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- Materials and methods
Background The efficacy of long-term adefovir dipivoxil monotherapy or combination of adefovir and lamivudine in hepatitis B e antigen (HBe-Ag)-negative lamivudine-resistant chronic hepatitis B (CHB) patients is still under investigation.
Aim To assess the safety and efficacy of the long-term adefovir treatment alone or in combination with lamivudine in HBe-Ag-negative CHB patients who had developed breakthrough because of lamivudine-resistant mutants.
Methods Fifty-nine patients received combination therapy, while 23 switched to adefovir alone after a 3-month course of combination therapy.
Results The median follow-up after adefovir’s onset was 31 (18–40) months. Baseline characteristics were similar between the two groups. At 12 and 24 months, 69% and 89% of patients receiving combination therapy and 73% and 82% of patients receiving adefovir monotherapy had serum HBV-DNA <104 copies/mL (P > 0.5). Normalization of alanine aminotransferase levels occurred in 81% and 79% of patients receiving combination vs. 61% and 53% receiving adefovir monotherapy at 12 and 24 months, respectively (P > 0.50). Virological breakthroughs because of adefovir-resistant mutants occurred in five patients under adefovir monotherapy and in none receiving combination therapy (P = 0.001). No one developed decompensated liver disease or hepatocellular carcinoma during follow-up. Re-introduction of lamivudine in adefovir-resistant patients achieved reduction in HBV-DNA and biochemical remission, but re-emergence of lamivudine mutants was observed in one patient after 7.5 months.
Conclusion In HBe-Ag-negative CHB patients with lamivudine resistance, adding adefovir to continuing lamivudine therapy maximizes anti-viral efficacy because of absence of viral resistance.
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- Materials and methods
Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) predominates in the Mediterranean area and Asia and has an increasing prevalence in Western Europe and North America.1–3 The HBeAg-negative CHB is characterized by intermittent periods of exacerbation and quiescence and often runs a more aggressive course resulting frequently in cirrhosis, liver failure and hepatocellular carcinoma (HCC).4, 5 The principal goal of therapy in patients with HBeAg-negative CHB is the sustained suppression of viral replication to arrest and reverse the progression of the liver injury.6
Current therapeutic approach of HBeAg-negative CHB includes treatment with interferon-alpha, lamivudine (LAM), adefovir dipivoxil (ADV), entecavir and telbivudine.7 The excellent safety and tolerability profile of LAM leads to long-term therapies which, however, have been associated with the emergence of viral resistance because of selection of resistant mutants. Clinical data showed that emergence of LAM-resistant mutations can be associated with hepatitis flares, hepatic decompensation and death.8, 9 ADV is active against not only wild, but also LAM-resistant HBV strains. In contrast to LAM, resistance to ADV seems to occur less frequently and later in the course of long-term treatment. Resistance to ADV is related mostly to the emergence of mutation at codon 236 (asparagine to threonine, rtN236T) or at codon 181 (alanine to valine, rtA181V) of the HBV polymerase gene. In naive patients receiving ADV, genotypic resistance is observed in 3% of patients at year 2, 11% at year 3, 18% at year 4 and 29% at year 5.10 Although in vitro susceptibility to ADV is reduced only by two- to 13-folds, ADV resistance can be associated with viral rebound and decompensation.11
Clinical studies have shown that virological and biochemical improvements are observed in LAM-resistant patients after the addition of ADV to ongoing LAM or with ADV monotherapy.12, 13 In a randomized-controlled trial in HBeAg-positive CHB, ADV alone and ADV plus LAM combination therapy were found to achieve similar reductions of serum HBV-DNA after 1 year of treatment.14 The efficacy of long-term ADV monotherapy or combination of ADV and LAM in HBeAg-negative CHB patients with LAM-resistant CHB is still under investigation.
