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Adefovir Resistance: clinical outcomes
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"Adefovir-resistant hepatitis B can be associated with viral rebound and hepatic decompensation"
Journal of Hepatology
Article in press July-2005, Oct 6 2005
See EDITORIAL I just distributed. All articles are posted to NATAP website.
Scott K. Funga, Pietro Andreoneb, Steve H. Hanc, K. Rajender Reddyd, Arie Regeve, Emmet B. Keeffef, Munira Hussaina, Carmela Cursarob, Pamela Richtmyera, Jorge A. Marreroa, Anna S.F. Loka
a Division of Gastroenterology, University of Michigan, Medical Center, 3912 Taubman Center, Ann Arbor, MI 48109-0362, USA
b Department of Internal Medicine and Hepatology, University of Bologna, Bologna, Italy
c University of California at Los Angeles, Los Angeles, CA, USA
d University of Pennsylvania, Philadelphia, PA, USA
e University of Miami, Miami, FL, USA
f Stanford University, Palo Alto, CA, USA
..... In this study, we have identified 8 patients with adefovir-resistant HBV mutations.... The aim of this report is to describe the clinical course of patients with adefovir-resistant HBV infection and to highlight that viral rebound and fatal hepatic decompensation can occur despite small (2-13-fold) reductions in sensitivity of adefovir-resistant mutations in in vitro studies..... In this series, viral rebound with serum HBV DNA levels >5log10 copies/ml was observed in 7 of 8 patients, and biochemical breakthrough with ALT >2 X upper limit of normal (ULN) was noted in 4 patients including 1 patient who had a hepatitis flare with ALT >10 X ULN.....4 patients had cirrhosis & a 4th had a liver transplant with recurrent HBV... Data from this case series demonstrate that viral rebound, hepatitis flares and hepatic decompensation can occur in patients with adefovir-resistant HBV despite minor changes in in vitro sensitivity to adefovir. Thus, surveillance for antiviral resistance is needed in patients receiving adefovir, so that salvage treatment can be implemented. Further studies are needed to determine the incidence of genotypic resistance to adefovir in clinical practice and the risk factors associated with adefovir resistance. Additional studies are also needed to delineate the clinical course of patients with adefovir resistance and the optimal management of these patients.......
".....Our experience suggests that prior lamivudine resistance may increase the risk of adefovir resistance, especially if lamivudine is discontinued. If our findings are confirmed, current treatment guidelines that include a switch from lamivudine to adefovir with a short overlap period in patients with lamivudine resistance need to be revised..... Our data also suggest that patients with a suboptimal response to adefovir have an increased risk of resistance and should be considered for additional therapy......
......There is no consensus on the management of patients with adefovir-resistant HBV infection. Data from our series as well as previous reports confirmed that lamivudine has in vivo efficacy [12,16], but the durability of viral suppression is not known in patients with prior lamivudine resistance. One of our patients (no. 8) who resumed lamivudine while adefovir was continued had 2log10 decrease in HBV DNA but rapid redetection of lamivudine-resistant mutations that differ (rtL180M+rtM204V) from those prior to adefovir therapy (rtM204I). In addition, this patient also had reversal of the adefovir-resistant mutation rtN236T but selection of a new adefovir-resistant mutation rtA181V after reintroduction of lamivudine. The simultaneous presence of lamivudine-resistant and adefovir-resistant mutations has been reported by other investigators [28], raising concerns that sequential monotherapy will lead to selection of multi-resistant HBV. Another patient (no. 3) had increasing HBV DNA 6 months after lamivudine was added. Two patients responded to entecavir, with a rapid reduction in HBV DNA and improvement in ALT levels. To our knowledge, this is the first report of in vivo efficacy of entecavir in patients with adefovir resistance. Both patients had discontinued lamivudine for 12-20 months with no detectable lamivudine-resistant mutation at the onset of entecavir therapy. Nevertheless, in view of recent reports that patients with prior lamivudine resistance who continued to receive lamivudine have an increased risk of entecavir resistance, these two patients need to be monitored for resistance to entecavir [29]. Three patients were switched to tenofovir but the short follow-up in two patients and the presence of confounders (liver transplantation, HBIG and lamivudine) in the other patient prevented meaningful interpretation of the efficacy of tenofovir. Tenofovir is structurally similar to adefovir and is not expected to be effective against adefovir-resistant HBV. However, in clinical practice, tenofovir appears to have more potent viral suppression possibly because of the use of a higher dose, and may provide temporary benefit......"
