Acute-phase serum amyloid A for early detection of hepatocellular carcinoma in cirrhotic patients with low AFP level

Abstract

Regular hepatocellular carcinoma (HCC) surveillance by ultrasonography in combination with the α-fetoprotein (AFP) examination is unsatisfactory in diagnostic sensitivity for early-stage HCC especially in cirrhotic patients. We conducted a prospective study in a tertiary medical center in Taiwan and consecutively collected serum samples from patients with chronic hepatitis, liver cirrhosis (LC), or HCC for new biomarker discovery. Overall, 166 patients were enrolled, including 40 hepatitis, 30 LC, and 96 HCC. Four acute-phase serum amyloid A (A-SAA) derived biomarkers including total A-SAA, A-SAA monomer and oligomer, and protein misfolding cyclic amplification (PMCA) signal were measured and compared between patients with and without HCC. A-SAA biomarkers significantly increased in the HCC group when compared to the hepatitis and LC groups, and generally increased in more advanced tumor stages. Among A-SAA biomarkers, the area under the receiver operator characteristic curves (AUROCs) for PMCA signal in discrimination of all-stage and early-stage HCC were 0.86 and 0.9 in cirrhotic patients, which is comparable to AFP. For cirrhotic patients with low AFP (< 7 ng/mL), PMCA signal maintained good capacity in prediction of early-stage HCC (AUROC: 0.94). Serum A-SAA and its prion-like property showed a potential to complement AFP in detection of early-stage HCC.

Figure 1

Comparison of A-SAA derived biomarker levels among hepatitis, LC, and HCC patients. A total of 166 serum samples from 40 hepatitis (indicated as H), 30 LC, and 96 HCC patients were measured and analyzed. (a) Total A-SAA. (b) A-SAA monomer. (c) A-SAA oligomer. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test, where P < 0.05 (*),  < 0.01 (**), and < 0.001 (***). NS not significant.

Figure 2

Comparison of PMCA signals among hepatitis, LC, and HCC patients. (a) Diagram illustrating the principle of PMCA technique. A standard PMCA procedure starts with incubation of oligomeric seeds with monomeric amyloid protein to allow growth of the polymers. The aggregates are then fragmented by sonication to increase the number of seeds for further aggregate growth. After repeating PMCA cycles, the concentration of initial preformed oligomeric protein can be amplified and thus easy to be detected. (b) Comparison of PMCA signals among hepatitis, LC, and HCC patients. A total of 146 serum samples from 30 hepatitis (indicated as H), 30 LC, and 86 HCC patients were measured and analyzed. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test, where P < 0.05 (*) and < 0.0001 (****). NS not significant.

Figure 3

The level of A-SAA derived biomarkers at different HCC BCLC stages. A total of 96 serum samples from stage 0 (n = 11), stage A (n = 31), stage B (n = 28), stage C (n = 21), and stage D (n = 5) were measured and analyzed. (a) Total A-SAA. (b) A-SAA monomer. (c) A-SAA oligomer. (d) PMCA signal. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test, where P < 0.05 (*) and < 0.01 (**).

Figure 4

ROC curves of A-SAA derived biomarkers in differentiating all-stage and early-stage HCC in all patients. (a) Detection of all-stage HCC. (b) Detection of early-stage HCC. AUROC value for each curve is shown in parentheses.

Figure 5

Comparison of A-SAA derived biomarkers in patients with cirrhosis. A total of 116 serum samples from 30 LC and 86 HCC patients were measured and analyzed. Early HCC indicates BCLC stage 0-A (n = 38). Intermediate HCC indicates BCLC stage B (n = 24). Advanced HCC indicates BCLC stage C-D (n = 24). (a) Total A-SAA. (b) A-SAA monomer. (c) A-SAA oligomer. (d) PMCA signal. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test, where P < 0.05 (*),  < 0.01 (**), < 0.001 (***), and < 0.0001 (****). NS not significant.

Figure 6

ROC curves of A-SAA derived biomarkers in discrimination of all-stage and early-stage HCC in cirrhotic patients with or without low AFP level. (a) Detection of HCC in all cirrhotic patients. (b) Detection of early-stage HCC in cirrhotic patients with low AFP. AUROC value for each curve is shown in parentheses.