When ADV was first available in an expanded access programme for patients with LAM resistance, two different strategies (adding vs. switching) were followed by the clinicians regarding the continuation of LAM. In this prospective nonrandomized study, we report the efficacy and safety of long-term LAM and ADV combination therapy compared with ADV monotherapy in HBeAg-negative CHB patients with LAM resistance. In addition, we determined the incidence and risk factors of resistance to ADV in this setting in clinical practice.
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- Materials and methods
Table 1 summarizes the baseline characteristics of patients in both groups. At ADV initiation, the demographic, clinical and virological characteristics did not significantly differ between two groups, except for a trend towards higher serum HBV-DNA and fibrosis levels in group 1. Seven (9%) patients had transaminase levels within normal ranges before starting ADV. Two patients were lost to follow-up during the second and third year. Both patients were receiving combination therapy and had undetectable serum HBV-DNA at the last visit. One patient receiving ADV monotherapy withdrew consent during the second year.
Table 1. Baseline characteristics of 82 patients with HBeAg-negative chronic hepatitis B and lamivudine resistance treated with adefovir plus lamivudine combination therapy or with lamivudine monotherapy
| ||Adefovir + lamivudine combination therapy (n = 59)|| Adefovir monotherapy (n = 23)|| P-value|
|Males (%) ||45 (76)||21 (91)||0.12|
|Age (years)||59 (33–79)||57 (25–71)||0.51|
|AST (IU/L)||90 (24–481) ||69 (19–453)||0.31|
|ALT (IU/L)||123 (35–935) ||125 (47–514)||0.62|
|Serum HBV-DNA (copies/mL) ||34 700 000 (44 300–1 040 000 000)||1 497 000 (1460–74 400 000)||0.07|
|Necroinflammatory activity*||9 (0–12)||9.5 (7–14)||0.22|
|Fibrosis stage*||4 (1–6)||2 (0–5)||0.06|
|Genotype D† (%)||14/14||12/13||0.7|
|Follow-up (months)||32 (18–40)||30 (18–40)||0.31|
No serious adverse effects were considered attributable to either study drug by the investigators in either group. No patient required a dose reduction.
At the time of this analysis, the median total duration of ADV treatment was 31 (range: 18–40) months. Prior to initiation of ADV, the median time of LAM monotherapy was 21 (range: 6–48) months.
At the initiation of ADV, serum HBV-DNA was >4 log10 copies/mL in 81 (99%), >5 log10 copies/mL in 80 (98%) and >6 log10 copies/mL in 53 (65%) of the 82 patients. Serum HBV-DNA concentrations did not differ between two groups at the end of the 3-month common treatment period (6.0 vs. 5.6 log10 copies/mL, P = 0.31). In the ADV and LAM combination group, median HBV-DNA reduction at 6 months was 2.9 log10 copies/mL (range: 0.34–5.59), at 12 months 3.2 (range: 0.86–6.15), at 18 months 4.2 (range: 1.6–6.4), at 24 months 4.6 (range: 1.9–6.4) and at 30 months 4.9 (range: 2–6). The proportion of patients with >4 log10 reduction of HBV-DNA at 12 and 24 months of therapy was 19 of 59 (32%) and 27 of 44 (61%), respectively. In the adefovir monotherapy group, median HBV-DNA reduction at 6 months was 2.5 log10 copies/mL (range: 0.56–4.6), at 12 months 3.2 (range: 0.56–5), at 18 months 3.4 (range: 0.17–4.9), at 24 months 3.1 (range: 0.56–5) and at 30 months 3.4 (range: 2.9–4.8). The proportion of patients with >4 log10 reduction of HBV-DNA at 12 and 24 months of therapy was three of 23 (13%) and four of 17 (24%), respectively. The effect of different treatment scheme on median serum HBV-DNA reduction became statistically significant at 18, 24 and 30 months (P = 0.01, P = 0.002, P = 0.03, respectively; Figure 1).
