ETV add-on Peg-interferon therapy plays a positive role in reversing hepatic fibrosis in treatment-naïve chronic hepatitis B patients: a prospective and randomized controlled trial

Background and Aim The efficacy of entecavir (ETV) add-on peg-interferon therapy compared with ETV monotherapy in treatment-naïve hepatitis B virus (HBV) patients remains controversial. We investigated whether adding Peg-interferon to ongoing ETV treatment leads to a better curative effect or not. Methods Eligible HBV patients (n=144) were randomly divided (1:1) to receive either ETV monotherapy (n=70) or peg-interferon add-on therapy from weeks 26 to 52 (n=74). Patients were followed-up for 2 years. We evaluated hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) seroconversion rate, sustained virologic response (SVR), transient elastography value, and histological scores. Results At week 26, no patient achieved HBsAg seroconversion in either group. At week 52, one patient in the monotherapy group was HBsAg-negative but there was none in the combination therapy group. The monotherapy group showed significantly better liver function recovery results than the combination therapy group. At week 78, one patient in combination group had HBsAg seroconverted. At week 104, only three patients in the combination therapy group were HBsAg-negative compared with one patient in monotherapy. The mean alanine aminotransferase (ALT)and aspartate aminotransferase (AST) levels and transient elastography values decreased significantly compared with baseline. Both group showed a favorable decrease in alpha fetoprotein(AFP) (monotherapy:23.4±77.3 vs 2.4±0.91, P=0.149; combination therapy: 33.3±96.9 vs 4.3±5.5,P=0.085) and an improved result of liver biopsy examination scores. The combination group showed a better improvement in histology compared with the monotherapy group(mean transient elastography value 7.5±3.4 kPa SD 1.9 vs. 12.8±13.9 kPa SD 1.9, P=0.037). But this research didn’t show significant difference in HBsAg conversion rate (1.79% (1/56)

There are approximately 240 million patients infected with Hepatitis B virus HBV worldwide, which represents a challenge to public health despite clinical application of new drugs and efficacious vaccines 1 . Nucleos(t)ide analogues (NAs) are used more commonly in HBV treatment protocols because they can suppress HBV-DNA replication, help to recover liver function, improve histology, and obtain a functional cure which defined as hepatitis B surface antigen(HBsAg)-negative regardless of the presence or absence of (hepatitis B surface antibody) HBsAb. Peg-IFNα-2a is an immunemodulating drug that can induce cytotoxic T-cells, which clears the HBV virus from infected cells through the immunomodulatory pathway and reduces the covalently closed circularDNA(cccDNA)levels 2,3 . Peg-IFNα-2a was shown to achieve a higher rate of HBeAg seroconversion and HBsAg seroclearance compared with entecavir (ETV) treatment 4,5 . Combination therapy of lamivudine (LAM) and peg-IFNα-2a showed a higher virologic response rate but there was no improvement in the post-therapy response compared with monotherapy 6 . However, peg-IFNα-2a has several disadvantages including a moderate antiviral effect, sustained risk of adverse events, inferior tolerability, and subcutaneous injections. What's more it requires regular clinical follow-up visits and a strict administration schedule. Therefore, NAs and peg-IFNα-2a combination therapy has been carried out to find an optimal efficacy, but the results of these trials are controversial because of the varying designs and evaluation criteria [7][8][9] . Some studies considered that combination therapy was better than monotherapy, but antiviral therapeutic options are largely influenced by the cost of drugs in China 10 . Most treatment-naïve patients generally start on NAs as an initial treatment because they are inexpensive and have a confirmed curative effect despite drug resistance. Therefore, we designed this prospective trial to evaluate the curative effect of adding peg-interferon to ongoing ETV therapy in treatment-naïve patients. Given the side effects of peg-IFNα-2a monotherapy and drug-resistance of ETV long-term therapy, we evaluated whether shortening the add-on course (26 weeks) of peg-IFNα-2a improve outcomes such as enhancing virologic response and sustained suppression of HBV DNA or not.

