Treatment outcomes after radiofrequency ablation in patients with non-B non-C hepatocellular carcinoma within Milan criteria: comparison with HBV-related hepatocellular carcinoma

Abstract Purpose This study aims to evaluate the treatment outcomes of radiofrequency ablation (RFA) for patients with non-B non-C hepatocellular carcinoma (HCC) (NBNC-HCC) within Milan criteria, as well as to compare them with those of patients with hepatitis B virus (HBV)-related HCC (HBV-HCC). Methods From January 2007 to February 2020, 303 patients with primary HCC who underwent RFA were retrospectively reviewed, including 259 patients with HBV-HCC (HBV-HCC group) and 44 patients with NBNC-HCC (NBNC-HCC group). The clinical characteristics and treatment survivals were evaluated and compared. Moreover, the propensity score matching was used to reduce selection bias. Results A significantly lower proportion of cirrhosis was observed in the NBNC-HCC group (p = .048). Before propensity score matching, local tumor progression, disease-free survival, and overall survival after RFA showed no significant differences between the two groups (all p > .05). After matching, the overall survival rates in the NBNC-HCC group were significantly better than those in the HBV-HCC group (p = .042). Moreover, for patients with NBNC-HCC, tumor size (hazard ratio = 8.749, 95% confidence interval, 1.599–47.849; p = .012) was the only independent predictor of local tumor progression. Conclusions Patients with NBNC-HCC within the Milan criteria after RFA had better long-term survival than patients with HBV-HCC, although larger, prospective and multicenter trials are required to validate these results.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common liver cancers, accounting for approximately 50% of the total number of cases and deaths in China [1].Although hepatitis B virus (HBV)-related HCC (HBV-HCC) is the largest proportion of patients with HCC in East Asia [2], the proportion of HCC cases negative for hepatitis B surface antigen (HBsAg) and hepatitis C antibody (HCVAb), which is called 'non-B non-C HCC (NBNC-HCC)', is rapidly growing [3].NBNC-HCC has special characteristics compared with viral-related HCC (HV-HCC), and its background and molecular mechanisms are unclear.Some investigations have suggested that alcohol consumption, metabolic factors and occult HBV infection should be considered potential pathogeneses for NBNC-HCC [4,5].
Liver resection, transplantation, and ablation are considered the initial curative treatment modalities for HCC within the Milan criteria [6,7].According to the Barcelona Clinic Liver Cancer (BCLC) staging system, ablation is recommended for early-stage HCC, with favorable results compared to liver resection [6].Radiofrequency ablation (RFA) is one of the most widely used thermal ablation treatments [8] and has been gradually and widely accepted owing to its advantages of favorable efficacy, minimal invasion, reproducibility and cost-effectiveness [9].However, although several studies have summarized the treatment efficacy and survival of liver resection for NBNC-HCC patients [10][11][12][13][14], few studies have focused on patients with NBNC-HCC after RFA.
The purpose of our study was to evaluate the treatment outcomes of patients with primary NBNC-HCC within the Milan criteria after RFA and to compare with those of patients with HBV-HCC.

Materials and methods
This retrospective study was conducted in accordance with the Declaration of Helsinki guidelines.Ethical approval for this study was obtained from the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University and exempted from informed consent.

Patients
Between January 2007 and February 2020, 529 consecutive patients with primary HCC underwent RFA at our hospital.HCC was diagnosed by percutaneous needle biopsy under the guidance of ultrasound or by noninvasive criteria according to the EASL (i.e., typical features of HCC or at least one positive finding on imaging study together with an a-fetoprotein (AFP) level of > 400 ng/mL) [15].NBNC-HCC is defined as HCC negative for both HBsAg and HCVAb, whereas patients with HBV-HCC are seropositive for HBsAg [11].
The inclusion criteria were as follows: a. Adults with primary HCC within the Milan criteria.A total of 303 patients were enrolled, including 44 with NBNC-HCC (NBNC-HCC group) and 259 with HBV-HCC (HBV-HCC group) (Figure 1).

