Hepatic resection is a well-accepted therapy for hepatocellular carcinoma (HCC), but many patients show cancer recurrence and the cumulative 5-year HCC recurrence rate exceeds 70%.1–3 This high incidence of tumor recurrence after hepatic resection remains a major drawback. Some benefits of interferon (IFN) therapy on tumor recurrence and survival have been reported.4–10 IFN suppresses replication of hepatitis C virus (HCV) and exerts a tumoricidal effect on a number of tumors, including HCC.10,11 However, several randomized controlled trials (RCTs) have revealed inconclusive results regarding the effects of IFN on survival and tumor recurrence after curative resection or ablation of HCC, either because the effects were not statistically significant or because they were considered only with respect to defined subpopulations.12–15

Recently, combination therapy consisting of pegylated interferon (peg-IFN) plus ribavirin (RBV) has been developed, and the effect of this combination has been reported to be higher than that of conventional IFN therapy.16,17 Peg-IFN has an extended serum half-life that provides viral suppression for 7 days, thus allowing weekly administration and enhanced clinical efficacy.17 Most Japanese patients infected with HCV are infected with HCV genotype Ib and have high viral load. Moreover, treatment with conventional IFN is complicated by a low sustained viral response (SVR) rate of 20–30%.18–20 However, peg-IFN plus RBV combination therapy has good tolerability in Japanese patients with HCV and resulted in an SVR rate of approximately 40–50%.21–23 The impact of adjuvant immunotherapy with IFN after curative resection of HCC is debatable, and few studies have investigated the effects of peg-IFN plus RBV combination therapy on survival and recurrence after curative resection of HCC.

In the present study, we aim to investigate the impact of peg-IFN plus RBV combination therapy on survival and HCC recurrence after curative resection in patients infected with HCV.

Patients and Methods

Patients and HCV Diagnosis

From June 2003 to June 2009, 370 HCC patients underwent hepatectomy as initial treatment at the Department of Gastroenterological Surgery, Hiroshima University Hospital, Japan. Of the 370 patients, 175 patients who were HCV RNA-positive/hepatitis B surface antigen-negative underwent curative hepatectomy. Of the 175 patients, 75 patients received IFN therapy after hepatectomy, and 100 patients did not receive any IFN therapy. Of the 75 patients who received IFN, 20 patients who received IFNs such as IFN-α or IFN-β were excluded. Of the 55 patients who received peg-IFN therapy, 43 patients who started peg-IFN within 9 months after curative resection were enrolled in this analysis. Twenty-four patients who had early recurrence of HCC within 9 months after surgery were excluded from the 100 patients who did not receive any IFN therapy, because these patients could lose the opportunity to receive IFN therapy for HCC recurrence if these patients were assigned to the peg-IFN therapy. Consequently, 119 patients were eventually enrolled in this study. Of these 119 patients, 43 received peg-IFN therapy within 9 months after hepatectomy, and 76 did not receive any IFN therapy.

Curative hepatectomy was defined as removal of all recognizable tumors. HCV RNA levels were measured by quantitative reverse-transcription polymerase chain reaction (RT-PCR; Amplicor, Roche Diagnostic Systems, CA, USA). HCV genotype was determined by PCR using a mixed primer set derived from the nucleotide sequences of the NS5 region. HCV negativity was evaluated by quantitative RT-PCR. The lower limit of the assay was 5 kIU/ml (equivalent to 5,000 copies/ml) in the quantitative method and 50 IU/ml (equivalent to 50 copies/ml) in the qualitative method. SVR was defined as undetectable HCV RNA at 24 weeks after completion of IFN therapy. The study was approved by the concerned institutional review boards. Written informed consent was obtained from all patients.

Preoperative Diagnosis and Evaluation of HCC

Hepatocellular carcinoma was diagnosed on the basis of routine imaging modalities such as Doppler ultrasonography (US), computed tomography (CT) during hepatic angiography (CTHA) and CT during arterial portography (CTAP), and magnetic resonance imaging. Tumor stage, liver damage classification, and surgical procedures were defined according to the General Rules for Clinical and Pathologic Study of Primary Liver Cancer, fifth edition, by the Liver Cancer Study Group of Japan.24

Hepatectomy

The surgical procedure was determined according to tumor extent and hepatic reserve function. Liver function was assessed by liver damage classification, Child–Pugh classification, and indocyanine green retention rate at 15 min (ICGR 15).25,26 If permitted by liver function, anatomic resection was performed.27,28 In patients with insufficient hepatic reserve, limited resection was performed. We divided the liver parenchyma by using an ultrasonic dissector.29 Postoperative complications were graded according to the method described by Clavien et al.30

Follow-Up

Follow-up evaluation after the surgery consisted of monthly blood chemistry tests and measurements of levels of tumor markers, including alpha-fetoprotein and des-gamma-carboxy prothrombin. Patients were examined by US every 3 months and by CT every 6 months. When recurrence was indicated by any of these examinations, patients were examined by CTAP and CTHA.