ABSTRACT
Background/Aims
The susceptibility of adefovir-resistant hepatitis B virus (HBV) mutants is only reduced by 3-10-fold in in vitro studies, suggesting that virologic breakthrough and clinical deterioration are unlikely. The aim of this study was to describe the clinical course of patients with adefovir-resistant HBV infection.
Methods
Testing for adefovir-resistant mutations was performed on patients who had a suboptimal response or virologic breakthrough on adefovir. Adefovir-resistant mutations were detected using a line probe assay and direct sequencing of the HBV P-gene.
Results
Eight male patients with pre-existing lamivudine resistance or breakthrough (mean age 47±13 years) were found to have adefovir-resistant mutations rtA181V/T or rtN236T. Baseline median ALT was 66IU/L (range, 27-1161) and median HBV DNA 7.9log10 copies/ml (range, 6-8.3). At the time of adefovir resistance (mean of 20±9 months), HBV DNA increased to ≥5log10 copies/ml in 7 patients. After detection of adefovir resistance, hepatic decompensation occurred in 2 patients, 1 of whom died. Salvage therapy with lamivudine, entecavir or tenofovir was given to 7 patients and a reduction in HBV DNA by ≥3log10 was seen in 3 patients.
Conclusions
In conclusion, adefovir resistance can be associated with significant viral rebound and hepatic decompensation which may be fatal.
Introduction
Selection of antiviral-resistant mutations during treatment of chronic hepatitis B virus (HBV) infection is an expected consequence due to the high rate of HBV replication, lack of proof reading during reverse transcription from pregenomic RNA to HBV DNA, and incomplete viral suppression [1,2]. Lamivudine-resistant mutations in the HBV polymerase gene have been detected in 15-30% after 1 year of treatment and in 70% after 5 years of lamivudine therapy [3-5]. In vitro studies showed that lamivudine-resistant mutations (methionine to isoleucine or valine rtM204I/V with or without leucine to methionine substitution rtL180M) are associated with a 1000-10,000-fold decrease in susceptibility [6]. Clinical studies showed that emergence of lamivudine-resistant mutations can be associated with marked viral rebound, increases in alanine aminotransferase (ALT) levels, hepatitis flares, and rarely hepatic decompensation and death from liver failure [7,8].
Resistance to adefovir is believed to be uncommon and to occur later in the course of treatment compared to lamivudine [9]. Extensive testing of 498 patients in phase III clinical trials failed to detect any adefovir-resistant mutation after 48 weeks of treatment [10]. Lower rates of resistance to adefovir may be related to a high degree of structural similarity between the adefovir molecule and the native substrate (dATP), thereby limiting steric discrimination by the viral polymerase. Additionally, adefovir contains an acyclic phosphonate bond that renders the molecule less susceptible to chain terminator removal once incorporated into nascent viral DNA [10]. The exact mechanism of adefovir resistance has not been elucidated [11].
Two novel mutations in the HBV polymerase gene, alanine to valine or threonine (rtA181V/T) and asparagine to threonine substitution (rtN236T), were recently reported to confer resistance to adefovir [12]. These mutations have been detected in 4-6% patients during years 2-3 of adefovir treatment [9]. In vitro testing indicated that susceptibility of rtA181V/T and rtN236T was only reduced by 2-4 and 7-13-fold, respectively, suggesting that marked increases in serum HBV DNA level and clinical deterioration are unlikely in patients with adefovir-resistant HBV [13]. Recently, other mutations in the HBV polymerase have been identified in patients who have had a suboptimal response to adefovir [11].