Figure 1. Median values of serum HBV-DNA in 82 patients with hepatitis B e antigen-negative chronic hepatitis B and lamivudine resistance during treatment with adefovir plus lamivudine combination therapy (n = 59) or with adefovir monotherapy (n = 23). The median follow-up after the onset of adefovir was 31 (18–40) months. No significant difference between the two groups was observed. However, the effect of different treatment scheme on median serum HBV-DNA reduction became statistically significant at 18, 24 and 30 months (P = 0.01, P = 0.002, P = 0.03, respectively).
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At 6 months, serum HBV-DNA was <10 000 copies/mL in 27 of 55 patients of group 1 and nine of 22 of group 2, while undetectable levels of serum HBV-DNA were reported in 15 and seven of the above patients, respectively. At 12 months, 40 of 58 patients of group 1 and 16 of 22 of group 2 had HBV-DNA <10 000 copies/mL, of whom undetectable levels of serum HBV-DNA were reported in 24 and 12 cases, respectively. At 24 months, 39 of 44 patients in group 1 and 14 of 17 in group 2 had HBV-DNA <10 000 copies/mL, of whom undetectable levels of serum HBV-DNA were reported in 33 and 12 of group 1 and 2, respectively (Table 2). Finally, 26 of 28 patients who continued combination therapy until month 30 had undetectable serum HBV-DNA. All patients with undetectable viraemia at 3, 6, 12, 18, 24 and 30 months of therapy had serum HBV-DNA ≤5 log10 copies/mL prior ADV initiation.
Table 2. Serum HBV-DNA levels at 3, 6, 12, 24 and 30 months of treatment with combination of adefovir plus lamivudine (n = 59) or with adefovir monotherapy (n = 23)
| ||Undetectable||<104 copies/mL ||>104 copies/mL ||Total|
|Adefovir + lamivudine combination therapy (n = 59; serum HBV-DNA)|
| 3 months||9||16||32||57|
| 6 months||15||12||28||55|
| 12 months||28||12||18||58|
| 24 months||33||6||5||44|
| 30 months||26||1||1||28|
|Adefovir monotherapy (n = 23; serum HBV-DNA)|
| 3 months||4||4||15||23|
| 6 months||7||2||13||22|
| 12 months||12||4||6||22|
| 24 months||12||2||3||17|
| 30 months||7||0||2||9|
Serum ALT levels declined in both treatment groups, with median ALT decreasing from 123 IU/L at baseline to 32.5, 26 and 28.5 IU/L at 6, 12 and 24 months in group 1 and from 125 IU/L at baseline to 33, 29 and 22.5 IU/L in group 2, respectively. At 6, 12 and 24 months, normal ALT was achieved in 41 of 59 (70%), 48 of 59 (81%) and 42 of 52 (79%) patients of group 1 and in 18 of 23 (78%), 14 of 23 (61%) and nine of 17 (53%) patients of group 2, respectively (Figure 2). ALT levels remained within normal levels in 27 of the 28 patients who continued combination therapy to month 30.
Figure 2. Percentage of patients with normal alanine aminotransferase levels during treatment with combination of adefovir plus lamivudine (n = 59) or with adefovir monotherapy (n = 23).
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Virological and biochemical breakthrough – Salvage therapy
Virological breakthrough after the onset of ADV developed in five patients (22%) of group 2 and in none of patients in group 1 (P = 0.001). The median time to VBR development was 15 (range: 12–33) months after ADV treatment was initiated. VBR were persistent in all patients during a median follow-up of 6 (0–12) months. At the onset of VBR, median ALT and serum HBV-DNA concentrations were 48 IU/L (28–162) and 1 726 000 copies/mL (25 000–26 535 468), respectively. All five patients who experienced VBR were men. There was no significant difference in age, gender, ALT levels and duration of LAM resistance period between group 2 patients with and without VBR (Table 3). However, patients in group 2 who developed VBR had significantly higher baseline HBV-DNA levels [40 000 000 (35 000 000–74 400 000) vs. 1 000 000 (1460–65 200 000), P = 0.005]. In addition, all patients with VBR had serum HBV-DNA >104 copies/mL at 6 months under ADV (median 4.9 log10, range: 4.7–5.6), while the reduction in HBV-DNA compared to baseline levels was <2.5 log10 in four of five patients. At the onset of VBR, specific ADV-resistant mutations were confirmed in all five patients. N236T was detected in three and A181V in two patients. Furthermore, no LAM-resistant strains were observed. The cumulative probability of ADV resistance was 9%, 13% and 17% at 12, 18 and 24 months of ADV therapy (Figure 3). VBR was followed by a rise in ALT in two of the five patients at a median of 3 months (range: 0–6) after onset of VBR. ALT levels increased by >3 times the upper limit of normal in one patient. No patient developed decompensated liver disease, while bilirubin levels increased to 4 mg/dL in one patient.
