1. Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia.
2. St Vincent’s Hospital, Fitzroy, Victoria, Australia
Hepatitis B virus was the first human virus unequivocally associated with malignancy. Long-term persistent infection with hepatitis B virus can result in the development of chronic liver disease, cirrhosis and hepatocellular carcinoma. Not surprising then, the main goal of antiviral therapy for chronic hepatitis B is to prevent the development of these life-threatening complications. The clinical trial treatment data now indicates that these goals are beginning to be achieved. Unfortunately, treatment failure due to the emergence of drug-resistant hepatitis B viruses compromises the success of antiviral therapy. Furthermore, the majority of drug-resistant hepatitis B viruses have an altered envelope which may even serve to accelerate the progression to hepatocellular carcinoma. The treating physician needs to ensure that current treatment regimens for chronic hepatitis B prevent active replication, interrupt the progression of liver disease and prevent the emergence of drug resistance.
Chronic hepatitis B (CHB) represents a significant public health issue in Australia. Despite a low overall prevalence of <2%, high risk population groups exist within the community.1 A national sero-survey in 1996-1999 estimated a CHB prevalence in Australia of up to 160,000.2 This figure is likely to be much higher now with ongoing active migration of individuals from high CHB prevalence countries into Australia.
The natural history of CHB can be divided into four phases of variable duration – immune tolerance, immune clearance, immune control (non-low replicative) and high-replicative immune escape (Hepatitis B e antigen [HBeAg] negative disease). These phases are determined by both host and viral factors, including HBeAg serostatus, HBV DNA level, serum alanine aminotransferase (ALT) and immunological status. The risk of progressive liver disease is highest during the immune clearance and immune escape phases, during which host immunological attack of hepatitis B infected hepatocytes is actively occurring.3
Without effective treatment, the natural history of CHB is that it can progress to liver failure and hepatocellular carcinoma (HCC). HCC is the fifth most common cancer and the third most common cause of cancer related mortality worldwide.4,5 Approximately 80% of HCC cases have been attributed to infection with either hepatitis B virus (HBV) or hepatitis C virus (HCV) and approximately half the total number of HCC cases can be attributed to chronic hepatitis B (CHB). The relative risk of HCC in patients with CHB is 100 fold compared to that in uninfected individuals.6
The primary treatment goal in the management of CHB is to prevent or delay the onset of clinical complications, especially HCC. Large natural history cohort studies clearly demonstrate that active viral replication drives the development of these complications.7,8 Consequently, the potential sequelae of untreated CHB can be minimised with antiviral therapy which effectively and durably suppresses viral replication.9
The two major treatment strategies in chronic hepatitis B are immune-modulatory therapy with pegylated interferon (Peg-IFN) or oral nucleos(t)ide analogue (NA) therapy. Currently licensed oral NA therapy for CHB includes lamivudine, adefovir, entecavir, telbivudine and tenofovir. The major licensing trials for these therapies have all demonstrated superior virological, biochemical and histological improvement in comparison to untreated controls.10 Oral antiviral therapy has also been shown to significantly reduce the incidence of hepatic decompensation in patients with advanced fibrosis or cirrhosis.9 Furthermore, treatment with either Peg-IFN or oral NA therapy has been associated with a reduction in the risk of HCC.11
The initial therapeutic endpoint in patients with HBeAg positive CHB is HBeAg seroconversion, as it is usually associated with suppression of viral replication and an improved prognosis.12 The HBeAg seroconversion rate following 48 weeks of therapy is approximately 20% with oral NA, and 32% with Peg-IFN.10,13 HBeAg seroconversion rates can increase with ongoing oral NA use, and the beneficial effects of Peg-IFN can persist after treatment. In HBeAg negative disease however, therapeutic endpoints are less predictive due to the high rate of relapse following drug cessation. Consequently, long-term oral NA is recommended to effectively suppress HBV viral replication, a strategy which increases the risk of antiviral resistance over time. While Peg-IFN therapy results in a 63% undetectable HBV rate at end of therapy, only 19% of patients continue to have adequate suppression of viral replication six months after treatment cessation.14
In CHB, HBsAg seroconversion is the preferred endpoint of therapy because it is believed to represent successful immunological control of the hepatitis B virus. In acute infection, HBsAg is cleared during recovery and following vaccination, an anti-HBs immune response is generally protective against possible subsequent infection. Although HBsAg seroconversion is associated with a favourable prognosis in CHB,15-17 a recent longitudinal study evaluating the clinical outcome of HBsAg seroclearance has identified the age of the patient at which HBsAg seroclearance occurs as an important factor.18 This study followed 298 patients and demonstrated that HBsAg seroclearance before the age of 50 was associated with both a lower risk of HCC development and a lower risk of significant fibrosis on transient elastography in comparison to later HBsAg seroclearance (>50 years).18 This will certainly impact on current treatment guidelines for CHB, especially in the Asia Pacific region.
