Soluble Programmed Cell Death-1 is a Novel Predictor of HBsAg Loss in Chronic Hepatitis B Patients When Long-Term Nucleos(t)ide Analog Treatment is Discontinued

Figures & data

Table 1 Clinical Characteristics of the Study Cohort

Characteristics of EOT	Total (n=115)	HBeAg-Positive (n=79, 68.7%)	HBeAg-Negative (n=36, 31.3%)	p-value
Age (years)	37 (29–42)	34 (28–40)	41 (37–47)	<0.001
Male (%)	93 (80.9%)	61 (77.2%)	32 (88.9%)	0.142
Interferon history	23 (20%)	17 (21.5%)	6 (16.7%)	0.550
Antiviral regiment
ETV/TDF (%)	58 (50.4%)	39 (49.4%)	19 (52.8%)	0.886
Others (%)	57 (49.6%)	40 (50.6%)	17 (47.2%)
Treatment duration, (years)	4.4 (3.0–6.0)	4.2 (3.0–5.9)	4.7 (2.8–6.6)	0.369
Consolidation treatment duration (years)	2.5 (1.5–3.6)	2.3 (1.3–3.4)	2.7 (1.8–4.0)	0.044
ALT level (ULN)	0.53 (0.43–0.75)	0.49 (0.42–0.63)	0.57 (0.46–0.82)	0.075
HBV DNA (log10 IU/mL)	LLOD	LLOD	LLOD	-
HBsAg (log10 IU/mL)	2.72 ± 0.81	2.81 ± 0.78	2.50 ± 0.83	0.061
<100 IU/mL (%)	23 (20%)	14 (17.7%)	9 (25%)	0.368
≥100 IU/mL (%)	92 (80%)	65 (82.3%)	27 (75%)
sPD-1 (log10 pg/mL)^a	2.25 (1.89–2.34)	2.25 (1.89–2.42)	2.22 (1.89–2.63)	0.866
Liver stiffness value (kPa)	5.4 (4.5–6.5)	5.3 (4.5–6.6)	5.5 (4.7–6.4)	0.384

Notes: Variables are expressed as means ± SD, median (interquartile range), or n (%). ^aForty-eight patients had undetectable sPD-1 level (<156 pg/mL), which had been signed as 78 pg/mL (1.89 log₁₀ IU/mL).

Abbreviations: EOT, end of treatment; ETV, entecavir; TDF, tenofovir; ALT, alanine aminotransferase; ULN, upper limit of normal (40 U/L); LOD, lower limit of the detection (< 20 IU/mL); HBeAg, hepatitis B envelope antigen; HBsAg, hepatitis B surface antigen; sPD1, soluble programmed cell death 1.

Figure 1 Plasma sPD-1 at different time points after NA treatment discontinuation. Plasma sPD-1 levels were measured from the end-of-treatment to week 48 or the clinical relapse time. Stratification of patients according to whether clinical relapse occurred or not.*p < 0.05 for the comparison of sustained response vs clinical relapse. Data are presented as compared between groups by Mann–Whitney U-test.

Figure 2 ROC curves of sPD-1 and it’s cut-off value to predict clinical relapse after discontinuation of NA therapy. (A) The AUROCs of EOT sPD-1 for clinical relapse at 2, 4, 6 and 8 year after NAs discontinuation were 0.71 (95% CI, 0.61–0.82), 0.67 (95% CI, 0.56–0.78), 0.64 (95% CI, 0.53–0.75) and 0.65 (95% CI, 0.54–0.76), respectively. (B) At EOT, 67 (58.3%) patients had undetectable sPD-1. The cumulative clinical relapse rates patients with detectable and undetectable sPD-1 levels were 48% and 67.7%, respectively, (Log rank test: p = 0.002).

Abbreviation: ROC, receiver operating characteristic.

