Michelle Martinot, INSERM U-773, CRB3, Hôpital Beaujon, 100 Bd. du Général Leclerc, 92110 CLICHY, France
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Since its discovery by Blumberg in 1965, the hepatitis B virus antigen (HBsAg) is used as the fingerprint of hepatitis B infection. The HBsAg level is a reflection of the transcriptional activity of cccDNA. It is an important marker that not only indicates active hepatitis B infection but can also predict clinical and treatment outcomes. Assays for HBsAg quantification are fully automated and have high output. HBsAg titres are higher in HBe antigen (HBeAg)(+) than in HBeAg(−) patients and are negatively correlated with liver fibrosis in HBeAg(+) patients. In HBeAg(−) chronic hepatitis B, an HBsAg level <1000 IU/ml and an HBV DNA titre <2000 IU/ml accurately identify inactive carriers. During PEG-IFN treatment, HBsAg quantification is used to identify patients who will not benefit from therapy as early as week 12 on therapy, so that treatment may be stopped or switched- ‘week 12 stopping rule’. With nucleos(t)ide analogues (NA), the role of HBsAg quantification must be clarified. Several studies show that baseline and on-treatment HBsAg levels might identify patients that can be treated with no subsequent risk of reactivation. In clinical practice, HBsAg quantification is a simple and reproducible tool that can be used in association with HBV DNA to classify patients during the natural history of HBV and to monitor therapy.
The hepatitis B virus (HBV) is a small DNA virus. Upon entry into the cell, HBV sheds its protein coat and the partially double-strand genome is transported into the nucleus of the hepatocyte where it is transformed into a fully double-strand covalently closed circular DNA (cccDNA). The cccDNA resides in the nucleus of infected hepatocytes, where it acts as a template for transcription of the viral gene and recycles in the nucleus to renew the cccDNA pool [1, 2]. Viral proteins of clinical importance include the envelope protein (HBsAg) whose synthesis during the HBV viral life cycle is complex. HBsAg production exceeds that required for virion assembly, and excess surface envelope proteins are covalently linked and secreted as empty non-infectious filamentous or spherical sub-viral particles . These empty particles may co-exist with anti-HBs as part of circulating immune complexes . Serum HBsAg is a result of the combination of these proteins (complete virion, filamentous or spherical sub-viral particles). HBsAg quantification measures all three forms of systemic HBsAg.
Several studies have shown the relationship between intrahepatic markers of HBV infection (cccDNA and integrated HVB DNA) and serum HBsAg [3, 5-7]. Differences in HBsAg levels during the different phases of the disease reflect the distribution of cccDNA during the respective infection phases. HBsAg levels are higher in HbeAg-positive (+) than in HBeAg-negative (−) patients [6-8].
HBsAg seroconversion (loss of HBsAg and development of anti-HBs) is rarely observed during the natural course of chronic HBV infection. The annual incidence is 1–2% world-wide . It is the ultimate goal of therapy. Recently, quantitative serum HBsAg assays have been developed [9, 10], and the importance of HBsAg quantification has been recognized as an important marker to monitor the natural history in chronic hepatitis and predict treatment outcome [11-13]. HBsAg levels decrease more in patients receiving interferon (IFN), an immune modulator, than in those receiving nucleos(t)ides analogues (NA), potent inhibitors of HBV DNA replication .
HBeAg positive chronic hepatitis B
In the initial 2–3 decades of life, HBV infection is characterized by an immune tolerance phase in which patients usually have positive HBeAg, very high HBV DNA, normal ALT levels and minimal histological damage . This is followed by an immune-clearance phase, which may lead to HBeAg seroconversion. Based on the results of cross-sectional studies, serum HBsAg levels were generally higher in patients in the immune tolerance phase than in the immune-clearance phase [3, 6-8, 13] (Fig. 1).
HBeAg negative chronic hepatitis B
Later in life, most of the HBV-infected population progresses to the immune active phase, loses HBeAg and seroconverts to anti-HBe (with various degrees of activity and fibrosis). Then, most of this HBeAg (−) chronic hepatitis B (CHB) population enters the inactive or low replicative phase, (characterized by wide fluctuations in serum HBV DNA levels and transaminases . The clinical spectrum of HBeAg (−) CHB ranges from ‘inactive carrier’ status to aggressive HBeAg (−) CHB that is generally differentiated from ‘inactive carriers’ by serial serum ALT and HBV DNA level determinations [16, 17]. However, in 45–65% of cases, ALT activity can fluctuate with long periods of normal ALT levels, resulting in misclassification (Fig. 2). Inactive carriers have no or mild histological lesions in the liver with an excellent prognosis for survival and a low incidence of cirrhosis and HCC, while patients with HBeAg (−) CHB with fluctuating activity have a more severe disease progression with frequent cirrhosis [17-19]. Differentiating the latter from inactive carriers is highly important as these patients could benefit from therapy. According to NIH and EASL guidelines [20, 21], the differentiation between inactive and active phases of HBeAg (−) CHB is based on an HBV DNA cut-off of 2000 IU/ml. This cut-off has led to several controversial studies [17, 22-25].