The annual rate of spontaneous HBsAg seroclearance is 1-2%. Treatment with standard interferon (IFN)-a or Peg-IFN results in HBsAg loss at a rate of 7.8% and 3% following 10-24 weeks and 48 weeks of therapy respectively.19 Furthermore, in long-term virologic responders to interferon, HBsAg loss can still occur after cessation of therapy, highlighting the ongoing immune-modulating effects of interferon.10,20 HBsAg loss has also been reported with potent oral antiviral agents such as tenofovir and entecavir. The rate of HBsAg loss following 96 weeks of tenofovir in HBeAg positive patients has been recently shown to be 6%,21 and similarly, 48 weeks of therapy with entecavir in HBeAg-positive patients results in a HBsAg loss of 5% at 120 weeks follow-up.22
A critical issue in treating patients with CHB is the evaluation of predictive markers of response to therapy. This is currently difficult with traditional serological and virological assays. While the HBV genotype may influence HBeAg seroconversion and response to Peg-IFN therapy, testing is not routinely performed. Recent clinical studies have shown that evaluating dynamic on-therapy changes in quantitative serum HBeAg and HBsAg titres may have promise as a biomarker in predicting responses to therapy.23,24 In HBeAg positive patients treated with Peg-IFN, a critical baseline HBeAg level of >31 PE IU/mL has been associated with an increased likelihood of HBeAg seroconversion. Furthermore, in this study, the negative predictive value of an HBeAg titre of >100 PE IU/mL at week 24 was greater than that of serum HBV DNA (96% compared to 86%).23 In HBeAg-negative patients also treated with Peg-IFN, an early reduction in HBsAg titre was shown to have a high predictive rate of sustained suppression of viral replication, and increased HBsAg seroclearance at four years post treatment.24 Ongoing research is required to validate these assays and to determine their feasibility for use in everyday clinical practice.
The development of HCC in chronic HBV infection is a multistep process proposed to be a consequence of the combination of at least three mechanisms: ongoing inflammation, liver damage, and regeneration; chromosomal instability due to integration of HBV DNA; and a direct effect of the virus or viral proteins.25
High levels of replicating HBV have been significantly associated with ongoing liver damage, inflammation, fibrosis and progression to HCC, particularly during the immune clearance and immune escape phases of CHB.8 Genotype C HBV has been reported to replicate to higher levels than other HBV genotypes and can cause more rapid progression to HCC.26-28 Infection with HBeAg negative strains of HBV has also been associated with more rapid progression to HCC.29,30 The host immune response to this higher level of replication may also contribute to HCC development.31
In addition, most HBV-associated HCCs harbour integrated HBV DNA, which can cause chromosomal instability,32,33 however integrated HBV DNA can also be found in non-tumourous tissue from patients with CHB.34 Integration of viral DNA into the host chromosome is not necessary for HBV replication, but does occur and may allow persistence of the viral genome. Integration can lead to the development of HCC due to deletion of cellular genes at the integration site, or transposition of viral and cellular genes.35 HBV DNA can integrate directly into, and modify, genes that regulate cell signalling, proliferation and viability.33 The protein products of some integrated HBV genes, notably HBx, one of the accessory proteins of HBV, and truncated L and M surface proteins, have also been implicated in the progression to HCC.36
HBV proteins expressed from either the HBV genome or integrated DNA may be involved in the development of HCC, and mediate their HCC-promoting effects via activation of pathways involved in cellular transformation either through direct transcriptional transactivation, or via other cellular responses including endoplasmic reticulum (ER) stress. These proteins include the widely studied HBx protein,36 the HBV splice protein,37 and C-terminally truncated HBV surface proteins which have been isolated from HCC samples and shown to have transcriptional transactivation activity due to their altered topology.38-40 Importantly, these truncated surface proteins are selected in the HBV genome during NA therapy (see below).