Table 2 Cox Proportional Hazard Model for Clinical Relapse (n=115)

Variables at EOT	Univariate Analysis			Multivariable Analysis
	HR	95% CI	p-value	HR	95% CI	p-value
Age (years)	1.035	1.007–1.065	0.015	1.045	1.012–1.078	0.007
Male vs Female	1.235	0.627–2.432	0.542
First-line vs Second-line drugs^†	0.865	0.519–1.443	0.579
HBeAg-positive at start of treatment	0.662	0.395–1.111	0.118
Treatment duration, (years)	1.127	0.980–1.297	0.093	1.101	0.986–1.230	0.087
Consolidation treatment duration (years)	0.963	0.825–1.126	0.639
ALT (U/L)	0.986	0.975–1.017	0.696
HBsAg (log10 IU/mL)	1.336	0.967–1.844	0.079	1.418	1.006–2.000	0.046
Detectable vs Undetectable sPD-1 (log10 pg/mL)	0.479	0.290–0.792	0.004	0.498	0.296–0.840	0.009

Note: ^†First-line: entecavir, tenofovir; second-line: lamivudine, adefovir, telbivudine, and combination treatment.

Abbreviations: ALT, alanine aminotransferase; HBeAg, hepatitis B envelope antigen; HBsAg, hepatitis B surface antigen; sPD1, soluble programmed cell death 1; HR, hazard ratio; 95% CI, 95% confidence interval. The minimum detection limit of sPD-1 was 156 pg/mL.

Table 3 Cox Proportional Hazard Model for HBsAg Loss (n=115)

Variables at EOT	Univariate Analysis			Multivariable Analysis
	HR	95% CI	p-value	HR	95% CI	p-value
Age (years)	1.043	0.979–1.112	0.195
Male vs Female	1.059	0.235–4.779	0.940
First-line vs Second-line drugs^a	1.526	0.528–4.408	0.435
HBeAg-positive at start of treatment	0.552	0.190–1.602	0.274
Treatment duration, (years)	0.988	0.778–1.253	0.919
Consolidation treatment duration (years)	0.961	0.706–1.309	0.801
ALT (U/L)	1.012	0.973–1.053	0.561
HBsAg (log10 IU/mL)	0.272	0.159–0.464	<0.001	0.233	0.121–0.450	<0.001
Detectable vs Undetectable sPD-1 (log10 pg/mL)^b	8.545	1.114–65.525	0.034	9.179	1.131–74.506	0.038
Develop SR vs CR	3.887	1.083–13.952	0.037

Notes: ^aFirst-line: entecavir, tenofovir; second-line: lamivudine, adefovir, telbivudine, and combination treatment. ^bThe minimum detection limit of sPD-1 was 156 pg/mL.

Abbreviations: ALT, alanine aminotransferase; HBeAg, hepatitis B envelope antigen; HBsAg, hepatitis B surface antigen; HBcrAg, hepatitis B core-relative antigen; ns, no significance; HR, hazard ratio; 95% CI, 95% confidence interval.

Figure 3 Cumulative rates of HBsAg loss and ROC curves in patients with different levels of HBsAg and sPD-1. (A) The EOT HBsAg level < 100 IU/mL (n=23) and ≥ 100 IU/mL (n=92) contributed 66.2% and 15.1% 8-year cumulative rates of HBsAg loss, respectively (Log rank test: p < 0.001). (B) The EOT undetectable and detectable sPD-1 levels exhibited 2.4% and 33.7% cumulative rates of HBsAg loss (Log rank test: p = 0.01). (C) The AUROCs of HBsAg < 100 IU/mL and detectable sPD-1 for HBsAg loss were 0.71 and 0.70, respectively.

Abbreviation: ROC, receiver operating characteristic.

Table 4 Spearman Correlation Between sPD-1 Level and ALT, HBsAg, HBV DNA at the End of Treatment and After Treatment Discontinuation

Spearman Correlation	r	p	r	p	r	p
	sPD-1 vs ALT		sPD-1 vs HBsAg		sPD-1 vs HBV DNA
EOT (n=115)	0.347	0.002	−0.125	0.182	-^a	-^a
4 weeks (n=112)	0.238	0.023	−0.227	0.021	−0.063	0.52
8 weeks (n=106)	0.408	<0.001	−0.202	0.044	−0.001	0.996
12 weeks (n=98)	0.282	0.005	−0.109	0.292	−0.043	0.671
24 weeks (n=90)	0.308	0.003	−0.081	0.448	−0.062	0.562
48 weeks (n=67)	0.184	0.048	−0.247	0.046	−0.037	0.762
Clinical relapse (n=51)	0.674	<0.001	−0.068	0.634	−0.101	0.482

Note: ^aNot computed as HBV DNA was undetectable at the end of treatment in all patients.