematological or biochemical parameters, tumor size, or number. Patients exhibiting high cit-GFAP expression demonstrated significantly poorer overall survival. Multivariate Cox analysis identified large tumor size (hazard ratio: 2.967; 95% confidence interval: 1.097-8.024; P = 0.032) and high cit-GFAP expression (hazard ratio: 2.753; 95% confidence interval: 1.015-7.464; P = 0.047) as independent predictors of poor postoperative survival. Although recurrence rates were high in patients with high cit-GFAP expression, the difference was not statistically significant.

CONCLUSION

Following curative resection in patients with HCC, high cit-GFAP expression may serve as a potential prognostic biomarker, although further validation through independent cohort studies is warranted.

Key Words: Citrulllination; Glial fibrillary acidic protein; Hepatocellular carcinoma; Biomarker; Survival; Hepatic resection

Core Tip: High citrullinated-glial fibrillary acidic protein (cit-GFAP) expression is significantly associated with increased mortality in patients with hepatocellular carcinoma following surgical resection. Multivariate Cox regression analysis identified larger tumor size and elevated cit-GFAP expression as independent predictors of poor postoperative survival. These findings suggest the potential prognostic value of cit-GFAP levels, although further validation through independent cohort studies is necessary to confirm its clinical utility as a biomarker.

INTRODUCTION

Liver cancer is the sixth most prevalent malignancy and the fourth leading cause of cancer-related mortality worldwide[1]. Hepatocellular carcinoma (HCC) accounts for approximately 75% of liver cancer cases, with over 90% of affected individuals having underlying chronic liver disease, predominantly cirrhosis of diverse etiologies[1-3]. The management of HCC is challenging compared to that of other malignancies for several reasons. First, approximately 90% of patients have impaired liver function due to preexisting liver conditions, such as chronic hepatitis B or C, alcoholic hepatitis, metabolic dysfunction-associated steatotic liver disease, and cirrhosis[3]. Second, while approximately 30% of patients with HCC are identified at an early stage and are eligible for potentially curative interventions such as hepatic resection, liver transplantation, or local ablative procedures, recurrence rates remain high even more than 5 years post-treatment, despite median overall survival exceeding 60 months in these patients. Third, while significant strides have been made in systemic therapies, such as immune checkpoint inhibitors, tyrosine kinase inhibitors, and monoclonal antibodies, clinical outcomes for patients with HCC remain suboptimal[4,5]. Consequently, identifying novel molecular targets is critical for developing effective therapeutics and improving current treatment strategies.

Citrullination is a post-translational modification characterized by the enzymatic conversion of arginine residues into citrulline through calcium-dependent peptidylarginine deiminases (PADs), thereby neutralizing the positive charge and inducing notable changes in protein structure and function[6,7]. Glial fibrillary acidic protein (GFAP), a type III intermediate filament crucial for maintaining the cytoskeletal integrity of glial cells, particularly activated astrocytes, undergoes structural alterations via PAD-mediated citrullination[8,9]. Among PAD isoforms, PAD2 preferentially targets GFAP[10], and aberrant citrullinated GFAP (cit-GFAP) has been implicated in the pathology of neurodegenerative diseases, including Alzheimer’s disease[10,11] and multiple sclerosis[12]. Notably, GFAP expression is upregulated in activated hepatic stellate cells (HSCs), along with the accumulation of GFAP-positive HSCs and myofibroblasts in fibrotic liver tissue[13,14]. Recent findings indicated that GFAP expression increases during liver fibrosis progression and is accompanied by an increase in cit-GFAP levels[15]. Okamura et al[16] reported significantly higher numbers of GFAP-positive stromal cells in intrahepatic cholangiocarcinoma than in scirrhous HCC. Wu et al[17] demonstrated a sixfold increase in GFAP expression in HCC, consistent with tumor progression. Citrullination has recently been recognized as a crucial post-translational modification involved in various cellular processes and is linked to several pathological conditions, including cancer[18]. Recent studies have suggested that matrisome citrullination promotes hepatic metastasis of colorectal cancer[19], and Katayama et al’s study[20] further demonstrated its role in enhancing liver metastatic growth. Beyond HCC, aberrant citrullination of extracellular matrix (ECM) proteins has been implicated in other malignancies. In breast cancer, citrullinated fibronectin promotes tumor initiation and migration, while in colorectal cancer, PAD4-mediated citrullination of ECM enhances liver metastases[19,20]. These findings indicate the broader oncogenic role of citrullinated proteins and provide a translational context for exploring cit-GFAP expression in HCC. Given the known involvement of GFAP in HSC activation and the emerging role of citrullination in ECM remodeling and cancer metastasis, we hypothesized that cit-GFAP expression might have clinical relevance in HCC. Although cit-GFAP expression in HCC has not been extensively investigated, its involvement in liver fibrosis and cirrhosis, along with evidence linking citrullinated proteins to tumor development in other cancers, supports the hypothesis that cit-GFAP may play a role in HCC. Therefore, we aimed to evaluate cit-GFAP expression in HCC tissues and elucidate its clinical significance.

