INTRODUCTION

In antiviral therapy for patients infected with hepatitis B virus (HBV), administration of nucleos(t)ide analogs (NAs) exert potent antiviral effects regardless of HBV genotypes and host factors. NAs inhibit the reverse transcription of HBV-DNA within hepatocytes, thereby suppressing the release of HBV-DNA into the bloodstream and leading to the resolution of hepatitis, as well as reducing the progression of liver fibrosis and development of hepatocellular carcinoma (HCC). However, NAs cannot eliminate covalently closed circular DNA from hepatocytes. As a result, patients with chronic hepatitis B require lifelong administration of NAs to prevent the risk for reactivation and/or severe exacerbation of hepatitis on discontinuation of treatment.^{1, 2}

Long-term administration of lamivudine (LAM) has been reported to lead to the emergence of drug-resistant mutations, such as rtL180M and rtM204V/I.^{3, 4} Several studies have demonstrated that entecavir hydrate (ETV) shows mild cross-resistance to LAM-resistant mutations, such as rtL180M and rtM204V/I,^{5, 6} whereas tenofovir (TFV) does not.⁷ However, it has been reported that the rtA194T mutation reduces TFV sensitivity by increasing the IC₅₀ level in in vitro analyses.^{8, 9} A recent study reported that the combination of quadruple mutations, namely rtS106C, rtH126Y, rtD134E, and rtL269I (CYEI) mutation, leads to a 15.3-fold increase in the amount of TFV required to inhibit HBV at the IC₅₀ level, and a 26.3-fold increase at the IC₉₀ level in in vitro analyses.¹⁰ Thus, in patients currently receiving combination therapy with ETV and tenofovir alafenamide fumarate (TAF) after the initial LAM administration, if LAM-resistant mutations were to emerge, it is possible that antiviral efficacy would still be favorable even with TAF monotherapy, provided that there exist no HBV clones harboring the rtA194T or CYEI mutations.

In clinical practice, analysis for drug-resistant mutations in HBV genome is challenging due to the difficulty of genome sequencing of HBV-DNA when there is a good antiviral response with undetectable serum HBV-DNA. Thus, we focused on HBV-RNA. Recently, serum HBV-RNA has been proposed as a novel surrogate biomarker of covalently closed circular DNA levels in hepatocytes, and serum HBV-RNA has been shown to be useful in various clinical contexts, such as the monitoring transcriptional activity of HBV and the discontinuation of NAs during long-term anti-viral therapy.^11-13 We devised a method to sequence the HBV pregenome RNA secreted in the sera during NAs administration. This approach enables the analysis of HBV genome sequences even when serum HBV-DNA is undetectable during antiviral therapy with NAs. Thus, the aim of the present study was to analyze the antiviral efficacy after switching to TAF monotherapy from the combination therapy of TAF and ETV in patients with chronic hepatitis B who initially had a poor antiviral response after LAM or ETV administration, but currently show a good antiviral response while receiving combination therapy with ETV and TAF, provided that no drug resistance mutations against TAF and tenofovir disoproxil fumarate (TDF) were detected by sequencing HBV pregenome RNA in the sera.

METHODS

RESULT

Viral response and ALT level in patients who switched from combination therapy with ETV and TAF to TAF monotherapy

TAF monotherapy was carried out in 20 patients whose pregenome RNA was sequenced in the sera. Two patients were excluded during follow-up: one due to development of HCC, and the other due to the initiation of pegylated interferon-α-2a therapy. Consequently, 18 patients were available for the analysis.

After the initiation of TAF monotherapy, all patients successfully maintained complete viral response for 48 weeks without viremia relapse (Figure 1a). Notably, even in the patient in whom rtA194T was detected at a frequency of 0.3% by deep sequencing. Furthermore, an additional analysis of HBV-RNA sequencing was conducted at 48 weeks after initiating TAF monotherapy, enabling HBV-RNA sequencing in a total of 10 patients. Among these, some nucleotide substitutions were observed in two patients harboring L180M + M204V mutations. However, no new amino acid substitutions were identified in any of the patients after initiating TAF monotherapy.

The median serum ALT levels (U/L) at 0, 4, 12, 24, and 48 weeks were 17 (IQR 13–24), 17 (14–21), 20 (18–26), 19 (14–23), and 17 (14–22), respectively. These levels remained consistent until 48 weeks after initiating TAF monotherapy (Figure 1b). Importantly, none of the patients experienced a serum ALT flare exceeding fivefold the upper limit of the standard values after initiating TAF monotherapy.