In vitro studies showed that adefovir-resistant HBV remains sensitive to lamivudine and entecavir [14,15]. There are very little data regarding the in vivo efficacy of these salvage therapies, particularly the durability of viral suppressive effects of additional lamivudine therapy in patients with prior lamivudine resistance. There is also a paucity of data on the clinical course of patients with adefovir-resistant HBV [12,16]. The aim of this report is to describe a case series of 8 patients found to have adefovir-resistant HBV mutations.
Patients
Since January 2003, testing for adefovir-resistant HBV mutation was performed in all patients who had received adefovir for ≥12 months and had evidence of a suboptimal virologic response (≦2log10 copies/ml reduction in HBV DNA or HBV DNA ≥4log10 copies/ml after 12 months of therapy) or virologic breakthrough (≥1log10 increase in HBV DNA after an initial virologic response). The study included patients who received adefovir for lamivudine resistance or breakthrough as well as patients who received adefovir as de novo treatment for hepatitis B. Twenty-eight patients met the criteria of suboptimal response or virologic breakthrough, but only 8 patients were found to have adefovir-resistant mutations. Four of the 8 patients (50%) were enrolled in the National Institutes of Health sponsored HBV liver transplant study which is conducted in 15 liver centers in the United States, 3 (38%) attended the University of Michigan Liver Clinic and 1 (12%) attended the University of Bologna in Italy. Among the 20 patients who were not found to have adefovir-resistant mutation, 6 patients were switched to tenofovir, the other 14 continued on adefovir and had no evidence of viral rebound. None of these 20 patients has developed hepatic decompensation or died.
Written informed consent was obtained from each patient and approval for this study was obtained from the Institutional Review Board at each site. Clinical and laboratory data of each patient were reviewed. Serial serum samples collected at the time of initiation of adefovir, every 3-6 months during adefovir treatment, and every 3 months after the detection of adefovir resistance were stored frozen at -80¡C, and tested in a central laboratory at the University of Michigan.
Results
Baseline characteristics
Eight male patients with a mean age 47±13 years were found to have adefovir-resistant mutations between September 2003 and January 2005. All had been previously treated with lamivudine for a mean of 29±12 months. One patient (no. 7) had previously received adefovir in a phase III trial for 1 year but genotypic testing revealed no evidence of adefovir-resistant mutation at the end of treatment. All patients were started on adefovir because of confirmed genotypic resistance to lamivudine or breakthrough infection during lamivudine therapy. At the start of adefovir, median ALT was 66IU/L (range, 27-1161IU/L) and median HBV DNA was 7.9log10 copies/ml (range, 6-8.3log10 copies/ml). Five (63%) patients were HBeAg-positive and 2 had received liver transplants. One transplant patient (no. 8) developed recurrent hepatitis B after liver transplantation despite receiving hepatitis B immune globulin (HBIG) and lamivudine prophylaxis. Seven (88%) patients were directly switched to adefovir with less than 1month of overlap with lamivudine, whereas 1 (12%) patient (no. 8) continued to receive lamivudine in combination with adefovir for 7 months. All patients received adefovir 10mg daily; no patient required a dose reduction.