Patient Selection for IFN Therapy

Patients with HCV genotype 1b in the IFN group received peg-IFNα-2b (Pegintron; Schering-Plough, NJ, USA) at weekly dosage of 1.5 μg/kg subcutaneously for 48 weeks. Daily RBV (Rebetrol, Schering-Plough) was administered orally for 48 weeks, and the dosage was adjusted according to weight (600 mg for patients weighing ≤60 kg, 800 mg for those weighing 60–80 kg). Patients with HCV genotype 2 received IFN monotherapy for 24 weeks. Blood samples were obtained every 4 weeks and analyzed for HCV RNA levels. All patients were informed about IFN therapy after hepatectomy, and only consenting patients received IFN therapy. The eligibility criteria for IFN therapy were as follows: (1) detectable serum HCV RNA level, (2) Eastern Cooperative Oncology Group (ECOG) performance score of 0 or 1, (3) platelet count ≥70,000/μl, (4) patients with no uncompensated cirrhosis (Child class C), and (5) hemoglobin concentration ≥10 g/dl. Peg-IFN therapy was commenced within 24 weeks of surgery or after the eligibility criteria were fulfilled.

Safety Assessments and Dose Modification of Peg-IFN Therapy

Adverse events were graded as mild, moderate, severe, or potentially life-threatening according to a modified World Health Organization grading system. The dose of peg-IFN was decreased by 50% and that of RBV was lowered to half in case of severe adverse events or when laboratory results revealed any of the following: hemoglobin concentration <10 g/dl in patients with no cardiac disease, decrease in hemoglobin concentration >2 g/dl in patients with cardiac disease, white blood cell count <3,000/mm3, or platelet count <50,000/mm3. Full dosage could be resumed on resolution of the adverse events. Treatment was permanently discontinued in case of life-threatening events or when laboratory results revealed hemoglobin concentration <7.5 g/dl after 4 weeks of dose reduction, white blood cell count <1,500/mm3, or platelet count <30,000 mm3.

Treatment for Recurrence

Patients with intrahepatic HCC recurrence were managed with ablative therapies such as radiofrequency ablation (RFA), percutaneous ethanol injection therapy, transarterial chemoembolization, or surgery including living-donor liver transplantation according to the tumor characteristics (number, size, and location of the tumors) and liver function.

Statistical Analyses

Categorical variables were compared using the chi-square test, and continuous variables were compared using the Mann–Whitney U-test. Overall survival and disease-free survival analyses were performed using Kaplan–Meier methods; comparisons between different groups were performed using the log-rank test. P value of less than 0.05 was considered significant. Calculations were performed using SPSS software (version 16; SPSS Inc., IL, USA).

Propensity analysis was performed using logistic regression to create a propensity score for the IFN and non-IFN therapy groups.31,32 Variables entered in the propensity model were age, sex, HCV genotype, liver function test, tumor factors, and operative factors. The model was then used to provide a one-to-one match between the two groups by using the nearest-neighbor matching method.33,34 Survival and disease-free survival analyses were performed in each matched subgroup to assess the impact of peg-IFN therapy on mortality after adjusting for the confounding factors.

Results

Characteristics and Postoperative Course of the Entire Population

Differences in the characteristics of patients who received peg-IFN therapy after hepatic resection and those who did not receive IFN therapy after hepatic resection are presented in Table 1. Patients who received peg-IFN therapy were younger (65 vs. 71 years; P = 0.0003). Regarding tumor characteristics, there was no significant difference between the two groups. Operation times tended to be longer in patients who received peg-IFN therapy than in those who did not receive IFN therapy (260 vs. 242 min; P = 0.05). There were no hospital-related deaths in this study. Postoperative complications did not differ between the two groups. In the entire population, the 3- and 5-year overall survival rates of patients who received peg-IFN therapy after hepatic resection were significantly higher than those of patients who did not receive IFN therapy (P = 0.0024) (Fig. 1a). However, there was no significant difference in disease-free survival between the two groups (P = 0.795) (Fig. 1b).

