Preoperative serum glutathione reductase activity and alpha-fetoprotein level are associated with early postoperative recurrence of hepatocellular carcinoma

Introduction

Liver cancer is one of the most prevalent malignant tumors, with over 900,000 individuals diagnosed in 2020. It is characterized by rapid progression and high mortality rates [1], 2]. Hepatocellular carcinoma (HCC) stands as the most common form of liver cancer. Although surgical resection remains the most effective approach for early-stage HCC treatment, it is associated with a considerable risk of postoperative recurrence [3]. Early detection of HCC recurrence and prompt implementation of radical treatments such as surgery and radiofrequency ablation can significantly enhance patients’ survival time.

Studies have demonstrated that hepatitis virus infection, liver cirrhosis, alcoholism, and smoking may act as intermediaries in carcinogenesis by stimulating chronic inflammation and increasing oxidative stress. The redox system is closely associated with the occurrence and progression of liver cancer [4], [5], [6]. However, the relationship between changes in oxidative stress and antioxidant capacity and the recurrence of liver cancer remains incompletely investigated. Glutathione reductase (GR) is a pivotal enzyme that catalyzes the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH). GR can prevent oxidative damage caused by reactive oxygen species (ROS) accumulation. Additionally, GSH, as a reduction product, can directly bind to and detoxify electrophilic heterobiomass through glutathione S-transferase (GST) [7], 8]. We hypothesize that the preoperative antioxidant status may impact the prognosis of HCC. Alpha-fetoprotein (AFP), a gold standard biomarker for advanced HCC stages and poorly differentiated tumors, plays a crucial role in HCC development and progression [9]. Continuous elevation of AFP levels is strongly correlated with HCC recurrence [10]. A study involving 852 patients who underwent HCC resection revealed that AFP levels at postoperative recurrence could predict survival after recurrence [11]. Nevertheless, there are limited studies investigating preoperative AFP levels as predictors for early postoperative HCC recurrence.

By studying the correlation between preoperative serum GR activity, AFP level and early postoperative recurrence in patients with HCC, the authors looked for a group of markers to predict early postoperative recurrence of HCC, so as to provide a new idea for clinicians to evaluate the risk of recurrence in individual patients.

Materials and methods

Results

Recurrence group had a significantly higher AFP levels (median: 226.7 vs. 99.7 μg/L, p<0.001) and significantly lower GR activity (median: 55.0 vs. 68.0 U/L, p<0.0001) compared with non-recurrent group (Figure 1).

Figure 1:

Differences in preoperative GR and AFP levels between the non-recurrence group and the recurrence group (A) recurrent group had a significantly lower GR activity compared with non-recurrent group; (B) recurrent group had a significantly higher AFP levels compared with non-recurrent group, ***p<0.001, ****p<0.0001.

In order to understand the correlation between GR activity and AFP levels. We took Spearman correlation analysis of HCC patients, which showed that GR activity was negatively correlated with the AFP levels (r=−0.4275, p<0.01; Figure 2).

Figure 2:

Correlation between GR activity and AFP levels. AFP, alpha-fetoprotein; GR, glutathione reductase.

We subsequently conducted a logistic regression analysis to explore the potential association between GR activity, AFP levels, and the risk of early postoperative recurrence in patients with HCC. Following adjustment for confounding factors (including tumor number and albumin levels), our findings revealed that low GR activity (OR=0.948; 95 % CI: 0.910–0.988; p=0.011) and high AFP levels (OR=1.003; 95 % CI: 1.000–1.006; p=0.036) independently contribute to an increased risk of early postoperative recurrence in HCC patients (Table 2).

The ROC curve analysis revealed that the area under the curve (AUC) of GR activity for predicting early postoperative recurrence of HCC was 0.790 (95 % CI: 0.691–0.890; p<0.01). The optimal cutoff value was determined to be 62.5 U/L, with a sensitivity of 80.0 % and specificity of 70.7 %. Additionally, the AUC for AFP levels in predicting early postoperative recurrence of HCC was found to be 0.708 (95 % CI: 0.600–0.816; p<0.01). Using a cutoff value of 127.3 μg/L, the sensitivity and specificity were calculated as 78.0 and 58.5 %, respectively (Figure 3).

Figure 3:

Receiver operating characteristic curve analysis to evaluate the predictive efficacy of GR and AFP in early postoperative recurrence of HCC. GR, glutathione reductase; AFP, alpha-fetoprotein.

The Kaplan-Meier survival analysis revealed that patients with GR>60U/L had a higher early postoperative non-recurrence rate than patients with GR ≤60U/L (71.4 % [30/42] vs. 22.4 % [11/49]; HR=4.026; 95 % CI: 2.254–7.188; p<0.01); Patients with AFP≤100 μg/L had a higher early postoperative non-recurrence rate than patients with AFP >100 μg/L (65.6 % [21/32] vs. 33.9 % [20/59]; HR=2.490; 95 % CI: 1.397–4.438; p<0.01) (Figure 4).

Figure 4:

Kaplan–Meier survival analysis of early postoperative non-recurrence in HCC patients with different GR activity and AFP levels. (A) Patients with GR >60U/L had a higher early postoperative non-recurrence rate than patients with GR ≤60U/L (B) Patients with AFP ≤100 μg/L had a higher early postoperative non-recurrence rate than patients with AFP >100 μg/L. GR, glutathione reductase; AFP, alpha-fetoprotein.

