Patient characteristics. HCC patients and BLD patients were predominantly middle-aged or older and male. The median age of HCC patients was 58 (49‒67) years, which was significantly higher than that of BLD patients (52 [44‒62] years) (P < 0.001). The serum levels of PIVKA-Ⅱ, AFP, and the PAB combination in HCC patients were higher than those in BLD patients (P < 0.001), while the serum levels of bilirubin in HCC patients were lower than that in BLD patients (P < 0.001). The proportion of HCC patients with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL was higher than that in BLD patients (P < 0.001). The serum levels of PIVKA-II, AFP and PAB combination were positively correlated with tumour size (r = 0.456, 0.315, and 0.150; P < 0.001) (Fig. 1). The correlation between the PAB combination and tumor size was significantly weaker than that of PIVKA-Ⅱ and AFP (P < 0.001). The clinical characteristics of the 3,481 patients are shown in Table 1.
Expression of bilirubin in HCC patients and BLD patients. Table 2 shows that the proportion of cases with serum levels of bilirubin ≥ 20.0 μmol/L, ≥ 50.0 μmol/L, ≥ 100.0 μmol/L, and ≥ 200.0 μmol/L cases in the BLD and HCC groups, and the corresponding positive predictive value (PPV) and negative predictive value (NPV). The proportions of cases with these different serum bilirubin levels were higher in the BLD group than in the HCC group (all P < 0.05). The proportions of cases in the BLD group with these different serum levels of bilirubin who also had serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL were significantly higher than those in the total BLD group for the corresponding serum bilirubin levels (all P < 0.001). The proportions of cases in the HCC group with serum levels of bilirubin ≥ 20.0 μmol/L, ≥ 100.0 μmol/L, and ≥ 200.0 μmol/L who also had serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL were not significantly different from that of the total HCC group for the corresponding serum bilirubin levels (all P > 0.05). However, the proportion of HCC cases with serum levels of bilirubin ≥ 50.00 μmol/L cases who also had serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL was different from that in the total HCC group for this level of serum bilirubin (P = 0.004). The proportions of BLD and HCC cases decreased with the increasing serum bilirubin levels (P < 0.001). As the serum bilirubin levels increased, the negative predictive value gradually increased, while the positive predictive value gradually decreased (both P < 0.001).
Serum levels of PIVKA-Ⅱ and AFP correlated positively with serum levels of bilirubin in BLD patients (r = 0.107, P < 0.001; r = 0.167, P < 0.001); their correlation coefficients were slightly higher than those in HCC patients (r = 0.084, P = 0.025; r = 0.120, P = 0.001) (Fig. 2), although these differences were not statistically significant (P > 0.05).
Performance of the PAB combination in the diagnosis of HCC. BLD cases as the control group, the AUC of PA combination in the diagnosis of HCC was 0.883 (95%CI: 0.867‒0.899), which was significantly higher than that of AFP (0.815 [95%CI: 0.795‒0.834]) (P < 0.001), but not significantly different from that of PIVKA-Ⅱ (0.883 vs. 0.884) (P = 0.934) (Fig. 3A). Using the PAB combination in the diagnosis of HCC, the AUC increased to 0.935 (95%CI: 0.923‒0.947) (Fig. 3A), which was significantly higher than that of the PA combination (P < 0.001). The sensitivity, specificity, PPV, and NPV of the PAB combination for HCC diagnosis were 82.45%, 95.77%, 83.51%, and 95.45%, respectively, which were better than those of the PIVKA-Ⅱ, AFP, and PA combination (Table 3).
There were 906 cases of BLD and 646 cases of HCC with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL. BLD cases with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL as the control group, the AUCs of PIVKA-Ⅱ, AFP, and PA combination in the diagnosis of HCC with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL were 0.870 (95%CI: 0.851‒0.888), 0.738 (95%CI: 0.712‒0.764), and 0.879 (95%CI: 0.861‒0.897) (Fig. 3B), which were slightly decreased as compared to the values of HCC. The AUC of PAB combination in the diagnosis of HCC with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL was further increased to 0.965 (95%CI: 0.956‒0.973) (Fig. 3B), which was significantly higher than that of PA combination (P < 0.001). The sensitivity, specificity, PPV, and NPV of PAB combination in the diagnosis of HCC with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL were 86.38%, 94.92%, 92.38%, and 90.72%, respectively, which were better than those of PIVKA-Ⅱ alone, AFP alone, and PA combination (Table 3).
Performance value of PAB combination in the diagnosis of HCC< 3.0 cm. There were 95 cases of HCC with tumour size < 3.0 cm (HCC < 3.0 cm). 2,763 BLD cases as control group, the AUC of PA combination in the diagnosis of HCC< 3.0 cm was 0.808 (95% confidence interval (CI): 0.763‒0.853), which was slightly higher than that of PIVKA-Ⅱ (0.756 [95%CI: 0.696‒0.817]) and of AFP (0.749 [95%CI: 0.697‒0.801]), but without statistically significant difference (P = 0.178 and P = 0.096) (Fig. 4A). The AUC of the PAB combination in the diagnosis of HCC< 3.0 cm was 0.862 (95%CI: 0.815‒0.910), which was slightly higher than that of the PA combination, but without statistically significant difference (P = 0.104) (Fig. 4A). The sensitivity, specificity, PPV, and NPV of PAB combination in the diagnosis of HCC< 3.0 cm were 71.58%, 93.45%, 27.31%, and 98.97%, respectively. Except that the sensitivity of the PAB combination was lower than that of the PA combination (81.05%), the other diagnostic performance parameter values were better for the PAB combination than for the PIVKA-Ⅱ, AFP, and PA combination (Table 4).
There were 80 cases of HCC < 3.0 cm with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL. 906 BLD cases with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL as control group, the AUCs of PIVKA-Ⅱ, AFP, and PA combination in the diagnosis of HCC< 3.0 cm were 0.695 (95%CI: 0.630‒0.759), 0.636 (95%CI: 0.570‒0.703), and 0.732 (95%CI: 0.673‒0.790) (Fig. 4B). The AUC of PAB combination in the diagnosis of HCC< 3.0 cm with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL was 0.910 (95%CI: 0.878‒0.943), which was higher than that of PA combination (P < 0.001) (Fig. 4B). The sensitivity, specificity, PPV, and NPV of PAB combination in the diagnosis of HCC< 3.0 cm with serum levels of PIVKA-II ≥ 40 mAU/mL and/or AFP ≥ 20 ng/mL were 86.25%, 85.10%, 33.82%, and 98.59% respectively. Except that the specificity of the PAB combination was lower than that of AFP (91.06%), the other diagnostic performance parameter values of the PAB combination were better than those of the PIVKA-Ⅱ, AFP, and PA combination (Table 4).