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This prospective study enrolled 91 antiviral naïve HBeAgnegative patients with CHB who were treated in Department of Hepatology Division 2, Beijing Ditan Hospital from May 2013 to May 2016. The patients with HBeAg-negative CHB were enrolled according the inclusion and exclusion criteria (Table 1). Enrolled patients received weekly subcutaneous injections of 180 μg PEG-IFNα-2a for tailoring course. The total course of treatment should not exceed 120 weeks. Treatment would be stopped if HBsAg loss with undetectable serum HBV DNA occurred and confirmed by twice test with an interval of 12 weeks. The patients with sustained HBsAg level decrease during treatment would be continually treated up to 120 weeks, and treatment would be discontinued if HBsAg level was not decreased as compared to the previous 6 months. The use of other immunosuppressive, regulatory, and/or antiviral drugs was prohibited during PEG-IFNα-2a treatment. Patients who discontinued PEG-IFNα-2a treatment would be on survey during study period. However, patients should receive treatment of entecavir (ETV) if they had hepatitis relapse after stopping PEG-IFNα-2a treatment, defined as HBV DNA load ≥ 2000 IU/mL and alanine aminotransferase (ALT) level > 40 U/L.
Inclusion criteria Exclusion criteria 1. Age > 18 years Active consumption of alcohol and/or drugs 2. HBsAg-positive and HBeAg-negative for > 6 months Co-infection with human immunodeficiency virus, hepatitis C virus, or other viruses 3. Detectable serum HBV DNA for > 3 months Clinical evidence of cirrhosis 4. Abnormal ALT, but ≤ 10 × ULN for > 3 month History of autoimmune hepatitis 5. No previous antiviral therapy or treatment with immunosuppressive or regulatory drugs Evidence of neoplastic diseases of the liver Psychiatric disease Severe cardiac or pulmonary disease HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B envelop antigen; HBV: hepatitis B Virus; ALT: alanine aminotransferease; ULN: Upper limit of normal. Table 1. Patient selection criteria.
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HBV DNA load and serological markers (HBsAg, HBeAg, and anti-HBe), liver function, renal function, blood glucose, alpha fetoprotein (AFP) and blood count were tested at baseline and every 3 months during PEG-IFNα-2a treatment. Liver fibrosis scan (FibroScan 502, EchosensTM, Paris, France), ultrasound (Acuson Sequoia, Siemens, Erlangen, Germany) or CT (Computed Tomography System, LightSpeed VCT, LightSpeed Pro32, Tokyo, Japan) imaging examination were performed at baseline and every 6 months. Anti-HBs was tested in patients who achieved HBsAg loss.
Liver function, renal function, blood sugar, and blood lipids were detected by Hitachi 7600 fully automatic biochemical analyzer (Wako Pure Chemical Industries, Ltd., Tokyo, Japan). Blood routine was examined using automatic blood cell analyzer (COURTER LH755, California, USA). HBV DNA load was detected by CobasTaqMan96 real-time quantitative PCR detection reagent (detection of off-line < 20 IU/mL) (Roche, Pleasanton, CA, USA). HBsAg, anti-HBs, and HBeAg were detected using Abbott Architect i2000 kits (Abbott Laboratories, Abbott Park, IL, USA). Serum HBsAg levels were determined by Abbott Architect HBsAg QT assay (range: 0.05–250 IU/mL). Samples were finally diluted 1:1000 with the Architect HBsAg diluent to expand the upper limit of the dynamic range from 250 to 250, 000 IU/mL. HBsAg loss was defined as an HBsAg level < 0.05 IU/mL. Anti-HBs levels were measured using an Architect i2000 kit (Abbott Laboratories), with a range of 0.00–1000 mIU/L. Anti-HBs levels ≥ 10 mIU/L is considered positive.
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The primary endpoint was HBsAg loss/seroconversion during treatment. The secondary endpoints included HBsAg decline, compared with baseline, and undetectable HBV DNA after treatment.
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Kidney function, and peripheral blood neutrophil and platelet counts were determined before treatment and every 1–3 months. Parameter of thyroid function, and anti-thyroglobulin and thyroid peroxidase antibodies were monitored every 3 months during treatment.
