Baseline characteristics
Table 1 summarizes the baseline characteristics of the patients. The median (range) age was 45 (23–79) years, and there were 78 (63.9%) males and 22 (18.0%) patients with cirrhosis. A total of 104 (85.2%) patients had HBV genotype C. Although the baseline characteristics were similar to those reported in our previous study,8 the median follow-up duration was extended from 4.4 (range, 1.0–10.7) to 6.2 (range, 1.1–13.3) years.
Cumulative rates of HCC development according to clinical factors at baseline
In our previous report,8 10 patients developed HCC during the follow-up period (median duration, 3.3 [range, 1.1–6.9] years); since then, an additional 3 patients have developed HCC (median duration, 4.3 [range, 1.1–7.6] years) (Table 2). In accordance with our previous report,8 liver fibrosis, the platelet count, the fibrosis-4 (FIB-4) index, age, type IV collagen, and α-fetoprotein (AFP) were significantly associated with HCC development according to the log-rank test. Moreover, hyaluronic acid and hepatitis B surface antigen (HBsAg) were associated with HCC development (Supplementary Fig. 1).
Cumulative rates of HCC development according to soluble immune checkpoint protein levels at baseline
The cumulative rates of HCC development according to the serum soluble inducible T-cell co-stimulator (sICOS) level are shown in Fig. 1A. The cumulative rates of HCC development at 3, 5, 7, and 10 years were 9.5%, 20.3%, 24.1%, and 27.9%, respectively, in patients with sICOS ≥ 164.71 pg/mL (n = 33), and 2.6%, 4.3%, 8.1%, and 8.1%, respectively, in those with sICOS < 164.71 pg/mL (n = 89) (p = 0.014).
The cumulative rates of HCC development according to the serum sPD-1 level are shown in Fig. 1B. The cumulative rates of HCC development at 3, 5, 7, and 10 years were 10.9%, 13.6%, 21.0%, and 25.7%, respectively, in patients with sPD-1 ≤ 447.27 pg/mL (n = 51), and 0%, 5.9%, 8.0%, and 8.0%, respectively, in those with sPD-1 > 447.27 pg/mL (n = 71) (p = 0.031).
The cumulative rates of HCC development according to the serum soluble (s) CD40 level are shown in Fig. 1C. The cumulative rates of HCC development at 3, 5, 7, and 10 years were 8.6%, 12.5%, 19.1%, and 21.9%, respectively, in patients with sCD40 ≤ 493.68 pg/mL (n = 64), and 0%, 5.4%, 5.4%, and 5.4%, respectively, in those with sCD40 > 493.68 pg/mL (n = 58) (p = 0.032).
The cumulative rates of HCC development according to the serum sHVEM level are shown in Fig. 1D. The cumulative rates of HCC development at 3, 5, 7, and 10 years were 5.3%, 11.7%, 23.1%, and 27.4%, respectively, in patients with sHVEM ≤ 2,470.83 pg/mL (n = 43), and 4.2%, 7.7%, 7.7%, and 7.7%, respectively, in those with sHVEM > 2,470.83 pg/mL (n = 79) (p = 0.038).
There were no significant associations between HCC development and the levels of soluble immune checkpoint proteins (sBTLA, sCD27, sCD28, sTIM-3, soluble glucocorticoid-induced TNFR-related [sGITR], soluble lymphocyte-activation gene 3 [sLAG-3], soluble toll-like receptor 2 [sTLR-2], soluble glucocorticoid-induced TNFR-related ligand [sGITRL], sCTLA-4, sCD80, sCD86, and sPD-L1) (Supplementary Fig. 2).
Predictive factors for HCC development during entecavir treatment
As we reported previously,8 the FIB-4 index, platelet count, cirrhosis status, age, and AFP were associated with HCC development during entecavir treatment in univariate analyses. In addition, levels of type IV collagen ≥ 200 ng/mL (p = 0.011; hazard ratio [HR], 5.300; 95% confidence interval [CI], 1.458–19.270), hyaluronic acid ≥ 143 ng/mL (p = 0.017; HR, 3.922; 95% CI, 1.281–12.008), HBsAg ≤ 3.53 log IU/mL (p = 0.027; HR, 5.507; 95% CI, 1.220–24.859), sICOS ≥ 164.71 pg/mL (p = 0.022; HR, 3.713; 95% CI, 1.210–11.393), sPD-1 ≤ 447.27 pg/mL (p = 0.042; HR, 3.393; 95% CI, 1.044–11.033), and sHVEM ≤ 2,470.83 pg/mL (p = 0.049; HR, 3.078; 95% CI, 1.006–9.416) were significantly associated with HCC development during entecavir treatment (Table 3).
From multivariate analysis, a serum sPD-1 level ≤ 447.27 pg/mL (p = 0.014; HR, 4.537; 95% CI, 1.363–15.103) and AFP level ≥ 6.4 ng/mL (p = 0.040; HR, 5.524; 95% CI, 1.084–28.164) were independently and significantly associated with HCC development during entecavir treatment (Table 3).
Cumulative rates of HCC development according to the combination of serum sPD-1 and AFP levels
When classified according to serum sPD-1 and AFP levels, the 10-year cumulative rates of HCC development were 0%, 6.5%, 12.8%, and 67.0% in the sPD-1 > 447.27 pg/mL + AFP < 6.4 ng/mL group (n = 30), sPD-1 ≤ 447.27 pg/mL + AFP < 6.4 ng/mL group (n = 34), sPD-1 > 447.27 pg/mL + AFP ≥ 6.4 ng/mL group (n = 41), and sPD-1 ≤ 447.27 pg/mL + AFP ≥ 6.4 ng/mL group (n = 17), respectively (p < 0.0001) (Fig. 2).
Relationships between the serum sPD-1 level and clinical/virological characteristics
The serum sPD-1 level at baseline was positively correlated with the HBV-DNA (p = 0.057, r = 0.17; Fig. 3A) and alanine aminotransferase (ALT) (p < 0.0001, r = 0.41; Fig. 3B) levels, and was similar between non-cirrhotic and cirrhotic patients (median 519.35 pg/mL vs. 534.62 pg/mL, p = 0.93; Fig. 3C).
Changes in serum sPD-1 level in patients who developed HCC during entecavir treatment
We analyzed the changes in serum sPD-1 level in 9 (cases 1, 2, 3, 7, 8, 9, 11, 12, and 13; Table 2) of 13 patients who developed HCC during entecavir treatment (Fig. 4). The serum sPD-1 level decreased rapidly after 6 months of entecavir treatment. Significant decreases were found at 6 (p = 0.028) and 12 (p = 0.028) months after the start of entecavir treatment, at 12 (p = 0.028) and 6 (p = 0.018) months before HCC development, and at the time of HCC development (p = 0.0077), compared with baseline values. Moreover, no re-increase in the serum sPD-1 level at HCC development was observed.