Baseline quantitative hepatitis B core antibody could strongly predict survival for patients with hepatocellular carcinoma undergoing transarterial chemoembolization

Abstract

The investigation regarding the clinical significance of quantitative hepatitis B core antibody (anti-HBc) in HCC patients after transarterial chemoembolization (TACE) is rare. The aim of this study was to determine the prognostic significance of anti-HBc in HCC patients after TACE. A total of 66 HCC patients with complete data and treated with TACE in Nanfang Hospital, Southern Medical University were retrospectively analyzed. Univariate analyses were performed for 20 pretreatment variables and those with a P value less than 0.05 by univariate analysis were subjected to Cox proportional hazards model. Among the 66 HCC patients, the 0.5-, 1-, and 2-year overall survival rates were 92.42%, 50.00%, and 21.21%, respectively. We defined 11.88 S/CO, with a maximum sum of sensitivity and specificity, as the optimal cut-off value of baseline anti-HBc level to predict the OS rate in HCC patients after TACE. Univariate and multivariate analyses revealed that baseline anti-HBc level, AFP level and vascular invasion were the only three independent significant prognostic factors of overall survival (P = 0.001, 0.020 and 0.010, respectively). We demonstrated that baseline anti-HBc level, combining AFP level and vascular invasion, might be a novel biomarker for predicting the survival of HCC patients after TACE.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death in the world, resulting in more than 500,000 deaths annually [1]. Due to rapid proliferation, increased intrahepatic spreading, and metastasis, the survival rate for unresectable HCC patients is typically poor [2]. In developing countries such as China, transarterial chemoembolization (TACE) remains the primary treatment for unresectable HCC patient [3]. Although the prognostic factors related to the survival of HCC patients after the treatment of TACE have been analyzed in many previous studies and review articles [4-10], there have been no studies for assessing the performance of quantitative anti-HBc level in patients with HCC undergoing TACE.

Anti-HBc, as one of the novel biomarkers of HBV infection, has been ignored in real-life clinical practice for a long time. It has been demonstrated that anti-HBc plays an important role in HBV infection, which is helpful for judging the prognosis of chronic hepatitis B (CHB) patients [11]. Moreover, anti-HBc is closely correlated with hepatic inflammatory activities and may serve as a surrogate marker for the host anti-HBV immune response [12]. Considering the above evidences, we hypothesized that anti-HBc levels may be correlated with the outcome of TACE. The aim of this study was to determine the prognostic significance of anti-HBc in HCC patients after TACE.

Patients and methods

Patients

This study enrolled patients who underwent TACE since 2005 in Nanfang Hospital, Southern Medical University. A diagnosis of HCC was made on the basis of pathology or the diagnostic criteria of the American Association of the Study of Liver Disease [13]. The crucial inclusion criteria of the current retrospective study were hepatitis B patients with cirrhosis. The exclusion criteria were as follows: (1) patients who underwent previous treatment before the first TACE, (2) World Health Organization (WHO) performance status > 2, (3) patients with significantly impaired liver function (Child-Pugh score class C), (4) patients who received only intra-arterial chemotherapy, or without embolization, (5) co-infection with hepatitis A, hepatitis C, hepatitis D, hepatitis E, or human immunodeficiency virus and (6) loss to follow-up within three months after treatment (In addition to the deaths). Finally, 66 HCC patients with complete data were enrolled. All patients provided informed consent before undergoing TACE.

Clinical and laboratory evaluation

Clinical and laboratory data were collected from all patients prior to TACE. Clinical data included age, sex, diabetes, Barcelona Clinic Liver Cancer (BCLC) stage and Child-Pugh score. In addition, imaging data were collected based on abdominal computed tomography (CT) or magnetic resonance imaging (MRI), including tumor size and vascular invasion. Laboratory data included platelet count (PLT), hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), hepatitis B e antibody (anti-HBe), hepatitis B core antibody (anti-HBc), HBV DNA, α-fetoprotein (AFP), albumin (ALB), total bilirubin (TBil), prothrombin time (PT), blood type, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). The serum HBV DNA level was measured with the Cobas Taqman HBV Kit (Roche Diagnostics; lower limit of detection, 15 IU/mL or 84 copies/mL). HBeAg, Anti-HBe, anti-HBc were detected using an Architect assay (Abbott Laboratories, Abbott Park, IL). Aminotransferases were measured according to standard procedures locally at the time of sampling.

