Abstract
Background:
Nucleot(s)ide analog treatment (entecavir (ETV) and tenofovir (TDF)) is reported to be associated with decreased tumor recurrence and death in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients, yet further work is needed to evaluate the different efficacies of these two agents on the prognosis of early-stage HBV-related HCC patients after curative liver resection.
Material and methods:
From July 2017 to January 2019, 148 patients with HBV-related HCC who underwent curative liver resection were randomized to receive TDF (n=74) or ETV (n=74) therapy. The primary end point was tumor recurrence in the intention-to-treat population. Overall survival and tumor recurrence of patients were compared by multivariable-adjusted Cox regression and competing risk analyses.
Results:
During the follow-up with continued antiviral therapy, 37 (25.0%) patients developed tumor recurrence, and 16 (10.8%) patients died (N=15) or received liver transplantation (N=1). In the intention-to-treat cohort, the recurrence-free survival for the TDF group was significantly better than that for the ETV group (P=0.026). In the multivariate analysis, the relative risks of recurrence and death/liver transplantation for ETV therapy were 3.056 (95% CI: 1.015–9.196; P=0.047) and 2.566 (95% CI: 1.264–5.228; P=0.009), respectively. Subgroup analysis of the PP population indicated a better overall survival and RFS of patients receiving TDF therapy (P=0.048; hazard ratio (HR) =0.362; 95% CI: 0.132–0.993 and P=0.014; HR =0.458; 95% CI: 0.245–0.856). Additionally, TDF therapy was an independent protective factor against late tumor recurrence (P=0.046; (HR)=0.432; 95% CI: 0.189–0.985) but not against early tumor recurrence (P=0.109; HR =1.964; 95% CI: 0.858–4.494).
Conclusion:
HBV-related HCC patients treated with consistent TDF therapy had a significantly lower risk of tumor recurrence than those treated with ETV after curative treatment.
Keywords: antiviral therapy, chronic hepatitis B, hepatectomy, hepatocellular carcinoma, tumor recurrence
Introduction
Highlights
Nucleot(s)ide analog therapy is known to decrease the risk of tumor recurrence while the superiority of tenofovir disoproxil fumarate (TDF) and entecavir for hepatitis B virus-related hepatocellular carcinoma (HCC) patients is still unclear.
In this study, we found that TDF as a favorable postoperative antiviral therapy can lead to a better prognosis for hepatitis B virus-related HCC patients after radical hepatectomy.
In a subgroup of patients with late HCC recurrence, those who received TDF treatment had significantly better recurrence-free survival than entecavir after curative treatment.
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the second leading cause of cancer-related death. In Asia, hepatitis B virus (HBV) infection is the main cause of HCC, especially in China1. At present, radical hepatectomy and liver transplantation are considered curative treatments, but the tumor recurrence rate still exceeds 70% at 5 years after curative resection2. In recent studies, many risk factors, such as alpha fetoprotein (AFP), microvascular infiltration (MVI), and high viral replication of HBV, have been reported to be associated with a higher recurrence rate of HCC. For HBV-related HCC patients, it is known that consistent viral replication of HBV is a major risk factor for HCC recurrence, and reducing virus loading via nucleos(t)ide analog therapy could significantly prolong the overall survival (OS) of HCC patients and reduce tumor recurrence of HCC3,4.
Entecavir (ETV) and tenofovir disoproxil fumarate (TDF), with high genetic barriers to drug resistance, are both recommended as first-line treatments for chronic hepatitis B by clinical practice guidelines5. Moreover, sequential clinical studies have compared the incidence of HCC between ETV and TDF, but the results are still controversial6–8. However, most of the results were based on retrospective studies with selection bias. Randomized controlled trials (RCTs) comparing the two drugs for tumor recurrence and OS are still lacking. Therefore, we conducted a RCT to evaluate the different efficacies of ETV and TDF on the OS and prevention of tumor recurrence after curative treatment for HBV-related HCC patients.
Material and methods
Patients
The study was a prospective, single-center, randomized, open-label study. From July 2017 to January 2019, consecutive patients in our hospital with newly diagnosed HBV-related HCC who had received R0 liver resection were eligible for enrollment. The preoperative diagnosis of HCC was based on the diagnostic criteria for HCC used by the European Association for the Study of the Liver9 and confirmed by a postoperative histopathologic examination. The study has been reported in line with the Consolidated Standards of Reporting Trials (CONSORT) Guidelines, Supplemental Digital Content 1, http://links.lww.com/JS9/A713, Supplemental Digital Content 2, http://links.lww.com/JS9/A714 10. The study has been registered in the Chinese Clinical Trials Registry.
The inclusion criteria for this study were as follows: Age 18–69 years; Positive test for hepatitis B surface antigen (HBsAg) and negative test for antibody to hepatitis C virus or HIV; (3) Serum HBV-DNA level greater than 200 IU/ml; (4) Barcelona clinic liver cancer stage 0 or A; (5) Platelet level greater than 50×10^9/l; (6) Good liver function with Child−Pugh Class A, no invasion into the major portal vein or the hepatic vein and two large branches, and no extrahepatic metastasis; (7) A serum creatinine clearance rate greater than 70 ml/min; (8) No previous long history of antiviral therapy; (9) No surgical treatments were performed before the operation; (10) The results of the postoperative histopathological biopsy were HCC; and (11) Eligible patients agreed to participate in the trial. Patients with poor compliance who did not adhere to the follow-up guidelines were excluded.
Study design and intervention
The study was designed to compare the efficacy of ETV and TDF for patients with HBV-related HCC undergoing curative liver resection. Eligible patients were randomly assigned in a 1:1 ratio via computer-generated allocation to either the ETV group or the TDF group after surgery. A block size and strata were used in the randomization. Patients in the ETV group received ETV tablets (RunZhong, ChiaTai Tianqing) 0.5 mg/d orally after the operation; those in the TDF group received TDF tablets (Viread, Aspen Port Elizabeth) 300 mg/d orally after surgery. Antiviral treatment was continued unless there was unacceptable toxicity or withdrawal of consent. The necessary sample size was determined by employing α=0.05 and β=0.9 according to our preliminary experimental results. The follow-up drop-out rate was 5%, owing to good compliance. The study was investigator-designed and investigator-driven, and it received no support from any pharmaceutical companies.
