Prognosticandpredictivevalueofphospho-c-Junexpressionin hepatocellular carcinoma patients treated with sorafenib

Authors

Abstract

AimHepatocellular carcinoma (HCC) is a highly lethal malignancy with limited treatment options at advanced stages. Sorafenib, a multikinase inhibitor, remains a key systemic therapeutic agent; however, treatment response varies significantly among patients. This study aimed to investigate phospho-c-Jun (PCJ) expression as a potential biomarker for sorafenib resistance and its association with survival outcomes in HCC patients.
Materials and Methods This retrospective study analyzed 32 HCC patients who received first-line sorafenib therapy between September 2020 and January 2021. Immunohistochemical analysis was performed to determine nuclear phospho-c-Jun expression levels. Patients were categorized based on H-score (≥87.5 vs <87.5) and staining intensity. Survival outcomes were analyzed using Kaplan-Meier curves and Cox regression analysis.
Results Patients with high PCJ expression (≥87.5 H-score) had significantly shorter median overall survival (5 months) compared to those with low PCJ expression (18 months, p=0.013). Similarly, patients with high staining intensity (+3) had worse prognosis (3.25 months) compared to those with low staining intensity (17.6 months, p=0.030). Univariate analysis confirmed the prognostic significance of PCJ expression; however, this significance was lost in multivariate analysis, likely due to limited sample size.
Discussion Elevated PCJ expression is associated with poor response to sorafenib and reduced survival in HCC patients. These findings suggest that PCJ expression could serve as a predictive biomarker for sorafenib response. Further validation in larger cohorts is necessary; however, targeting the c-Jun N-terminal kinase pathway may be a potential therapeutic strategy to overcome sorafenib resistance. This study highlights the importance of molecular profiling in HCC.

Keywords

Introduction

Hepatocellular carcinoma (HCC) is a common cancer with a high mortality rate and increasing prevalence. According to the current GLOBOCAN data, it is the 6th most frequently diagnosed cancer type in the world and the 3rd cancer type with the highest mortality rate [1]. HCC is the most common primary liver cancer, and the vast majority of cases occur in patients with chronic hepatitis or chronic liver disease, regardless of etiology [2]. Although treatment is determined by the disease stage, patients often lose the chance to receive curative treatment because they are at an advanced stage when diagnosed. The 5-year survival rate for advanced HCC is <10%; however, when the liver mass is ≤2 cm, which corresponds to the ideal stage for treatment, the 5-year survival rate reaches 100% [3].
Systemic therapies play a central role in advanced stages of HCC. Among these, the use of chemotherapy declined following the demonstrated efficacy of sorafenib. This pivotal development marked a turning point in the treatment of advanced-stage disease. Sorafenib, a multikinase inhibitor, effectively targets vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and stem cell growth factor receptor (c-Kit). Inhibition of approximately 40 kinases, including the serine/threonine protein kinases BRAF, RAF1, and KIT, suppresses the proliferation and angiogenesis of HCC cells [4]. Although the efficacy of sorafenib has been well documented, some patients experience limited therapeutic benefits and develop adverse effects such as weight loss, diarrhea, hand-foot syndrome, hypertension, and abdominal pain. Although candidate biomarkers, such as basal extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and VEGF-A, have been proposed to predict sorafenib resistance, no reliable biomarker has been identified to accurately determine which patients will benefit from treatment or to what extent [5, 6]. Several hypotheses have been proposed to explain this resistance, primarily based on the mechanisms underlying the development of HCC.
The activation of signaling pathways that drive malignant transformation of hepatocytes is primarily attributed to the effects of chronic inflammation, which disrupts intracellular signaling networks and alters the molecular microenvironment of the liver [7]. Chronic inflammation is believed to upregulate the synthesis of anti-apoptotic molecules, thereby promoting the activation of mitogen-activated protein kinases (MAPKs), which play a pivotal role in HCC progression [8]. These protein kinases include ERK, JNK, and p38 [9]. Notably, studies have demonstrated that the ERK pathway is frequently upregulated in patients with HCC, and therapeutic inhibition of this pathway with sorafenib has been shown to confer clinical benefits, further supporting the critical role of ERK signaling in HCC pathogenesis [10]. Upon activation of the JNK signaling pathway, a variety of transcription factors involved in the regulation of apoptosis, oncogenesis, inflammation, and cellular proliferation are phosphorylated and subsequently activated [9]. Notably, preclinical animal studies have demonstrated that the transcription factors c-Jun and c-myc are overexpressed in HCC and that inhibition of the JNK pathway effectively suppresses tumor cell proliferation [11]. C-Jun is an intermediate component of the JNK signaling pathway. Phosphorylation takes on the phospho-c-Jun (PCJ) form, which helps to sustain the active state of the JNK pathway [12]. Notably, sorafenib does not directly target the JNK pathway, raising the possibility that its therapeutic efficacy may be reduced in HCC patients with hyperactivation of this signaling cascade. Therefore, our study aimed to investigate the potential relationship between sorafenib resistance and JNK pathway activation with the objective of identifying novel predictive biomarkers for treatment response.

