The Ethics Committee of Sun Yat-sen University Cancer Center approved this retrospective study (B2019061), and the requirement to obtain informed consent was waived. The study consists of 2915 patients with primary intermediate-stage HBV-HCC who underwent TACE at the Sun Yat-sen University Cancer Center between January 2012 and October 2018. The inclusion criteria were as follows: (a) aged 18 years or above; (b) with intermediate-stage HCC according to the Barcelona Clinic Liver Cancer staging system; (c) no previous antiviral or anti-tumor treatment; (d) underwent TACE as initial treatment and achieved complete response; (e) HBV DNA level ≥2000 IU/ml before TACE; (f) liver function scored by Child-Pugh A; (g) patients’ performance status scored by Eastern Cooperative Oncology Group: 0-1; (h) absence of simultaneous carcinoma. Among them, 1650 patients were excluded for the following reasons: HBV DNA levels <2000 IU/ml (n = 843), coinfection with hepatitis C virus or other viral hepatitis (n = 46) and human immunodeficiency virus (n = 16), presence of chronic alcohol intake (n = 89), metabolic syndrome (n = 13), Child-Pugh B or C (n=412), Eastern Cooperative Oncology Group performance status score >1 (n=153) and presence of simultaneous carcinoma (n = 78). The workflow of the present study was shown in Figure 1 .

HCC was diagnosed by imaging techniques according to the American Association for the Study of Liver Diseases guideline (8). Data of the following variables were collected at baseline before TACE treatment (within one week): age; sex; history of diabetes, arterial hypertension, obesity, viral hepatitis, and human immunodeficiency virus. Routine examination included complete blood count (white blood cells, hemoglobin, platelet count); serum liver function tests (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, albumin, total bilirubin) and kidney function tests; alpha fetoprotein; prothrombin time and serum HBV DNA level. Serum HBV DNA level was quantified using a real-time polymerase chain reaction assay with a limit of detection of 100 IU/ml. Thus, an HBV DNA level lower than 100 IU/mL was considered undetectable in our study.

According to the European Association for the Study of the Liver clinical practice guidelines (22), VR was defined as an undetectable HBV DNA level on two consecutive measurements during NA treatment. The VR group was allocated with patients who had achieved a VR after NA therapy throughout the follow-up period. Meanwhile, those who never achieved a VR were assigned to the non-VR group. MVR was defined as a persistently undetectable HBV DNA level after achieving a VR during the follow-up period (23). Non-MVR was defined as intermittent or persistent period of detectable HBV DNA levels after achieving a VR throughout the follow-up period.

Cirrhosis was clinically defined by ultrasonographical features, including small-sized liver, nodular liver surface or caudate lobe hypertrophy, and splenomegaly (by imaging) or by the presence of varices (by upper endoscopy or imaging test) (24). Tumor burden was evaluated by the up-to-seven criteria (25).

TACE was performed by 2 radiologists with at least 5 years of interventional therapy experience as described in the previous research (26, 27). Briefly, visceral angiography of the superior mesenteric and hepatic artery was performed to assess arterial blood supply of the liver and to confirm patency of the portal vein via inserting a selective catheter into the segmental or subsegmental tumor-feeding arteries. And 300 mg of carboplatin (Bristol-Myers Squibb, New York, NY) was used in hepatic artery infusion chemotherapy. Then, chemolipiodolization was conducted via using 30-50 mg epirubicin (Pharmorubicin; Pfzer, Wuxi, China) and 6-8 mg mitomycin C (Zhejiang Hisun Pharmaceutical, Taizhou, China) mixed with 5-20 mL lipiodol (Lipiodol Ultra-Fluide; André Guerbet Laboratories, Aulnay-Sous-Bois, France). Finally, embolization was performed using absorbable gelatin sponge particles of 1-2 mm in diameter (Gelfoam, Hangzhou Alc, China) or with polyvinyl alcohol particles of 350-560 μm in diameter (Alicon Pharmaceutical, Hangzhou, China) until achieving blood static for more than 10 successive cardiac beats. Angiography was conducted again to detect the extent of vascular occlusion and to evaluate blood flow in other arterial vessels after embolization. TACE therapies were performed until achieving complete response. Tumor response to TACE was evaluated 4-week after each TACE cycle. The modified Response Evaluation Criteria in Solid Tumors were applied at contrast-enhanced computed tomography or magnetic resonance imaging to evaluate treatment response (28). NAs using entecavir (0.5mg per day; Sino-American Squibb, Shanghai, China) were applied within 1 week prior to the first TACE treatment.