MATERIALS AND METHODS

Study population

This retrospective cohort study included 169 patients with HCC who underwent curative hepatic resection at the Hallym University Sacred Heart Hospital, South Korea, between January 2011 and December 2015. Patients were followed for a median of 33 months (range, 1-183 months) to monitor disease recurrence and overall survival. The eligibility criteria were as follows: (1) No prior treatment for HCC before surgical resection; (2) Histopathological confirmation of HCC in resected tissue; (3) Complete tumor resection with negative margins; (4) A single tumor < 10 cm in diameter; and (5) Up to three tumors, none exceeding 3 cm in diameter. Postoperative surveillance began 1 month after surgery and continued every 3 months. Routine assessments included complete blood count, liver function tests, prothrombin time, and serum alpha-fetoprotein (AFP) measurements. Imaging studies, including dynamic liver computed tomography and magnetic resonance imaging, were performed every 3 months to monitor recurrence. In the event of recurrence, patients received appropriate treatment, such as transarterial chemoembolization, radiofrequency ablation, systemic chemotherapy, or other indicated therapies. Survival data were collected from outpatient medical records, death certificates, or telephone interviews with patients or family members. Owing to the retrospective nature of the study and the use of anonymized data, the requirement for informed consent was waived. The study protocol was approved by the Institutional Review Board of Hallym University Sacred Heart Hospital (No. HALLYM202105009002-HE002).

Immunohistochemical staining and tissue microarray construction

Tissue microarrays were generated from paraffin-embedded HCC tissue blocks. Two 2-mm cores were extracted from representative tumor areas and embedded in recipient paraffin blocks. Sections of 4 μm thickness were cut from the tissue microarray blocks. Antigen retrieval was performed by microwaving the slides in citrate buffer (pH = 6.0) for 15 minutes. After cooling, the sections were treated with a peroxidase-blocking reagent (Agilent Dako, Copenhagen, Denmark) to inhibit endogenous peroxidase activity. Non-specific antibody binding was minimized using a protein-blocking solution (Agilent Dako, Copenhagen, Denmark), incubated for 60 minutes at room temperature. Slides were incubated overnight at 4 °C with a mouse monoclonal anti-cit-GFAP antibody (1:1000 dilution; Clone CTGF-1221). This antibody has been previously validated, demonstrating specificity in detecting cit-GFAP[15]. Immunoreactivity was detected using the Dako EnVision Detection System (Agilent Dako, Copenhagen, Denmark), according to the manufacturer’s protocol. Images were captured using a digital camera attached to a light microscope.