Outcomes and ffollow-up
In the study, the primary outcome measures included HCC recurrence of patients from the date of the operation. Secondary outcome measures included OS of the patients, patient virologic, biochemical, and serologic response to antiviral therapy, tolerance of antiviral treatment, and additional cytokine levels such as IFN-λ1 IFN-λ2, IFN-λ3, IL-1β, tumor necrosis factor (TNF)-α, IL-6, IL-8, and IL-10 during the follow-up period. All patients received follow-up monitoring 1 month after the operation, every 3 months thereafter during the first 3 years, and then every 6 months in subsequent years. Physical examination, blood cell and differential counts, renal and liver function tests, tumor markers, HBV serology and HBV-DNA levels, serum cytokine levels, and imaging examinations were included in the follow-up examinations when necessary.
Tumor recurrence was diagnosed based on the identification of one or more new lesions on at least 2 radiological examinations (contrast-enhanced ultrasound, contrast-enhanced computed tomography or MRI) following the same criteria of the diagnosis of HCC11,12. Patients with tumor recurrence were actively treated with salvage liver transplantation, repeat hepatic resection, radiofrequency ablation, transcatheter arterial chemoembolization, sorafenib, and/or chemotherapy, depending on the extent of the disease, liver function, and general condition of the patients.
Statistical analysis
Continuous variables were compared between groups using the t-test or the Mann–Whitney U test for variables. Categorical data were compared using the χ 2-test or Fisher’s exact test. Overall and recurrence-free survival (RFS) rates were analyzed using the Kaplan–Meier method. The Cox proportional hazard model was used for univariate and multivariate analyses of prognostic factors after surgery. Competing risk analyses were conducted to evaluate the cumulative incidence of tumor recurrence. Risk factors were examined by computing hazard ratios (HRs) in the multivariate analysis. Two-tailed P values of 0.05 were considered statistically significant, and in the multivariate analysis, potential risk factors (P<0.1 in the univariate analysis) were included. Calculations were performed using the SPSS package (SPSS, Inc.) and R statistical software, version 3.6 (R Foundation for Statistical Computing; https://www.R-project.org/). The intention-to-treat population consisted of all patients undergoing randomization. The ITT population was used for demographics as well as for the primary efficacy analysis. The per protocol (PP) set comprised a subset of the ITT that excluded patients with major protocol deviations, such as incomplete study treatment. The PP set was used as a secondary efficacy analysis. For more details of the measurement of cytokine levels, see the supplementary materials, Supplemental Digital Content 3, http://links.lww.com/JS9/A715.
Results
Patients
A total of 528 patients underwent curative liver resection for HBV-related HCC at our institution. Thirteen patients who fulfilled the inclusion criteria refused to participate. Finally, 148 consecutive patients were randomized to the two treatment groups (Supplementary Figure S1, Supplemental Digital Content 3, http://links.lww.com/JS9/A715). In the ITT analysis, 74 patients were randomized to both the TDF and ETV groups. The median follow-up was 46.61 months (range 18.0–62.0 months). The PP population comprised 72 patients in the TDF group and 70 who received ETV therapy. Six patients were lost to follow-up (four patients in the ETV group and two patients in the TDF group) because they had stopped taking antiviral treatment without authorization from the clinicians at months 18, 25, 30, 32, 38, and 42 after antiviral therapy. The remaining patients continued antiviral treatment until the end of the study. The median follow-up in the PP population was 45.91 months (range 10.0–62.0 months).
The baseline characteristics before the operation and the operative data of the patients in the ITT and PP populations are presented in Table 1. There was no significant difference in the parameters between the two groups except for the history of hypertension in the ITT population. The serum phosphate level and history of hypertension were significantly different between the two groups in the PP population. During the antiviral period, no patients developed primary nonresponse or viral resistance to both ETV and TDF, and no patients who underwent antiviral therapy developed adverse effects during follow-up. A previous study13 showed that long-term use of TDF can induce renal dysfunction. The results showed that there were no patients with renal dysfunction or electrolyte disturbance during the follow-up period, and no significant difference was found in blood urea nitrogen, creatinine level, estimated glomerular filtration rate, serum phosphate, and serum calcium level between the TDF and ETV groups in the ITT population (Supplementary Table S1, Supplemental Digital Content 3, http://links.lww.com/JS9/A715).
Table 1.
Patient demographics and disease characteristics before operation and operation data of the patients in the ITT and PP populations.