TACE procedures

Superior mesenteric and celiac arteriography was initially performed to assess anatomy, tumor burden, and the tumor-feeding artery. Two or three kinds of chemotherapeutic agents including epirubicin, platinum, mitomycin C were infused. Next, an emulsion of chemotherapeutic agents and 5-20 mL lipiodol was administered (the amount of lipiodol was selected depending on the tumor size and vasculature). Chemotherapeutic agents and doses were adjusted based on liver function and white blood cell counts. The catheter was selectively inserted into the tumor-feeding artery as close as possible to the tumor. Then gelatin sponge particles were used for further embolization. For patients with difficult intubation due to hepatic artery distortion, micro catheters were adopted.

Statistical analysis

All statistical analyses were conducted using SPSS 18.0 software (SPSS Inc., Chicago, IL). Data were expressed as counts and percentages for categorical variables and as median (minimum, maximum) for continuous variables (Because none of them are subject to normal distribution). Qualitative differences between subgroups were analyzed using χ² tests for categorical parameters. For analyze of performance of quantitative anti-HBc level in predicting treatment outcome, area under the receiver operator characteristic curve (AUROC) was calculated. The AUROCs were compared by Delong test. Sensitivity, specificity, positive likelihood ratio (LR+) and negative likelihood ratio (LR-) of several cut-off values of anti-HBc levels were calculated to explore the best cut-off value in predicting the survival of HCC patients after TACE.

The observed points in this study were 0.5-, 1-, 2-year overall survival (OS) rates. OS was calculated from the time of the first chemoembolization to the date of death regardless of cause. Univariate and multivariate Cox regression analyses were performed to assess factors related to patient survival. All statistical analyses were based on two-tailed hypothesis tests with a significance level of P < 0.05.

Results

Clinical characteristics

This study included 66 patients with HCC who had undergone TACE during the study period, for whom complete data were available. The demographic, virological and clinical characteristics of the patients are summarized in Table 1. The median age was 52.00 (23.00, 80.00) years, predominantly men (92.42%) and 27.27% of patients were HBeAg positive. The median of baseline HBV DNA, HBsAg, HBeAg, anti-HBe, anti-HBc levels was 3.04 (0, 8.72) log10 copies/mL, 205.63 (2.72, 3715.30) IU/mL, 0.10 (0.04, 28.61) PEIU/ml, 0.01 (0, 3.02) PEIU/ml, and 11.50 (9.09, 14.99) S/CO, respectively.

Table 1.

Baseline characteristics of 66 HCC patients underwent TACE

Characteristics	Number/median (minimum, maximum)	Percentage (%)
Gender
M/F	61/5	92.42/7.58
Age (year)	52.0 (23.0, 80.0)
HBsAg (IU/mL)	205.63 (2.72, 3715.30)
HBeAg (PEIU/ml)	0.10 (0.04, 28.61)
Anti-HBe (PEIU/ml)	0.01 (0, 3.02)
Anti-HBc (S/CO)	11.50 (9.09, 14.99)
HBV DNA level, lg copies/mL	3.04 (0, 8.72)
AFP (ng/mL)
≤ 200/> 200	17/49	25.76/74.24
ALT (U/L)
≤ 40/> 40	35/31	53.03/46.97
AST (U/L)
≤ 40/> 40	19/47	28.79/71.21
PLT (109/L)
< 125/≥ 125	29/37	43.94/56.06
ALB (g/L)
< 40/40-55	49/17	74.24/25.76
TBIL (µmol/L)
3.42-20.5/> 20.5	41/25	62.12/37.88
PT (S)
≤ 13/> 13	16/50	24.24/75.76
Blood type
A/B/AB/O	23/9/5/29	32.85/13.63/7.58/43.94
BCLC stage
A/B/C	2/19/45	3.03/28.79/68.18
Child-Pugh
A/B	25/41	37.88/62.12
Diabetes
Yes/No	23/43	34.85/65.15
Tumor size (cm)
< 3/3-5/> 5	12/10/44	18.18/15.15/66.67
Vascular invasion
Yes/No	50/16	75.76/24.24