Discussion

Surgical resection is one of the current radical treatments for HCC, but the recurrence rate is very high, with a 3-year recurrence rate of 52 %, and a 5-year recurrence rate of 70 % [13], 14]. Reducing the high recurrence rate of HCC is still a great challenge in clinic. At present, the prediction of postoperative recurrence of HCC is mainly based on the characteristics of the tumor, stage, lymphatic invasion and so on [15], [16], [17], but these indicators are difficult to be used as targets to improve prognosis. Yahya et al. have shown that the levels of lipid peroxidation products and reactive oxygen species metabolites in patients with HCC are significantly higher than those in patients with hepatitis and healthy people [18]. High oxidative stress and low antioxidant capacity in HCC tissue lead to high mortality and recurrence rate of HCC [19], 20]. There is a negative correlation between serum total antioxidant capacity (TAC) and one-year mortality after liver transplantation (OR=0.27595 % CI:0.135–0.562, p<0.001) [21]. Redox-related enzymes may play an important role in preventing or delaying the development of hepatocellular carcinoma. GSSG-GSH redox system is very important to maintain the stability of the internal environment. GR is the key enzyme that catalyzes the transformation of GSSG to GSH, which can inhibit the cytotoxicity produced by oxidative stress and reduce the level of oxidative stress. AFP is synthesized by liver cells and yolk sac cells, and it will be synthesized in large quantities when the liver is damaged. AFP can also diagnose tumor recurrence and metastasis by detecting the ability of hepatocyte regeneration [22]. There is a significant correlation between AFP doubling time and survival rate in patients with HCC, those slowest doubling times surviving longest [23]. It can be seen that AFP is an important immunological index for routine monitoring and prognosis evaluation of HCC at present.

Our study demonstrated a significant decrease in GR activity and a significant increase in AFP levels in the early postoperative recurrence group of HCC patients compared to the non-recurrence group. The reduced GR activity observed in the recurrent group suggests an elevated state of oxidative stress and diminished antioxidant capacity, highlighting redox imbalance as a crucial factor contributing to early HCC recurrence. Furthermore, low GR activity and high AFP levels independently serve as risk factors for early postoperative recurrence. Specifically, each 1 U/L increment in GR activity reduces the risk of early postoperative recurrence by 5.2 %, while every 1 μg/L rise in AFP level increases this risk by 0.3 %. Notably, our findings indicate that preoperative AFP levels are closely associated with early postoperative recurrence among HCC patients, complementing previous research on postoperative AFP levels and HCC recurrence [11]. Additionally, both GR activity and AFP levels hold predictive value for identifying early postoperative recurrence of HCC. The AUC for predicting HCC early postoperative recurrence using GR activity is 0.790, surpassing traditional indicators such as tumor characteristics regarding sensitivity and specificity [24]. Survival analysis also showed that the early postoperative recurrence rate of HCC patients with low GR activity and high AFP levels was higher than that of patients with high GR activity and low AFP levels, which is consistent with the research results of Hsiao et al. [25], Ma et al. [26] and Chan et al. [27].

Oxidative stress is fatal to normal cells. In contrast, it plays a key role in cell survival and proliferation of several types of cancer [28]. Unlimited proliferation of cancer cells, to some extent, relies on intracellular oxidants such as ROS [29]. Our research indicates that Oxidative stress is closely related to early postoperative prognosis of HCC. The higher the preoperative antioxidant indicators such as GR activity in HCC, the lower the probability of early postoperative recurrence. Although many biomarkers have been studied in the past few decades, serological detection of HCC recurrence mainly relies on the traditional marker AFP. From this study, it can be seen that the traditional indicator AFP is less effective in predicting early postoperative recurrence of HCC than the redox indicator GR (AUC: 0.708 vs. 0.790). Redox related enzymes seem to play an important role in preventing postoperative recurrence of HCC, which also provides new ideas for the treatment of HCC.

Although the results of this study indicate the adverse effects of high AFP levels on early postoperative recurrence of HCC, but factors such as tumor size, number of tumors, vascular invasion and impaired liver function, can all affect AFP levels [30]. Larger tumors may produce more AFP, although this correlation is not always straightforward. In some cases, very large tumors may produce less AFP due to necrosis or reduced metabolic activity. However, as a general trend, larger tumors tend to have higher AFP levels [31]. Patients with multiple tumors may have higher AFP levels compared to those with a single tumor, especially if these tumors are actively producing AFP. Vascular invasion, where cancer cells invade blood vessels, is associated with more aggressive tumor behavior and higher AFP levels. Tumors with vascular invasion are more likely to produce AFP and tend to have poorer prognoses compared to tumors without vascular invasion [32]. Patients with impaired liver function, such as those with cirrhosis or advanced liver disease, may have higher baseline AFP levels due to reduced clearance of AFP by the liver. When evaluating the prognosis of HCC patients and making treatment decisions, the impact of the above factors on AFP should be comprehensively considered.

The strength of this study was the combination of its cross-sectional and follow-up design, which enabled an exploration of preoperative serum GR activity and AFP level in relation to HCC recurrence. There were some limitations to our study. Our study is a single-center study, and the oxidative stress and antioxidant capacity indicators used are relatively single. The inclusion of multi-center and more antioxidant indicators will be our next work.

Conclusions

Our study suggests that low GR activity and high AFP levels are related to early postoperative recurrence in patients with HCC. GR activity and AFP levels can be used as predictive indicators and treatment targets for early postoperative recurrence in patients with HCC.