Renal function and serum phosphorus levels in patients who switched from combination therapy with ETV plus TAF to TAF monotherapy

The median eGFR (mL/min/1.73 m²) at 0, 4, 12, 24, and 48 weeks was 66.3 (IQR 56.5–81.5), 72.2 (57.9–82.4), 68.4 (58.1–83.7), 66.2 (57.5–76.9), and 67.3 (54.1–79.0), respectively (Figure 2a). There was no significant change in eGFR after the initiation of TAF monotherapy. However, the changes in eGFR were −3.7 (−8.0 to −2.5) mL/min/1.73 m² over the 48 weeks of TAF and ETV combination therapy, and by −2.1 (−3.8 to +3.1) mL/min/1.73 m² over the 48 weeks of TAF monotherapy. Notably, the reduction in eGFR was significantly smaller during the TAF monotherapy period compared with the TAF and ETV combination therapy period (p = 0.0295, Wilcoxon signed-rank test; Figure 2b).

Additionally, the median serum phosphorus level (mg/dL) at 0, 4, 12, 24, and 48 weeks was 3.0 (2.6–3.9), 3.0 (2.4–3.5), 3.1 (2.8–3.7), 3.5 (3.0–4.1), and 3.1 (2.7–3.5), respectively (Figure 2c). No significant changes were observed in the serum phosphorus level, and there was no significant difference in the changes between the TAF monotherapy period and the TAF and ETV combination therapy period (Figure 2d).

DISCUSSION

In the present study, we introduced an innovative method to identify drug-resistant mutations, encompassing minor clones, through direct and/or deep sequencing. This approach facilitates the analysis of HBV genome sequences even when serum HBV-DNA becomes undetectable during antiviral therapy with NAs. By utilizing this approach, the patients receiving combination therapy with ETV plus TAF for chronic HBV infection due to experiencing BTH or PR after initial LAM or ETV administration were able to safely transition to TAF monotherapy based on nucleotide sequences of HBV pregenome RNA in the sera. The monitoring serum HBV-DNA is available to confirm antiviral response and medication adherence during antiviral therapy. Cultured cells infected with HBV, as well as serum and tissue samples obtained through liver biopsy, show encapsulated RNA, which correspond to HBV pregenome RNA.^{11, 15} In the present study, however, not only encapsulated RNA, but also other forms of RNA, were extracted and subjected to the analysis. The achievement of HBV pregenome RNA amplification was associated with HBeAg, HBsAg, and HBcrAg levels. These observations supported that HBV pregenome RNA levels reflected the covalently closed circular DNA levels and HBV transcriptional activity under NAs administration. We examined in detail the drug-resistance profile of Pol/RT domain by direct sequencing and deep sequencing using HBV pregenome RNA from patients with persistent under detection sensitivity of serum HBV-DNA for a long period of time while receiving NAs. Furthermore, we reported for the first time the favorable antiviral response when switching to TAF monotherapy from the combination therapy of TAF and ETV in patients with chronic hepatitis B. Notably, we demonstrated that TAF monotherapy yields favorable viral response even in patients harboring drug-resistance mutations against LAM and/or ETV in the real-world cohort. This is because TFV shows no cross-resistance to LAM or ETV, demonstrating that TAF has high antiviral efficacy against HBV strains that are drug resistant to LAM and ETV in vivo.

The current Japan Society of Hepatology Guidelines for the Management of HBV Infection (4th edition) present the treatment strategy for good and poor responders to NA therapies in patients with chronic hepatitis B.¹⁶ In patients showing good response to ETV monotherapy, the guidelines recommend either continuing with ETV or considering a switch to TAF, taking into account the emergence of drug-resistant mutations. Furthermore, in patients showing poor response to LAM, ETV, TDF, or TAF monotherapy, the guidelines recommend changing the combination therapy with ETV and TAF. However, the guidelines lack a clear therapeutic strategy for patients currently undergoing the combination therapy of ETV and TAF. In the present study, we demonstrated that switching to TAF monotherapy from combination therapy with ETV and TAF is sufficient for maintain viral response in patients harboring drug-resistance mutations against LAM and/or ETV. Furthermore, five patients who did not show HBV-RNA amplification also dropped out of this prospective study, but TAF monotherapy was initiated based on the presumption of low transcriptional activity of the virus, and all of them are progressing well with good antiviral effects in TAF monotherapy. Although the sample size is limited, these findings imply that in clinical settings, patients who received ETV + TAF due to poor viral response against previous NAs might be transitioned to TAF monotherapy without necessitating HBV-RNA sequencing.

