| BACKGROUNDChronic hepatitis B (CHB) infection is a serious global public health problem. Approximately 2 billion people have been infected by the hepatitis B virus (HBV), and approximately 5% of these people (approximately 300-400 million)were chronically infected. About 1 million people died per year due to HBV-related complications such as liver failure, liver cirrhosisand hepatocellular carinoma, and in China CHB it’s the most common cause of liver cirrhosis and liver cancer.Antiviral therapy is the most fundamental andimportant treatment for patients with CHB. The short-term goal of antiviral therapy is to inhibit the replication of HBV, for patients with hepatitis B e antigen (HBeAg) positive also require HBeAg seroconversion. The long-term aim of antiviral therapy is to delay or prevent liver disease from developing into cirrhosis and liver cancer, thus improving the patients’ survival rate and quality of life.Anti-HBV drugs are currently divided into two groups: injectable interferon and oral nucleos(t)ide analogues (NAs). NAs could inhibit the activity of RNA-dependent DNA polymerase, thereby inhibiting viral replication and reducing the virus load. NAs are administrated once daily. Compared to injectable interferon, the administration of NAs is more convenient, safe, and suitable for long-term use. In China, there are five types of oral NAs available widely, including lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT) and tenofovir disoproxil (TDF).Given oral NAs should consider the acceptance, compliance, economic burden, and long-term side effects experienced by patients receiving long-term antiviral therapy, as well as the effect on childbearing among patients of childbearing age. Another major concern is long-term use of NAs might lead to the evolution of resistant viral mutants. There is currently no definitive scientific evidence that might guide the reasonable application of NAs, and there are also no reliable laboratory parameters that could be used to indicate when the medication can be safely discontinued. Although European Association for the Study of the Liver (EASL) andAmerican Association for the Study of Liver Disease (AASLD) guidelines recommended that discontinuation could be considered when HBsAg seroconversion occurs, the possibility of HBsAg seroconversion induced by NAs therapy is rare. In addition, the Asian Pacific Association for the Study of the Liver (APASL) Guideline and the 2nd Edition of the Chinese chronic hepatitis B prevention and treatment guidelines recommend:for HBeAg-positive patients, after reached a complete response (CR; when HBV DNA is undetectable and HBeAg seroconversion and alanine aminotransferase (ALT) levels have normalized) and consolidation treatment had last for at least 12 months, discontinuation can be considered. Similarly, for HBeAg-negative patients, after CR has been reached (HBV DNA is undetectable and ALT levels have normalized), consolidation had last for at least 18 months, treatment discontinuation can be considered. Actually this recommendation is not widely practiced and remains a controversial. One reason is the lack of high quality evidence. Another reason is that there are high relapse rates after discontinuation according to theserecommendations.Although NAs adequately suppresses the viral replication, it does not eradicate the covalently closed circular DNA(cccDNA). Therefore, most patients have reactivated viral replication once NAs is discontinued. Moreover,NAs discontinuation is not widely practiced and remains a controversial but highly relevant with the clinical practice.Then,we carried out this prospective study to identify predictors of a sustained response, as well as the optimal treatment endpoint for minimizing the risk of recurrence.METHODS1. Study design and populationThis was a prospective, single-center, observational study of well-suppressed Asian CHB patients (HBsAg positive>6 months) who discontinued NAs, and consented to prospective study follow-up. Patients were recruited from Nanfang Hospital (Guangzhou, China) fromNovember 2012 until January 2015. The analysis included follow-up data until July 2015and all patients have completed at least 6 months of follow-up.Patients who were HBeAg-positive at NA initiation were eligible if they fulfilled the stopping criteria of international guidelines:HBeAg