Study design
All patients have been recruited from Shanghai Public Health Clinical Center and Zhongshan Hospital.
Eligible participants were randomly divided into two groups at the start of the study by using a computerized randomization program. 144 eligible patients were randomly divided, in a 1:1 ratio, to receive either ETV monotherapy (n=70) or Peg-interferon add-on therapy (n=74). Both groups were given ETV for 2 years, but after 26 weeks, the combination therapy group received additional peg-IFN once weekly from week 26 to week 52. The efficacy of NAs monotherapy and combination therapy was compared every 3 months until the end of the study. Liver histology was also evaluated when participants were enrolled into the trial and after 2 years of therapy. They underwent a liver biopsy at the beginning and end of study.

Inclusion and exclusion criteria
This randomized controlled trial was approved by legally instituted ethics committees at Shanghai Public Health Clinical Center and Zhongshan Hospital. All chronic HBV patients were positive for HBsAg and had an HBV-DNA viral load >500 IU/mL with two consecutive tests at least 6 months apart, and the patients were treatment-naïve. Patients who were either HBeAg-positive or negative were included. Exclusion criteria were as follows: 1) co-infected with hepatitis A virus, hepatitis C virus, hepatitis D virus, or human immunodeficiency virus ; 2) decompensated liver disease(such as hepatocyte dysfunction and portal hypertension) or a history of esophageal varices; 3) hepatocellular carcinoma; 4) pregnancy or lactation, or liver transplantation; 5) alcoholic hepatitis, drug hepatitis, autoimmune disease, or metabolic liver disease; 6) comorbidities such as diabetes mellitus, arterial hypertension, dyslipidemia, coronary artery diseases, thyropathy. All patients signed an informed consent for indicating that their participation was voluntary and that their samples could be used for research. They were also informed of adverse events. The trial design is shown in Fig. 1.

Measurements
Before participants were enrolled in the trial, laboratory tests and routine examinations were performed at 3-month intervals for 6 months. Serum HBsAg and HBeAg levels were tested by Architect i2000 analyzer (Abbott Diagnostics, Abbott Park, IL, USA; HBsAg range of 0.05 to 52,000 IU/mL and HBeAg cut-off index <10 m IU/mL), HBV DNA were amplified using the iCycler device (Bio-Rad, USA; lower limit of quantification: 500 IU/mL).Liver biopsies were assessed at Zong Shan Hospital and Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.

Evaluation criteria
HBsAg/HBeAg, serum HBV-DNA viral load, and liver function were assessed at the beginning of treatment. The rate of patients with HBsAg loss was defined as the primary endpoint.. The proportion of patients with sustained suppression of HBV DNA below 500 IU/mL, HBeAg seroconversion loss or seroconversion, histology improvement, and liver function were defined as secondary endpoints.
HBsAg loss refers to undetectable serum HBsAg <0.05 IU/mL, and HBsAg/HBeAg seroconversion means the detectable HBeAb /HBsAb antibodies. HBV-DNA< 500 IU/mL was defined as an undetectable level. Ishak score was evaluated by professional pathologist. A reduction in the Ishak score or liver stiffness measurement (LSM)between the first day of treatment and the end of therapy was defined as the histology improvement criterion. The liver cirrhosis diagnosis were based on pathological stages and grades of liver histology and transient elastography standard (LSM >12.4 kPa in CHB patients with normal bilirubin and 1×ULN<ALT<2×ULN considered the progression of liver fibrosis) 11 . We Analyzed HBsAg/HBeAg loss or seroconversion, viral load, and liver function every 3 months and the end of treatment between the two groups. Adverse events were also recorded in both groups.

Statistical analysis
All statistical analyses were processed using the SPSS version 19 (SPSS Inc. Chicago, IL, USA).
Continuous variables were compared using the Student's t-test and categorical data were presented as n (%). The HBsAg/HBeAg seroconversion rate between the two groups was compared using the Chi-square test. P value of <0.05 was defined as statistically significant.

Results
All baseline characteristics of the two groups are shown in Table 1. Almost all clinical data from the two groups were comparable. All patients were HBsAg positive, with an HBV viral load >500 IU/ml, no participant had decompensated liver cirrhosis. Data are shown as the mean± standard deviation (SD) or n (%) and they were compared using the independent-sample test or a chi-square test.