Table 1 Baseline characteristics and operative data on patients who underwent hepatectomy: data are reported for whole study and for the matched study population after propensity score analysis
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Fig. 1
figure 1

Overall survival (a) and disease-free survival (b) of the entire study population of 175 patients with hepatitis C-related HCC with respect to IFN therapy after hepatic resection. Overall survival (c) and disease-free (d) survival of the matched study population of 76 patients with hepatitis C-related HCC with respect to IFN therapy after hepatic resection

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Results After Propensity Score Matching

Characteristics of the patients after propensity score analysis are presented in Table 1. Thirty-eight of the 43 patients who received peg-IFN therapy after hepatic resection and an equal number of the 76 patients who did not receive IFN therapy were matched after covariate adjustment. The study group of 76 patients was well matched; in particular, all covariates that significantly affected recurrence and postoperative liver failure in the entire study group were equally distributed between the two matched groups. Matched patients who received peg-IFN therapy after hepatic resection had similar total bilirubin and serum albumin levels and similar platelet counts to matched patients who did not receive IFN therapy. Similarly, the tumor characteristics, the surgical procedure, operation times, and blood loss during the operation in matched patients who received peg-IFN therapy were almost similar to those in patients who did not receive IFN therapy. There were no hospital-related deaths in the matched groups. Postoperative complications also did not differ between the two groups. The median follow-up period for patients who received peg-IFN and those who did not receive IFN therapy was 3.8 (1.2–6.9) and 3.5 (1.3–6.8) years, respectively. In the matched study groups, the 3- and 5-year overall survival rates of patients who received peg-IFN therapy after hepatic resection were significantly higher than those of patients who did not receive IFN therapy (P = 0.00135) (Fig. 1c). However, there was no significant difference in disease-free survival between the two matched groups (P = 0.886) (Fig. 1d).

In the matched 38 patients of the peg-IFN group, peg-IFN therapy was initiated at a median of 4.3 (0.9–9.6) months after hepatic resection. Thirty-one of 38 HCC patients began peg-IFN therapy within 6 months after hepatectomy. Seven patients required more than 6 months to commence peg-IFN therapy. Two patients required a longer time to recover platelet counts of more than 70,000/μl. Five patients required a longer time to decide to receive peg-IFN therapy. Sixteen (42.1%) of the matched 38 patients who received peg-IFN therapy after hepatectomy attained SVR. Among 16 patients who attained SVR, 10 patients received full-dose peg-IFN therapy without dose reduction, whereas 6 patients received a reduced dose of peg-IFN and/or RBV until completion of treatment. Nine patients discontinued peg-IFN therapy because of adverse events such as thrombocytopenia and neutropenia (n = 2), skin eruption (n = 1), depression (n = 2), and severe malaise (n = 4). Three patients discontinued peg-IFN therapy because of HCC recurrence. Adherence to peg-IFN therapy was 68.4% in this study. No life-threatening adverse events were observed, and none of the total 15 deaths in both sets of matched patients were related to the IFN treatment or to surgical procedures. The 3- and 5-year overall survival rates of patients (n = 16) who attained SVR after peg-IFN therapy were 100% and 100%, respectively; those of patients who did not attain SVR (n = 22) were 100 and 85.7%, respectively; and those of patients who did not receive IFN therapy were 76.6 and 50.6%, respectively. There was a statistically significant difference in overall survival among the three groups (P = 0.005) (Fig. 2a). However, there was no statistically significant difference in disease-free survival among the three groups (P = 0.90) (Fig. 2b).

Fig. 2
figure 2

Overall survival and disease-free survival of patients with hepatitis C-related HCC with respect to SVR after IFN therapy

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Table 2 presents the patterns of cancer recurrence and the treatment details of the recurrences in both groups. Twenty-one (55.3%) of the patients who received peg-IFN therapy after hepatic resection and 17 (44.7%) of the patients who did not receive IFN therapy had HCC recurrences after hepatic resection. Regarding the pattern of recurrence, the proportion of patients who had multiple intrahepatic recurrences (more than four nodules) was significantly lower in the peg-IFN group than in the non-IFN group (P = 0.0047). The proportion of patients in whom surgery or RFA was selected for treatment was significantly higher in the peg-IFN group than in the non-IFN group (P = 0.0346). Furthermore, regarding re-recurrence of HCC after treatment of the first-recurrent HCC, the 1-year disease-free survival rates of patients after treatment of the first-recurrent HCC was 48.5% in patients (n = 21) who received peg-IFN therapy and 12.5% in patients (n = 17) who did not receive IFN therapy. There was a statistically significant difference in disease-free survival between the two groups (P = 0.0012) (Fig. 3).