Based on longitudinal studies, HBsAg levels are higher in patients with active HBeAg (−) CHB than in ‘inactive carriers’ [22-26].
A longitudinal Asian study in 68 HBeAg (−) CHB patients predominantly infected with genotype C reported that the patients with inactive disease tend to have lower HBsAg levels than those with active disease; 2.24 ± 1.61 log10 IU/ml vs. 2.98 ± 0.88 log10 IU/ml respectively (P = 0.054). No cut-off value can confidentially differentiate ‘inactive carriers’ . During long-term follow-up (median 10 years), the authors report that HBsAg <1000 IU/ml predicts seroclearance (91% specificity: 75% sensitivity) . A study performed by Brunetto et al.  in Italian genotype D patients reported that HBsAg levels are higher in patients with HBeAg (−) CHB than in ‘inactive carriers’ and that a single point quantification of HBV DNA <2000 IU/ml and HBsAg <1000 IU/ml identifies inactive carriers with a positive predictive value (PPV) of 88%. However, this observation must be validated across all HBV genotypes. Similar results were reported in a recent study from France  in 165 patients with HBeAg (−) CHB (genotypes A–E). HBsAg levels in this study were lower in the 76 ‘inactive carriers’ than in the 89 patients with an HBeAg (−) CHB; 3.25 ± 0.96 log10 IU/ml vs. 3.67 ± 0.70 log10 IU/ml respectively (P < 0.001). The combination of a single measurement of HBsAg <1000 IU/ml and HBV DNA <2000 IU/ml identifies ‘inactive carriers’ with a PPV of 86%. More recently, the authors reported that the combination of a single measurement of HBsAg >1000 IU/ml and HBV DNA >200 IU/ml identifies patients with a ‘high risk of reactivation’ with a negative predictive value (NPV) of 96%, and sensitivity 92% . The authors conclude that combination of HBsAg and HBV DNA levels at a single time point may accurately indentify HBeAg (−) CHB patients, during remission with a high probability of reactivation and who are good candidates for treatment.
HBsAg and liver histology
Seto et al.  found an association between higher HBsAg (≥25 000 IU/ml) serum levels and mild fibrosis (stage F ≤ 1) in a cohort of 140 Asian HBeAg (+) CHB patients (no indication of HBV genotype) with ALT ≤ 2 times the upper limit of normal. Similar results are reported in a cohort of 406 chronic hepatitis B patients with genotypes A–E . A negative correlation was found between the level of HBsAg and the stage of fibrosis, so that patients with no or mild fibrosis (F 0–1 Metavir score) could be distinguished from those with moderate or severe fibrosis (≥ F2), in HBeAg (+) patients, with a high NPV (91%). Furthermore, the authors describe a specific serum HBsAg cut-off (3.85 log10 IU/ml) to identify moderate to severe fibrosis (≥ F2) in HBeAg (+) CHB patients infected with HBV genotypes B or C. This association was not observed in HBeAg (−) patients . There was no association between HBsAg titre and histological grade found in either study.
HBsAg level and therapy
Only a small proportion of patients (3–7%) experience HBsAg loss during 48 weeks of interferon-based (IFN) therapy . The efficacy of pegylated-IFN (PEG-IFN) has been confirmed in two large pivotal studies [29, 30]. The HBeAg seroconversion rate was observed in 32% in HBeAg-positive patients  and an HBV DNA <400 IU/ml in 19% in HBeAg-negative patients . In the early 1990's, HBsAg quantification was already considered to be a promising, simple and inexpensive method to monitor viral replication in chronic hepatitis B patients who were receiving IFN treatment . More recently, the availability of well-standardized commercial assays has renewed interest in the quantitative serum HBsAg as a biomarker for treatment response in chronic hepatitis B [9, 10].