| ITT population | PP population | |||||
|---|---|---|---|---|---|---|
| Characteristics | TDF Group (n=74) | ETV Group (n=74) | P | TDF Group (n=72) | ETV Group (n=70) | P |
| Basic characteristics | ||||||
| Sex (male/female) | 63/11 | 66/8 | 0.461 | 61/11 | 62/8 | 0.501 |
| Age, year | 50.97±12.17 | 49.78±11.95 | 0.550 | 50.79±12.23 | 49.20±11.62 | 0.428 |
| BMI, n, % | 0.166 | 0.302 | ||||
| ≥25 | 8 (10.8%) | 14 (18.9%) | 8 (11.1%) | 12 (17.1%) | ||
| <25 | 66 (89.2%) | 60 (81.1%) | 64 (88.9%) | 58 (82.9%) | ||
| History of Smoke, n, % | 1.000 | 0.956 | ||||
| Yes | 7 (9.5%) | 7 (9.5%) | 7 (9.7%) | 7 (10.0%) | ||
| No | 67 (90.5%) | 67 (90.5%) | 65 (90.3%) | 63 (90.0%) | ||
| Hypertension, n, % | 0.034 | 0.049 | ||||
| Yes | 4 (94.6%) | 12 (83.8%) | 4 (5.6%) | 11 (15.7%) | ||
| No | 70 (92.3%) | 62 (84.6%) | 68 (94.4%) | 59 (84.3%) | ||
| Diabetes, n, % | 0.166 | 0.211 | ||||
| Yes | 8 (10.8%) | 14 (18.9%) | 8 (11.1%) | 13 (18.6%) | ||
| No | 66 (89.2%) | 60 (81.1%) | 64 (88.9) | 57 (81.4%) | ||
| Cardiovascular Disease, n, % | 0.347 | 0.483 | ||||
| Yes | 4 (5.4%) | 7 (9.5%) | 4 (5.6%) | 6 (8.6%) | ||
| No | 70 (94.6%) | 67 (90.5%) | 68 (94.4%) | 64 (91.4%) | ||
| Alcohol Abuse, n, % | 0.797 | 0.952 | ||||
| Yes | 8 (10.8%) | 9 (12.2%) | 8 (11.1%) | 8 (11.4%) | ||
| No | 66 (89.2%) | 65 (87.8%) | 64 (88.9%) | 62 (88.6%) | ||
| Liver cirrhosis, n, % | 0.852 | 0.927 | ||||
| Yes | 55 (74.3%) | 54 (73.0%) | 53 (73.6%) | 52 (74.3%) | ||
| No | 19 (25.7%) | 20 (27.0%) | 19 (26.4%) | 18 (25.7%) | ||
| Ishak Fibrosis Score | 0.587 | 0.510 | ||||
| ≥4 | 20 (27.0%) | 23 (31.1%) | 20 (27.8%) | 23 (32.9%) | ||
| <4 | 54 (73.0%) | 51 (68.9%) | 52 (72.2%) | 47 (67.1%) | ||
| Laboratory Test | ||||||
| TBIL, μmoI/l | 15.09±6.89 | 14.93±6.42 | 0.833 | 15.08±6.98 | 14.66±6.35 | 0.709 |
| ALT, IU/I | 34.42±13.95 | 34.59±11.34 | 0.933 | 34.19±14.08 | 34.5±11.25 | 0.887 |
| AST, IU/I | 33.73±11.08 | 35.11±11.23 | 0.454 | 33.60±11.21 | 35.17±11.38 | 0.408 |
| ALB, g/l | 40.93±5.16 | 40.81±4.70 | 0.888 | 40.83±5.13 | 40.77±4.65 | 0.940 |
| BUN, mmol/L | 5.29±1.89 | 5.06±1.73 | 0.448 | 5.25±1.86 | 5.04±1.70 | 0.487 |
| CREA, μmoI/l | 73.40±14.67 | 74.35±14.45 | 0.692 | 73.84±12.83 | 75.17±12.64 | 0.533 |
| eGFR, ml/min | 103.81±13.47 | 101.99±13.40 | 0.410 | 104.08±13.48 | 101.60±13.50 | 0.277 |
| P, mmol/l | 1.17±0.27 | 1.25±0.29 | 0.092 | 1.18±0.27 | 1.27±0.28 | 0.044 |
| Ca, mmol/l | 2.21±0.26 | 2.19±0.27 | 0.654 | 2.21±0.26 | 2.20±0.28 | 0.740 |
| PLT, 109/l | 98.05±46.64 | 92.43±51.69 | 0.488 | 98.60±47.08 | 93.14±52.90 | 0.517 |
| WBC, 109/l | 5.44±3.20 | 4.86±2.46 | 0.218 | 5.46±3.24 | 4.91±2.49 | 0.258 |
| Serum IFN-λ3 level, pg/ml | 8.92±5.49 | 8.54±5.78 | 0.683 | 9.02±5.47 | 8.43±5.83 | 0.533 |
| Virologic characteristics | ||||||
| HBV genotype(A:B:C:D), n | 0:58:15:1 | 0:60:12:2 | 0.523 | 0:57:14:1 | 0:57:11:2 | 0.560 |
| HBV-DNA,IU/ml | 0.702 | 0.773 | ||||
| <2×10^3 IU/ml | 55 (74.3%) | 57 (77.0%) | 53 (73.6%) | 53 (75.7%) | ||
| ≥2×10^3IU/ml | 19 (25.7%) | 17 (23.0%) | 19 (26.4%) | 17 (24.3%) | ||
| HBeAg, n, % | 0.259 | 0.285 | ||||
| Positive | 16 (21.6%) | 22 (29.7%) | 15 (20.8%) | 20 (28.6%) | ||
| Negative | 58 (78.4%) | 52 (70.3%) | 57 (79.2%) | 50 (71.4%) | ||
| Tumor characteristics | ||||||
| AFP, ng/ml, n, % | 0.736 | 0.760 | ||||
| ≥20 | 46 (62.2%) | 44 (59.5%) | 45 (62.5%) | 42 (60.0%) | ||
| <20 | 28 (37.8%) | 30 (40.5%) | 27 (37.5%) | 28 (40.0%) | ||
| Tumor number, n, % | 0.259 | 0.625 | ||||
| >1 | 21 (28.4%) | 16 (21.4%) | 19 (26.4%) | 16 (22.9%) | ||
| 1 | 53 (71.6%) | 58 (78.6%) | 53 (73.6%) | 54(77.1%) | ||
| Tumor diameter, cm | 2.91±0.82 | 3.05±0.89 | 0.318 | 2.89±0.82 | 3.07±0.91 | 0.234 |
| BCLC stage, n, % | 0.059 | 0.050 | ||||
| 0 | 32 (43.2%) | 21 (28.4%) | 32 (44.4%) | 20 (28.6%) | ||
| A | 42 (56.8%) | 53 (71.6%) | 40 (55.6%) | 50 (71.4%) | ||
| Differentiation, n, % | 0.620 | 0.487 | ||||
| High | 39 (52.7%) | 42 (56.8%) | 38 (52.8%) | 41 (58.6%) | ||
| Moderate or low | 35 (47.3%) | 32 (43.2%) | 34 (47.2%) | 29 (41.4%) | ||
| Satellite nodule, n, % | 0.699 | 0.084 | ||||
| Yes | 3 (4.1%) | 4 (5.4%) | 3 (4.2%) | 0 (0%) | ||
| No | 71 (95.9%) | 70 (94.6%) | 69 (95.8%) | 70 (100%) | ||
| MVI, n, % | 0.702 | 0.625 | ||||
| Yes | 19 (25.7%) | 17 (23.0%) | 19 (26.4%) | 16 (22.9%) | ||
| No | 55 (74.3%) | 57 (77.9%) | 53 (73.6%) | 54 (77.1%) | ||
| Operation data | ||||||
| Transfusions, n, % | 0.439 | 0.316 | ||||
| Yes | 10 (13.5%) | 7 (9.5%) | 10 (13.9%) | 6 (8.6%) | ||
| No | 64 (86.5%) | 67 (90.5%) | 62 (86.1%) | 64 (91.4%) | ||
| Operation time, h | 2.83±0.33 | 2.82±0.26 | 0.806 | 2.84±0.33 | 2.83±0.27 | 0.925 |
| Hospital stay, days | 7.67±1.13 | 7.67±1.15 | 0.999 | 7.69±1.13 | 7.61±1.16 | 0.678 |
AFP, alpha fetoprotein; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BCLC, Barcelona staging; BMI, body mass index; BUN, blood urea nitrogen; Ca, serum calcium; CREA, creatinine; eGFR, estimated glomerular filtration rate; ETV, entecavir; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; IFN-λ3, interferon-λ3; ITT, intention-to-treat; MVI, microvascular infiltration; P, serum phosphate; PLT, platelet; PP, per protocol; TBIL, total bilirubin; TDF, tenofovir disoproxil fumarate; WBC, white blood cell.