Followed up assessments were performed every 3 months during the first 2 years, every 6 months for years 3-5 and then every 12 months thereafter. The follow-up examinations included the above-mentioned biochemical tests, HBV DNA level, alpha fetoprotein, ultrasonography and contrast-enhanced computed tomography or magnetic resonance imaging.

The endpoint of the study was overall survival (OS), which was measured from the date of initiation of TACE to the date of patient death or the last follow-up (December 31, 2020).

The study variables were presented as median (range) for continuous data and numbers or percentage for categorical data. Differences in medians were compared using Mann-Whitney U test, and differences in percentages were evaluated by the Chi-square test. Survival rates were plotted using Kaplan-Meier method and differences between various VR status or MVR duration groups were analyzed using log-rank test. The associations between clinicopathological factors and survival outcomes were assessed by univariate and multivariate analyses using the Cox proportional hazard regression models. The variables significant on univariate analysis (P<0.05) were subjected in the multivariate Cox regression model. The proportional hazards assumption was checked based on the scaled Schoenfeld residuals. All statistical analyses were performed using Statistical Product and Service Solutions software version 19.0. A value of two-sided P<0.05 was considered statistically significant.

A total of 1265 HBV-HCC patients treated with TACE receiving NA therapy were included in final analyses. The baseline characteristics of study patients were shown in Table 1 . Overall, the median follow-up time was 18 months (range, 2-78 months) and the median cycles of receiving TACE was 3 (range, 1-6) for all subjects. VR was observed in 744 patients (58.8%), while the remaining 521 subjects (41.2%) did not achieve VR. For patients achieving VR, a total of 250 patients undergoing other treatment including sorafenib (n=174), radiofrequency ablation (n=38), hepatectomy (n=19), radiotherapy (n=13) and chemotherapy (n=6). While 151 patients received other treatment in non-VR group, including sorafenib (n=104), radiofrequency ablation (n=24), hepatectomy (n=11), radiotherapy (n=8) and chemotherapy (n=4). There was no significant difference in demographic and clinical characteristics, such as median age, sex, proportion of patients with cirrhosis, ascites, splenomegaly, or tumor capsule and percentage of participants beyond the up-to-seven criteria, as well as laboratory tests including blood routine examination and liver and kidney function tests, and other treatment after TACE between the VR and non-VR groups (P>0.05). As shown in Table 2 , splenomegaly, up-to-seven criteria, alpha fetoprotein, total bilirubin, platelet count and sorafenib treatment after TACE were associated with OS in VR group. In the multivariable Cox regression analysis, splenomegaly (hazard ratio (HR), 1.340; 95% confidence interval (CI), 1.095-1.641; P=0.005), up-to-seven criteria (HR, 1.298; 95% CI, 1.048-1.607; P=0.017), total bilirubin (HR, 1.505; 95% CI, 1.104-2.052; P=0.010), platelet count (HR, 1.326; 95% CI, 1.088-1.617; P=0.005) and sorafenib treatment after TACE (HR, 0.605; 95% CI, 0.494-0.742; P<0.001) were identified as independent factors of OS in patients with NA therapy achieving VR.