Current data [32-37] indicate that on-treatment HBsAg quantification could help identify either patients with a high probability of sustained virological response (SVR) or non-responders. In a study by Chan et al. , SVR rates were 62 and 11% in patients with HBsAg ≤300 IU/ml and in those with HBsAg >300 IU/ml at 24 weeks of therapy respectively. The authors also showed that patients with a combined response of HBsAg ≤300 IU/ml and a decrease of ≥1 log10 IU/ml at 24 weeks had higher SVR rates than those without the combined response (75% vs. 15%), with a PPV and NPV of 75 and 85% respectively. Tangkijvanich et al.  report that baseline HBsAg levels were lower in patients with HBeAg seroconversion at the end of therapy than in non-responders. In the NEPTUNE study [35, 37] the highest HBeAg seroconversion rates were observed in patients with HBsAg levels ≤ 1500 IU/ml at 12 or 24 weeks of therapy with a PPV of 57 or 54% and a NPV of 72 or 76% respectively. These results confirm those of the phase III PEG-IFN alpha 2a treatment registration trial on . Sonneveld et al.  showed that patients who received a combination of PEG-IFN plus lamivudine had a more pronounced on-treatment decrease in HBsAg patients than those who received PEG-IFN alone, although the former relapsed. The absence of decline at week 12 had a NPV of 97% for SVR and no chance of HBsAg loss. The phase III registration trial  by Lau et al. reported a lower NPV (82%) than the study by Sonneveld et al. The discrepancies between the two studies [34, 36] might be owing to the different populations studied. Indeed, in the study by Sonneveld et al., most of the patients had genotypes A and D, whereas the Lau study included genotypes B and C Asian patients. It is interesting to note that certain studies have reported a more significant decrease in HBsAg and more frequent HBsAg in patients with HBV genotype A who receive PEG-IFN [38-40].
The response rate to PEG-IFN is low (<20%) in HBeAg (−) patients [41-44]. These patients are difficult to monitor. Indeed, most of them achieve undetectable serum HBV DNA at the end of therapy, but relapse after treatment is stopped [30, 43, 44]. Therefore, predictive factors of on-treatment response may help define more appropriate treatment strategies in certain patients. The most recent evidence suggests that HBsAg quantification is a worthwhile marker for monitoring PEG-IFN therapy [45-47] (Fig. 3).
Rijckborst et al.  reported that the combination of HBsAg and a decrease in HBVDNA is the best predictor of an SVR in patients receiving PEG-IFN. A lack of decrease in HBsAg and a serum HBVDNA decline of less than 2 log10 IU/ml have a NPV of 100% for SVR. Another  study has shown that a serum HBsAg decrease of ≥ 0.5 log IU/ml at week 12 had a high PPV of SVR (PPV 89%) and a lack of decrease of ≥1 log10 IU/ml at week 24 had a high NPV of non-SVR (NPV 92%).
Studies have investigated end- and post-treatment HBsAg titres to predict SVR and loss of HBsAg during post-treatment follow-up. In a cohort of 127 HBeAg-negative genotype D patients who received 48 weeks of PEG-IFN treatment, Brunetto et al.  showed that an on-treatment ≥1 log10 IU/ml HBsAg decrease and an HBsAg level <10 IU/ml at the end of therapy were highly predictive for a SVR at the 24-week post-treatment follow-up and for HBsAg loss at the 3-year post-treatment follow-up. In this study, at the 3-year post-treatment follow-up, an HBsAg loss was observed in 52% of the patients with an HBsAg <10 IU/ml at week 48 of treatment, compared with only 2% of the patients with an HBsAg>10 IU/ml at week 48. The authors did not find any association between serum HBV DNA response (<400 IU/ml) at the end of therapy and HBsAg loss. A French retrospective study  also reported that most of the patients who lost HBsAg during post-treatment follow-up had a ≥1 log10 IU/ml decrease in HBsAg levels at the end of 48 weeks of IFN treatment followed by a steady decrease until seroclearance.
Week 12 stopping rule
Most studies report that the absence of a decrease in HBsAg or a HBV DNA decline of < 2 log10 after 12–24 weeks of a 48-week course of PEG-IFN is associated with a NPV of 84–100% for SVR  (Table 1). Rijckborst et al.  confirmed these results in patients in the PARC study  and performed external validation in the phase III registration study  and the PegBeLiver study , both of which included patients with HBV genotypes A–D. There was a NPV ≥95% for SVR in both validation studies in the absence of a decrease in HBsAg or a decline in HBV DNA <2 log10 IU/ml at week 12 of treatment. The authors propose a response-guided therapy algorithm based on HBsAg kinetics at week 12 of treatment. Early identification of non-responders would allow discontinuation of therapy and/or changing the treatment strategy (Table 1).
Table 1. Sustained virological response (SVR) as predicted by serum HBsAg at week 12 and 24 of treatment
Associated with < 2 log decrease in HBV DNA from baseline.