Evaluation of immunohistochemical staining

Nuclear staining for cit-GFAP was considered positive. Both the intensity and proportion of cit-GFAP-positive cells were assessed using a semi-quantitative four-point grading scale: Grade 0 (undetectable), grade 1 (weak), grade 2 (moderate), and grade 3 (strong). Light microscopy (BX51, Olympus, Tokyo, Japan) was used for evaluation (Figure 1). For clinical correlation, samples were classified as cit-GFAP-low (< 50%; grade 0 or 1) or cit-GFAP-high (> 50%; grade 2 or 3). Although the anti-cit-GFAP antibody has been previously validated[15], potential cross-reactivity with other citrullinated intermediate filament proteins cannot be completely excluded. This is a methodological limitation of this study. Two experienced pathologists (Cho YA and Choe JY) independently scored the samples. Discrepancies were resolved through joint re-evaluation and consensus.

Figure 1 Representative cases of citrullinated glial fibrillary acidic protein expression according to intensity. A: Weak staining (1 +); B: Moderate staining (2 +); C: Strong staining (3 +). Nuclear localization of citrullinated glial fibrillary acidic protein within tumor cells is clearly observed in all representative images. Original magnification × 100.

Statistical analysis

To clarify the prognostic value of cit-GFAP in HCC recurrence and overall survival, we established the following criteria: First, HCC recurrence was defined solely based on radiological confirmation. Second, overall survival analysis included only deaths from hepatic causes, excluding non-hepatic-related deaths. Using these definitions, multivariate analyses of recurrence and survival were performed using Cox proportional hazards or logistic regression models. Continuous variables are presented as means ± SDs or as medians with ranges, as appropriate. Group comparisons were performed using Student’s t-test for continuous variables and the χ² test for categorical variables. The Kaplan-Meier method was used to estimate the cumulative recurrence and survival rates, and differences were assessed using the log-rank test. Multivariate analyses were performed using the Cox proportional hazards model or logistic regression to identify the independent predictors of recurrence and survival. Statistical significance was defined as P < 0.05. All analyses were performed using SPSS software (version 27.0; SPSS Inc., Chicago, IL, United States).

RESULTS

Baseline characteristics and differences according to cit-GFAP expression

The baseline clinical and pathological characteristics of the 169 patients included in this study are summarized in Table 1 (mean age, 62.49 years; 81.1% patients were male). Hepatitis B surface antigen (HBsAg) positivity was observed in 68.5% of patients. Most patients had preserved liver function, with only 7.1% (12 patients) classified as Child-Pugh class B. The mean tumor number was 1.24 ± 0.612, and 122 patients (72.6%) harbored tumors smaller than 5 cm in maximum diameter. Of the 169 HCC tissue samples available for immunohistochemical analysis, 168 (99.4%) exhibited cit-GFAP staining, while one sample was negative. Cit-GFAP expression was predominantly localized in the nuclei of tumor cells, with minimal or no staining in the surrounding stromal or non-neoplastic hepatic cells. Among the positively stained samples, 86 were graded as 1 (weak expression), 46 as 2 (moderate expression), and 36 as 3 (strong expression). Based on staining intensity, 82 patients (48.3%) were categorized into the high cit-GFAP expression group (grade 2 or 3), whereas 87 patients (51.7%) were categorized into the low expression group (grade 0 or 1). Analysis of clinical characteristics showed that high cit-GFAP expression was significantly associated with male sex, older age, and HBsAg positivity (P < 0.05). Furthermore, a higher Edmonson-Steiner histological grade (grade 3 or 4) was significantly more frequent in the high cit-GFAP group (P = 0.038). However, cit-GFAP expression was not significantly associated with other clinical parameters, including diabetes mellitus, hypertension, liver cirrhosis, Child-Pugh class, tumor size, tumor number, perivascular invasion, complete blood count, prothrombin time, or AFP level (P > 0.05).

Table 1 Baseline characteristics according to citrullinated glial fibrillary acidic protein expression, mean ± SD/n (%).