Recently, Park et al. reported that rtS106C, rtH126Y, rtD134E, and rtL269I show resistance to TFV. The IC₅₀ against TFV of HBV strain harboring these quadruple mutations, as CYEI mutation, showed 15.3-fold higher than that of wild-type strain in vitro study.¹⁰ In contrast, Liu et al. reported that among 3886 patients with chronic hepatitis B enrolled in the clinical trial, two patients showed a CYEI mutation and one had a triple mutation of CYE, and these patients showed a good response to TDF or TAF monotherapy over 96 weeks.¹⁷ These observations suggested no significant clinical concerns about CYEI mutation. In the present study, none of the patients showed the quadruple CYEI mutation, and only one patient had the CEI mutation. Nevertheless, all patients with these mutations, including rtS106C, rtH126Y, rtD134E, and rtL269I, showed favorable response to TAF monotherapy.

It has been reported that combination therapies, such as LAM + ADV or ETV + ADV, have been effective in patients with poor response against ETV monotherapy.^{18, 19} Additionally, in a phase III trial of TDF in Japan, ETV + TDF combination therapy exerted a good response to patients who showed resistance to ETV monotherapy or ETV + ADV combination therapy. Furthermore, for patients with a resistance mutation against ETV, there was no significant difference in the antiviral response at the 48-week mark between the TDF monotherapy group and the TDF + ETV combination therapy group (71% vs. 73%).²⁰ In the present study, among 18 patients showing poor response to initial therapy with LAM or ETV, three patients showed drug-resistance mutations to ETV and LAM, and four patients showed drug-resistance mutations to LAM. However, the antiviral efficacy remained favorable, even after switching to TAF monotherapy, in these patients. These observations provide scientific evidence for the effectiveness of TAF monotherapy against HBV strains harboring ETV resistance in real clinical practice.

Regarding drug-resistance mutations against TFV, a previous study reported that the coexistence of rtA194T with LAM-resistant mutations, such as rtL180M + rtM204V, in patients co-infected with HBV and HIV results in decreased susceptibility to TFV.⁸ In the present study, deep sequencing analysis detected coexistence of rtA194T with rtL180M, rtM204M, rtV173L, and rtT184A as 0.3% of minor clones in patient showing BTH during LAM administration. In this patient, TAF monotherapy showed a sustained antiviral response over a 48-week period. These observations revealed that rtA194T, as well as CYEI mutation, does not contribute to resistance to TAF.

In a long-term observation of 1298 patients who initiated TAF treatment enrolled in a phase III trial, no patient developed drug resistance mutation over 5 years, and the high resistance barrier of TAF was reported.²¹ In the present study, among 18 patients in whom HBV pregenome RNA was analyzed, a direct sequencing of the Pol/RT domain of HBV-DNA had been conducted for nine patents at a time when they showed a poor antiviral response to the initial therapy with LAM or ETV (a median of 8 years earlier), and nucleoside sequences of HBV-DNA at the time of viremia relapse and nucleotide sequences of HBV pregenome RNA during the combination therapy with ETV and TAF were found to be identical (data not shown). No amino acid substitutions, including drug resistance mutations, developed during the period of additional TAF administration after viremia relapse, confirming a high resistance barrier of TAF.

We previously conducted a prospective study on switching to TAF from ETV in patients who had achieved a good antiviral response.^{22, 23} That study showed a favorable antiviral response after switching to TAF. In that study, we conducted a questionnaire survey to assess medication adherence and satisfaction among patients who switched from ETV to TAF, because ETV must be taken on fasting stomach, whereas TAF can be taken regardless of meals. In the questionnaire survey, approximately 35% of patients mentioned that their dosing schedule had changed, approximately 30% reported a reduced frequency of forgetting to take their medication, and approximately 60% expressed satisfaction with the switching from ETV to TAF.²² Consequently, in our current study, the possibility of discontinuing ETV, which must be taken on an empty stomach, may have a positive impact on patient satisfaction with their medication.