Virological, biochemical, and serological responses and changes in serum cytokines during antiviral treatment
The pretreatment HBV-DNA levels and liver function of the ETV and TDF groups were comparable in the ITT and PP populations (Table 1). TDF showed higher virologic responses at months 30 (P=0.006) and 36 (P=0.003) (Fig. 1A). The rates of biochemical responses were similar between the two groups, but a slightly higher tendency was observed in the ETV group than in the TDF group (Fig. 1B). Additionally, a significant difference was observed in the rates of reduction in the HbsAg levels. At 18 months and longer, the TDF group had significantly higher rates of HbsAg reduction than the ETV group (P<0.05) (Fig. 1C).
Figure 1.
Virologic, biochemical and serological responses to TDF and ETV in HBV-related HCC patients. (A) Virological response; (B) biochemical response; (C) reduction in HBsAg level. (*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001).
Nevertheless, we compared the serum cytokine levels of patients treated with ETV or TDF therapy and the changes in related cytokines before and after antiviral therapies. Among the cytokines, including IFN-λ1 IFN-λ2, IFN-λ3, IL-1β, TNF-α, IL-6, IL-8, and IL-10, the level of IFN-λ3 significantly increased after the application of antiviral therapy (P=0.041), while there was no significant difference in the other cytokines (Supplementary Figure S2, Supplemental Digital Content 3, http://links.lww.com/JS9/A715).
Recurrence-free and overall survival
In the ITT population, during the follow-up with continued antiviral therapy, 37 (25.0%) patients developed tumor recurrence, and 16 (10.8%) patients died (N=15) or received liver transplantation (N=1). Of the patients who had died, 10 were in the ETV group (1 had received liver transplantation), and 6 were in the TDF group. The 1-, 3-, and 5-year OS rates for the TDF group and ETV group were 100.0 and 98.6%, 95.9 and 91.1%, and 90.8 and 78.5%, respectively. The corresponding RFS rates at 1, 3, and 5 years for the two groups were 95.9 and 95.9%, 82.38 and 70.4%, and 75.3 and 55.2%, respectively. The RFS for patients who received TDF antiviral therapy was significantly better than that for patients who received ETV antiviral therapy (P=0.026, HR=0.497 and 95% CI=0.269–0.922; Fig. 2B). However, there was no significant difference in OS between the two drugs, while TDF antiviral therapy also had a potential better benefit in OS (P=0.062; HR =0.385; 95% CI: 0.143–1.051; Fig. 2A). In the PP population, both better OS (P=0.048; HR =0.362; 95% CI: 0.132–0.993; Fig. 2C) and RFS (P=0.014; HR =0.458; 95% CI: 0.245–0.856; Fig. 2D) were found in the TDF group than in the ETV group.
Figure 2.
Overall and recurrence-free survival in HCC patients receiving TDF and ETV in the ITT and PP populations. Comparison of overall survival and recurrence-free survival in the ITT (A, B) and PP populations (C, D) for patients with HBV-related HCC treated with TDF or ETV after curative resection. HCC, Hepatocellular Carcinoma; HBV, Hepatitis B Virus; ETV, Entecavir; TDF, Tenofovir Disoproxil fumarate; ITT, Intention-to-treat; PP, Per protocol.
In the multivariate Cox analysis, compared to the TDF group, the ETV group was associated with a significantly higher risk of death or liver transplantation in the ITT population (P=0.047 HR =3.056, 95% CI: 1.015–9.196), and the predictive ability of ETV therapy status was independent of other predictive factors. Other significant factors associated with a higher risk of death or liver transplantation after curative treatment were positive HBeAg level (P=0.023, HR =3.335, 95% CI: 1184–9.390), multiple tumor number (P=0.003, HR =4.699, 95% CI: 1.691–13.055), tumor diameter of more than 3 cm (P=0.019, HR =4.317, 95% CI: 1.278–14.588), and MVI (P=0.010, HR =3.962, 95% CI: 1.389–11.302) (Table 2). The subgroup analysis of the PP population suggested worse survival for HCC patients alone when they had multiple tumor number (P=0.011, HR =4.076, 95% CI: 1.384–12.001), a tumor diameter of more than 3 cm (P=0.043, HR =3.627, 95% CI: 1.023–13.385), MVI (P=0.046, HR =2.459, 95% CI: 1.009–8.433), liver cirrhosis (P=0.033, HR =3.662, 95% CI: 1.183–16.706) and ETV antiviral therapy (P=0.041, HR =2.607, 95% CI: 1.031–8.53) (Supplementary Table 2, Supplemental Digital Content 3, http://links.lww.com/JS9/A715).
Table 2.