Variables	Total (n = 169)	Low (n = 87)	High (n = 82)	P value
Age (years)	62.49 ± 12.15	63.03 ± 10.76	61.83 ± 13.47	0.520
Sex (male)	137 (81.1)	76 (87.4)	61 (74.3)	0.032
HBsAg-positive (+)	115 (68.5)	66 (75.9)	49 (60.5)	0.032
DM	53 (31.4)	28 (32.2)	25 (30.4)	0.812
HTN	78 (46.2)	40 (46.0)	38 (46.3)	0.962
LC	92 (54.4)	53 (60.9)	39 (47.5)	0.081
Child-Pugh class (B/A)	13/156	5/82	8/74	0.328
Tumor size (≥ 5 cm/< 5 cm)	47/122	24/63	23/59	0.947
Tumor number	1.24 ± 0.612	1.22 ± 0.53	1.26 ± 0.08	0.230
PVI	65 (38.7)	28 (32.2)	37 (45.7)	0.073
Edmonson-Steiner (1, 2/3, 4)	52/116	33/54	19/63	0.038
WBC	5800 (1100-24400)	5700 (1100-19600)	6742 (1700-24400)	0.271
Platelet	167 (22-401)	157 (22-401)	175 (34-401)	0.169
Serum ALT (IU/L)	28 (8-235)	28 (11-140)	28 (8-235)	0.276
Total bilirubin	0.64 (0.20-9.30)	0.64 (0.30-2.80)	0.65 (0.20-9.30)	0.662
Albumin	4.2 (2.3-5.5)	4.2 (2.7-5.5)	4.3 (2.3-5.2)	0.504
PT-INR	1.04 (0.87-1.63)	1.06 (0.88-1.63)	1.02 (0.87-1.56)	0.079
AFP > 400	29 (17.3)	14 (16.1)	15 (18.2)	0.705

HBsAg: Hepatitis B surface antigen; DM: Diabetes mellitus; HTN: Hypertension; LC: Liver cirrhosis; PVI: Perivascular invasion; WBC: White blood counts; ALT: Alanine aminotransferase; PT-INR: Prothrombin time and international normalized ratio; AFP: Alpha-fetoprotein.

Association between cit-GFAP expression and overall survival in patients with HCC

During a median follow-up of 32 months, 147 patients (86.9%) survived without tumor recurrence. Among patients with high cit-GFAP expression, 67 patients (81.7%) were alive at the end of follow-up, compared with 80 (91.9%) in the low-expression group, indicating a statistically significant difference (P = 0.048). Kaplan-Meier survival analysis further demonstrated that 1-year, 3-year, and 5-year cumulative overall survival rates were significantly lower in patients with high cit-GFAP expression than in those with low expression (91.9%, 82.2%, and 72.0% vs 97.7%, 93.9%, and 89.2%, respectively; P = 0.033) (Figure 2A).

Figure 2 Cumulative survival rates and recurrence rates. A: Cumulative survival rates were significantly higher in patients with hepatocellular carcinoma and low glial fibrillary acidic protein (GFAP) expression compared with those with high citrullinated GFAP (cit-GFAP) expression (P = 0.033). Patients with low cit-GFAP expression had 1-year, 3-year, and 5-year cumulative survival rates of 97.7%, 93.9%, and 89.2%, respectively. In contrast, the corresponding rates for patients with high cit-GFAP expression were 91%, 82.2%, and 72.0%, respectively; B: Cumulative recurrence rates were higher in patients with hepatocellular carcinoma and high cit-GFAP expression compared with those with low cit-GFAP expression. Patients with low cit-GFAP expression had 1-year, 3-year, and 5-year cumulative recurrence rates of 13.3%, 41.6%, and 56.1%, respectively. In contrast, the corresponding rates for patients with high cit-GFAP expression were 235%, 47.3%, and 73.2%, respectively (P = 0.125). cit-GFAP: Citrullinated glial fibrillary acidic protein.

Prognostic factors for overall survival in patients with HCC

Univariate Cox regression analysis identified larger tumor size (≥ 5 cm), elevated AFP levels (≥ 400 ng/mL), and high cit-GFAP expression as potential risk factors for reduced overall survival (P < 0.05). Subsequent multivariate analysis revealed that larger tumor size [hazard ratio (HR) = 2.967; 95% confidence interval (CI): 1.097-8.024; P = 0.032] and high cit-GFAP expression (HR = 2.753; 95%CI: 1.015-7.464; P = 0.047) were independent prognostic factors for poor survival after curative surgery (Table 2).