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Continuous variables were expressed as mean ± standard deviation or median (range), as appropriate. HBV DNA (IU/mL) and HBsAg (IU/mL) were logarithmically transformed for analysis. For patients with negative HBsAg levels, the results were taken as the lower limit of detection (0.05 IU/mL for HBsAg) for the purposes of calculation. To investigate factors associated with HBsAg seroclearance, continuous variables including HBV DNA and HBsAg were compared by Mann–Whitney U tests, and categorical variables were analyzed using Fisher's exact tests. The area under the receiver operating characteristic (ROC) curve was used to analyze the predictive value of HBsAg kinetics for HBsAg seroclearance, and the best cutoff value was determined from the coordinates of the ROC curve. All statistical tests were two-sided. Statistical significance was taken as P < 0.05. Statistical analysis was performed using SPSS statistical software version 13.0 (Chicago, IL, USA).
Patients and Treatment
Laboratory Measurements
Efficacy Endpoints
Drug Safety
Statistical Analysis
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Among the 91 patients enrolled at the beginning, 3 patients withdrew before 12 weeks of treatment because of side effects of PEG-IFNα-2a, 5 patients lost continuous followup observation, and 2 patients withdrew because of unexpected pregnancy. So 81 patients were included in the study for analysis in the end (Fig. 1).
Figure 1. Flow diagram of patient enrollment, allocation, treatment, and follow-up. CDT, cumulative discontinuation of treatment.
Serum HBsAg levels decreased continually during treatment. Patients were grouped as complete response, partial response, and poor response according to their HBsAg level reached loss < 100 IU/mL or not during PEG-IFNα-2a treatment (Chan et al. 2011a, b). There were 12 (14.81%) patients who achieved HBsAg loss and undetectable HBV DNA [considered as the complete-response group (group 1)], 20 (24.69%) patients had HBsAg positive but < 100 IU/mL [defined as the partial-response group (group 2)], and 49 patients had HBsAg level ≥ 100 IU/mL [defined as the poor-response group (group 3)]. The demographics and baseline clinical characteristics in three groups are shown in Table 2. No patient developed decomposition of liver function and hepatic cell carcinoma during study period. No hepatitis relapse occurred in patients of group 1 and group 2, but eight patients in poor response group had hepatitis relapse after PEG-IFN treatment was discontinued and received ETV therapy.
Overall Complete response (Group 1) Partial response (Group 2) Poor response (Group 3) F/P value Number (%) 81(100) 12(14.81) 20(24.69) 49(60.49) Age (yr) 35.99 ± 9. 68 35.00 ± 8.08 30.35 ± 8.24 38.53 ± 9.56 F = 5.760 P = 0.005 ≥40 22(27.16%) 2(16.67%) 2(10.00%) 18(36.73%) < 40 59(72.84%) 10(83.33%) 18(90.00%) 31(63.27%) Male 65(80.25%) 9(75.00%) 17 (85.00%) 39(79.60%) χ2 = 0.061 P = 0.729 Family history of HBV 39(48.15%) 3(25.00%) 9(45.00%) 27(55.10%) χ2 = 3.604 P = 0.165 Baseline HBsAg (log10 IU/mL) 3.24 ± 0.54 3.18 ± 0.54 3.00 ± 0.60 3.35 ± 0.49 F = 3.17 P = 0.047 Baseline HBV DNA log10 copies/mL 5.20 ± 1.33 4.82 ± 1.35 5.19 ± 1.53 5.30 ± 1.24 F = 0.609 P = 0.546 ≥1.0 × 4 log10 65(80.24%) 7(58.33%) 16(80.00%) 42(85.71%) < 1.0 × 4 log10 16(19.75%) 5(41.67%) 4(20.00%) 7(14.29%) Baseline ALT U/L 179.49 ± 186.98 269.35 ± 279.55 157.06 ± 146.27 167.98 ± 170.82 F= 1.492 P = 0.231 1-2 ULN 21(25.93%) 2(16.67%) 6(30.00%) 13(26.53%) 2-5 ULN 41(50.62%) 6(50.00%) 10(50.00%) 25(51.02%) > 5 ULN 19(23.46%) 4(33.33%) 4(20.00%) 11(24.45%) Albumin, g/L 46.74 ± 4.46 46.54 ± 3.72 46.66 ± 6.27 46.82 ± 4.46 F = 0.021 P = 0.979 Granulocytes × 109/L 2.85 ± 1.18 3.01 ± 1.46 2.61 ± 1.26 2.89 ± 1.10 F = 0.377 P = 0.688 Platelets × 109/L 166.74 ± 49.23 154.52 ± 59.74 148.81 ± 48.08 176.31 ± 45.45 F = 2.040 P = 0.139 HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B envelop antigen; HBV: hepatitis B virus; DNA: deoxyribonucleic acid; ALT: alanine aminotransferease; ULN: Upper limit of normal; log: logarithmic. Table 2. Demographics and characteristics of patients with different HBsAg responses to treatment.