The introduction of nucleotide analogue (NA) therapy has also witnessed the emergence of antiviral drug resistance, which has become the main factor limiting the long-term treatment of patients with CHB. Several major NA-resistance pathways for HBV (rtM204I/V, rtN236T and rtA181T/V) have now been characterised. The first pathway, rtM204V/I, is responsible for resistance to the L-nucleosides such as lamivudine and telbivudine, and also entecavir which is also used as rescue therapy in lamivudine-experienced patients. This pathway is associated with clusters of secondary mutations (rtT184G, rtS202I) that can affect subsequent treatment with NAs such as entecavir. The second pathway, rtN236T, accounts for adefovir and tenofovir resistance. The third pathway, rtA181T/V, is associated with resistance to lamivudine and adefovir and is a potential multi-drug resistance pathway that will probably impact on tenofovir sensitivity, either alone or with the rtN236T. In naïve patients treated with entecavir, a fourth pathway has been described where at least three mutations need to be selected out at the same time – rtL 80M+rtM204V plus either one of rtT184 or rtS202 or rtM250 codon changes. Finally, in highly experienced NA treated patients, other multi-drug resistance pathways are being increasingly recognised such as rtA181T+rtN236T+rtM250L. Sequential monotherapy treatment with NAs promotes the selection of multi-drug resistant HBV.
Antiviral drug resistance in CHB is not surprising when the viral life-cycle of HBV is taken into consideration. Viral genome replication revolves around two key processes: generation of HBV covalently closed circular DNA from genomic relaxed circular DNA and its subsequent processing by host enzymes to produce viral RNA; and reverse transcription of the pregenomic RNA within the viral nucleocapsid to form relaxed circular DNA. Active replication of HBV is marked by a high frequency of mutational events resulting from an enormous viral turnover rate combined with the error prone reverse transcriptase/polymerase. In the patient, this produces a large quasispecies pool of HBV at any one point in time.
As shown in figure 1, the viral surface gene overlaps completely with the reverse transcriptase gene, hence nucleotide mutations encoding NA resistance in the reverse transcriptase can result in changes in the surface proteins. Other viruses that are treated with NAs, including HIV and HSV, do not have the added complexity of poor proof-reading ability and overlapping reading frames in their polymerase regions, hence treatment with NA for those viruses is straightforward and directly affects only the polymerase. In contrast, NA treatment for HBV has more far-reaching consequences. The reverse transcriptase-surface gene overlap in HBV is important since it has been shown that common LMV resistant HBVs such as rtV173L+rtL180M+rtM204V have important and significant changes in HBsAg (sE164D+sI195M) which significantly reduce anti-HBs (vaccine-associated) binding in vitro.41
Recent studies have shown that these NA selected S mutants may also enhance the progression to HCC. In particular, the mutation encoding the multi-drug resistant rtA181T change also results in a stop codon in the overlapping surface gene at position s172 (sW172*). The C-terminally truncated surface proteins expressed from this variant are very similar to those isolated from patients with HCC and have been shown to be transactivators.38-40 Studies from our group and others have shown that the surface proteins expressed by this variant accumulate within the cell,42 transactivate cellular promoters, and cause tumours when injected into nude mice, whereas the wt full-length surface proteins do not.43,44 Several other C-terminally truncated S variants have been selected in patients who developed HCC during NA-therapy.44
The selection of HBV encoding truncated surface proteins also presents a challenge for the clinical detection of drug resistance, as they have a dominant negative effect on virion secretion.42 The virological case definition of drug resistance, >1.0 log IU/ml from nadir in two consecutive samples taken one month apart,45-47 does not hold up if this mutant is (co)-selected out. The viral load, following first appearance of rtA181T, only very gradually increases from nadir over 12 months. The practical implication of this finding will be the need for HBV genotyping and polymerase sequencing, as well as HBV viral load monitoring in patients undergoing antiviral therapy.
Hence, although NA therapies significantly decrease viral load and improve patient survival in the short-term,9 they can also select for HBV variants that are potentially oncogenic, negating the overall efficacy of NAs in preventing hepatocarcinogenesis, the main long-term goal of antiviral therapy in CHB. It is critical to ensure that when NA therapy is commenced for CHB, that resistance is prevented through the use of effective drugs which ensure as complete inhibition of HBV replication as possible.
Significant progress has been made in our understanding of the natural history of CHB. Treatment outcomes are improving with more efficacious antiviral therapy and the development of algorithms to minimise antiviral resistance. However, there remains a need for the development of additional antiviral therapies which target different steps in the hepatitis B viral replication cycle or the host immune response. The key goals for these future novel classes of antiviral agents should be to alter the natural history of CHB, and in particular, to improve HBeAg and HBsAg seroconversion rates, ensure total suppression of active replication and adopt strategies that prevent the emergence of resistance.
12. Niederau C, Heintges T, Lange S, Goldmann G, Niederau CM, Mohr L, et al. Long-term follow-up of HBeAg-positive patients treated with interferon alfa for chronic hepatitis B. N Engl J Med. 1996;334:1422-7.
13. Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S, Cooksley G, et al. Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med. 2005;352:2682-95.
14. Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, et al. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med. 2004;351:1206-17.