Pre-procedure evaluation
Pre-procedure evaluation included detailed medical, physical, psychological, metabolic, and nutritional assessments.The final selection of therapy strategies was determined through multidisciplinary team discussion, including interventional radiologists, oncologists, hepatologists, and pathologists.The following data from peripheral blood specimens were collected and recorded: viral markers (HBsAg, HBeAg, HBsAb, HBeAb, and HBcAb) and other laboratory values with potential for prognosis evaluation.Diagnostic imaging was used to evaluate the tumor variables.

RFA
All RFA procedures were performed percutaneously by interventional operators with > 15 years of experience in tumor ablation (G.L. H., X. Y. X., and X. H. X.).The entire procedure was performed via the ultrasound guidance.During treatments, 0.1 mg of fentanyl, 5 mg of droperidol, and 0.1 mg of tramadol hydrochloride were intravenous administered for conscious analgesic sedation.Local anesthesia with 5 ml of 1% lidocaine was applied to the puncture site.Electrocardiograms and vital signs were monitored continuously during the procedure.Three RFA devices, included LeVeen electrodes (Boston Scientific, Natick, MA, Starburst XL electrodes (RITA Medical Systems, Mountain View) and Cooltip electrodes (Valleylab, Boulder, CO), were used.Device selection was based on tumor size and location.The number of electrodes was determined by the tumor size, shape, and location with the aim of achieving an ablative margin at least 0.5 cm beyond the tumor boundary.At the end of the procedure, the needle track was carefully ablated with the electrode withdrawn in 1 cm increments to prevent bleeding and tumor seeding.
Patients were hospitalized for 1-2 days after RFA, unless their complications necessitated longer hospitalization.Contrast-enhanced ultrasound (CEUS) was performed the next day, which might allow an initial evaluation of ablated zone.Complications were classified according to the SIR classification [16].

Treatment assessment and follow-up
Four weeks after ablation, the technical success of RFA was assessed using contrast-enhanced computed tomography (CT).Complete ablation (CA) was defined as the absence of contrast uptake within or at the periphery of the treated tumor on contrast-enhanced CT.For patients with incomplete ablation (ICA), in which the residual viable tumor was found, additional ablation was performed.Technical failure was defined as imaging confirmation of the ICA after an additional RFA procedure, and patients were recommended for other treatments.
Thereafter, contrast-enhanced ultrasound (CEUS), liver function tests, and serum AFP tests were performed once every 3 months for the first 2 years, once every 6 months from 2-5 years and every 12 months after 5 years.If recurrence was suspected on CEUS, additional investigations such as contrast-enhanced CT or magnetic resonance imaging (MRI) were performed.
We evaluated potential outcome predictors for NBNC-HCC patients using three primary endpoints: local tumor progression (LTP), disease-free survival (DFS), and overall survival (OS).The secondary endpoints were assessed based on treatment-related major complications and ICA.LTP was defined as the reappearance of tumor enhancement adjacent to or within the ablation zone after complete ablation.Time-to-LTP was defined as the time between the starting time and LTP per tumor treated [17].
To minimize the effect of these differences in clinical characteristics and account for potential bias due to the absence of randomization between the NBNC-HCC and HBV-HCC groups, propensity score matching (PSM) was applied based on the following variables: age, sex, liver cirrhosis, Child-Pugh classification, albumin-bilirubin (ALBI) score, AFP, total bilirubin, albumin, alanine aminotransferase, platelet count (PLT), prothrombin time (PT), tumor number, and tumor size.