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All patients in completed response group, 90.0% (2/20) of patients in partial response group, and 75.5% (37/49) of patients in poor response group, archived undetectable HBV DNA levels at 36 weeks (Fig. 2). Among total patients, there were 95.06% (77/81) achieved HBV DNA negative conversion at 48 weeks. In complete response group, all patients maintained undetectable HBV DNA level during the study period.
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After 12-week treatment, the decline of HBsAg levels from baseline were 0.534 ± 0.559, 0.386 ± 0.644, and 0.053 ± 0.316 log10IU/mL in group 1, group 2, and group 3, respectively. After adjustment for differences at baseline, the degree of HBsAg decline was significantly higher in groups 1 and 2 than in group 3 (F = 11.39, P < 0.0001). After treatment of 24 weeks, the degree of HBsAg decline became significant different among three groups (F = 30.30, P < 0.0001), and the magnitude of deviation enlarged during subsequent treatment. HBsAg level in group 1 continued to decrease through the treatment course; however, the decline reached a plateau after 72 weeks in groups 2 and 3 (Fig. 3).
Figure 3. Dynamic changes of serum HBsAg levels in different patient groups during 120-weeks. HBsAg level declined slightly in poor response patients, in the partial-response group those declined continually early treatment time but reached a plateau after 72 weeks, and in complete response group those decreased sustainably.
Among 12 patients who achieved HBsAg loss, 11 patients obtained HBsAg < 100 IU/mL at 48 weeks. 41.66% (5/12) of HBsAg loss occurred within 72 weeks after treatment, and 58.33% occurred between 72 and 120 weeks (Fig. 4). During the 48-week standard PEG-IFN treatment, HBsAg loss rate was only 3.7%, while it reached to 6.2%, 11.1% and 14.8% when the treatment was extended to 72, 96, and 120 weeks, respectively.
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The cut-off value for HBsAg levels at 12 weeks predicting HBsAg loss at 96 weeks and at 120 weeks was 400 IU/mL and 750 IU/mL respectively, while the cutoff value at 24 weeks predicting HBsAg loss at 96 weeks and at 120 weeks were 174 IU/mL and 236 IU/mL. To predict HBsAg loss at 96 weeks, the area under the ROC curve (AUC) of 24 weeks was 0.925 (95% CI, 0.853–0.990), and that of 12 weeks was 0.725 (95% CI, 0.505–0.940). For predicting HBsAg loss at 120 weeks, the AUC of 24 weeks was 0.922 (95% CI, 0.853–0.984), and that of 12 weeks was 0.722 (95% CI, 0.547–0.888). The predictive abilities at 24 weeks were thus stronger than those at 12 weeks to predict for HBsAg loss at both 96 and 120 weeks (χ2 = 3.880, P = 0.049 and χ2 = 4.412, P = 0.036, respectively) (Fig. 5).
Figure 5. Predictive abilities of HBsAg level at 12 weeks and 24 weeks for HBsAg loss during PEG-IFN treatment. Regardless of predicting HBsAg loss occurred during the treatment of 96 weeks (A) or HBsAg loss occurred within 120 weeks (B) after treatment, the cutoff value of HBsAg levels at 12 weeks had lower AUC and NPV than the cutoff value of HBsAg levels at 24 weeks.
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It's reported that 58 patients (71.6%) had influenza-like syndrome, and 56 patients (69.1%) had neutrophils decrease which reached abnormal levels (< 2 × 109/L) in 21 patients. Platelet counts decrease occurred in 55 cases (67.9%), and it reached lower than the low detection limit (< 100 × 109/L) in 32 patients. Abnormal thyroid function was observed in three patients, of whom two developed hyperthyroidism and one developed thyroid hypofunction; but all recovered at the end of PEG-IFN therapy after specific treatments. However, drug doses were not necessarily adjusted in patients with decreased neutrophil and/or platelet counts, or in those with thyroid dysfunctions. Serum creatinine and urea nitrogen levels remained normal throughout treatment in all patients.