Introduction
Hepatocellular carcinoma (HCC) is a major health problem worldwide. It is the fifth most common cancer in men and the seventh in women and the third most common cause of cancer-related deaths (1–4). In Japan, as well as in other countries, most cases of HCC are associated with viral infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), although in our country, the number of HCC patients with etiologies other than HBV and HCV has recently been increasing (2,5). In general, the prognosis for untreated HCC is poor, and the curative treatments consist of surgical resection (SR) and liver transplantation (1,3,4). However, HCC frequently recurs after curative SR, leading to high mortality, although recurrence only occurs at intrahepatic sites in 68–96% of patients (6,7). Stringent follow-up of HCC patients following SR is therefore essential.
Serum antibody to hepatitis B core antigen (anti-HBc) positivity, which indicates a past history of HBV infection, has recently been attracting attention as a predictor of liver carcinogenesis in patients with HCV-related liver diseases (8–12). HBV DNA may be present in a latent form, even after seroclearance of HB surface antigen (HBsAg), which is referred to as occult HBV infection (8,13). Anti-HBc is reported to be a surrogate marker for such latent carriers (14). Moreover, previous studies have indicated that so-called occult HBV infection, reflected by anti-HBc positivity, is highly prevalent in a number of patient subgroups, including those with HCV infection and HCC, and may play an important role in hepatocarcinogenesis through expression of oncogenic viral protein (10,15).
There have been several reports regarding the effect of anti-HBc positivity on carcinogenesis in patients with HCV-related liver disease, and most of these studies have reported that the presence of anti-HBc is a risk factor for the development of HCC in individuals with HCV infection (8,10,15,16). However, it remains unknown whether anti-HBc positivity constitutes an additional risk factor in terms of survival after curative SR for HCV-related HCC. The aim of the present study was, therefore, to examine the relationship between anti-HBc positivity and survival in HCV-related HCC patients who underwent curative SR.
Patients and methods
Patients
Patients were selected for SR based on assessment of tumor characteristics, remnant liver volume, and general condition, through discussion with experienced surgeons, radiologists and physicians.
SR was performed on 405 treatment-naïve HCC patients at the Department of Surgery, Osaka Red Cross Hospital, Japan, between December 2001 and June 2012. There were 265 HCV-related HCC patients (64.7%), negative for HBsAg and positive for the HCV antibody (HCVAb). Of these, we excluded patients operated on without curative intent (n=24), with surgery-related death (n=3) and those for whom anti-HBc was not tested (n=16). Curative surgery was defined as the resection of all tumors detectable using imaging modalities. A total of 222 HCV-related HCC patients were thus analyzed in the present study (Fig. 1). Patients were classified into two groups: anti-HBc-positive (n=119, 53.6%) and anti-HBc-negative (n=103, 46.4%). Overall survival (OS) and recurrence-free survival (RFS) rates were compared between the two groups.
All the protocols were approved by the Ethics Committee of our institution. Written informed consent was obtained from all patients prior to surgery, and the study protocol complied with all of the provisions of the Declaration of Helsinki. The present study comprised a retrospective analysis of patient records registered in our database and all treatments were conducted in an open-label manner.
HCC diagnosis
HCC was diagnosed using abdominal ultrasound and dynamic computed tomography (CT) scans (hyperattenuation during the arterial phase in all or some part of the tumor and hypoattenuation in the portal-venous phase) and/or magnetic resonance imaging (MRI), based mainly on the recommendations of the American Association for the Study of Liver Diseases (17). Arterial- and portal-phase dynamic CT images were obtained at ~30 and 120 sec, respectively, after the injection of the contrast material. HCC stage was determined using the Liver Cancer Study Group of Japan staging system (18). HCC was confirmed pathologically in specimens at surgery.
Serological studies
HBsAg and anti-HBc were detected using commercial enzyme immunoassay kits (Dainabot, Tokyo, Japan) (13). The results of the anti-HBc assays were expressed as the percentage of inhibition, and the specimens were considered to be anti-HBc-positive when the percentage of inhibition was >50% (19). HCVAb was assessed using second-generation assays (Dainabot) (13). In the present study, serum HCV RNA levels were tested in 182 (82.0%) out of 222 patients by using a competitive reverse transcription-polymerase chain reaction assay.
