Assessment of combined serum sST2 and AFP levels in the diagnosis of hepatocellular carcinoma

Participants

A total of 273 serum samples were collected from three distinct groups: healthy controls (HCs group), patients with chronic hepatitis B (CHB group), and patients with diagnosed hepatocellular carcinoma (HCC group). This study was approved by the Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University ([2020]339) and the ethics committee waived the need for informed consent.

Individuals in the HCs group tested negative for routine blood examinations, biochemical parameters, and HBV-related biomarkers. The diagnosis of chronic hepatitis B (CHB) was confirmed by the presence of hepatitis B surface antigen (HBsAg) for at least 6 months (Terrault et al., 2018). Patients with HCC were clinically or pathologically diagnosed according to the American Association for the Study of Liver Diseases guidelines (Marrero et al., 2018).

Exclusion criteria included patients with other types of tumors, liver damage due to drugs, autoimmunity, alcohol, parasites, or other microorganisms, other hepatitis virus infections, chronic diseases affecting other body systems, or human immunodeficiency virus (HIV) infection.

Detection method

Statistical analysis

All the detected data represent the mean   ±  SD from three independent experiments for each condition. Statistical analyses were conducted using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). A P-value of less than 0.05 was considered statistically significant. All data were determined non-normally distributed by the D’Agostino-Pearson omnibus normality test. Kruskal-Wallis test was applied for analyses involving more than two groups. Categorical variables were compared using the Chi-squared test. ROC curves were generated, and AUC values were calculated. Youden’s index identified optimal cut-off points. Spearman correlation analyses were used to explore relationships between sST2 and other variables.

Characteristics of HCC group

The characteristics of the HCC group are presented in Table 1. In HCC group, 87.1% patient were male and over half of the patient (49, 52.7%) were at age of ≥60. Based on the widely used and clinically accepted cutoff values (Galle et al., 2019), serum levels ≥20µg/L for AFP was considered positive. At the 20µg/L cutoff, 54 (58.1%) HCC samples were AFP positive. Nearly 75% (69, 74.2%) of the patients were with tumor sizes more than 3 centimeter in diameters. 34 (36.6%) of the patients were stratified into the TNM stage II, III, IV. The portal vein tumor thrombus (PVTT) and lymph node metastases were observed in 24 (25.8%) and 17 (18.3%) patients, respectively. Liver cirrhosis and hepatitis B virus infection were detected in 42 (45.2%) and 72 (77.4%) of patients, respectively.

HCC group displayed higher sST2

As shown in Fig. 1A, the ages (years) in HCs (56.42 ± 8.74), CHB (58.14 ± 9.19) and HCC (59.23 ± 11.58) group were similar (P > 0.05). There was no significant difference in gender (male) percentage among HCs (70, 77.8%), CHB (71, 78.9%) and HCC (81, 87.1%) group (P > 0.05) (Fig. 1B). AST showed an elevated level in HCC group (36.00 [28.00–62.00] U/L) compared with HCs group (19.00 [16.00–25.00] U/L, P <0.0001) and CHB group (26.00 [18.00–35.00] U/L, P <0.01) (Fig. 1C). ALT level was higher in HCC group (29.00 [19.00–47.00] U/L) than HCs group (15.5 [13.00–24.00] U/L, P <0.0001) and CHB group (28.00 [21.00–36.00] U/L, P <0.05) (Fig. 1D). LogAFP was also significantly higher in HCC group (71.72 [5.16–839.04] µg/L) than in HCs group (3.94 [2.86−5.40] µg/L, P <0.0001) and CHB group(4.53 [3.12−5.47] µg/L, P <0.0001) (Fig. 1E). The levels of sST2 in HCC group (24.00 [15.20–49.90] ng/mL) increased significantly compared with HCs group (7.65 [5.20–10.53] ng/mL, P <0.0001) and CHB group (19.55 [15.23–24.95] ng/mL, P <0.0001) (Fig. 1F). Besides, AFP levels (Fig. S1a) and sST2 levels (Fig. S1b) between the TNM stage I and II–IV in HCC group showed no significant differences (P > 0.05), and sST2 levels didn’t differ between the patients aged ≥60y and aged <60y (Fig. S1c) or between male and female (Fig. S1d) in HCC group (P > 0.05).

sST2 had no significant correlation with other indicators in HCC group

Further investigation of whether sST2 is associated with other indicators was conducted using Spearman correlation analysis in patients with HCC. As is shown in Table 2, sST2 had no significant correlation with age, AFP, TS, WBC count, Hb, PLT count, ALB, TBIL, ALT, AST, GGT, HBV-DNA copies, PT, APTT, or INR in the whole HCC group (P > 0.05). After dividing the HCC group into AFP-negative (AFP-neg) group and AFP-positive (AFP-pos) group, it was found that sST2 was positively correlated with age in the AFP-neg group (P < 0.01). There was no significant correlation between sST2 and the aforementioned indicators in AFP-pos group (P > 0.05).

sST2 had a good diagnostic performance which was significantly improved with combination of AFP

To assess the predictive value of sST2 for HCC, the ROC curves were evaluated (Fig. 2 and Table 3). Based on the comparison between the HCC group and the HCs group, the AUC for AFP alone was 0.883 (95% CI [0.830–0.935]) with the optimal cut-off value of 4.575 ng/ml (76.30% sensitivity and 96.50% specificity). The AUC for sST2 alone was 0.861 (95% CI [0.810–0.913]) with the optimal cut-off value of 13.95 µg/L (82.80% sensitivity and 72.10% specificity), indicating similar performance (Fig. 2A). The combination of sST2 and AFP yielded an area under the curve (AUC) of 0.963 (95% CI [0.939–0.987]) with a sensitivity of 89.2% and a specificity of 94.2% for distinguishing HCC group from HCs group. When AFP was negative(AFP ≤20µg/L), the AUC for sST2 alone was 0.868 (95% CI [0.795–0.930]), with a threshold concentration of 14.50µg/L (sensitivity 82.10%, specificity 73.30%) (Fig. 2B).

In comparisons between the HCC group and the CHB group, the AUC for AFP alone was 0.8478 (95% CI [0.784–0.911]) with the optimal cut-off value 6.48 ng/ml(sensitivity 69.90% and specificity 97.50%), and for sST2 along the AUC was 0.7094 (95% CI [0.619–0.800]) with the optimal cut-off value 8.45 µg/L(sensitivity 80.6% and specificity 52.5%), as depicted in Fig. 2C. The AUC increased to 0.896 (95% CI [0.845–0.947]) with a sensitivity of 72.00% and a specificity of 100.00% after the combination of AFP and sST2, which distinguished HCC group from CHB group. The AUC for sST2 when AFP was negative was 0.707 (95% CI [0.593–0.821]), with a threshold concentration of 14.45 µg/L (sensitivity 82.10%, specificity 34.60%) (Fig. 2D).