In long-term administration of NAs, renal dysfunction often occurs due to mitochondrial impairment in the proximal renal tubules.²⁴ In particular, the administration of ADV or TDF is associated with an increased risk of kidney damage, as evidenced by reduced eGFR and hypophosphatemia, necessitating close monitoring of renal function and serum phosphorus levels during prolonged use of these drugs.²⁵ Conversely, there is minimal reporting of renal function impairment during long-term ETV administration. The advantage of ETV lies in its safety profile, especially in patients with pre-existing renal dysfunction. However, when administering ETV to patients with impaired kidney function, dose adjustment of ETV and continuous renal function monitoring are necessary. Although there is no report about renal toxicity directly attributed to ETV, it is possible that ETV could induce renal toxicity through various mechanisms, including renal tubular injury, apoptosis, and mitochondrial toxicity.^{25, 26} In the present study, the reduction in eGFR over 48 weeks after discontinuing ETV was significantly lower than that observed over the 48 weeks before discontinuation. These findings suggest that discontinuing ETV may contribute to the preservation of residual renal function. Furthermore, previous studies have reported an association between chronic HBV infection and chronic kidney disease.^27-31 Specifically, it has been noted that glomerular diseases, such as membranous nephropathy and mesangiocapillary glomerulonephritis, could be underlying causes of renal dysfunction in patients with HBV infection.^{32, 33} Therefore, in the long-term management of chronic hepatitis B, it is imperative to closely monitor renal function. From this perspective, discontinuing ETV in patients on long-term administration with TAF and ETV may be beneficial for the patients. However, comparing changes in eGFR necessitates consideration of variables, such as age and alterations in muscle mass attributable to health conditions or sarcopenia. Therefore, further investigation is required to ascertain the renal benefits of transitioning from ETV + TAF to TAF monotherapy.

There were two limitations in the present study. The first limitation was the difficulty in proving the accuracy of the sequencing of HBV pregenome RNA. In the present study, HBV-DNA sequencing was not feasible, because all patients had HBV-DNA levels below the detection threshold. Consequently, we conducted only HBV-RNA sequencing. To mitigate these limitations, we confirmed that the sequences of HBV-DNA and HBV-RNA were consistent in nine patients at a time when they showed a poor antiviral response to the initial therapy with LAM or ETV. Additionally, in another cohort of patients with chronic hepatitis B before NAs treatment, we verified that the HBV-RNA sequences, when HBV-DNA became undetectable under NAs administration, aligned with the HBV-DNA sequences obtained before NA administration. These findings support the reliability of HBV-RNA sequencing in our study. The second limitation of this study was the small cohort size. Thus, it was not possible to establish a control group for comparing clinical outcomes, particularly antiviral effects, eGFR, and serum phosphate levels after ETV discontinuation. Given the limited number of patients, a comparison was conducted over a 48-week period before and after ETV discontinuation. In recent years, the occurrence of a drug-resistance mutation during NAs administration has become less frequent, making it difficult to conduct a study with a large number of patients within a single-center analysis. The findings of this study need validation in a multicenter larger cohort in the future.

In conclusion, it was possible to analyze drug resistance profiles in the Pol/RT domain of HBV by sequencing serum pregenome RNA in patients for whom serum HBV-DNA was undetectable during NAs administration. In patients developing drug-resistance mutations against LAM or ETV, the additional administration of TAF did not lead to the emergence of drug-resistance mutations against TAF. Furthermore, these patients showed a favorable antiviral effect, even when changing to TAF monotherapy. As a result, patients who are being treated with ETV plus TAF can safely change to TAF monotherapy.

CONFLICT OF INTEREST STATEMENT

Satoshi Mochida has received speaking fees or honoraria from AbbVie GK, Gilead Sciences Inc., Otsuka Pharmaceutical Co., Ltd., Toray Medical Co. Ltd., Eisai Co., Ltd. and ASKA Pharmaceutical Co., Ltd., has received research grants from EPS Corporation, Gilead Sciences Inc., MSD K.K. and intellim Corporation, and scholarship grants from AbbVie GK., EA Pharma Co. Ltd., Eisai Co., Ltd. and Sumitomo Pharma Co., Ltd. Nobuaki Nakayama is an Editorial Board member of Hepatology Research. The other authors declare no Conflict of Interests for this article

ETHICS STATEMENTS

Written informed consent was obtained from all patients. The study adhered to the ethical guidelines of the Declaration of Helsinki and was conducted with the approval of the Institutional Review Board of Saitama Medical University Hospital (2022-017). Additionally, the study was registered with the Japan Registry of Clinical Trials as jRCT1031220145.