Clinical and virological impact of adefovir-resistant mutations
The clinical course of 4 representative patients with adefovir resistance is illustrated in Fig. 1. Based on HBV DNA ≥4log10 copies/ml after 12 months of adefovir, 6 patients met the criteria of a suboptimal response. Three patients had initial viral suppression of >3log10 copies/ml, 2 of whom were noted to have virologic breakthrough; the other patient (no. 2) did not have virologic breakthrough but continued to have HBV DNA >4log10 copies/ml after 1 year of treatment. Adefovir-resistant mutations including rtA181V/T and rtN236T were first detected at a mean of 20±9 months after starting treatment (Table 2). Median ALT was 66IU/L (range, 6-906IU/L) and median HBV DNA was 6.7log10 copies/ml (range 4.4-9.8log10 copies/ml). During a median follow-up of 6 months (range, 2-12 months) after the detection of adefovir-resistant mutation, viral rebound with HBV DNA ≥5log10 copies/ml was observed in 7 (88%) patients, and ALT flares ≥2 times upper limit of normal (X ULN) occurred in 4 (50%) patients. One patient (no. 5, Fig. 1A) had a flare in ALT to 906 IU/L but was asymptomatic and had normal bilirubin level. Decompensation of liver disease occurred in 2 (25%) patients (nos. 1, 6). Patient no. 6 with known esophageal varices had a variceal hemorrhage, from which he recovered after a brief hospitalization. The other patient had a rise in ALT and bilirubin levels while being treated for osteomyelitis. Despite switching to tenofovir, he had worsening liver failure evidenced by rising bilirubin levels, worsening coagulopathy and renal failure and died 2 months later.
Fig. 1. Clinical course and response to salvage therapy.
The clinical course and response to salvage therapy is illustrated for four representative patients: A, patient 5; B, patient 6; C, patient 7; and D, patient 8. HBV DNA levels in solid lines and ALT levels in dashed lines are plotted against time in months from the start of antiviral therapy. HBV polymerase gene mutations are displayed in the panel above each graph. S gene changes are shown for patient 8 only. Antiviral therapy is also shown above each graph; rectangles indicate therapy of defined duration, while arrows indicate previous or ongoing treatment. The upper limit of normal for ALT is 40IU/L. LAM, lamivudine; ADV, adefovir; TDF, tenofovir; ETV, entecavir; HBIG, hepatitis B immune globulin; rt, reverse transcriptase; LT, liver transplantation; PCR, polymerase chain reaction
HBV S-gene changes included leucine to phenylalanine substitution (sL173F) in 5 patients who had adefovir-resistant mutation rtA181V. In one transplant patient (no. 8), glycine to arginine substitution (sG145R) was detected while receiving HBIG (Fig. 1D). HBIG- and lamivudine- resistant mutations were no longer detected when adefovir resistance was diagnosed, 42 and 35 months after HBIG and lamivudine was discontinued, respectively. Serial testing revealed reemergence of lamivudine-resistant mutations, rtL180M+ rtM204V and the replacement of rtN236T by rtA181V, 6 months after the reintroduction of lamivudine.
Response to salvage therapy
Salvage therapy was initiated in 7 patients using lamivudine (2 patients), entecavir (2 patients), tenofovir (2 patients), and lamivudine plus tenofovir (1 patient) (Table 3: in two patients receiving entecavir HBV DNA was reduced by -5.5 & -4.3 logs, resptively. Patient 6 had prior Lamivudine experience & mutation rtN236T; therapy with Lamivudine+Tenofovir+LT resulted in -6.1 log HBV DNA reduction). Salvage therapy was withheld in 1 patient (no. 2), in whom ALT levels have remained near normal and HBV DNA levels have stabilized. One patient (no. 4) who received a liver transplant 1month after the detection of adefovir resistance is receiving prophylaxis with HBIG, lamivudine and tenofovir and has no evidence of recurrence of HBV 6 months post-transplant. Of the 5 patients in whom ≥6 months of follow-up was available, 3 (60%) patients (2 treated with entecavir and 1 with lamivudine plus tenofovir) have had ≥3log10 reduction in HBV DNA levels and improvement in ALT. One patient who was re-started on lamivudine in addition to ongoing adefovir continued to have elevated serum HBV DNA levels at the last follow-up.
Discussion
In this study, we have identified 8 patients with adefovir-resistant HBV mutations. Because of the heterogeneity in underlying liver disease and patient management as well as referral bias, we are unable to determine the rate of adefovir resistance. Further studies of the rate of adefovir resistance are currently underway in two cohorts of patients followed at the University of Michigan Liver Clinic and the National Institutes of Health sponsored HBV Liver Transplant Study. The aim of this report is to describe the clinical course of patients with adefovir-resistant HBV infection and to highlight that viral rebound and fatal hepatic decompensation can occur despite small (2-13-fold) reductions in sensitivity of adefovir-resistant mutations in in vitro studies.