Univariate and multivariate analyses for death/liver transplantation and tumor recurrence in the ITT population.
| Death or liver transplantation | Tumor recurrence | |||||||
|---|---|---|---|---|---|---|---|---|
| Univariate analysis | Multivariate analysis* | Univariate analysis | Multivariate analysis* | |||||
| Factors | P | HR | 95% CI | P | P | HR | 95% CI | P |
| Age (≥60 vs. <60, years) | 0.157 | 0.649 | ||||||
| Sex (male/female) | 0.841 | 0.616 | ||||||
| HBV genotype (Type C/nontype C) | 0.982 | 0.213 | ||||||
| Increasing serum IFN-λ3 level at 3 months (Yes vs. No) | 0.783 | 0.132 | ||||||
| HBeAg (Positive vs. Negative) | 0.013 | 3.335 | 1.184–9.390 | 0.023 | 0.001 | 3.919 | 1.917–8.010 | <0.001 |
| HBV-DNA (≥2*10^3 vs. <2*10^3, IU/ml) | 0.025 | 0.026 | 2.411 | 1.145–5.074 | 0.020 | |||
| AFP (≥20 vs. <20, ng/ml) | 0.107 | 0.030 | 2.158 | 0.998–4.665 | 0.051 | |||
| TBIL (≥20 vs. <20, umol/l) | 0.835 | 0.093 | ||||||
| ALT (≥40 vs. <40, U/l) | 0.708 | 0.810 | ||||||
| ALB (<40 vs. ≥40, U/l) | 0.583 | 0.140 | ||||||
| CREA (≥90 vs. <90, mmol/l) | 0.364 | 0.269 | ||||||
| Differentiation (Moderate or Low vs. High) | 0.400 | 0.013 | ||||||
| Tumor number (Multiple vs. Single) | 0.004 | 4.699 | 1.691–13.055 | 0.003 | 0.007 | 3.233 | 1.627–6.425 | 0.001 |
| Tumor diameter, cm (≥3 vs. <3) | 0.047 | 4.317 | 1.278–14.588 | 0.019 | 0.037 | 2.315 | 1.144–4.684 | 0.020 |
| BCLC stage (A vs. 0) | 0.248 | 0.147 | ||||||
| MVI (Yes vs. No) | 0.006 | 3.962 | 1.389–11.302 | 0.010 | 0.005 | 2.169 | 1.025–4.591 | 0.043 |
| Satellite nodule (Yes vs. No) | 0.571 | 0.540 | ||||||
| Liver cirrhosis (Yes vs. No) | 0.071 | 0.031 | 3.306 | 1.134–9.636 | 0.028 | |||
| Ishak Fibrosis Score (≥4 vs. <4) | 0.076 | 0.071 | ||||||
| Transfusion (Yes vs. No) | 0.392 | 0.111 | ||||||
| BMI (≥25 vs. <25) | 0.981 | 0.130 | ||||||
| History of smoke (Yes vs. No) | 0.087 | 0.150 | ||||||
| Hypertension (Yes vs. No) | 0.190 | 0.143 | ||||||
| Diabetes (Yes vs. No) | 0.065 | 0.414 | ||||||
| Cardiovascular disease (Yes vs. No) | 0.815 | 0.895 | ||||||
| Alcohol abuse (Yes vs. No) | 0.081 | 0.787 | ||||||
| Antiviral therapy (ETV vs. TDF) | 0.068 | 3.056 | 1.015–9.196 | 0.047 | 0.046 | 2.57 | 1.264–5.228 | 0.009 |
AFP, Alpha Fetoprotein; ALB, Albumin; ALT, Alamine Aminotransfera; BCLC, Barcelona staging; CREA, Creatinine; ETV, Entecavir; HBeAg, Hepatitis B e Antigen; HBV, Hepatitis B Virus; HR, Hazard Ratio; ITT, Intention-to-treat; MVI, Microvascular Infiltration; TBIL, Total Bilirubin; TDF, Tenofovir Disoproxil Fumarate.
Multivariate Analysis: Multivariate analysis was based on the potential risk factors (P<0.1 in the univariate analysis).
In the multivariate analysis of tumor recurrence, seven significant factors were associated with poorer tumor recurrence: positive HBeAg level (P<0.001, HR =3.919, 95% CI: 1.917–8.010), high viral replication of HBV (P=0.026, HR =2.411, 95% CI: 1.145–5.074), multiple tumors (P=0.001, HR =3.233, 95% CI: 1.627–6.425), tumor diameter of more than 3 cm (P=0.020, HR =2.315, 95% CI: 1.144–4.684), MVI (P=0.043, HR =2.169, 95% CI: 1.025–4.591), liver cirrhosis (P=0.028, HR =3.306, 95% CI: 1.134–9.636) and ETV antiviral therapy (P=0.009, HR =2.566, 95% CI: 1.264–5.228) (Table 2). The subgroup analysis of the PP population suggested a higher risk of tumor recurrence for HCC patients alone when they had a positive HBeAg level (P=0.001, HR =3.399, 95% CI: 1.659–6.963), high viral replication of HBV (P=0.003, HR =3.004, 95% CI: 1.466–6.157), moderate or low differentiation (P=0.012, HR =2.534, 95% CI: 1.227–5.233), multiple tumor number (P<0.001, HR =3.982, 95% CI: 1.971–8.043), a tumor diameter of more than 3 cm (P=0.002, HR =3.199, 95% CI: 1.552–6.595), MVI (P=0.003, HR =2.243 95% CI: 1.042–5.198), liver cirrhosis (P=0.027, HR =3.288, 95% CI: 1.143–9.46), and ETV antiviral therapy (P=0.006, HR =2.687, 95% CI: 1.332–5.420) (Supplementary Table 2, Supplemental Digital Content 3, http://links.lww.com/JS9/A715).
Competing risk analysis
During the study period, six patients died before the recurrence of HCC in the entire cohort. Of the six patients, four (66.7%) patients (three in the ETV group and one in the TDF group) died from liver failure, and two (33.3%) patients died from upper gastrointestinal hemorrhage (one in the ETV group and one in the TDF group). With the competing risk analysis, TDF therapy was also associated with a significantly lower risk of tumor recurrence than ETV therapy (HR=0.507, 95% CI=0.263–0.976, P=0.042; Supplementary Figure S3, Supplemental Digital Content 3, http://links.lww.com/JS9/A715).
