BACKGROUND: Circulating HBsAg, HBV RNA, and hepatitis B core-related antigen (HBcrAg) are potential biomarkers for the response to nucleos(t)ide analog (NA) treatment discontinuation in patients with chronic hepatitis B (CHB). We retrospectively investigated the long-term kinetics of HBsAg, HBV RNA, and HBcrAg in HBeAg-negative patients treated with NA for up to 14 years in a prospective cohort study. METHODS: Ninety-six patients (mean age 65 y, 77% male, 52% with cirrhosis, all HBV genotype D) who were undergoing first (n=33, group A) or second-line (n=63, group B) treatment with tenofovir disoproxil fumarate were included. HBV biomarkers collected during tenofovir disoproxil fumarate treatment were measured in 384 serum samples stored at -20 °C. The combined biomarker endpoints associated with functional cure following NA discontinuation included HBsAg <
1000 IU/mL, HBV RNA <
54 copies/mL, and HBcrAg <
2 log U/mL. RESULTS: Before NA treatment, HBV RNA and HBcrAg were detectable in 85% (mean 3.9±2.3 [range, 0-9.2] log10 copies/mL) and 80% (mean 4.3±1.9 [2-8.9] log10 U/mL), respectively, of the patients in group A. In groups A and B, the percentages of patients with detectable HBV RNA levels decreased to 53% and 34%, respectively, during years 8-10 of NA treatment, and to 29% in group B during years 11-14 to 29%. HBcrAg could be quantified in 2% of patients in group B NA treatment years 8-10. Combined biomarker endpoints were met at baseline and at years 1-4, 5-7, 8-10, and 11-14 of treatment by 3.3%, 12% and 14%, 13% and 38%, 26% and 29%, and 41% of patients, respectively. CONCLUSIONS: HBV biomarker endpoints are associated with functional cure after the discontinuation of NA increase during long-term NA treatment.