In this series, viral rebound with serum HBV DNA levels >5log10 copies/ml was observed in 7 of 8 patients, and biochemical breakthrough with ALT >2 X upper limit of normal (ULN) was noted in 4 patients including 1 patient who had a hepatitis flare with ALT >10 X ULN. In two patients, adefovir resistance was associated with hepatic decompensation. One patient developed variceal bleeding; the other patient died from hepatic failure, although sepsis may have contributed to his demise. Thus, as is the case with lamivudine resistance, patients with adefovir-resistant HBV, particularly those with cirrhosis or immunosuppression need to be monitored very closely for hepatitis flares and hepatic decompensation.
Clinical trials of nucleoside-naive patients receiving adefovir monotherapy have shown that adefovir resistance is uncommon: 0% in year 1, 3% in year 2 and 6% in year 3, while studies of patients with prior lamivudine resistance have reported even lower rates of adefovir resistance, but >90% patients in these studies continued to receive lamivudine [9,20-23]. In a study of 324 transplant patients with lamivudine resistance, no resistance to adefovir was detected after 48 weeks of treatment [24]. In another report, in 167 patients who had lamivudine resistance and most of whom continued to receive lamivudine in combination with adefovir, the cumulative probability of adefovir resistance after years 1, 2, 3, and 4 were 0, 3, 11 and 18%, respectively, and all cases of resistance arose in patients who received adefovir monotherapy [25]. Adefovir resistance was detected in all patients after the first year (mean, 20 months) but variable sampling in some patients may have led to delays in the first detection of adefovir-resistant mutations. In our case series, all 8 patients had received prior lamivudine treatment with documented breakthrough infection or confirmed genotypic resistance. One patient continued to receive lamivudine in combination with adefovir but the overlap only lasted 7 months; the other 7 patients had 0-1month of overlapping treatment. To date, we have not found adefovir-resistant mutation in lamivudine-naive patients or in patients with prior lamivudine resistance who continued to receive lamivudine and adefovir (unpublished data). Our experience suggests that prior lamivudine resistance may increase the risk of adefovir resistance, especially if lamivudine is discontinued. If our findings are confirmed, current treatment guidelines that include a switch from lamivudine to adefovir with a short overlap period in patients with lamivudine resistance need to be revised.
Suboptimal virologic response has been reported to be associated with an increased risk of antiviral resistance. In a retrospective analysis of 159 nucleoside-naive chronic hepatitis B patients who received lamivudine, lamivudine-resistant mutations were detected in 86/136 (63%) patients who had serum HBV DNA level above 3log10 copies/ml at month 6 compared to 3/23 (13%) patients who had serum HBV DNA <3log10 copies/ml [26]. In our current series, 6 patients had serum HBV DNA >4log10 copies/ml after at least 12 months of adefovir treatment and only 3 (38%) patients had a maximum viral suppression >3log10 copies/ml. A retrospective analysis of HBeAg-positive patients who participated in a phase III clinical trial of adefovir found that suboptimal virologic response was common, after 48 weeks of treatment serum HBV DNA was suppressed by <2.2log10 in 25% and by 2.2-3.5log10 in another 25% of patients [27]. Our data also suggest that patients with a suboptimal response to adefovir have an increased risk of resistance and should be considered for additional therapy.
