This is a retrospective study of 111 patients with HCC who received combination therapy from January 1, 2013 to December 31, 2016 at Beijing Youan Hospital (Beijing, China). Most of the patients were early-stage small HCC and all initial treatment patients. For the subsequent diagnosis and treatment plan and prognosis, all patients voluntarily underwent needle biopsy. Biopsy is generally performed under the guidance of ultrasound. Contraindications for percutaneous liver biopsy mainly include patients with bleeding tendency, patients with severe cardiopulmonary disease, massive ascites, severe extrahepatic obstructive jaundice, lack of consciousness, inability to cooperate, suspected hemangioma or other vascular tumors, and suspected echinococcus cyst in the liver. All patients participating in the study were required to meet the following inclusion criteria: 1) age between 18 and 75 years old; 2) the combination of TACE plus ablation is the primary treatment method; 3) Child-Pugh class A or B; 4) no other malignancies that may affect prognosis; 5) all patients underwent core needle biopsy before combined therapy; and 6) HBV-associated HCC. The exclusion criteria were described below: 1) imaging evidence of invasion of the main branches of the portal/hepatic veins; 2) presence of extrahepatic metastases; 3) severe coagulation disorders; 4) incomplete ablation; 5) secondary liver cancer; 6) co-infection with HCV; and 7) missed follow-up examinations Figure 1 .

Demographic information, pathological indicators and clinical laboratory data were collected: 1) demographic and etiology indicators, such as age, sex, history of hypertension and somking, serum HBsAg; 2) tumor-related indices, such as the number and size of tumors, and alpha-fetoprotein (AFP) level; 3) liver function indices, including cirrhosis, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total serum bilirubin (TBIL), serum albumin, globulin, and γ-glutamyl transpeptidase(γ-GT); 4) routine blood examinations, such as neutrophil count (NEU), lymphocyte count (LYM), platelet count (PLT); 5) blood coagulation indicators: including prothrombin time (PT), prothrombin activity (PTA), fibrinogenic (Fib); and 6) pathological indicators: Ki67, P53, HBsAg, Glypican-3 (GPC3), the degree of tumor differentiation. The study was conducted in accordance with the 1964 Declaration of Helsinki and approved by the Ethics Committee of You’an Hospital in Beijing. Information on these patients was kept confidential. As a minimal risk study, we dropped the requirement for informed consent.

Combined therapy was performed by three qualified radiologists and hepatologists with more than 5 years of experience. The femoral artery was punctured using a modified Seldinger method, in which a microcatheter was inserted into the supplying artery of the tumor and doxorubicin (Pfizer, USA) and lipiodol (Gabcod, France) were injected. The interruption of blood flow in the tumor supplying artery was considered complete embolization.Local ablation was performed within 2 weeks after TACE. After the patient has underwent local anesthesia,the procedure is performed percutaneously by hepatologists under the guidance of triphasic computed tomography (CT) or magnetic resonance imaging (MRI). According to the number and size of tumor, overlapping ablation, multi-site ablation and fractional ablation were performed respectively. To ensure complete ablation, a safe distance of 0.5-1.0cm should be maintained around the tumor.

In order to ensure complete ablation of large tumors and multiple tumors, we use the combined treatment of TACE plus ablation, first reducing the target lesion by TACE to facilitate the subsequent complete ablation.On the one hand, TACE can block the blood supply and mark the tumor; on the other hand, TACE is beneficial for subsequent ablation; ablation therapy can further inactivate residual lesions, and ablation treatment is a minimally invasive, efficient and reproducible treatment, so for patients difficult to complete primary ablation, repeated ablation is used to achieve complete ablation.