Table 3. HBeAg-negative chronic hepatitis B patients with lamivudine resistance treated with adefovir monotherapy
| ||ADV-R||No ADV-R||P-value|
|Age (years)||54 (39–71)||57.5 (25–68)||0.9|
|Duration of LAM resistance||11 (8–14)||69 (10–48)||0.1|
| ALT (IU/L)||198 (47–287)||108 (54–714)||0.4|
|Baseline serum HBV-DNA (copies/mL) ||40 000 000 (35 000 000–74 440 000)||1 000 000 (1460–65 200 000) ||0.005|
Figure 3. Cumulative probability of virological breakthrough in 82 patients with hepatitis B e antigen-negative chronic hepatitis B and lamivudine resistance treated with adefovir plus lamivudine combination therapy (n = 59, solid line) or with adefovir monotherapy (n = 23, dashed line) (P = 0.001). Numbers at risk at the beginning of each interval time are displayed.
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Salvage LAM therapy was initiated in four patients at a median of 6 (0–12) months after the diagnosis of VBR. We did not offer salvage treatment in only one patient with VBR (A181V mutation) who had no evidence of baseline cirrhosis, because of persistently normal levels of ALT and a stabilized serum HBV-DNA at or around 104 copies/mL. Two (one with A181V) patients were followed up for ≥6 months after salvage treatment. After a median follow-up of 7.5 months, combination of LAM plus ADV resulted in a >2.5 log10 reduction in serum HBV-DNA and normalization of transaminase levels in both patients. Sequencing analysis at the last visit revealed the re-emergence of the LAM-resistant mutant in one patient (L180M + M204V), while the second patient remained with the A181V ADV-resistant mutant strain.
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- Materials and methods
Lamivudine is an effective and well-tolerated treatment for patients with CHB, but has the major limitation of the development of drug-resistant mutants occurring at a rate of 16–32% during the first year of treatment and increasing by 15% with each year of additional treatment. Resistance development is equivalent to treatment failure.18 Therefore, it is clear that an alternative treatment with an agent without cross-resistance to LAM, such as ADV, is necessary to maintain anti-viral efficacy against wild, precore and YMDD mutant strains of HBV. The role, however, of continuing LAM treatment in patients with LAM-resistant HBV who are started ADV is a matter of controversy. A previous study by Peters et al. suggested that there may be no benefit of ADV plus LAM combination therapy compared to ADV monotherapy regarding ALT flares and viral suppression after 1 year in patients with HBeAg-positive CHB.14 In contrast to the above study, we have shown that in HBeAg-negative CHB patients with LAM resistance, adding on ADV is superior to adefovir monotherapy, as it is associated with no evidence of viral resistance and VBR up to 40 months. According to our data, VBR as a result of ADV-resistant mutants were observed in 22% of patients on ADV monotherapy arm compared to none of those receiving combination therapy. In accordance with our results, two recent studies from Italy and Greece reported that in HBeAg-negative patients who developed LAM resistance, ADV monotherapy is associated with a higher risk of ADV-resistant mutant strains development and ADV-related treatment failure.19, 20 The Italian study involving 588 patients from 31 centres reported ADV-related mutations in 5% in the ADV monotherapy group, while Rapti et al. involving 42 patients in a randomized study found a higher rate (21%) of ADV-related treatment failure because of development of ADV resistance mutations. The enhanced anti-viral efficacy of LAM plus ADV combination therapy in LAM-resistant patients may be explained by the fact that LAM hampers the emergence of ADV-resistant strains.