seroconversion, undetectable HBV DNA, and normalized ALT with subsequent consolidation therapy>12 months. Patients who were HBeAg-negative at NA initiation were eligible if they fulfilled the Asian-Pacific stopping criteria:undetectable HBV DNA with subsequent continued therapy>18months. Furthermore eligibility criteria were at least 18 years of age; HBsAg positive and undetectable HBV DNA at moment of NA discontinuation; and written informed consent with motivation to attend follow-up visits. Exclusion criteria included co-infection with hepatitisC virus, hepatitis D virus, or human immunodeficiency virus; immunocompromised patients or patients with malignancy; autoimmune liver disease; alcohol abuse; prior history of liver transplantation; other severe or active disease; history or presence of decompensated liver disease (ultrasound confirmed ascites, bleeding esophageal varices, overt encephalopathy, total bilirubin level>2x the upper limit of normal (ULN), albumin<32 g/L, or prothrombin time prolongation of>3 seconds compared to reference); radiological suspicion of HCC or alpha-fetoprotein>20 ng/mL; biopsy-proven cirrhosis; or liver stiffness>9 kPa with fibroscan (Echosens, Paris, France).This study was conducted in compliance with the Declaration of Helsinki, and approved by the ethics committee with the following study ID:NFEC-201209-K3.2. Follow-upAfter NA treatment discontinuation, patients were followed every month in the first 3 monthsand every 3 months till year 2. Hereafter, the patients were evaluated every 6 months. At each visit, biochemical (ALT, AST, total bilirubin, albumin) and virological (quantitative HBsAg, HBeAg, anti-HBe, HBV DNA) tests were performed.Blood routine,coagulation function, AFP, Ultrasound and liver stiffness measurement were also performed at treatment discontinuation, year 1, and year 2.3. Endpoints, retreatment criteria, and definitions.The primary endpoint was clinical relapse defined as HBV DNA>2,000 IU/mL combined with ALT>2x ULN. Patients with clinical relapse were retreated and discontinued from this prospective study follow-up.Otherendpoints included virological relapse (HBV DNA>2,000IU/mL), biochemical relapse (ALT>2x ULN), off-treatment HBsAg kinetics,HBsAg seroclearance, ultrasonographic signs of cirrhosis at year 1 and 2,and liver stiffness measurements at year 1 and 2.Ultrasonographic signs of cirrhosis were defined as nodules within the hepatic parenchyma, splenomegaly, or portal vein>16 mm. As described by practice guidelines, consolidation therapy was defined as continued NA therapy after HBeAg seroconversion, undetectable HBV DNA and ALT normalization in HBeAg-positive patients, and after undetectable HBV DNA in HBeAg-negative patients. Persistent HBV DNA elevation was defined as at least two tests of HBV DNA>2,000 IU/mL within 3 months.4. Laboratory tests.Local standardized automated techniques were used to perform biochemical tests. The ULN of ALT was 40 U/L for males and 35 U/L for females. Quantitative HBsAg (lower limit of detection [LLOD]:0.05IU/mL), HBeAg, and anti-HBe were determined using the ARCHITECT (Abbott Laboratories, Chicago, Il, USA). Data of pre-treatment serum HBV DNA levels were retrospectively collected and measured with a polymerase chain reaction HBV assay with a LLOD of 1,000 copies/mL (Daan Gene Co, Ltd; Sun Yat-sen University; Guangzhou, China). Serum HBV DNA levels at NA stop and follow-up were determined using the CobasTaqMan polymerase chain reaction HBV assay with a LLOD of 20 IU/mL (Roche Diagnostics, Basel, Switzerland).5. Data analysis.The date of NAs stop was defined as baseline. Follow-up was calculated from baseline until a particular study endpoint, loss of follow-up, censorship (retreatment), or last follow-up when appropriate.The count data were expressed as mean±standard deviation or median and range, while the measurement data were expressed as percentages. The categorical variables were evaluated using the x2 test, and the Student’s t-test or Mann-Whitney test for continuous variables when appropriate. Associations between factors and study endpoints were examined by Cox proportional hazards regression. In order to investigate the predictors of biochemical relapse after an initial HBV DNA elevation>2,000 IU/mL, a time-reset approach was used in which the time of first HBV DNA elevation was considered