Statistical analysis
Continuous variables were presented as means ± standard deviation with range and 95% confidence interval (CI) and compared using the unpaired t-test or Mann-Whitney U test, as appropriate.The normality of continuous data was evaluated using the Shapiro-Wilk test.Categorical variables were presented as numbers and percentages, and compared using the v 2 test or Fisher's exact test, as appropriate.Propensity scores were estimated to match the patients with a caliper at 0.04 in ratios of 1:1 using a logistic regression model.Survival analysis of LTP, DFS, and OS rates was performed using the Kaplan-Meier method, and survival comparisons were performed using the log-rank test.Multivariate regression analysis was performed using the Cox proportional hazard model with forward elimination, in which the risk factors were determined by univariate analysis using the logrank test.Differences were considered statistically significant at a two-sided p-value < .05.All analyses were conducted using SPSS software (version 23.0; SPSS Inc., Chicago, IL).

Baseline characteristics
Among 303 patients, there were 266 male and 37 were female.The mean age was 56.3 ± 11.7 years (range, 24-88 years) (Table 1).NBNC-HCC was diagnosed in 44 (14.5%) patients and the remaining 259 (85.5%) had HBV-HCC.A significantly lower proportion of patients with cirrhosis was observed in the NBNC-HCC group (p ¼ .009).The ALBI scores were significantly different between the two groups (p ¼ .008).The HBV-HCC group had a higher ALT value and lower PLT value than the NBNC-HCC group (p ¼ .026and .027,respectively).There were 256 patients with solitary HCC tumors, 38 with two tumors, and nine with three tumors.Therefore, 359 tumors were included in this study.The mean tumor size was 2.49 ± 0.85 cm (range, 0.8-5.0cm).Tumor size did not differ between the NBNC-HCC and HBV-HCC groups (p ¼ .814).

Treatment outcomes and complications
The mean ablation time was 22.9 ± 9.7 min (6-68 min), without significant differences (p ¼ .826).After the first RFA session, residual tumors were detected in five patients (all in the HBV-HCC group).Additional treatment of RFA was performed for all patients and four tumors successfully achieved CA.Therefore, the treatment effectiveness was 99.7% (302/303), without significant difference between two groups (p ¼ 1.000).
Seven patients (7/303, 2.3%) were detected major complications after RFA, including one patient with bile duct obstruction who underwent liver resection, one patient with bleeding who underwent RFA for hemostasis, three patients with bleeding using hemostatic drugs, one patient with biloma requiring drainage, and one patient with pleural effusion needing drainage (Table 2).Among them, six patients were in the HBV-HCC group and one patient was in the NBNC group (p ¼ .731).Pain and fever were the two most common minor complications.No treatment-related deaths occurred during this period.

OS
At the end of the follow-up period, 41 patients died of tumor progression or liver function failure.The 1-, 3-, 5-, and 10-year cumulative OS rates were 99.3%, 95.3%, 84.3%, and 69.6%.The OS rates at 1, 3, and 5 years after RFA were 100%, 97.1%, and 93.2% in the NBNC-HCC group and 99.2%, 95.0%, and 82.8% in the HBV-HCC group, respectively (p ¼ .306)(Figure 2(C)).was the only independent predictor of LTP.PT was associated with DFS in the univariate analysis (p ¼ .045).However, the multivariate analysis revealed that PT was not an independent risk factor for DFS in.Moreover, according to the univariate and multivariate analyses, no independent risk factors for OS were detected (all p > .05).