Clinical efficacy of two groups
The clinical efficacy was different between the two groups at different time points, as shown in Table   2. Baseline and endpoint treatment variables were also compared in each group (Table 3   Note: All data are presented as the mean±SD, or median (IQR: 1st, 3rd quartiles) as appropriate. *P value of <0.05 was defined as statistically significant.

HBsAg/HBeAg clearance or seroconversion results
At week 26, patients in both groups only received ETV for treatment, and none of them showed HBsAg loss, whereas some patients began to lose and/or seroconvert HBeAg.
At week 52, one patient in the monotherapy group but none in the combination therapy group had lost HBsAg. In both groups, another three patients had lost and/or seroconverted HBeAg. The patient who had lost HBsAg in the monotherapy group also had an undetectable HBV viral load, but his liver function test results remained abnormal during the treatment. Additionally, before receiving ETV monotherapy treatment, the patient had already been followed-up in our department for 2 years with slightly high ALT and AST levels and HBV viral load.
At week 78, one patient from combination group had lost and/or seroconverted HBsAg.
At the end of our study, there was altogether one patient who lost HBsAg in the monotherapy group while three patients in the combination therapy group showed HBsAg clearance (1.79% (1/56) vs 4.11% (3/73), P=0.632). All data are presented in Table 4. Additionally, Figure 2A Figure 2C shows the change of HBsAg levels. Figure 2D presents the results of sustained suppression of HBV DNA. There was no significant difference in sustained suppression of HBV DNA between the two groups. Note: Data are presented as the number of patients.

Adverse events
During treatment, three patients showed thyroid dysfunction (one patient in monotherapy) and two patients had granulopenia (both in the combination therapy group). Six patients had a fever in the combination therapy group after they received their treatment. One patient felt fatigue in the ETV monotherapy group.