Materials and Methods

Forty patients who were diagnosed with HCC at our tertiary referral hospital between September 2020 and January 2021 and who were administered sorafenib as first-line treatment for metastatic or unresectable disease were retrospectively analyzed. 32 patients for whom complete and pathological data were available were included in the study.
Pathological Evaluation
Histopathological re-evaluation was performed by two expert pathologists using archived hematoxylin-eosin slides for histological confirmation. We then checked whether the paraffin blocks contained sufficient amounts of tissue. PCJ staining was performed using the streptavidin-biotin triple indirect immunoperoxidase method with an automatic immunohistochemical device (Ventana XT, TX, US). Anti- phospho-c-Jun (Ser63) II antibody (rabbit monoclonal, 1:100 dilution, Cell Signaling Technology, MA, US) was used to investigate the presence of PCJ expression in tumor cells. Its expression was analyzed by measuring the staining intensity and prevalence (H-score). Nuclear PCJ expression in tumor cells was graded according to the staining intensity as negative (0), mild (+1), moderate (+2), or strong (+3). The prevalence of staining was calculated by multiplying the percentage of area corresponding to each score (Figure 1).
The sum of these prevalence rates corresponds to the criterion of 300. Breast cancer tissues were used as positive controls.
Statistical Analysis
All data were entered into SPSS 22.0 for statistical analysis. Descriptive statistics were used to summarize the demographic and clinical characteristics of the patients. Continuous variables are presented as mean ± standard deviation or median (interquartile range), depending on the data distribution, while categorical variables are expressed as frequencies and percentages. The Shapiro-Wilk W test was used to assess the normality of the data. One-way ANOVA was conducted to compare the numerical variables. A 95% confidence interval was used, and a p-value ≤0.05 was considered statistically significant.
Ethical Approval
This study was approved by the Ethics Committee of Gazi University (Date: 2020-09-07, No: 541).
Patient Consent
This study involved a retrospective review of previously gathered (archived) records. As the study design did not require direct patient interaction, the requirement for informed consent was waived by the Local Ethics Committee. All information was de-identified to protect privacy. This study was conducted in accordance with the institutional ethical standards and followed the guidelines of the Declaration of Helsinki and its updates.

Results

Of the 32 patients included in the study, 3 (9.4%) were female and 29 (90.6%) were male. All the patients were diagnosed with HCC after cirrhosis. Demographic data and patient characteristics are shown in Table 1.
Sorafenib treatment was started at 400 mg in two patients and increased to 800 mg, while it was started at 800 mg in 30 patients. Local treatment was administered in addition to sorafenib to 23 patients. The drug dose was reduced from 800 mg to 600 mg in only one patient (3.1%) due to side effects. Adverse effects were observed in 15 (46.9%) patients after sorafenib initiation. New hypertension developed in one (3.1%), diarrhea in one (3.1%), cytopenia in three (9.4%), and skin toxicity in 10 (31.3%) patients. The median overall survival (mOS) was 13 months (IQR 4.25-32), and the median progression-free survival (mPFS) was 7.5 months (IQR 3.75-26.75). Progression was observed in 31 patients (97%) receiving first-line sorafenib, whereas one patient showed no progression.
When the relationship between H-Score and mOS was examined, it was found that mOS was 17.6 months (IQR 0–35.9) in patients with PCJ staining intensity of 0/+1/+2; whereas it was 3.25 months (IQR 0–6.74) in patients with +3 (p=0.030) (Figure 2).
With a median H-score of 87.5, patients scoring below this threshold were classified as having low PCJ expression, whereas those with a score of ≥87.5 were classified as having high PCJ expression. Of the total patients, 16 (50%) exhibited low H-scores and 16 (50%) exhibited high H-scores. The mOS was 18 months (IQR 0–35.9) in the low PCJ staining group compared to five months (IQR 1.13–4.3) in the high group, showing a statistically significant difference (p=0.013) (Figure 3).
The univariate analyses of patient-related and disease-related parameters that may affect survival outcomes are shown in Table 2.
In the multivariate analysis, factors such as differentiation, PCJ H-score, and staining intensity, which were significant in the univariate analysis, were not associated with survival. The PCJ H-score had a p-value of 0.08 (HR: 4.5; 95% CI: 0.83–24.00), and staining intensity had a p-value of 0.68 (HR: 0.72; 95% CI: 0.15–3.41) (Table 3).