Table 2 Univariate and multivariate Cox regression analyses for mortality following surgical treatment in hepatocellular carcinoma patients.

Variables	Univariate analysis			Multivariate analysis
	HR	95%CI	P value	HR	95%CI	P value
Age (years)	1.021	0.981-1.062	0.312
Sex (male/female)	1.274	0.363-4.475	0.705
DM	0.871	0.308-2.461	0.794
HTN	0.869	0.349-2.164	0.762
HBsAg-positive (+)	0.494	0.199-1.221	0.127
LC	0.954	0.387-2.349	0.918
Child-Pugh class (B/A)	2.192	0.504-9.542	0.296
Tumor size (≥ 5 cm/< 5 cm)	3.323	1.315-8.398	0.011	2.967	1.097-8.024	0.032
Tumor number (single vs multiple)	2.270	0.882-5.842	0.089
Edmonson-Steiner (1, 2/3, 4)	2.318	0.759-7.083	0.140
Platelet	1.003	0.995-1.010	0.493
Serum ALT (IU/L)	1.010	0.995-1.025	0.208
AFP > 400	2.736	1.025-7.303	0.045	1.696	0.5691-4.863	0.326
cit-GFAP (high/Low)	2.707	1.006-7.290	0.049	2.753	1.015-7.464	0.047

DM: Diabetes mellitus; HTN: Hypertension; HBsAg: Hepatitis B surface antigen; LC: Liver cirrhosis; ALT: Alanine aminotransferase; AFP: Alpha-fetoprotein; cit-GFAP: Citrullinated glial fibrillary acidic protein; HR: Hazard ratio; CI: Confidence interval.

Association between cit-GFAP expression and recurrence in patients with HCC

Overall, 68 patients (40.2%) experienced recurrence during follow-up. Among them, 32 (39.0%) were in the high cit-GFAP group and 36 (41.3%) were in the low cit-GFAP group. The recurrence rates did not differ significantly between groups (P = 0.875). Kaplan-Meier analysis indicated that patients with high cit-GFAP expression exhibited higher 1-year, 3-year, and 5-year cumulative recurrence rates compared with those with low expression (23.5%, 47.3%, and 73.2% vs 13.3%, 41.6%, and 56.1%, respectively); however, the difference was not statistically significant (P = 0.125) (Figure 2B). Patients in the high-expression group demonstrated a consistently higher recurrence rate across the follow-up period than did those in the low-expression group; however, the difference was not statistically significant.

Prognostic factors for recurrence in patients with HCC

In univariate analysis, male sex, tumor size ≥ 5 cm, and elevated serum alanine transaminase levels were significantly associated with recurrence (P < 0.05). Multivariate Cox regression analysis determined that only tumor size ≥ 5 cm remained an independent predictor of recurrence (HR = 2.378; 95%CI: 1.010-4.075; P = 0.048) (Table 3).

Table 3 Univariate and multivariate Cox regression analyses for recurrence following surgical treatment in hepatocellular carcinoma patients.

Variables	Univariate analysis			Multivariate analysis
	HR	95%CI	P value	HR	95%CI	P value
Age (years)	1.002	0.982-1.022	0.845
Sex (male/female)	0.932	0.487-1.787	0.833
DM	0.864	0.512-1.458	0.583
HTN	1.237	0.768-1.994	0.381
HBsAg-positive (+)	0.965	0.576-1.617	0.892
LC	0.959	0.592-1.552	0.863
Child-Pugh class (B/A)	2.196	0.947-5.090	0.067
Tumor size (≥ 5 cm/< 5 cm)	2.493	1.503-4.136	< 0.001	2.378	1.426-3.965	< 0.001
Tumor number (single vs multiple)	1.173	0.650-2.120	0.596
Edmonson-Steiner (1, 2/3, 4)	1.223	0.735-2.037	0.493
Platelet	0.999	0.996-1.003	0.646
Serum ALT (IU/L)	1.009	1.001-1.017	0.032	1.007	0.999-1.015	0.081
AFP > 400	1.643	0.824-2.793	0.181
cit-GFAP (high/Low)	1.456	0.895-2.369	0.130