Among patients with VR, MVR was present in 542 patients (72.8%), while the other 202 patients (27.2%) were in the non-MVR group. No significant differences in baseline characteristics were found between the MVR and non-MVR groups ( Table 3 ). Additionally, splenomegaly, up-to-seven criteria, sorafenib treatment after TACE and radical therapy after TACE were correlated with OS in the univariable Cox regression analysis. Similarly, in the multivariable analysis, the prognostic factors included splenomegaly (HR, 1.339; 95% CI, 1.059-1.692; P=0.015), tumor burden beyond up-to-seven criteria (HR, 1.430; 95% CI, 1.110-1.844; P=0.006), receiving sorafenib treatment after TACE (HR, 0.668; 95% CI, 0.533-0.838; P<0.001) and receiving radical therapy after TACE (HR, 0.586; 95% CI, 0.392-0.877; P=0.009) ( Table 4 ).

For patients achieving VR, the 1-, 3-, and 5-year OS rates were 69.8%, 17.7%, and 13.6%, respectively. In comparison, in the non-VR group, the corresponding OS rates were 51.0%, 14.1%, and 8.0%, respectively ( Figure 2A ). Thus, patients in the VR group had better OS than patients in the non-VR group (median OS: 21 vs 16 months; HR, 0.707; 95% CI, 0.622-0.804; P<0.001). As shown in Table 5 , without controlling for other factors, VR was associated with increased OS in patients undergoing TACE (P <0.001). After adjusting splenomegaly, up-to-seven criteria, alpha fetoprotein and platelet count as confounders, VR remained as an independent factor for OS in patients received NA therapy undergoing TACE (HR, 0.772; 95% CI, 0.615-0.916), P=0.003).

The 1-, 3-, and 5-year OS rates were 75.1%, 19.2%, and 15.3%, respectively, for the MVR group and 55.3%, 15.4%, and 11.2%, respectively, for the non-MVR group ( Figure 2B ). Thus, patients achieving MVR to NA therapy had significantly longer OS than patients in the non-MVR group (median OS: 23.2 vs 18 months; HR, 0.736; 95% CI, 0.612-0.885; P=0.001).

Moreover, survival analyses were separately performed for each subgroup of interest stratified by presence of splenomegaly and up-to-seven criteria for HCC. As a result, MVR again showed significantly improved OS than the non-MVR group, irrespective of patients with or without splenomegaly (HR, 0.758; 95% CI, 0.616-0.931; P=0.005 and HR, 0.553; 95% CI, 0.363-0.843; P=0.008, respectively) ( Figures 3A, B ). In addition, a consistent survival benefit of achieving MVR was observed among patients within the up-to-seven criteria. Among these patients with relative low tumor burden, the 1-, 3-, and 5-year OS rates for patients with MVR were 77.0%, 20.5%, and 16.9%, compared to 57.3%, 13.6%, and 0% among patients in the non-MVR group (HR, 0.707; 95% CI, 0.575-0.869; P=0.001). For patients beyond the up-to-seven criteria, there was no significant difference in OS between the MVR and non-MVR groups (median OS, 17.7 vs 11.5 months; HR, 0.906; 95% CI, 0.598-1.373; P=0.639) ( Figures 3C, D ).

In the MVR cohort, patients were classified into three subgroups (1 year, 1-2 years, ≥2 years) according to the duration of MVR status. There were 123 (22.7%), 190 (35.1%), and 229 (42.2%) patients in the above three subgroups, respectively. As shown in Figure 4 and Table 6 , there was a significant difference in OS among different MVR duration subgroups (log-rank P<0.001). Particularly, patients with MVR status more than two years showed a better OS than those with just one-year (HR, 0.719; 95% CI, 0.650-0.797; P<0.001) or one-to-two-year MVR (HR, 0.612; 95% CI, 0.471-0.795; P=0.024). Meanwhile, patients in the one-to-two-year MVR duration subgroup had a significantly improved OS when compared to those did not achieve one-year MVR. Furthermore, in the MVR group, patients with high tumor burden showed obviously worse OS than those with low tumor burden, with respective OS at 1, 3 and 5 years (65.5%, 12.4%, 8.3% vs. 77.0%, 20.5%, 16.9%; HR, 1.491; 95% CI, 1.158-1.920; P=0.002). In addition, patients with splenomegaly had a worse OS compared to those without enlarged spleen (HR, 1.287; 95% CI, 1.109-1.626; P=0.034). The results of the subgroup survival analyses were summarized in Table 6 .