Discussion
Our results showed that liver function based on the ALBI score was better, and the proportion of cirrhosis was lower in the NBNC-HCC group.After matching, NBNC-HCC patients had better OS after RFA, although there were no significant differences in LTP, DFS and OS between the two groups.
Tumor size > 3 cm was an independent predictor of LTP in patients with NBNC-HCC.Larger, prospective, and multicenter trials are required to validate the differences between NBNC-HCC and HBV-HCC, and further molecular and pathological studies are needed to investigate the etiology of NBNC-HCC and to improve prognosis.
Recently, several studies have investigated the clinic pathological features and treatment outcome after curative treatments in patients with NBNC-HCC [11-13, 18, 19].In 2015, Hiwatashi et al. [18] performed a large nationwide study on patients with HCC who underwent liver resection.Their results indicated that patients with NBNC-HCC had a significantly better OS than those with HV-HCC.Meanwhile, patients with NBNC-HCC had a significantly lower risk of HCC recurrence.Many other studies have also demonstrated similar results [14], while some studies reported that there were no differences in long-term survival after liver resection between patients with NBNC-HCC and HV-HCC [11,13,19].Although RFA is an important treatment strategy for earlystage HCC, to our knowledge, no studies have focused on the treatment outcomes and survivals of patients with NBNC-HCC after RFA.NBNC-HCC: non-B, non-C hepatocellular carcinoma; LTP: local tumor progression; DFS: disease-free survival; OS: overall survival; HR: hazard ratio; CI: confidence interval; M: male; F: female; ALBI: albumin-bilirubin; AFP: alpha fetoprotein; TBIL: total bilirubin; ALB: albumin; ALT: alanine aminotransferase; PLT: platelet count; PT: prothrombin time..219†Data are given as mean ± SD.NBNC-HCC: non-B, non-C hepatocellular carcinoma; HBV-HCC: hepatitis B virus (HBV)-related hepatocellular carcinoma; ALBI: albumin-bilirubin; AFP: alpha fetoprotein; TBIL: total bilirubin; ALB: albumin; ALT: alanine aminotransferase; PLT: platelet count; PT: prothrombin time.
In the present study, the proportion of NBNC-HCC cases was relatively lower than that previously reported in a largescale series.A possible explanation is that NBNC-HCC patients received less attention and regular follow-up for liver disease, and thus patients reached the advanced stage at the time of diagnosis, which is unsuitable for RFA.A previous study had demonstrated that NBNC-HCC tumors were larger [11].Moreover, the clinical backgrounds of the NBNC-HCC and HBV-HCC patients who underwent RFA were compared.The different mechanisms of NBNC-HCC compared to HBV-HCC, including metabolic factors and alcohol consumption, which may lead to differences in the clinicopathological characteristics and prognosis of HCC [4].Moreover, most patients with habitual alcohol consumption are male [18,20].
The precise reasons for age and sex differences between the two groups remain to be determined.
Due to the relatively low number of NBNC-HCC patients and the differences in baseline characteristics between the groups in our study, using a propensity score to adjust for factors driving bias became more suitable.Although there were no significant differences in OS after RFA between the two groups, NBNC-HCC patients had better OS than HBV-HCC patients after matching.Indeed, previous studies have reported that patients with NBNC-HCC have better prognosis than those with HV-HCC [14,18], which was consistent with our results.Therefore, our study provides evidence that patients with HBV-HCC require closer follow-up after RFA due to their poor prognosis when compared with NBNC-HCC.The DFS after RFA in NBNC-HCC patients was comparable to that in HBV-HCC patients, consistent with some studies on NBNC-HCC after liver resection [11,12,19].Moreover, LTP was comparable.Nevertheless, contrary to our results, other studies demonstrated that NBNC-HCC had a significantly lower risk of HCC recurrence after liver resection than HBV-HCC [18].These inconclusive results suggest that further larger scale studies are needed to evaluate and compare tumor recurrence after curative treatment in NBNC-HCC patients, especially for NBNC-HCC patients after RFA.