DM: Diabetes mellitus; HTN: Hypertension; HBsAg: Hepatitis B surface antigen; LC: Liver cirrhosis; ALT: Alanine aminotransferase; AFP: Alpha-fetoprotein; cit-GFAP: Citrullinated glial fibrillary acidic protein; HR: Hazard ratio; CI: Confidence interval.

DISCUSSION

In this study, we confirmed the presence of cit-GFAP expression in liver tissues from patients with HCC who underwent surgical resection and found that its expression was closely correlated with survival outcomes. Except for one patient, cit-GFAP expression was detected in the liver tissues of all patients. Notably, survival rates varied significantly based on the degree of GFAP staining. Furthermore, although not statistically significant, a correlation was also observed between recurrence rates and cit-GFAP expression levels. This discrepancy between overall survival and recurrence-free survival is likely attributable to the study’s limited sample size and statistical power, rather than to a true biological dissociation. The consistent directional trend toward higher recurrence in the high-expression group suggests that cit-GFAP levels may influence recurrence. However, this needs to be validated through larger multicenter cohort studies. It is also possible that cit-GFAP partly impacts the overall survival through mechanisms, such as modulation of hepatic functional reserve or the immune microenvironment, beyond recurrence.

Most HCC cases arise in fibrotic livers. Activated HSCs function similarly to fibroblasts, synthesizing ECM proteins and playing a central role in the progression of liver fibrosis[21]. The tumor microenvironment in HCC involves a complex interplay between cancer cells and the surrounding ECM, along with various stromal cells and their secreted proteins. These components collectively drive the carcinogenic processes. Malignancy is not solely driven by tumor cells; rather, an aberrantly activated stroma supports and enhances their malignant potential[22]. Thus, the tumor microenvironment and its stromal components have emerged as promising therapeutic targets[22]. In HCC, fibroblasts play a pivotal role in shaping the tumor microenvironment by secreting ECM components and proinflammatory cytokines[22-24]. Increased expression of alpha-smooth muscle actin (α-SMA), a marker of HSC activation in tumor tissues, has been associated with unfavorable clinical outcomes in patients with HCC[25,26]. Myojin et al[23] revealed that HSCs secrete growth differentiation factor 15 via an autophagy-dependent mechanism in hepatoma cells, thereby promoting their proliferation and accelerating tumor progression. They also confirmed the presence of growth differentiation factor 15-producing HSCs in human HCC tissues, contributing to a tumor-supportive microenvironment[23].

GFAP is predominantly found in astrocytes and regulates ECM architecture and astrocyte-ECM interactions, especially in gliomas and central nervous system (CNS) damage[9]. Although GFAP is considered an astrocyte-specific intermediate filament, its distribution has been reported widely in non-glial and even non-CNS cells, such as chondrocytes, fibroblasts, myoepithelial cells[27], lymphocytes[28], and HSCs[13,14]. In the CNS, GFAP expression is essential for blood-brain barrier restoration following injury[29]. Given functional similarities between astrocytes and HSCs, GFAP expression in HSCs may contribute to vascular remodeling in injured liver tissue, where neovascularization increases markedly during liver fibrosis progression[30]. The early accumulation of GFAP/desmin-positive HSCs[13], their proliferation[31], and induction of ECM-related genes and proteins[32], which are key features of HSC activation, suggest that elevated GFAP is involved in the early activation and phenotypic transition of HSCs. Carotti et al[14] reported a positive association between GFAP-expressing HSCs and worsening fibrosis in patients with hepatitis C virus recurrence following liver transplantation. Additionally, they reported that GFAP immunostaining revealed a distinct HSC population more evident in early fibrosis, in contrast to α-SMA, supporting GFAP as an early activation marker. Their findings also linked GFAP expression to vascular remodeling, offering novel perspectives on the involvement of GFAP in HSC activation[14].