Follow-up
Follow-up after surgery consisted of periodic blood tests and monitoring of tumor markers, including α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), using chemiluminescent enzyme immunoassays (Lumipulse PIVKA-II Eisai; Eisai Co., Ltd., Tokyo, Japan). Dynamic CT scans and/or MRI were obtained every 2–4 months after surgery. Chest CT, whole abdominal CT, brain MRI, and bone scintigraphy were performed when extrahepatic HCC recurrence was suspected.
Statistical analysis
Data were analyzed using univariate and multivariate analyses. Continuous variables were compared using unpaired t-tests and categorical variables were compared using Fisher’s exact tests. Time to recurrence was defined as the interval between each therapy and first confirmed recurrence. For analysis of RFS, follow-up ended at the time of first recurrence; other patients were censored at their last follow-up visit or the time of death from any cause without recurrence. For analysis of OS, follow-up ended at the time of death from any cause, and the remaining patients were censored at the last follow-up visit. The cumulative OS and RFS rates were calculated using the Kaplan-Meier method and tested using the log-rank test. Factors with a P-value <0.05 in univariate analysis were subjected to multivariate analysis using the Cox proportional hazards model. These statistical methods were used to estimate the interval from initial treatment. Data were analyzed using SPSS for Windows (SPSS Inc., Chicago, IL, USA). Data are expressed as means ± standard deviation (SD). Values of P<0.05 were considered to be statistically significant.
Results
Baseline characteristics
The baseline characteristics of the patients in the two groups are shown in Table I. The median observation periods were 3.4 years (range, 0.3–10.9 years) in the anti-HBc-positive group and 3.2 years (range, 0.5–10.9 years) in the anti-HBc-negative group. In terms of maximum tumor size (P=0.046), aspartate aminotransferase (AST) value (P=0.027) and DCP value (P=0.046), significant differences were observed in the two groups. The proportion of HCC patients with cirrhotic liver in the anti-HBc-positive group tended to be higher than that in the anti-HBc-negative group (P=0.068).
Cumulative OS and RFS rates
The 1-, 3- and 5-year cumulative OS rates were 88.8, 70.2 and 50.0%, respectively, in the anti-HBc-positive group and 95.8, 77.1 and 61.7% in the anti-HBc-negative group (P=0.300) (Fig. 2). The corresponding RFS rates were 68.7, 33.0 and 20.0%, respectively, in the anti-HBc-positive group and 74.4, 38.5 and 16.5% in the anti-HBc-negative group (P=0.482) (Fig. 3).
Univariate and multivariate analyses of factors contributing to OS
Univariate analysis identified the following factors as significantly associated with OS for all cases (n=222): HCC stage (P<0.001); maximum tumor size ≥4 cm (P=0.016); tumor number (P=0.002); interferon (IFN) therapy after surgery (P=0.044); serum albumin ≥3.8 g/dl (P=0.023); and microscopic vascular invasion (P<0.001) (Table II). The hazard ratios (HRs) and 95% confidence intervals (CIs) calculated using multivariate analysis for the 6 factors that were significant in univariate analysis are detailed in Table III. Serum albumin ≥3.8 g/dl (P=0.005) and microscopic vascular invasion (P<0.001) were found to be significant predictors linked to OS in multivariate analysis.
Univariate and multivariate analyses of factors contributing to RFS
Univariate analysis identified the following factors as significantly associated with RFS for all cases (n=222): HCC stage (P<0.001); maximum tumor size ≥4 cm (P=0.023); tumor number (P<0.001); IFN therapy after surgery (P=0.019); total bilirubin ≥1.0 mg/dl (P=0.022); AST ≥60 IU/l (P=0.027); alanine aminotransferase ≥50 IU/l (P=0.008); AFP ≥40 ng/ml (P=0.001); DCP ≥100 mAU/ml (P=0.034); and microscopic vascular invasion (P<0.001) (Table III). The HRs and 95% CIs calculated using multivariate analysis for the 10 factors that were significant in univariate analysis are detailed in Table IV. IFN therapy after surgery (P=0.011), AFP ≥40 ng/ml (P=0.030) and microscopic vascular invasion (P<0.001) were found to be significant prognostic factors linked to RFS.