The treatment paradigm for HBV has shifted in the past decade from a finite duration of treatment with IFN to long-term HBV suppression with nucleos(t)ides analogues (NA). Indeed, the newer agents such as tenofovir or entecavir are more effective in suppressing HBV and are less likely to be associated with drug resistance than original NA. In addition, they are orally administered and have excellent safety profile. However, they must probably be taken indefinitely because withdrawal is generally associated with viral reactivation, and HBsAg seroconversion is rarely reported. Although HBVDNA becomes rapidly undetectable, studies have clearly shown that the decline in HBsAg titres is significantly lower with NA therapy than with IFN-based treatment [57, 58] (Fig. 4). Several studies have reported that baseline HBsAg levels and on-treatment HBsAg quantification are good predictive markers of the end of treatment response and SVR [57-66]. Zoutendijk et al.  investigated HBsAg kinetics in patients who were successfully treated with long-term entecavir (ETV) or tenofovir (TDF). The authors used linear mixed regression analysis of individual HBsAg declines to estimate the duration of therapy required to achieve an HBsAg decline of 1 log10 UI/ml from baseline and HBsAg clearance. They showed that the median durations of therapy to achieve a 1 log10 IU/ml decrease were as follows: 6.6 [1.7–18] years and 8 [0.5–15] years in HBeAg (+) and HBeAg (−) patients respectively. Median durations for HBsAg clearance were as follows: 36 [10–93] years and 39 [1.3–90] years in HBeAg (+) and HBeAg (−) patients respectively. These results show the importance of determining HBsAg cut-offs to discontinue NA therapy with lowest risk of reactivation.
In the two pivotal studies of TDF [68, 69], study 102 (HBeAg −) and study 103 (HBeAg +) patients, the authors confirm that HBsAg kinetics is deeper in HBeAg (+) patients than in HBeAg (−) and in patients receiving TDF monotherapy. The only patients with HBsAg loss were HBeAg (+) patients with a ≥2 log10 IU/ml HBsAg decrease from baseline at 24 weeks of therapy, a higher baseline HBsAg level and genotypes A or D. In a cohort of 162 HBeAg (+) patients with HBV DNA <60 IU/ml after 3 years of telbuvidine therapy, Wursthorn et al.  showed that 25% of patients with ≥1 log10 IU/ml HBsAg decrease after 1 year of therapy had an HBsAg loss compared with 1.4% of patients with <1log10 IU/ml HBsAg decrease. The study by Reijnders et al.  in HBeAg (+) or (−) patients, who received 48 weeks of either PEG-IFN or ETV, an HBsAg decline was only observed in HBeAg (+) patients after 48 weeks of either PEG-IFN or ETV therapy. In HBeAg (−) patients, no decline in HBsAg levels was observed in patients receiving ETV. Recent studies including treatment-naive patients receiving NA report that low baseline HBsAg levels and an early decline in HBsAg (24-week therapy) are good predictors of SVR [63-66]. Furthermore, studies suggest that an HBsAg cut-off ≤2–3 log10 IU/ml could be used for treatment discontinuation [70-73]. In the study by Liang et al. , 121 consecutive patients were prospectively recruited after treatment was stopped (lamivudine, adefovir, entecavir). The authors report that higher HBsAg levels at the end of treatment were associated with higher risk of relapse. End of the treatment, HBsAg levels ≤ 3 log10 IU/ml and ≤ 2 log10 IU/ml were associated with a 31 and 9% relapse respectively. Similar results are reported by Cai et al.  in patients receiving 2 years of telbuvidine therapy. An HBsAg level ≤2 IU/ml at the end of treatment cessation had a PPV and NPV for the prediction of SVR of 100 and 93% respectively.
Recently, it has been proposed that in HBe negative-patients the combination of HBsAg level < 1000 IU/ml and HBV DNA < 2000 IU/ml can be considered as a probable criteria to define the minimal risk for hepato-cellular carcinoma (74).
Recent studies on serum HBsAg monitoring show that HBsAg levels change during the natural course of chronic hepatitis B and during ongoing therapy. These results can be used to determine the best management strategy for patients. During the natural history of HBV, HBsAg quantification can be used to differentiate between inactive carriers (no need for treatment) and HBeAg (−) chronic hepatitis B patients, who are likely to reactivate (closer monitoring) and who can benefit from therapy. During PEG-IFN therapy, early HBsAg monitoring could be used to develop a response-guided algorithm: to stop or switch therapy at week 12 in poor responders, to continue standard 48-week treatment in most patients with a favourable response and to extend therapy for intermediate on-treatment responders to improve the chances of response. The role of HBsAg monitoring during NA therapy must be clarified. The development of stopping rules should be determined in these life-long therapies. Several studies suggest that baseline and on-treatment HBsAg levels might help identify patients who can stop therapy with no risk of reactivation.
Tarik Asselah is a speaker and investigator for BMS, Boehringer-Ingelheim, Tibotec, Janssen, Gilead, Roche and Merck. Patrick Marcellin is a speaker and investigator for BMS, Boehringer-Ingelheim, Tibotec, Janssen, Gilead, Roche and Merck.