16. Fattovich G, Giustina G, Sanchez-Tapias J, Quero C, Mas A, Olivotto PG, et al. Delayed clearance of serum HBsAg in compensated cirrhosis B: relation to interferon alpha therapy and disease prognosis. European Concerted Action on Viral Hepatitis (EUROHEP). Am J Gastroenterol. 1998;93:896-900.
18. Yuen MF, Wong DK, Fung J, Ip P, But D, Hung I, et al. HBsAg Seroclearance in chronic hepatitis B in Asian patients: replicative level and risk of hepatocellular carcinoma. Gastroenterology. 2008;135:1192-9.
19. Lai CL, Dienstag J, Schiff E, Leung NW, Atkins M, Hunt C, et al. Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis. 2003;36:687-96.
20. Buster EH, Flink HJ, Cakaloglu Y, Simon K, Trojan J, Tabak F, et al. Sustained HBeAg and HBsAg loss after long-term follow-up of HBeAg-positive patients treated with peginterferon alpha-2b. Gastroenterology. 2008;135:459-67.
21. Lee SS, Heathcote EJ, Sievert W, Trinh HN, Kaita KD, Younossi ZM, et al. Tenofovir disoproxil fumarate (TDF) versus adefovir dipivoxil (ADV) in asians with HBeAg positive and HBeAg negative chronic hepatitis B participating in studies 102 and 103. Hepatology. 2008;48:Suppl 4:746A Abstract 980.
23. Fried MW, Piratvisuth T, Lau GK, Marcellin P, Chow WC, Cooksley G, et al. HBeAg and hepatitis B virus DNA as outcome predictors during therapy with peginterferon alfa-2a for HBeAg-positive chronic hepatitis B. Hepatology. 2008;47:428-34.
24. Marcellin P, Brunetto MR, Bonino F, Hadziyannis E, Kapprell H, McCloud P, et al. In patients with HBeAg-negative chronic hepatitis B HBsAg serum levels early during treatment with peginterferon alfa-2a predict HBsAg clearance 4 years post-treatment. Hepatology. 2008;48:Suppl 4:718A Abstract 919.
28. Orito E, Ichida T, Sakugawa H, Sata M, Horiike N, Hino K, et al. Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology. 2001;34:590-4.
29. Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ, Makris A, et al. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet. 1989;2:588-91.
30. Akahane Y, Yamanaka T, Suzuki H, Sugai Y, Tsuda F, Yotsumoto S, et al. Chronic active hepatitis with hepatitis B virus DNA and antibody against e antigen in the serum. Disturbed synthesis and secretion of e antigen from hepatocytes due to a point mutation in the precore region. Gastroenterology. 1990;99:1113-9.
33. Paterlini-Brechot P, Saigo K, Murakami Y, Chami M, Gozuacik D, Mugnier C, et al. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene. 2003;22:3911-6.
34. Brechot C, Thiers V, Kremsdorf D, Nalpas B, Pol S, Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely „occult“? Hepatology. 2001;34:194-203.
35. Dandri M, Burda MR, Burkle A, Zuckerman DM, Will H, Rogler CE, et al. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation. Hepatology. 2002;35:217-23.
37. Soussan P, Tuveri R, Nalpas B, Garreau F, Zavala F, Masson A, et al. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis. J Hepatol. 2003;38:343-8.
38. Caselmann WH, Meyer M, Kekule AS, Lauer U, Hofschneider PH, Koshy R. A trans-activator function is generated by integration of hepatitis B virus preS/S sequences in human hepatocellular carcinoma DNA. Proc Natl Acad Sci USA. 1990;87:2970-4.
40. Schluter V, Meyer M, Hofschneider PH, Koshy R, Caselmann WH. Integrated hepatitis B virus X and 3‘ truncated preS/S sequences derived from human hepatomas encode functionally active transactivators. Oncogene. 1994;9:3335-44.
41. Torresi J, Earnest-Silveira L, Deliyannis G, Edgtton K, Zhuang H, Locarnini SA, et al. Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy. Virology. 2002;293:305-13.
42. Warner N, Locarnini S. The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound. Hepatology. 2008;48:88-98.
44. Lai MW, Huang S, Hsu C, Chang M, Liaw YF, Yeh CT. Identification of nonsense mutations in hepatitis B virus S gene in patients with hepatocellular carcinoma developed after lamivudine therapy. Antiviral Therapy. 2009;In press.
46. Lok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, Pawlotsky JM, et al. Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology. 2007;46:254-65.
47. Pawlotsky JM, Dusheiko G, Hatzakis A, Lau D, Lau G, Liang TJ, et al. Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach. Gastroenterology. 2008;134:405-15.