Causes of death in the two groups
Fifty-two patients in the anti-HBc-positive group (43.7%) died during the follow-up period. The causes of death were HCC recurrence in 39 patients, liver failure in 9 and other causes in 4. Forty patients in the anti-HBc-negative group (38.8%) died during the follow-up period, and the causes of death were HCC recurrence in 25 patients, liver failure in 10, and other causes in 5.
HCC recurrence
In the present study, 85 anti-HBc-positive patients (71.4%) and 68 anti-HBc-negative patients (66.0%) had HCC recurrence during the follow-up period. The patterns of HCC recurrence after surgery in the anti-HBc-positive group were: single HCC recurrence in the liver in 35 patients; multiple HCC recurrences in the liver in 42 patients; multiple HCC recurrences in the liver with lung metastases in 3 patients; multiple HCC recurrences in the liver with lymph node metastases in 2 patients; multiple HCC recurrences in the liver with peritoneal dissemination in 1 patient; multiple HCC recurrences in the liver with portal vein tumor invasion in 1 patient; and single brain metastasis in 1 patient. The patterns of HCC recurrence after surgery in the anti-HBc-negative group were: single HCC recurrence in the liver in 28 patients; single HCC recurrence in the liver with portal vein invasion in 1 patient; multiple HCC recurrences in the liver in 36 patients; multiple HCC recurrences in the liver with lymph node metastases in 1 patient; multiple HCC recurrences in the liver with inferior vena cava invasion in 1 patient; and multiple HCC recurrences in the liver with bone metastases in 1 patient.
Treatment methods for HCC recurrence
Treatment methods for the first HCC recurrence in the anti-HBc-positive group were: SR in 9 patients; radiofrequency ablation (RFA) in 35; transcatheter arterial chemoembolization (TACE) in 26; percutaneous ethanol injection (PEI) in 3; systemic chemotherapy in 3; radiotherapy in 1; and no specific treatment in 8 patients. The treatment methods used in the anti-HBc-negative group were: SR in 3 patients; RFA in 40; TACE in 20; PEI in 3; radiotherapy in 1; and no specific treatment in 1 patient.
IFN therapy after surgery
In the present study, 16 patients [11 patients (9.2%) in the anti-HBc-positive group and 5 (4.9%) in the anti-HBc-negative group] received IFN therapy after surgery. They included stage I HCC in 2 patients, stage II in 9, stage III in 4 and stage IV in 1. Whether IFN therapy after surgery was performed was mainly determined by decision of the attending physicians. Of these, all patients had high viral load as defined by the guidelines before IFN therapy (20,21). Fourteen patients received peginterferon and ribavirin combination therapy and 2 received long-term low-dose IFN maintenance therapy. Seven patients (43.8%) achieved sustained virological response as defined by undetectable HCV RNA 24 weeks after completion of IFN treatment. Seven patients (43.8%) had HCC recurrence and 2 (12.5%) died during the follow-up period.
Subgroup analyses according to maximum tumor size
In terms of maximum tumor size, there was a significant difference in baseline characteristics between the anti-HBc-positive and anti-HBc-negative groups. We therefore performed subgroup analyses according to maximum tumor size. In patients with maximum tumor size ≥4 cm [51 (42.9%) in the anti-HBc-positive group and 43 (41.7%) in the anti-HBc-negative group], no significant difference was observed in OS (P=0.661) and RFS (P=0.790) (Fig. 4A and 4B). In patients with maximum tumor size <4 cm [68 (57.1%) in the anti-HBc-positive group and 60 (58.3%) in the anti-HBc-negative group], there was no significant difference in OS (P=0.319) and RFS (P=0.419) (Fig. 5A and 5B).
Subgroup analyses according to background liver disease
Marginal significance was observed between the two groups in terms of background liver disease (P=0.068), and we therefore performed subgroup analyses accordingly. In patients with cirrhotic liver [83 (69.7%) in the anti-HBc-positive group and 59 (57.3%) in the anti-HBc-negative group], there was no significant difference in OS (P=0.226) and RFS (P=0.634) (Fig. 6A and 6B). In patients with non-cirrhotic liver disease [36 (30.3%) in the anti-HBc-positive group and 44 (42.7%) in the anti-HBc-negative group], there was no significant difference in OS (P=0.922), whereas there was a significant difference in RFS (P=0.043) (Fig. 7A and 7B).