Patients follow-up were performed on outpatient clinic. Follow-up consisted of physical examination, blood tests, and imaging examination, which included abdominal ultrasound every 3-6 months and ontrast-enhanced CT/MRI every 6 months. The follow-up involved a physical examination and blood tests, as well as imaging, included an abdominal ultrasound every 3–6 months and contrast-enhanced CT/MRI every 6 months. Recurrence was defined as intrahepatic local progression, distal intrahepatic recurrence and extrahepatic metastases. RFS was calculated as the time between the date of ablation and the patient’s first evidence-based recurrence or death in patients without evidence of disease recurrence, whereas OS was defined as the time between the date of ablation and tumor-related death or the date of the last visit. The cut-off date for this study is July 1, 2020. Patients were treated with radiofrequency ablation or TACE when they were found to have tumor recurrence. Contrast-enhanced CT and/or MRI is used to determine if a patient has recurrence. Recurrence is considered when a patient’s imaging shows enhancement of areas within or around the primary tumor.

Data were analyzed using SPSS 26.0 software (IBM, Armonk, NY, USA), and all figures were created by SPSS and Graphpad Prism 8.0(Graphpad software Inc). Continuous variables were expressed as the mean ± standard deviation (SD), and categorical data were presented as the frequency. Univariate and multivariate Cox regression analyses were performed to assess the independent risk factors of prognosis in HCC patients undergoing combined therapy. The RFS and OS rates were calculated with the Kaplan-Meier method, and the differences between groups were compared by using the Log-rank test. Statistical significance was considered when the P value < was 0.05.

The present study consisted of 90 men (81.1%) and 21 women (18.9%) with a mean age of 56 years ± 9 years. Additionally, 27 patients (24.3%) had hypertension, 55 patients (49.5%) were received antiviral therapy before they underwent combined therapy. There were 50 patients (45.0%) who had a history of smoking and 33 patients (29.7%) with a history of drinking. By the end of follow-up, 88 (79.3%) recurred and 43 (38.7%) died. The median follow-up duration was 52.2 (36.4-64.9) months. The 1-,3- and 5-year RFS rates were 60.4% (67/111), 29.7% (33/111),and 20.7%(23/111). Moreover, the 1-, 3-, and 5-year cumulative OS rates were 100%(111/111), 73.0% (81/111) and 61.3% (68/111), respectively. There were differences in age (P=0.040) and lymphocyte count (P=0.015) between the recurrence group and the non-recurrence group ( Table 1 ).

Correlations between demographic and pathological indicators and clinical laboratory data and RFS were assessed with univariate and multifactorial analyses. The univariate analysis demonstrated that RFS was significantly associated with age, intrahepatic HBsAg, albumin, total serum bilirubin, PT and PTA. The multivariate analysis showed that intrahepatic HBsAg expression (HR: 1.965; 95%CI: 1.169-3.304) was an independent predictor of HCC recurrence (P<0.05) ( Table 2 )

Univariate and multivariate analyses were performed to assess the relationship between demographic, pathological indicators and clinical laboratory data and OS. Univariate analysis showed that OS was significantly correlated with cirrhosis, antiviral, intrahepatic HBsAg expression, GGT, PT and PTA. The multivariate analysis showed that intrahepatic HBsAg expression (HR:2.320;95%CI:1.093~4.925)and antiviral(HR:3.272;95%CI:1.480~7.234) were an independent predictor of HCC survival status (P<0.05) ( Table 3 ).

To investigate the influence of intrahepatic HBsAg expression on patient prognosis, patients were divided into two groups according to the expression of intrahepatic HBsAg; one group was positive intrahepatic HBsAg expression, and the other one was negative intrahepatic HBsAg expression. Statistical analysis showed that the age (P=0.004), cirrhosis (P=0.040) and neutrophil count (P=0.020) were significant difference between the two group ( Table 4 ; Figure 2 ).

Kaplan-meier analysis confirmed that positive intrahepatic HBsAg expression was a negative predictor of RFS and OS. The cumulative 1-, 3-, and 5-year RFS rates for patients with negative HBsAg expression after combined therapy were 78.3%, 43.5% and 30.4%; while for patients with positive HBsAg expression were 58.5%, 24.5% and 17.0%, respectively (P=0.018).( Figure 3 ) Meanwhile, the cumulative 1-, 3-, and 5-year OS rates for patients with negative HBsAg expression after combined therapy the were 100%, 89.1% and 80.4%, while, for patients with positive HBsAg expression were 100%, 75.5% and 58.5%, respectively (P=0.008) ( Figure 4 ).