LAM is the first approved oral antiviral agent for the treatment of chronic hepatitis B and has been therefore widely used as the first line therapeutic agent in the past decades. Despite the availability of other NAs carrying lower risk of drug resistance (such as ADV and ETV), LAM remains to be a popular anti-HBV agent in many parts of the world because of its low cost, excellent safety profile in long term use, and absence of oncogenic potential in animal studies. However, because of widespread use, an increasing number of LAM-resistant patients have emerged.
Currently, the most widely accepted strategy for rescue therapy is ADV add-on treatment . The de novo development of the cross-resistant mutant, rtA181T, was about 4% in a 4-year period of combination treatment with LAM plus ADV . Previous studies also suggested effective viral suppression in LAM-resistant patients by adopting ADV or ETV monotherapy. However, it was found that the risk of developing ADV or ETV drug resistance was high with this strategy . Recently, it was shown that tenofovir is superior to ADV in treating LAM-resistant patients with a better viral suppression and no detectable genotypic resistance [18, 19].
Despite the availability of rescue therapy, severe disease outcomes still occurred in a substantial percentage of patients. Previous studies indicated that successful viral suppression in ADV add-on therapy did not prevent HCC occurrence in patients with liver cirrhosis [22, 24]. This study demonstrated that age > 50 years, cirrhosis, BCP mutation, and the presence of the rtA181T mutation, prior to initiation of rescue therapy were significantly associated with severe liver consequences. Interestingly, neither the choice of rescue drugs nor the duration of antiviral drug use seemed to affect the clinical outcomes of these patients. In fact of the 21 patients receiving LAM monotherapy, only 1 developed HCC. It is possible that rescue therapy was not initiated in these patients because they had mild hepatitis despite development of drug resistance. Notably, two important variables, HBV-DNA level and pre-S internal deletion mutants, which were frequently identified as prognostic indicators in treatment-naïve patients were not found to be associated with clinical outcomes in LAM-resistant patients. It is likely that during rescue therapy in these patients, HBV replication was suppressed to a low level, resulting in reduction in the development of the pre-S internal deletion mutations. In this view, the pre-S internal deletion mutants are unlikely to play an important role in hepatocarcinogenesis in this subset of patients.
The most striking finding in this study was that pre-existing rtA181T mutant in LAM-resistant patients was independently associated with severe liver consequences, especially occurrence of HCC. The rtA181T mutant has been shown to confer ADV-resistant [28–30]. In ADV add-on studies, it was found that rtA181T mutants could be either pre-existing in LAM-resistant patients or de novo-developed after ADV add-on therapy . Furthermore, because the reading frame of HBV polymerase overlaps with that of the S gene, some resistant mutations (such as rtA181T) in the polymerase gene coexist with S gene mutations, including stop codon mutations . In some patients with rtA181T mutants, a concomitant sW172* mutation was observed in the overlapping S reading frame, resulting in truncation of the pre-S/S proteins . Several previous studies have reported the transactivation activities of a truncated pre-S2 mutant isolated from the chromosomal integrated HBV genome of hepatoma cells. Such mutants were capable of transactivating several promoters including those of oncogenic proteins . Transactivation activity occurred only when the premature stop codons are located within a region of the S gene, named "transactivity-on-region" . The premature stop codon of the S gene in the rtA181T/sW172* mutant was located near the border of the transactivity-on-region and its transactivating activity was recently confirmed [35, 36]. Additionally, owing to secretory defect and a dominant negative effect on wild-type HBV virion secretion, emergence of rtA181T/sW172* largely reduced the typical extent of virological breakthrough in serum, resulting in difficulty to recognize drug resistance . In this study, a hypersensitive ACRES method was used to detect small percentages of the rtA181T mutants. Of the 10 rtA181T-positive patients, the mutants constituted < 5%, 10-30% and > 90% of the viral population in 6, 2, and 2 patients, respectively. We speculated that despite a small percentage of the mutants was detected in the serum, a larger percentage could exist in the liver, owing to the secretory defect of the sW172* mutant. Presumably, intrahepatic accumulation of this mutant partially contributed to its oncogenic effect.
Finally, in our study, 23 patients had two unfavorable conditions: liver cirrhosis and older than 50 years of age. During the follow-ups, 5 of the 23 patients (21.7%) developed HCC and 7 of the 23 (30.4%) patients developed severe liver consequences. The present data indicated that in this special subgroup of patients, careful selection of antiviral agent and closely monitoring for viral mutants (including BCP and rtA181T mutations) are mandatory.