There is no consensus on the management of patients with adefovir-resistant HBV infection. Data from our series as well as previous reports confirmed that lamivudine has in vivo efficacy [12,16], but the durability of viral suppression is not known in patients with prior lamivudine resistance. One of our patients (no. 8) who resumed lamivudine while adefovir was continued had 2log10 decrease in HBV DNA but rapid redetection of lamivudine-resistant mutations that differ (rtL180M+rtM204V) from those prior to adefovir therapy (rtM204I). In addition, this patient also had reversal of the adefovir-resistant mutation rtN236T but selection of a new adefovir-resistant mutation rtA181V after reintroduction of lamivudine. The simultaneous presence of lamivudine-resistant and adefovir-resistant mutations has been reported by other investigators [28], raising concerns that sequential monotherapy will lead to selection of multi-resistant HBV. Another patient (no. 3) had increasing HBV DNA 6 months after lamivudine was added. Two patients responded to entecavir, with a rapid reduction in HBV DNA and improvement in ALT levels. To our knowledge, this is the first report of in vivo efficacy of entecavir in patients with adefovir resistance. Both patients had discontinued lamivudine for 12-20 months with no detectable lamivudine-resistant mutation at the onset of entecavir therapy. Nevertheless, in view of recent reports that patients with prior lamivudine resistance who continued to receive lamivudine have an increased risk of entecavir resistance, these two patients need to be monitored for resistance to entecavir [29]. Three patients were switched to tenofovir but the short follow-up in two patients and the presence of confounders (liver transplantation, HBIG and lamivudine) in the other patient prevented meaningful interpretation of the efficacy of tenofovir. Tenofovir is structurally similar to adefovir and is not expected to be effective against adefovir-resistant HBV. However, in clinical practice, tenofovir appears to have more potent viral suppression possibly because of the use of a higher dose, and may provide temporary benefit [30].
Data from this case series demonstrate that viral rebound, hepatitis flares and hepatic decompensation can occur in patients with adefovir-resistant HBV despite minor changes in in vitro sensitivity to adefovir. Thus, surveillance for antiviral resistance is needed in patients receiving adefovir, so that salvage treatment can be implemented. Further studies are needed to determine the incidence of genotypic resistance to adefovir in clinical practice and the risk factors associated with adefovir resistance. Additional studies are also needed to delineate the clinical course of patients with adefovir resistance and the optimal management of these patients.
HBV DNA Quantification
All samples were shipped to the central laboratory and tested for HBV DNA using a PCR assay, COBAS Amplicor HB Monitor Assay (Roche, Branchburg, NJ), with a lower limit of detection of 200 copies/ml.
Detection of antiviral-resistant mutations
All samples shipped to the central laboratory that tested positive for HBV DNA were tested for antiviral-resistant HBV mutations. HBV DNA was extracted using methods previously described [17,18]. The presence of lamivudine- and adefovir-resistant mutations was determined by a line probe assay (InnoLiPA DR2 assay; InnoGenetics Inc, Ghent, Belgium) [19]. The DR2 assay is similar to the DR1 assay that detects lamivudine-resistant mutations at positions rt180 and rt204, but contains additional probes on the test strip to detect the presence of lamivudine-resistant mutations at rt80 and rt173 as well as adefovir-resistant mutations at rt181 and rt236. All samples were also tested using bidirectional automated sequencing at the DNA sequencing core facility (University of Michigan Medical Center, Ann Arbor, MI) using the standard protocol for the Applied Biosystems DNA Sequencer 377 (Perkin-Elmer Corp., Foster City, CA). Specific primers used for amplification of the HBV polymerase gene were SS3076, 5'-TTGGGGTGGAGCCCTCAG-3'(nucleotides 3076-3093) and SAS1019, 3'-GCAAAGCCCAAAAGACCCA-5' (nucleotides 1019-1000) for the first round of PCR; SS61, 5'-TGGTGGCTCCAGTTCCGG-3'(nucleotides 61-78) and SAS991, 3'-GACATACTTTCCAATCAATAG-5'(nucleotides 991-970) for the second round. The amplicon includes domains A, B, C, D, and E of the reverse transcriptase region of the HBV polymerase.
Sequencing results of serial samples from the same patient were compared to identify changes after the initiation of adefovir therapy. For patients with no available baseline samples, sequences from samples taken during treatment were compared with published genotype-specific sequences of the HBV polymerase gene to identify any discrepancies and to determine the presence of previously reported lamivudine- and adefovir-resistant HBV mutations. Changes in the overlapping S gene were also analyzed.
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