Early and late recurrence
Among the 37 patients with tumor recurrence, 18 (48.6%) patients had early recurrence (<2 years), and 19 (51.4%) patients had late recurrence (≥2 years). In the patients with early recurrence, seven (38.9%) patients received TDF as antiviral therapy, and 11 (61.1%) patients received ETV. For late recurrence, eight (42.1%) patients were in the TDF group, while 11 (57.9%) patients were in the ETV group. As shown in Figure 3, there was no significant difference in early tumor recurrence between the two groups in either the ITT population (P=0.109; HR =1.964; 95% CI: 0.858–4.494) or the PP population (P=0.267; HR =1.632; 95% CI: 0.687–3.876). In the multivariate analysis, type C HBV genotype (P=0.001, HR =2.276, 95% CI: 1.897–4.674), positive HBeAg (P<0.001, HR =1.658, 95% CI: 1.505–3.907), MVI (P=0.001, HR =2.701, 95% CI: 1.707–5.474), and liver cirrhosis (P=0.046, HR =3.827, 95% CI: 1.038–6.042) were associated with a higher risk of early tumor recurrence in the ITT population (Supplementary Table S3, Supplemental Digital Content 3, http://links.lww.com/JS9/A715). Additionally, type C HBV genotype (P=0.007, HR =3.049, 95% CI: 1.640–6.318), positive HBeAg (P=0.027, HR =3.797, 95% CI: 1.166–8.368), multiple tumors (P=0.044, HR =3.980, 95% CI: 1.035–10.304), MVI (P=0.002, HR =2.762, 95% CI: 1.902–5.455) and liver cirrhosis (P=0.015, HR =2.329, 95% CI: 1.387–4.478) were associated with a higher risk of early tumor recurrence in the PP population (Supplementary Table S4, Supplemental Digital Content 3, http://links.lww.com/JS9/A715).
Figure 3.
Early and late recurrence-free survival of HCC patients who underwent curative liver resection in the ITT and PP populations. The different effects of TDF and ETV on early and late recurrence-free survival in ITT (A, B) and PP (C, D) populations for HBV-related HCC patients following radical hepatectomy. HCC, Hepatocellular Carcinoma; HBV, Hepatitis B Virus; ETV, Entecavir; TDF, Tenofovir Disoproxil fumarate; ITT, Intention-to-treat; PP, Per protocol.
However, TDF was associated with a lower risk of late RFS than ETV in both the ITT (P=0.045; HR =0.432; 95% CI: 0.189–0.985) and PP (P=0.022; HR =0.373; 95% CI: 0.160–0.870) populations (Fig. 3C and D). In the multivariate analysis, ETV was also an independent risk factor for preventing late tumor recurrence (P=0.012; HR =2.088; 95% CI: 1.285–4.422) in the ITT population, together with a high level of HBV-DNA (P<0.001; HR =3.180; 95% CI: 2.784–7.519), AFP level of more than 20 ng/ml (P=0.038; HR =2.942; 95% CI: 1.063–6.143), moderate/low differentiation of HCC (P=0.048; HR =2.461; 95% CI: 1.008–6.006), multiple tumors (P<0.001; HR =1.509; 95% CI: 1.329–3.196), tumor diameter of more than 3 cm (P=0.001; HR =2.083; 95% CI: 1.884–7.716), obesity (P=0.022; HR =3.187; 95% CI: 1.387–6.998), and diabetes (P=0.014; HR =2.582; 95% CI: 1.299. The subgroup analysis of the PP population suggested a higher risk of late tumor recurrence for HCC patients alone in the following: high level of HBV-DNA (P=0.002; HR =3.557; 95% CI: 1.832–6.955), AFP level of more than 20 ng/ml (P=0.044; HR =2.663; 95% CI: 1.007–4.085), moderate/low differentiation of HCC (P=0.023; HR =1.943; 95% CI: 1.154–3.768), multiple tumor number (P=0.042; HR =3.058; 95% CI: 1.011–6.796), tumor diameter of more than 3 cm (P=0.036; HR=3.023; 95% CI: 1.028–6.655), MVI (P=0.003; HR=2.075; 95% CI: 1.851–3.944), and diabetes (P=0.028; HR=3.077; 95% CI: 1.198–7.138), and ETV antiviral therapy (P=0.032, HR =3.007, 95% CI:
Discussion
This RCT evaluated the different efficacies of ETV and TDF on the survival and tumor recurrence of patients with early-stage HBV-related HCC (barcelona clinic liver cancer stage 0 or A) after radical hepatectomy. In this study, we found that patients treated with TDF had a lower risk of tumor recurrence and potentially better OS after curative liver resection than those treated with ETV therapy, especially for late recurrence.
To date, many studies have been conducted to evaluate the different effects of ETV and TDF on the incidence of HCC in chronic hepatitis B patients, but the conclusions are debatable, let alone for HCC patients following curative liver resection14–17. In a nationwide cohort study in Korea, researchers found that TDF treatment had a significantly better benefit in HCC recurrence and OS for patients with HBV-related HCC after curative liver resection compared to ETV therapy15, which is in line with another study based on the population of Taiwan16. In contrast, Kao et al. 17 reported in their observational study that there was no difference between the two drugs. However, these studies were all retrospective studies that had a relatively high risk of selection bias and low heterogeneity. We believe this study is the first prospective randomized study on this topic.