as t=0. Persistent HBV DNA elevation was analyzed as a time-dependent covariate allowing patients to switch to the persistent HBV DNA elevation group during follow-up.Statistical tests were two-sided and a p-value< 0.05 was considered as statistically significant. All tests were performed with SPSS version 21.0 (IBM Corp., Armonk, NY, USA).RESULTS1. Patient characteristicsIn total,87 patients gave informed consent and were enrolled. Five patients were negative for HBsAg at week 0, and were excluded. Therefore, this analysis included 82patients who were HBsAg-positive and HBV DNA negative at the moment of NA discontinuation. Among these 82 patients,73 were male and 9 were female.63 patients were treatment-naive, whereas 19 had a history of NA treatment and were re-treated due to recurrence. And 18 patients had a history of IFN treatment. At the end of treatment,32 received the first-line antiviral durg (ETV), and the other 50 receivedthe second-line durgs(included LAM, ADV, LdT and LAM+ADV). The average age of the 82 patients were 35.23±8.85 years, and had received a total treatment duration for a median of 3.96 years (interquartile range 2.48-3.96). The median of consolidation time was 2.25 years (interquartile range 1.40-3.87).At the initiation of NAs treatment,60 (73%) patients were HBeAg-positive and 22 (27%) were HBeAg-negative. Pre-treatment HBeAg-negative patients were older and have received longer period of consolidation therapy than that of pre-treatment HBeAg-positive patients.Overall, only 2 of the 82 (2%) patients were lost to follow-up at week 24 and 60, respectively. Patients who did not develop a clinical relapsewere followed for a median of 1.9years (interquartile range 1.4-2.5).2. Virological relapseDuring the follow-up,58 of the 82 patients developed HBV DNA elevation>2,000 IU/ml (virological relapse) at least once resulting in cumulative rates of 65%,69%and 73%for pre-treatment HBeAg-positive patients and 55%, 64%and 77%for pre-treatment HBeAg-negative patients at year 0.5,1and 2, respectively (p=0.86).The COX hazard rate regression was carried out to assess the predictors that were associated with virological relapse. We found that only the antiviral drug was associated with virological relapse. Patients treated with the non-ETV were almost 2 times more likely to develop a virological relapse compared to patients treated with ETV (HR 1.96; 95%CI 1.07-3.59; p=0.028). At year 2, the cumulative rates of virological relapse for patients treated with ETV (64.60%) was significant lower than that of patients treated with non-ETV(80.27%)(p=0.0135).3. Clinical relapse.Twenty-eight patients developed a clinical relapse of whom 16 (57%) with an ALT elevation>5xULN. The cumulative rates of clinical relapse in pre-treatment HBeAg-positive patients were 14%,26%and 31%, at year 0.5,1and2, respectively. For pre-treatment HBeAg-negative patients, the rates were 14%,27%and 53%, at year 0.5,land 2,respectively. These rates were not statistically different between pre-treatment HBeAg-positive and pre-treatment HBeAg-negative patients (p=0.099).Only age was significantly associated with clinical relapse.Patients with end-of-treatment HBsAg levels≤200 IU/mL tended to be less likely to develop a clinical relapse. At year 2,48% of the patients>35 years developed a clinical relapse compared to 25% of the patients≤35 years (p=0.007), while 22% of the patients with end-of-treatment HBsAg levels≤200 IU/mL developed a clinical relapse compared to 44% of the patients with higher levels (p=0.042).4. The prediction of biochemical relapseIn total,58 of the 82 patients developed virological relapse. Ofthese 58 patients, 31 developed biochemical relapse giving a cumulative rate of 65% at year 2. None of the 24 patients with sustained off-treatment HBV DNA≤2,000 IU/mL developed biochemical relapse during a median follow-up of 2 years (interquartile range 1.3-2.5).When assessing the level of HBV DNA at the first elevation above 2,000 IU/mL, biochemical relapse was very likely to develop if the level of HBVDNA was>200,000 IU/mL at the initial elevation compared to>2,000-≤200,000 IU/mL (HR 8.42; 95% CI 3.07-23.10; p<0.001). Biochemical relapse developed in all of the 7 patients with HBV DNA>200,000 IU/mL compared to 5 of the 11 patients with HBV DNA>20,000-≤200,000 IU/mL and 19 of the 40 patients with HBV