TACE transarterial chemoembolization, HBsAg hepatitis B surface antigen, HBeAg hepatitis B e antigen, Anti-HBe hepatitis B e antibody, Anti-HBc hepatitis B core antibody, AFP α-fetoprotein, ALT alanine aminotransferase, AST aspartate aminotransferase, PLT platelet count, ALB albumin, TBIL total bilirubin, PT prothrombin time, BCLC stage Barcelona Clinic Liver Cancer stage.

Selection of anti-HBc cutoff value

To evaluate the correlation between the OS and the quantitative baseline HBsAg, HBeAg, anti-HBe, anti-HBc, HBV DNA, respectively, areas under the receiver operator characteristic curve (AUROC) were calculated in Figure 1. The AUROC of anti-HBc level was the highest (0.755, P = 0.011) while other 4 values showed no statistical significance.

Figure 1.

Receiver operating characteristics curves for overall survival in HCC patients underwent TACE.

Table 2 shows the sensitivity and specificity of baseline anti-HBc level in predicting the OS. Obviously, the sum of sensitivity and specificity would achieve the highest when the cut-off value is 11.88 S/CO. Therefore, we adopted 11.88 S/CO as the optimal cut-off value of baseline anti-HBc level in the following analyses. In this study, 15 patients had baseline anti-HBc > 11.88 S/CO and 51 patients had baseline anti-HBc ≤ 11.88 S/CO, among which 66.67% (10/15) and 7.84% (4/51) remained alive at the end of observation (χ² = 20.607, P < 0.001).

Table 2.

Performance of baseline anti-hbc level in predicting the os after TACE

Cut-off values	Sensitivity (%)	95% CI	Specificity (%)	95% CI	+LR	-LR
< 9.09	0	0.0-6.8	100	76.8-100.0		1
≤ 9.09	1.92	0.05-10.3	100	76.8-100.0		0.98
≤ 9.57	1.92	0.05-10.3	85.71	57.2-98.2	0.13	1.14
≤ 10.63	25	14.0-38.9	85.71	57.2-98.2	1.75	0.88
≤ 10.67	25	14.0-38.9	78.57	49.2-95.3	1.17	0.95
≤ 11.56	65.38	50.9-78.0	78.57	49.2-95.3	3.05	0.44
≤ 11.63	65.38	50.9-78.0	71.43	41.9-91.6	2.29	0.48
≤ 11.88*	90.38	79.0-96.8	71.43	41.9-91.6	3.16	0.13
≤ 11.98	90.38	79.0-96.8	64.29	35.1-87.2	2.53	0.15
≤ 12.04	92.31	81.5-97.9	64.29	35.1-87.2	2.58	0.12
≤ 12.06	92.31	81.5-97.9	57.14	28.9-82.3	2.15	0.13
≤ 12.15	96.15	86.8-99.5	57.14	28.9-82.3	2.24	0.067
≤ 12.57	96.15	86.8-99.5	42.86	17.7-71.1	1.68	0.09
≤ 12.7	98.08	89.7-100.0	42.86	17.7-71.1	1.72	0.045
≤ 13.77	98.08	89.7-100.0	0	0.0-23.2	0.98
≤ 14.99	100	93.2-100.0	0	0.0-23.2	1

^*The cut-off value has the highest sum of sensitivity and specificity;

OS Overall Survival, TACE transarterial chemoembolization, CI confidence Interval, +LR positive likelihood ratio, -LR negative likelihood ratio.

Overall survival

At the median follow-up of 13 months (range, 1-66 months), 26 patients (39.10%) remained alive. The 0.5-, 1-, and 2-year OS rates for all patients were 94.42%, 50.00%, and 21.21%, respectively (Figure 2).