In the present study, tumor size was also independently associated with LTP in the NBNC-HCC group, whereas no independent risk factors for DFS and OS were detected.To date, several studies have focused on risk predictors of liver resection in the NBNC-HCC group [11,13,18].Various factors, such as tumor size, capsule, differentiation, and vascular invasion, have been considered prognostic factors for NBNC-HCC [11,13].Meanwhile, some previous reports on HCC identified tumor size as an independent predictor of LTP after RFA [21,22], although no studies were limited to patients with NBNC-HCC.The therapeutic efficacy of RFA has been reported to decrease with increasing tumor size [23].The therapeutic efficacy of RFA decreases as the tumor size increases owing to micrometastases that may exist up to 1 cm away from the main tumor, even with encapsulated tumors 3 cm or smaller [24].A sufficient ablative margin around the tumor to ensure the satellite lesion or microscopic invasion around the periphery of a tumor to be eradicated for large tumors may be more difficult than for small ones.Therefore, for NBNC-HCC > 3 cm, sufficient safety margin is important to reduce LTP after RFA.
To date, many ablative techniques, not only RFA but also others such as percutaneous ethanol injection (PEI), microwave ablation (MWA), and laser ablation (LA), have been applied for patients with early HCC within Milan criteria.PEI is the first used ablative technique, able to induce coagulative necrosis of the target lesion.However, the local tumor progression rate for lesions >2 cm was high, which limits its use [25].Subsequently, thermal ablative therapies emerged, including RFA, MWA, and LA.Several studies have showed a similar efficacy and safety profile between RFA and MWA, although MWA seems to decrease the rate of long-term recurrences [26].LA is considered a safer option for HCC patients with 'critical lesions' which are at high-risk locations where RFA and MWA are considered to be aggressive [27].
Patients who develop chronic fibrotic liver disease, caused by viral or non-viral etiologies, are at a high risk of developing HCC.Moreover, the additional therapies for chronic fibrotic liver disease may influence the outcomes of HCC after curative treatments.For viral-related HCC, a series of studies have demonstrated that the antiviral therapy such as nucleotide analogous (NA) for HBV could significantly improve treatment survivals after RFA [28].Moreover, RFA plus NA is more costeffective than RFA monotherapy [28].For patients with HCC in non-viral etiology, metabolic etiology is one of the most common causes.It was displayed that treatments such as statins, anti-diabetic drugs and bariatric surgery are associated with a decreased risk of HCC [29][30][31] and the addition of statins and insulin therapies might improve HCC survival after liver resection or RFA [32,33].Therefore, further studies will be performed to evaluate the effects of these additional treatments for NBNC-HCC after RFA.
The study had some limitations.First, the sample size in the NBNC-HCC group was relatively small and we did not include hepatitis C virus (HCV)-related HCC patients, because HBV infection is the major cause of HCC in China.Second, the data collected in this study came from a single center, which might have led to enrollment bias.Thus, a multicenter prospective study is needed to further validate the results.Third, we did not perform a subgroup analysis between HBcAb-positive and HBcAb-negative NBNC-HCC patients, although there might be some differences between the two subgroups.
In conclusion, NBNC-HCC patients within the Milan criteria had better long-term survival than HBV-HCC patients after matching, although LTP and DFS did not differ.Tumor size was the only independent predictor of LTP in patients with NBNC-HCC, whereas no independent risk factors for DFS and OS were detected.Larger prospective multicenter trials are required to validate these results.
b.No previous treatment for HCC.c. Patients tested with a preoperative examination of serological viral markers within 1 week before RFA.d.Child-Pugh grade of liver function was class A or B. e. East Coast Oncology Group (ECOG) performance score of 0 or 1. f.No severe coagulopathy.The exclusion criteria included: a. Patients positive for HCVAb.b.HCC tumors were treated with RFA combined with other treatments such as TACE or ethanol injection.c.Follow-up period < 3 months or loss to follow-up.

Table 1 .
In the HBV-HCC group, prognostic factors related to LTP, DFS and OS in the univariate and multivariate analyses are shown in Table3.Univariate analysis showed that sex (p ¼ .026)and tumor size (p ¼ .003)were significantly associated with LTP after RFA.Multivariate analysis demonstrated that tumor size (hazard ratio [HR] ¼ 8.749, 95% CI, 1.599-47.849;p ¼ .012)Baseline characteristics of the study patients.

Table 3 .
Prognostic factors of LTP, DFS, and OS in the NBNC-HCC group.

Table 4 .
Baseline characteristics after propensity score matching between NBNC-HCC and HBV-HCC patients.