A recent study including 95 HBeAg-positive CHB patients showed that that ADV resistance is more common in patients with prior LAM resistance compared with naive patients after 48 weeks of therapy.21 We and others previously showed that clinical deterioration and death may occur very soon after development of BBR because of emergence of YMDD mutants under long-term LAM or ADV monotherapy in patients with HBV cirrhosis.8, 11 Furthermore, our data confirmed previous observations showing that salvage therapy with ADV, in patients with LAM resistance, achieves more rapid and higher rates of virological response, if ADV initiates at an early phase of LAM resistance with low levels of viral replication.9, 19, 22 Thus, close monitoring of serum HBV-DNA using a sensitive technique is crucial in patients under long-term anti-viral treatment to detect resistance and initiate salvage therapy promptly.
We found that combination of ADV and LAM was associated with a significantly higher reduction in serum HBV-DNA after 18 months of treatment, although the proportion of patients with undetectable HBV-DNA or viraemia <4 log did not differ between the two groups. The lower baseline serum HBV-DNA concentrations in group 2 compared with group 1 and the viral rebounds because of ADV-resistant mutations in group 2 might be responsible for the differences in the reduction in viraemia between the two groups.
Another important finding of this study is the association between baseline serum HBV-DNA, suboptimal response to ADV and breakthrough because of ADV-resistant mutations. Patients who developed VBR had higher HBV-DNA levels at the initiation of ADV and HBV-DNA >4 log10 copies/mL at 6 months of treatment; only one of them had serum HBV-DNA reduction of >2.5 log10 copies/mL during treatment. Previous retrospective analysis of patients with CHB receiving LAM or ADV have shown similar results indicating that high levels of viral replication may reduce the magnitude of HBV-DNA suppression and thus facilitate the emergence of drug resistance.10, 16 Therefore, an early initiation of additional therapy in patients at high risk of drug resistance could prevent resistance and may be a promising future therapeutic strategy.
Studies evaluating the treatment of patients who have developed resistance to ADV are lacking. In vivo and in vitro data have shown that LAM and entecavir may be promising drugs.23–25 According to our data, addition of LAM in patients who developed ADV-resistant mutants achieved biochemical response and reduction in viraemia. However, reintroduction of LAM was followed by re-emergence of LAM-resistant mutants in one patient after 7.5 months. Our results are compatible with the observations of Fung et al.11 stressing the problem in the treatment of patients who have acquired resistance to more than one nucleos(t)ide analogue. Although longer follow-up is required to establish the significance of our finding, in the era of newer anti-viral drugs, LAM may not be an optimal therapeutic option for LAM-experienced patients with ADV resistance.
In this study, our aim was to present the long-term efficacy of two different therapeutics strategies in patients with HBeAg-negative CHB and LAM resistance. However, it might be argued that as our study was not randomized, our results are prone to selection bias. The similarity, however, of the baseline characteristics of the patients between the two groups may overcome the weak points of this comparison. The fact that a trend towards higher baseline HBV-DNA levels was observed in patients under combination therapy could not feeble our results, because high HBV-DNA prior treatment is associated with higher risk of drug-induced mutations.16
In conclusion, our results clearly demonstrate that in HBeAg-negative CHB patients with LAM resistance, adding ADV to continuing LAM therapy maximizes anti-viral efficacy because of prevention of viral resistance. Baseline high viraemia levels and suboptimal response to ADV are associated with an increased risk of subsequent ADV resistance. Although our data suggest that combination of LAM plus ADV is the best therapeutic choice for HBeAg-negative CHB patients with LAM resistance, the optimal type and timing of combination therapy should be carefully assessed within well-designed studies, particularly in the forthcoming era of new stronger anti-viral agents.