DNA>2,000-≤20,000 IU/mL (year 2 rates:100%,65%, and 51%, respectively).According to the study protocol, patients with virological relapse but ALT<2 x ULN would continue to follow-up. We found the HBV DNA elevation in some patients were transient. At the last visit, only 44 patients still had HBV DNA over 2000 IU/ml. In order to assess the effect of persistent HBV DNA elevation on the development of biochemical relapse, the 7 patients with an initial HBV DNA level>200,000 IU/mL were excluded and 4 additional patients due to concurrent occurrence of biochemical relapse with the first HBV DNA elevation. In the remaining 47 patients, a persistent HBV DNA elevation was found in 21 (45%) patients, whereas transient HBV DNA elevation was observed in 26 (55%) patients. Seventy-three percent of the patients developed biochemical relapse after a persistent elevation within 2 years compared to 17%of the patients after a transient elevation (p<0.001). Based on these data, we propose an easy-to-use algorithm which uses the off-treatment HBV DNA levelsto guide the off-treatment management of discontinued patients.5. Serum HBsAg kineticsFive patients cleared serum HBsAg during off-treatment follow-up resulting in a cumulative HBsAg seroclearance rate of 10% at year 2. Patients with end-of-treatment HBsAg levels≤100 IU/mL were 7-fold more likely to clear serum HBsAg (HR 7.08; 95% CI 1.17-42.81; p=0.033).The HBsAg change was significantly different between sustained responders and clinical relapsers (mean HBsAg change per year:-0.48 vs.+1.02 log IU/mL; p=0.004). After exclusion of the 5 patients with HBsAg seroclearance, the HBsAg decline in sustained responders was more modest, but still significantly different compared to clinical relapsers (-0.16 vs.+1.02 log IU/mL; p=0.016).6. Longitudinal liver stiffness and ultrasound assessments.As patients who developed a clinical relapse were retreated and not followed further in this study protocol, liver stiffness and ultrasound assessments were only available during off-treatment follow-up. At year 1,56 patients were still in off-treatment follow-up versus 32 patients at year 2. Paired liver stiffness measurements were available in 51 of the 56 (91%) patients for baseline and year 1, and in 25 of the 32 (78%) patients for baseline and year 2. Compared to baseline, the liver stiffness measurements were not significantly different at year 1 (5.7 vs.6.0 kPa; p=0.33) or year 2 (5.6 vs.5.8 kPa; p=0.57).Ultrasound was performed in 49 of the 56 (88%) patients at year 1 and in 23 of the 32 (72%) patients at year 2. At year 1 and 2, none of the patients developed ultrasonographic signs of cirrhosis.CONCLUSION1. We showed that HBV DNA elevation were common after NAs discontinuation in HBsAg positive patients.On multivariate analysis by COX harzard ratemodel, only the antiviral drugs were significantly associated with virological relapse. Patients treated with ETV had a lower risk of virological relapse.2. The pre-treatment HBeAg status had no effects on the development of virological and clinical relapse.3. On multivariate analysis by Cox proportional hazard model, only age was significantly assoiated with clinical relapse. Although the ent-of-treatment HBsAg levels had no significant association with clinical relapse, patients with end-of-treatment HBsAg levels≤200 IU/mL trended to be less likely to develop a clinical relapse.4. Our results shows,there were no signs of histological progression by the means of liver stiffness and ultrasound assessments after NAs discontinuation.5. The off-treatment HBV DNA levels and the persistent HBV DNA elevationg were the risk factors of biochemical relapse. Patients with an HBV DNA level>200,000 IU/mL should be retreated immediately regardless of the ALT level due to the high risk of biochemical relapse. Patients with an HBV DNA level between>2,000-≤200,000 IU/mL combined with ALT>2x ULN should be retreated in order to prevent further liver damage. In case of HBV DNA level between>2,000-≤200,000 IU/mL and ALT level≤2x ULN, consider retesting the HBV DNA level within 1-3 months. If HBV DNA is still>2,000 IU/mL at retest (persistent elevation), retreatment should be started due to the high risk of biochemical relapse, whereas patients with HBV DNA<2,000 IU/mL may continue off-treatment follow-up. |
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