A growing body of evidence suggests that ECM proteins within tumor tissues undergo post-translational modifications through citrullination[19,33,34]. Fibronectin, a major ECM protein involved in cell adhesion, migration, and tissue architecture, is notably susceptible to citrullination under pathological conditions, which affects cellular signaling, matrix remodeling, and tumor cell behavior. In particular, citrullinated fibronectin promotes tumor initiation and progression, highlighting its potential role in the pathogenesis of malignancies[35]. Wang et al[34] demonstrated that fibronectin citrullination enhances proliferation, migration, colony formation, and reduces apoptosis in MDA-MB-231 breast cancer cells. Similarly, Yuzhalin et al[19] showed that citrullinated type I collagen facilitates metastatic spread of liver tumors, suggesting a broader role for citrullinated ECM proteins in cancer progression. Thus, citrullinated ECM components are crucial for promoting tumor cell growth and metastasis. GFAP, also considered an ECM component in HCC, is highly susceptible to citrullination[10] and is expressed in GFAP-positive stromal cells in HCC tissues[16], with one report noting a six-fold increase in its expression[17]. These findings imply that cit-GFAP is likely involved in HCC development, progression, and metastasis.

In our prior study, cit-GFAP colocalized with α-SMA-positive and cytokeratin 19-positive cells in bile duct-ligated mouse livers, indicating its localization within HSCs and cholangiocytes. Conversely, cit-GFAP expression was not observed in Kupffer cells or hepatic sinusoidal endothelial cells. Collectively, these findings suggest that upregulated GFAP expression and aberrant cit-GFAP accumulation are closely associated with HSC and portal fibroblast activation during hepatic fibrogenesis in the bile duct ligated model. Extending these findings to human diseases, the present study demonstrated that cit-GFAP expression is significantly associated with survival outcomes in patients with HCC who underwent surgical resection. Although cit-GFAP was detected in 99.4% of resected HCC tissues, survival outcomes varied markedly according to the intensity of cit-GFAP expression. These results imply that cit-GFAP contributes to hepatocarcinogenesis by modulating ECM remodeling processes, particularly through its effects on HSCs and portal fibroblasts. Although direct cell-level experiments were beyond the scope of this study, previous reports suggest that protein citrullination can remodel ECM architecture, alter integrin-mediated signaling, and shape immune recognition in the tumor microenvironment. Furthermore, HSCs, which express GFAP, are central players in fibrogenesis and tumor-stroma crosstalk. These mechanisms provide biologically plausible pathways linking cit-GFAP expression to HCC progression[36,37].

Importantly, to the best of our knowledge, this is the first study to implicate ECM citrullination - specifically cit-GFAP - as a prognostic factor in HCC, highlighting a novel pathophysiological mechanism. We identified significant associations between cit-GFAP expression and key clinicopathologic factors such as sex, HBsAg positivity, and high Edmonson-Steiner grade; in contrast, no significant associations were observed with baseline liver function, comorbidities, or tumor burden, suggesting a unique biological role independent of conventional risk factors. Although cit-GFAP expression was more frequently observed in male patients, sex was not associated with overall survival based on the multivariate analysis, suggesting that the observed sex difference with regard to its expression may not reflect a direct impact on patient prognosis. Age distribution was also similar between high and low cit-GFAP groups. Given the limited existing literature on cit-GFAP expression patterns across demographic subgroups, the underlying biological or clinical relevance of these associations remains unclear. Further research is warranted to determine whether age- or sex-related factors contribute to citrullinated protein accumulation or modify the prognostic implications of the underlying disease.