Discussion
In present, various therapies have been proceeded for CHB, but the optimal regimen remains unclear.
The clinical cure rate of combined treatment with NAs and peg-IFN is not sufficient to treat naïve chronic hepatitis B patients. peg-IFN monotherapy was also found to be efficient for HBsAg loss and seroconversion, but combination therapy was thought to cause more adverse events [12][13] . However, other studies have reported that the therapeutic efficacy of NAs combined with peg-IFN was better than monotherapy 14 .NAs are thought to directly inhibit HBV DNA replication, while peg-IFNα-2a as an immunomodulator can enhance the innate and adaptive immune responses to play a synergistic antiviral role 15,16 . In one study 10 , addition of NAs to peg-IFNα-2a therapy enhanced the virologic response in chronic hepatitis B patients who did not have an early response to peg-IFNα-2a. This suggests that in patients with an early poor virologic response to Peg-IFNα-2a, addition of NAs could inhibit viral replication. An trial directed by Ning also found that patients who switched from entecavir to peg-IFNα-2a significantly had increased rates of HBeAg seroconversion and HBsAg loss 17 .
Presently a new switching study 18 showed that HBeAg-positive chronic hepatitis B patients who switched from NAs to pegylated interferon achieved 12.5% and 16.2% HBeAg seroconversion and HBsAg loss, respectively. In a recent study, Patients on long-term NA who are unlikely to meet therapeutic goals can achieve high rates of HBsAg loss by switching to Peg-IFN alfa-2a 19 . It seems add-on therapy resulted in more viral decline and appeared to prevent relapse after stopping ETV compared with monotherapy. Therefore, based on this synergistic mechanism, combination therapy may be an ideal method for chronic HBV patients. However, we did not observe these results. In our study, we did not find a significant difference in clinical efficacy between the two groups, which is similar to other studies that reported that combination therapy failed to improve clinical efficacy 20 .
However, a recent meta-analysis also showed that combination therapy increased the virologic response and SVR 7 . Adding peg-IFNα-2a to ADV adefovir dipivoxil or ETV showed better clinical efficacy in HBeAg-negative patients 21 .
In this randomized controlled trial, our results showed that combination therapy did not have better antiviral efficacy than ETV monotherapy for sustained virologic suppression, HBsAg/HBeAg clearance, and seroconversion, but its histologic results showed improvement in the combination group.
Additionally, the rate of HBsAg/HBeAg clearance and seroconversion was not different from combination therapy compared with ETV monotherapy after adding peg-IFNα-2a. But a recent retrospective cohort study 22 showed that patients in the peg-IFN add-on therapy group were more likely to achieve HBeAg seroconversion (44% vs. 6%; P < 0.0001) compared with those in the ETV monotherapy group. The reason may be that the duration of the added peg-IFNα-2a administration was too short, and the different was not significant. Although some guidelines recommend 48 weeks of Peg-IFN administration for patients with chronic hepatitis B, we administered 26 weeks of peg-IFN add-on therapy because of the side effects and cost of the drug. Additionally, Brouwer WP 23 indicated that peg-IFN add-on therapy led to a higher proportion of HBeAg response compared with ETV monotherapy for HBeAg-positive chronic hepatitis B. The inclusion and exclusion criteria for patients may lead to differences in the outcomes.
In generally speaking, the sustained benefit of combination therapy requires further investigation. In our 2-year study, we compared the sustained efficacy between the two groups at different time points and we found that there was no significant difference between sustained suppression of HBV DNA in the two groups. However, this was not in consistent with the results of a recent study, which showed that combination therapy enhanced the virologic response and sustained suppression of HBV DNA 7 .
The ideal result of anti-HBV therapy is HBsAg seroconversion, which often means a viral clearance.
Achieving full viral clearance still remains a challenge. During the trial, three patients (two in the monotherapy group and one in the combination group) had hepatitis relapses even though their HBsAg was cleared and their viral load was undetectable during treatment. This phenomenon requires further study. Additionally, it improved histology following long-term treatment. We also found that the AFP levels in the monotherapy group were lower than that in the combination group during the treatment. Lower post-treatment AFP levels were reported to be significantly correlated with liver fibrosis regression 25 .
This phenomenon may be explained by the original baseline AFP levels in the combination therapy that were higher than those in the monotherapy group, although there is no significant difference between the two groups. Liver biopsy histological scores were evaluated at the end of the study, and the combination therapy was likely to have improved histology results compared with monotherapy, but the difference was not significant.
The most frequent adverse events reported in combination group were fatigue, headache, fever, and myalgia [26][27][28] . These adverse events were mostly related to adding Peg-IFNα-2a. As expected, more chronic hepatitis B patients taking combination therapy had side effects compared with those taking monotherapy in our study. Three patients had thyroid dysfunction, two patients had granulopenia, and one patient had fatigue in the ETV monotherapy. Therefore, attention must be paid to side effects of combination treatment.
There are some limitations in this trial, such as the small sample size, especially because some patients were lost to follow-up during treatment resulting from pregnancy, economic conditions, or the patient was unwilling to continue in the trial. However, the advantage of this study was that it was a randomized controlled perspective study. We evaluated the changes in ALT, AST, HBsAg/HBeAg clearance and seroconversion rate, and SVR at regular intervals, and we observed liver histological examination results. We also studied the effect of reducing the use time of interferon Peg-IFNα-2a.

Conclusion
For treatment-naïve patients with chronic hepatitis B, both monotherapy and combination therapy successfully improved liver function and histology. However, combination therapy did not show a better effect on HBsAg and HBeAg clearance and seroconversion or satisfactorily reduce HBV-DNA to an undetectable level. Our data also showed that combination therapy played a more positive role in reversing hepatic fibrosis than did monotherapy, but the safety of combination therapy requires further study. The curative effect of combination therapy and monotherapy also requires further study.

Availability of data and material
I'm sorry the data and material were not applicable , because this study is still going on.

Acknowledgements
We are grateful to the patients whom enrolled in this study. Authors are very appreciative for the cooperation extended by all the team members.  There was no significant difference in the HBsAg/HBeAg conversion rate, HBsAg levels and SVR between two groups at the end of the trial. But there is a tendency that combination therapy has a lower HBsAg levels than ETV monotherapy.

Figure 3
The transient elastography value is presented as the mean and standard deviation (SD). P < 0.05 indicates a significant difference.