Figure 2.

Survival curve of patients treated with TACE.

Correlation between baseline variables and OS

Univariate analysis identified the following factors as affecting OS: low anti-HBc level (≤ 11.88 S/CO, P = 0.001), elevated AFP level (> 200 ng/mL, P < 0.001), large tumor (3-5 cm or > 5 cm, P < 0.001), BCLC stage (B or C, P = 0.009), Child-Pugh stage (B, P = 0.013) , diabetes (P = 0.013), and vascular invasion (P < 0.001). We then entered these significant factors in multivariate analysis by using Cox’s proportional hazard model, and we found that along with AFP and vascular invasion, baseline anti-HBc was an independent predictive factor for OS (hazard ratio [HR] = 0.066, 95% confidence interval [CI] = 0.013-0.340, P = 0.010) (Table 3) .

Table 3.

Univariate and multivariate analysis of baseline variables affecting OS in patients receiving TACE

Factors	Univariate			Multivariate
	HR	95% CI	P Value	HR	95% CI	P Value
Gender: M/F	1.098	0.395-3.053	0.858
Age: ≥ 40	0.557	0.258-1.201	0.135
Baseline HBsAg (IU/mL)	1	1-1	0.977
Baseline HBeAg (PEIU/ml)	1.003	0.991-1.014	0.665
Baseline Anti-HBe (PEIU/ml)	1.032	0.927-1.147	0.567
Baseline HBV DNA level, lg copies/mL	1.053	0.904-1.226	0.506
Anti-HBc: > 11.88 (S/CO)	0.183	0.074-0.484	0.001	0.066	0.013-0.340	0.010
AFP (ng/mL): > 200	16.674	5.021-55.38	0.000	7.763	1.367-43.797	0.020
ALT (U/L): > 40	1.408	0.817-2.428	0.218
AST (U/L): > 40	1.519	0.780-2.960	0.218
PLT (109/L): < 125	0.788	0.452-1.374	0.401
ALB (g/L): < 40	1.250	0.654-2.388	0.499
TBIL (µmol/L): > 20.5	1.053	0.603-1.837	0.856
PT (S): > 13	0.809	0.429-1.525	0.512
Blood type: A/B/AB/O	-	-	0.863	-	-
BCLC stage: B/C	-	-	0.009	-	-	0.706
Child-Pugh: B	2.217	1.181-4.164	0.013	0.648	0.317-1.325	0.234
Diabetes	2.023	1.159-3.530	0.013	0.669	0.357-1.257	0.212
Tumor Size (cm): 3-5/> 5	-	-	0.000	-	-	0.055
Vascular invasion	11.787	3.627-38.302	0.000	7.022	1.589-31.032	0.010

TACE transarterial chemoembolization, HR hazard ratio, CI confidence Interval, HBsAg hepatitis B surface antigen, HBeAg hepatitis B e antigen, Anti-HBe hepatitis B e antibody, Anti-HBc hepatitis B core antibody, AFPα-fetoprotein, ALT alanine aminotransferase, AST aspartate aminotransferase, PLT platelet count, ALB albumin, TBIL total bilirubin, PT prothrombin time, BCLC stage Barcelona Clinic Liver Cancer stage.

Discussion

The prognostic factors related to the survival of HCC patients after the treatment of TACE have been analyzed in many previous studies and review articles [4-10]. However, to the best of our knowledge, there have been no studies for assessing the performance of quantitative anti-HBc level, a novel immunological biomarker, in patients with HCC undergoing TACE. In the present report, we chose chronic hepatitis B patient with cirrhosis, who finally developed HCC and underwent TACE as research object. Our results demonstrated that a baseline anti-HBc level > 11.88 S/CO is associated with higher rates of Overall Survival in patients with HCC undergoing TACE.