Discussion

Although combination therapies are now considered first-line treatment options for advanced HCC according to international guidelines, the potential use of sorafenib in advanced HCC should not be overlooked and remains a reasonable treatment option. Therefore, identifying biomarkers that can identify the patients most likely to benefit from sorafenib is a valuable endeavor. Our focus on PCJ as a biomarker is based on its role in cell survival pathways, which may influence sorafenib resistance [13]. In our study investigating the relationship between PCJ expression and efficacy of sorafenib treatment in patients with HCC, we identified results that replicated the demographic findings of previous studies. Our study group consisted mostly of male patients, with an average age of 65 years, all of whom had HCC secondary to cirrhosis. This aligns with the demographic trends observed in the global incidence of HCC, highlighting the higher prevalence of this disease in older males [14].
Laboratory parameters, such as albumin, INR, and total bilirubin, are important for calculating the Child-Pugh score, which is part of the BCLC (Barcelona Clinic Liver Cancer) staging system. However, in contrast to the literature, our study did not find a significant relationship between survival and laboratory values such as albumin, INR, total bilirubin, alpha-fetoprotein (AFP), hemoglobin, or platelet counts in HCC patients. We attributed this finding to the relatively preserved liver function in our cohort, as most patients had Child-Pugh class A (68.7%) or B7 (31.3%) [15]. In addition, some studies have shown that elevated AFP levels (>400) as well as hemoglobin and platelet counts have prognostic value [16, 17, 18]. We believe that our findings differ from those in the literature because most of our patients had normal or near-normal laboratory results and were in Child-Pugh class A, along with the limited size of our study group.
Adverse effects were observed in 46.9% of the patients, with dermatological reactions being the most common, occurring in 31.3% of the cases. The incidence of dermatological side effects in our study was lower than the rates reported for the sorafenib group in Asia-Pacific (81.9%) and SHARP (80%) studies. Other common side effects, such as diarrhea, skin lesions, new-onset hypertension, and cytopenia, were reported at much lower rates in our study, likely because of the smaller cohort size [19, 20]. In contrast, the association between tumor differentiation and survival outcomes is consistent with the established understanding that tumor biology significantly affects HCC prognosis and treatment response [21]. This underscores the importance of incorporating histological analysis into the clinical evaluation of HCC patients to facilitate the development of more tailored therapeutic strategies.
Notably, patients with diminished PCJ expression experienced a median overall survival of 18 months, compared to only 5 months for those with higher expression, indicating the potential of PCJ as a prognostic marker for sorafenib treatment in HCC. Our findings revealed that lower PCJ expression correlated with significantly improved survival outcomes in patients with HCC receiving sorafenib. This observation is consistent with recent studies suggesting that the JNK pathway, in which c-Jun is a key component, plays a pivotal role in cancer progression and response to therapy [22, 23].
The importance of our study lies in the fact that although there are publications on the JNK pathway and c-Jun in various cancer types, it is one of the few studies in the literature showing a relationship between this pathway and resistance to sorafenib treatment [24]. Additionally, data from a cell line study showed that inhibition of the c-Jun pathway, in addition to sorafenib treatment, enhances the effect of sorafenib [25]. While univariate analyses yielded promising results, the significance did not persist in multivariate analysis. We attribute this primarily to the low number of patients in our study and anticipate that studies with larger cohorts could sustain this significance.

Limitations

This study has several important limitations that should be acknowledged. The primary limitation is the small sample size of 32 patients, which may have contributed to the loss of statistical significance in multivariate analysis and limited our ability to perform meaningful subgroup analyses. The retrospective design introduces potential selection bias and limits the quality of data collection. Additionally, the homogeneous patient population, consisting predominantly of Child-Pugh class A patients with relatively preserved liver function, may limit the generalizability of our findings to patients with more advanced liver dysfunction. The limited diversity in HCC etiology within our cohort also restricts the broader applicability of our results. Furthermore, we were unable to perform dose modification analyses or detailed treatment response evaluations due to insufficient sample size.

Conclusion

Our study demonstrates that elevated phospho-c-Jun expression is associated with significantly shorter overall survival in HCC patients treated with sorafenib, suggesting its potential utility as a predictive biomarker for treatment response. These findings support the hypothesis that JNK pathway activation may contribute to sorafenib resistance and highlight the importance of molecular profiling in HCC management. While the clinical significance was evident in univariate analysis, larger prospective studies are needed to validate these findings and establish PCJ expression as a reliable biomarker for clinical decision-making. The identification of such biomarkers represents a crucial step toward personalized treatment approaches in HCC, potentially improving patient outcomes through better treatment selection strategies.

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