Even though our conclusion was similar to some of the other studies, there were some differences. First, in Choi’s study, patients were enrolled from 2010 to 2018. However, TDF was approved in South Korea in 2012, which was 6 years later than ETV, and the difference in follow-ups might distort their results. Second, due to the potential influences of TDF on renal function, elderly and renal dysfunctional patients need to be particularly evaluated for antiviral therapy by clinicians, which might affect the reliability of the results. In the present study, we created thresholds for age and renal function to reduce the influences of side effect bias. The results showed that there was no significant difference in renal function between the two groups (Supplementary Table S1, Supplemental Digital Content 3, http://links.lww.com/JS9/A715), which made the result more reliable. Third, although propensity score matching was applied in some of the studies, not all factors (such as HBV genotype, obesity, and alcohol abuse), which could be potential risk factors for tumor recurrence and death18,19, were included in the analysis. In our study, these factors were comparable between the two groups. Moreover, we also found that obesity had a potential risk associated with HCC recurrence, especially for late tumor recurrence. Although the underlying mechanism is still unclear, adipokine-related hepatocarcinogenesis and disordered inflammation might be involved. Regarding the HBV genotype, numerous studies have reported that HBV genotype C is associated with an increased risk of liver cirrhosis and HCC because of the longer duration of HBeAg seroconversion and higher viral load than other genotypes20,21. Even though most of the patients involved in our study belonged to genotype C, which met the demographic characteristics of the Asian population, HBV genotype C was more likely to be associated with early tumor recurrence for HBV-related HCC patients. The results indicated that HBV genotype C infection seemed to be associated with poorer disease progression and remission.
The potential mechanism of the different effects of the two drugs on the prevention of HCC recurrence is still unclear. In our opinion, this difference could be associated with HBV-DNA suppression by the two drugs. Numerous studies, including RCTs, in recent years have indicated that active HBV replication, even at a low level, increases the risk of HCC incidence, HCC recurrence and death in HBV-related HCC patients22,23. Active HBV replication initiates hepatocarcinogenesis by inhibiting tumor immune surveillance and promoting the proliferation and stemness of HCC cells to enhance tumor development and metastasis24,25. Nucleot(s)ide analog (NUC) treatment was proven to be effective in preventing HBV reactivation and reducing liver inflammation, but the superiority between ETV and TDF is still debatable. To reveal the association between NUC treatment, HBV replication, and HCC recurrence, we enrolled only NUC-naive patients into this trial. Notably, we found no significant difference between TDF and ETV in terms of virological and biochemical responses before 30 months. TDF showed a better virological response at 30 months and resulted in a greater reduction in HBsAg levels at 18 months, which was similar to other RCTs indicating the noninferiority of TDF to ETV8. The difference in HBsAg reduction with TDF might be explained by a recent study that indicated that nucleotide analogs (such as TDF and adefovir dipivoxil) rather than nucleoside analogs (such as ETV and lamivudine) had novel additional pharmacological effects of inducing IFN-λ3 to inhibit viral mRNA translation and HBsAg production26. Additionally, another in vitro study reported that TDF had additional antitumor effectiveness through the inhibition of enteric lipopolysaccharide-mediated IL-10 and induction of IL-12p70 and TNF-α27. However, in our study, we also found a slightly increasing level of IFN-λ3 in the TDF group at 3 months after antiviral treatment, but there was no significant difference in antitumor-related cytokine factors between the two drugs. The difference might be due to the different measurement of detection and greater fold of drug concentration of in vitro experiments than given to humans. Furthermore, we did not find a relationship between IFN-λ3 levels and HCC recurrence, while HBV-DNA replication and different types of antiviral treatment were both independent risk factors for late recurrence of HCC. Therefore, these results indicated that the differences in HCC recurrence were possibly due to altered HBV-DNA suppression and anti-inflammatory effects, but the antitumor effect of TDF needs to be proven in future studies.
Our study also had several limitations. First, the study was a single-center trial, which could have limitations in geographic distribution. Second, in this study, most patients were infected with genotype C HBV, which is mainly detected in Asia and is known as one of the risk factors for HCC28. Although the HBV genotype between the two groups was comparable, the difference between TDF and ETV in HCC patients with genotypes A and D was still unclear. The results should be validated in a future multicenter clinical trial. Nevertheless, TDF therapy appears to be of better benefit in the prevention of tumor recurrence and prolong survival for HBV-related HCC patients following curative liver resection.
Conclusion
Early-stage HBV-related HCC patients receiving consistent postoperative TDF antiviral treatment had better OS and a lower risk of HCC recurrence (especially late recurrence) than patients receiving ETV.
Ethical approval
This study was approved by the ethics committee of West China Hospital, Sichuan University(reference number [2018]435).
Sources of funding
This study was supported by the Scientific and Technological Support Project of Sichuan Province (21ZDYF1542), the Sichuan Province Science and Technology Support Program (2021YFS0166), the Postdoctoral Sustentation Fund of West China Hospital, Sichuan University, China (2020HXBH057), the National Natural Science Foundation of Sichuan province (2023NSFSC1880) and the National Natural Science Foundation of China (81900576).
Author contribution
All authors have full access to all data used in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. T.F.W. and C.L. proposed the study. L.Y.H., Z.J.X., and Y.J.Z. performed the research. X.Y.Z. and L.Y.H. analyzed the data. L.Y.H. and Z.J.X. wrote the first draft. C.L. and Z.H.L. reviewed the paper.
Conflicts of interest disclosure
All authors have no conflicts of interests.
Research registration unique identifying number (UIN)
Name of the registry: Chinese Clinical Trial Registry chictr.org.cn.
Unique Identifying number or registration ID: ChiCTR1800014629.
Hyperlink to your specific registration (must be publicly accessible and will be checked): http://www.chictr.org.cn/showproj.aspx?proj=24963.
Guarantor
Chuna Li, MD,PHD.
Data availability statement
The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
Provenance and peer review
Not commissioned, externally peer-reviewed.
Supplementary Material
Footnotes
L.H. and Z.X. contributed equally to this manuscript.
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal’s website, www.lww.com/international-journal-of-surgery.