Discussion
To the best of our knowledge, this is the first reported comparative study to examine the relationship between anti-HBc positivity and survival in patients with HCV-related HCC who underwent curative SR, although our study was retrospective in nature. Several previous studies have reported that anti-HBc positivity influences carcinogenesis in patients with HCV-related liver disease; however, as far as we are aware, there has been no report regarding the effect of anti-HBc positivity on survival after curative SR for HCC. Hence the reason for the present study.
In our analyses, anti-HBc positivity was not a significant factor in terms of OS and RFS. Moreover, in subgroup analyses according to maximum tumor size and background liver disease, RFS did not differ significantly in all subgroups except in patients with non-cirrhotic liver disease. These results suggest that anti-HBc positivity cannot be a useful predictor for patients with HCV-related HCC who undergo curative SR, although in those with non-cirrhotic liver disease, it may be associated with HCC recurrence after surgery.
There were 119 patients (53.6%) with anti-HBc positivity in the present study. Marusawa et al(12) reported that 363 (59.4%) out of 611 HCV-related HCC patients had anti-HBc positivity, and have suggested that HBV infection, including latent infection, plays an important role in carcinogenesis in patients with HCV-related liver disease. Our results were similar to that study, although in terms of HCC recurrence, anti-HBc positivity was not demonstrated to be a prognostic factor. In contrast, Mazzaferro et al(22) reported that 70 (46.7%) out of 150 patients with HCV-related HCC were anti-HBc positive, which was slightly lower than the result in our study. Racial and geographical factors may have been associated with these results.
In our study, the proportion of patients with cirrhotic liver in the anti-HBc-positive group tended to be higher than that in the anti-HBc-negative group. Several studies have demonstrated that the prevalence of anti-HBc positivity is closely correlated with the clinical stage of liver disease (9,10,12). Our results were consistent with these reports, which suggest that anti-HBc should be closely monitored in patients with advanced HCV-related liver disease.
The baseline AST level in the anti-HBc-positive group was significantly higher than that in the anti-HBc-negative group in our study. Although the reasons for this are unknown, in patients with HCV-related to liver disease, anti-HBc positivity may correlate with higher activity of background liver disease.
HCC often recurs after curative surgery, leading to high mortality (6). Indeed, in the present study, 85 anti-HBc-positive patients (71.4%) and 68 anti-HBc-negative patients (66.0%) had HCC recurrence during the follow-up period. Stringent follow-up of HCC patients following SR is therefore essential. In the present study, microvascular invasion was the strongest prognostic factor in terms of both OS and RFS. In the postoperative management of HCC, preoperative factors such as degree of liver damage, radiological findings, and tumor markers as well as factors based on postoperative status should be considered (23).
In the present study, IFN therapy after surgery was a significant prognostic factor in terms of RFS, and was a marginally significant factor associated with OS, although the number of patients who received IFN therapy after surgery was small. Several investigators have reported that IFN therapy after curative SR in patients with HCV-related HCC improves clinical outcome (24–26). Our results are consistent with these reports; additional IFN therapy should be taken into account in patients with HCV-related HCC who undergo curative SR.
Serum albumin levels were significantly associated with OS in our multivariate analysis. Patients with liver cirrhosis and low serum albumin levels can develop protein-energy malnutrition with increased catabolism (27). Protein-energy malnutrition is associated with high morbidity and mortality because of an increased risk of life-threatening complications, resulting in poor survival and reduced quality of life (28). In the present study, 142 patients (64.0%) had cirrhotic liver, indicating that a high proportion of patients with HCV-related HCC have concurrent liver cirrhosis. Branched chain amino acid treatment may optimize clinical outcome in these patients (29,30).
There were several limitations to the present study. First, it was a single-center retrospective study. Second, the median observation periods in the two groups were relatively short for survival analysis. Third, patients in whom anti-HBc was not tested were excluded from our analysis, leading to bias. Larger prospective studies with longer observation periods are thus required to confirm these results. However, the current study demonstrated that anti-HBc positivity was not associated with survival in patients with HCV-related HCC who underwent curative surgery. In conclusion, anti-HBc positivity need not be taken into account when assessing clinical outcome in patients with HCV-related HCC after curative surgery.