In the current study, the 0.5-, 1-, and 2-year overall survival rates were 92.42%, 50.00%, and 21.21%, respectively, which were basically in accordance with other studies [14-16], indicating the survival rate of HCC patients after TACE is still low. For patients with unresectable HCC, the goal of TACE as a palliative treatment is to prolong survival and to enhance quality of life. However, unlike other neoplasms, the survival of HCC depends not only on tumor status, but also on hepatic functional reserve, performance status, and response to treatment [17-19]. Therefore, it would be necessary to find better predicting factors for HCC treatment outcomes before deciding a treatment option.

Anti-HBc, as one of the old biomarkers of HBV infection, has been ignored in real-life clinical practice for a long time. Recently, Fan R et al [15] proved that a baseline anti-HBc level is associated with HBeAg seroconversion in CHB patients treated with both Peg-IFN and NUC in two well-controlled and large cohorts. This study demonstrated that qAnti-HBc levels are closely correlated with hepatic inflammatory activities and can serve as a new treatment response marker. Therefore, in the current study, we evaluated the correlation between the OS of HCC patients and the quantitative baseline HBsAg, HBeAg, anti-HBe, anti-HBc, HBV DNA, respectively, by calculating the areas under the receiver operator characteristic curve (AUROC). The AUROC of anti-HBc level was the highest (0.755, P = 0.011) while other 4 values showed no statistical significance. Indeed, we also defined a unified optimal cut-off value of 11.88 S/CO with a maximum sum of sensitivity and specificity for predicting the OS, which will be convenient for its application in real-life clinical practice.

Although the baseline anti-HBc level has been proved to be independently associated with the OS in patients with HCC undergoing TACE, the mechanism underlying the predictive value of anti-HBc level is still unknown. As we know, anti-HBc plays an important role in HBV infection. It reflects the presence of serum Dane particle and intra-hepatic HBV replication, which is helpful for judging the prognosis of chronic hepatitis B [20]. Besides, many studies have shown that B lymphocytes played a crucial role in mediating immune response against HBV [21,22], and it was speculated that native HBcAg is released from damaged hepatocytes and provide potent antigenic stimulation to B-cells [12,23]. Furthermore, other studies also revealed that high levels of anti-HBc are related to the high serum IL-2R levels, which is mainly released from sensitization of T lymphocytes, and the production of anti-HBc is greatly enhanced by HBcAg-specific Th cell activation [24,25]. The above evidences reveal that anti-HBc is closely correlated with hepatic inflammatory activities and may serve as a surrogate marker for the host anti-HBV immune response. Therefore, the results indicated that HCC patients with higher qAnti-HBc levels may have a higher host immune response, resulting in a higher likelihood of achieving a better outcome after TACE.

In addition, we also demonstrated that elevated AFP level, large tumor, BCLC stage, Child-Pugh stage, diabetes, and vascular invasion affected OS. However, when we entered these factors in multivariate analysis, we found that only baseline anti-HBc, AFP level and vascular invasion were the independent predictive factors for OS. Many studies have strongly supported the feasibility of AFP level and vascular invasion before treatment as useful markers in predicting treatment response and patients’ survival after therapies including TACE, although the AFP cutoff value was different in those studies [4,6,7,26-28]. In the present study, a cutoff level of 200 ng/ml was used for predicting the OS because AFP over 200 ng/mL lasting for more than 8 weeks is a crucial evidence for the diagnosis for HCC in real-life clinical practice [29,30]. The role of AFP in HCC progression has been studied recently and it has been demonstrated that AFP promotes cell proliferation and angiogenesis, and that it increases the resistance of cells toward tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis [31,32]. However, the mechanism of AFP in HCC development and prognosis is still a mystery and further study is needed.

Our study had included a sufficient number of indicators and the results are important for deciding on a treatment option for HCC patients. What’s more, this is the first study to indicate that anti-HBc might be an independent predictor of OS for HCC patients after TACE. Nonetheless, there are some limitations of the current study. First, there are limited numbers of patients. Second, this is a retrospective study, which may cause selective bias. Therefore, better controlled, prospective and larger sample size cohorts would be needed.

In conclusion, baseline anti-HBc level, in addition to AFP level and vascular invasion, might be a novel biomarker for predicting the survival of HCC patients after TACE.

Disclosure of conflict of interest

None.