Published online 16 June 2023
Contributor Information
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References
- 1.Llovet JM, Kelley RK, Villanueva A, et al. Hepatocellular carcinoma. Nat Rev Dis Primers 2021;7:6. [DOI] [PubMed] [Google Scholar]
- 2.Chen SL, Xiao H, Xie ZL, et al. The presence of microvascular invasion guides treatment strategy in recurrent HBV-related HCC. Eur Radiol 2020;30:3473–3485. [DOI] [PubMed] [Google Scholar]
- 3.Huang G, Lau WY, Wang ZG, et al. Antiviral therapy improves postoperative survival in patients with hepatocellular carcinoma: a randomized controlled trial. Ann Surg 2015;261:56–66. [DOI] [PubMed] [Google Scholar]
- 4.Wang ZY, Tao QF, Wang ZH, et al. Antiviral therapy improves post-operative survival outcomes in patients with HBV-related hepatocellular carcinoma of less than 3cm - A retrospective cohort study. Am J Surg 2020;219:717–725. [DOI] [PubMed] [Google Scholar]
- 5.Cornberg M, Lok AS, Terrault NA, et al. , EASL-AASLD HBV Treatment Endpoints Conference Faculty. Guidance for design and endpoints of clinical trials in chronic hepatitis B - Report from the 2019 EASL-AASLD HBV treatment endpoints conference. Hepatology. 2019; doi: 10.1002/hep.31030 [DOI] [PubMed]
- 6.Choi J, Kim HJ, Lee J, et al. Risk of hepatocellular carcinoma in patients treated with entecavir vs tenofovir for chronic hepatitis B: a Korean nationwide cohort study. JAMA Oncol 2019;5:30–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Kim SU, Seo YS, Lee HA, et al. A multicenter study of entecavir vs. tenofovir on prognosis of treatment-naive chronic hepatitis B in South Korea. J Hepatol 2019;71:456–464. [DOI] [PubMed] [Google Scholar]
- 8.Koike K, Suyama K, Ito H, et al. Randomized prospective study showing the non-inferiority of tenofovir to entecavir in treatment-naive chronic hepatitis B patients. Hepatol Res 2018;48:59–68. [DOI] [PubMed] [Google Scholar]
- 9.Sacco R, Gadaleta-Caldarola G, Galati G, et al. EASL HCC summit: liver cancer management. Future Oncol 2014;10:1129–1132. [DOI] [PubMed] [Google Scholar]
- 10.Schulz KF, Altman DG, Moher D. CONSORT Group. CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. BMJ 2010;340:c332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Xie DY, Ren ZG, Zhou J, et al. 2019 Chinese clinical guidelines for the management of hepatocellular carcinoma: updates and insights. Hepatobiliary Surg Nutr 2020;9:452–463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.European Association for the Study of the Liver. EASL clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol 2018;69:182–236. [DOI] [PubMed] [Google Scholar]
- 13.Makamu P, Bezuidenhout S, Matlala M. Prevalence of kidney injury in patients taking tenofovir based antiretroviral therapy at a primary health care clinic, in East Rand, Gauteng Province. Hosp Pract 19952021;49:88–94. [DOI] [PubMed] [Google Scholar]
- 14.Cheung KS, Mak LY, Liu SH, et al. Entecavir vs tenofovir in hepatocellular carcinoma prevention in chronic hepatitis B infection: a systematic review and meta-analysis. Clin Transl Gastroenterol 2020;11:e00236. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Choi J, Jo C, Lim YS. Tenofovir versus entecavir on recurrence of hepatitis B virus-related hepatocellular carcinoma after surgical resection. Hepatology 2021;73:661–673. [DOI] [PubMed] [Google Scholar]
- 16.Tsai MC, Wang CC, Lee WC, et al. Tenofovir is superior to entecavir on tertiary prevention for BCLC stage 0/A hepatocellular carcinoma after curative resection. Liver Cancer 2022;11:22–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Kao WY, Tan EC, Lee HL, et al. Entecavir versus tenofovir on prognosis of hepatitis B virus-related hepatocellular carcinoma after curative hepatectomy. Aliment Pharmacol Ther 2023;57:1299–1312. [DOI] [PubMed] [Google Scholar]
- 18.Conci S, Cipriani F, Donadon M, et al. Hepatectomy for metabolic associated fatty liver disease (MAFLD) related HCC: propensity case-matched analysis with viral- and alcohol-related HCC. Eur J Surg Oncol 2022;48:103–112. [DOI] [PubMed] [Google Scholar]
- 19.Lee YT, Wang JJ, Luu M, et al. State-level HCC incidence and association with obesity and physical activity in the United States. Hepatology 2021;74:1384–1394. [DOI] [PubMed] [Google Scholar]
- 20.Lin CL, Kao JH. Natural history of acute and chronic hepatitis B: the role of HBV genotypes and mutants. Best Pract Res Clin Gastroenterol 2017;31:249–255. [DOI] [PubMed] [Google Scholar]
- 21.Tseng TC, Liu CJ, Chen CL, et al. Higher lifetime chance of spontaneous surface antigen loss in hepatitis B carriers with genotype C infection. Aliment Pharmacol Ther 2015;41:949–960. [DOI] [PubMed] [Google Scholar]
- 22.Huang G, Li PP, Lau WY, et al. Antiviral therapy reduces hepatocellular carcinoma recurrence in patients with low HBV-DNA levels: a randomized controlled trial. Ann Surg 2018;268:943–954. [DOI] [PubMed] [Google Scholar]
- 23.Lee SW, Kwon JH, Lee HL, et al. Comparison of tenofovir and entecavir on the risk of hepatocellular carcinoma and mortality in treatment-naive patients with chronic hepatitis B in Korea: a large-scale, propensity score analysis. Gut 2020;69:1301–1308. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Ding WB, Wang MC, Yu J, et al. HBV/pregenomic RNA increases the stemness and promotes the development of HBV-related HCC through reciprocal regulation with insulin-like growth factor 2 mRNA-binding protein 3. Hepatology 2021;74:1480–1495. [DOI] [PubMed] [Google Scholar]
- 25.Ho DW, Tsui YM, Chan LK, et al. Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma. Nat Commun 2021;12:3684. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Murata K, Asano M, Matsumoto A, et al. Induction of IFN-lambda3 as an additional effect of nucleotide, not nucleoside, analogues: a new potential target for HBV infection. Gut 2018;67:362–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Murata K, Tsukuda S, Suizu F, et al. Immunomodulatory mechanism of acyclic nucleoside phosphates in treatment of hepatitis B virus infection. Hepatology 2020;71:1533–1545. [DOI] [PubMed] [Google Scholar]
- 28.Chiu SM, Kuo YH, Wang JH, et al. Associations of HBV genotype B vs C infection with relapse after cessation of entecavir or tenofovir therapy. Clin Gastroenterol Hepatol 2020;18:2989–2997.e3. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.