To enhance contextual relevance, we included AFP, a well-established HCC biomarker, in our multivariate survival analysis. In our study, cit-GFAP remained a statistically significant predictor of overall survival, whereas AFP did not, suggesting that cit-GFAP may have independent and potentially superior prognostic value. Other biomarkers, such as glypican-3 or protein induced by vitamin K absence or antagonist-II, were not assessed in this study. In addition, antiviral therapy use was not assessed, and liver fibrosis staging was not available, although 54.4% of patients had cirrhosis, and most had preserved liver function. The absence of an external validation cohort also limits the reproducibility and generalizability of our findings. While cit-GFAP expression showed a significant association with overall survival, the lack of statistical significance regarding recurrence-free survival may reflect insufficient statistical power rather than a true absence of effect. Further studies are needed to validate these findings in independent cohorts and to clarify the biological mechanisms underlying these associations. Although our current study focused on histological evaluation, future integration of transcriptomic or public gene expression data may help elucidate the molecular pathways underlying cit-GFAP expression and its prognostic relevance.

This study has some limitations. First, the sample size was relatively small and limited to patients who underwent surgical resection, introducing potential selection bias. Further, the relatively small sample size may have limited our ability to detect subtle demographic associations, such as age- or sex-related differences in cit-GFAP expression or its prognostic impact. Larger, adequately powered studies are required to clarify whether these demographic factors exert meaningful biological or clinical influence. Second, the retrospective design may have limited the strength of causal inferences. Third, mechanistic studies elucidating the functional effect of cit-GFAP on hepatocarcinogenesis have not been performed. Fourth, the 50% threshold used to dichotomize cit-GFAP expression levels was based on the distribution of immunohistochemical staining scores and practical interpretability, rather than biological or statistical validation. Although this cutoff improved the clarity of statistical analysis and clinical interpretation, we acknowledge its arbitrary nature and potential impact on generalizability. Fifth, although cit-GFAP expression was independently assessed by two board-certified pathologists, no formal interobserver agreement analysis (e.g., kappa statistic) was performed. Discrepant cases were resolved through consensus review, but the absence of a quantitative reproducibility assessment limits methodological robustness. The proportional hazards assumption for Cox models was not formally tested, which may affect the robustness of the survival analysis. However, no visual evidence of non-proportionality was noted in the Kaplan-Meier curves. Finally, intraoperative parameters such as operative time and blood loss were not uniformly available; however, as all resections were performed by a single hepatobiliary surgeon, variability in surgical technique was minimal. Future multicenter studies incorporating perioperative factors may further strengthen the generalizability of our findings.

To strengthen the clinical relevance of our findings, future studies should pursue both mechanistic and translational directions. First, mechanistic investigations are warranted to elucidate the role of cit-GFAP in ECM remodeling and tumor biology. Second, validation in prospective, multicenter, and etiologically diverse HCC cohorts is essential to confirm its prognostic value and generalizability. Third, comparative analyses incorporating established biomarkers such as AFP, glypican-3, and protein induced by vitamin K absence or antagonist-II will clarify the added utility of cit-GFAP in existing diagnostic frameworks. Finally, identifying optimal cutoff values for cit-GFAP expression through data-driven approaches - such as receiver operating characteristic curve analysis, classification algorithms, or machine learning models - should be a key objective of future research. Furthermore, a translational roadmap should include assay standardization to ensure reproducibility across centers, careful definition of patient populations most likely to benefit (e.g., patients with resectable and non-resectable HCC), and integration of cit-GFAP testing into clinical workflows. Liquid biopsy approaches may further expand the applicability of cit-GFAP to patients who are not surgical candidates, thereby enhancing its clinical utility. Collectively, these efforts will determine whether cit-GFAP can serve as a clinically useful biomarker and offer novel insights into the contribution of ECM citrullination to HCC progression.

CONCLUSION

Our study demonstrated that high cit-GFAP expression in HCC tissues is closely associated with poor postoperative survival outcomes. Thus, cit-GFAP may serve as a novel prognostic biomarker in patients with HCC undergoing curative hepatic resection. Further research is warranted to investigate the broader roles of citrullinated ECM proteins in HCC pathogenesis and explore their potential as therapeutic targets for improving clinical outcomes.