Table 2 Recurrence and treatments for recurrence after hepatic resection
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Fig. 3
figure 3

Comparison of disease-free survival rate after treatment of first-recurrent HCC in patients who received peg-IFN therapy or in those who did not receive IFN therapy

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A comparison of results of the preoperative liver function test with those of postoperative 1-year liver function tests is presented in Table 3. In patients who received peg-IFN therapy, total bilirubin levels 1 year after surgery were significantly decreased compared with preoperative total bilirubin levels (P = 0.018), whereas in patients who did not receive IFN therapy, the total bilirubin level at 1 year after surgery was similar to the total bilirubin level before surgery (P = 0.107).

Table 3 Comparison of preoperative liver function with 1-year liver function after hepatic resection
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Discussion

Our results revealed that peg-IFN therapy after hepatic resection improved the outcomes of HCV patients, although the interval of disease-free survival was not prolonged. Peg-IFN therapy after hepatectomy improved hepatic reserve function and suppressed multiple HCC recurrences (more than four nodules). Furthermore, re-recurrence after treatment of first-recurrent HCC after hepatic resection was significantly suppressed in the peg-IFN group compared with that in the non-IFN group. IFN has been reported to exert antitumor effects. IFN increases natural killer cell activity and exhibits antiangiogenic properties.35,36 IFN has also been reported to be effective in eradicating HCV RNA from serum and hepatic tissue, thereby preventing deterioration of liver function in patients with HCV infection.37 IFN prevents worsening of compensated cirrhosis.18,37 Our results are compatible with those reported in those studies. In the peg-IFN group, most patients with HCC recurrence could undergo curative treatments such as repeat hepatectomy or RFA as a recurrence treatment, because the number of recurrent tumors was usually limited to three. IFN therapy appears to increase survival not only by improving residual liver function and increasing the possibility of radical treatment of recurrences but also by suppressing re-recurrence after the first recurrence of HCC.

The current study also revealed that the overall survival of patients with SVR was significantly better than that of patients without SVR. This result suggests that IFN prolongs the outcomes of patients with HCC after hepatic resection by causing remission of active hepatitis and eradication of HCV RNA in patients who attained SVR after hepatic resection.

In this study, to clarify the impact of peg-IFN therapy on outcomes of HCV-related HCC after hepatic resection, patients who received IFNs such as IFN-α or IFN-β were excluded. RCTs investigating adjuvant effects of IFN after resection or ablation of HCC were performed using IFN-α. Few studies have investigated the effects of peg-IFN plus RBV combination therapy on survival and recurrence after curative resection of HCC. Combination therapy with peg-IFN and RBV has recently been developed, and peg-IFN therapy has resulted in significantly higher SVR rates and better tolerability than treatment with IFN-α.21,23 In our study, incidence of SVR after hepatic resection was 42.1%, which was higher than that in previous studies that reported an SVR rate of 0–10%.12–14 The compliance of patients to peg-IFN therapy observed in the present study (68.4%) was higher than that reported elsewhere (approximately 40%).14 This enhanced efficacy of the peg-IFN formulations might contribute to the prolonged survival of HCC patients after hepatic resection.

In this study, HCC patients who received peg-IFN therapy within 9 months after surgery were enrolled, and HCC patients who experienced recurrence of HCC within 9 months after hepatic resection were excluded from the non-IFN group, because these patients could lose the opportunity to receive IFN therapy for HCC recurrence on being assigned to the peg-IFN therapy group.

Before matching by using the propensity score, the clinical characteristics of the entire study population that can strongly influence outcomes differed significantly between the peg-IFN group and non-IFN group. The proportion of older patients was higher in the non-IFN group than in the peg-IFN group, whereas the proportion of patients who had longer operation times tended to be lower in the non-IFN group than in the peg-IFN group. To overcome bias due to the different distribution of the severity of liver function impairment between the two groups, a one-to-one match was created using propensity score analysis. After matching by propensity score, prognostic variables were appropriately handled, and there was no significant difference in prognostic factors between the two matched groups. This study had a limitation related to the small sample size after propensity score matching. To overcome this, further examination with larger sample sizes is necessary, and the potential efficacy of peg-IFN therapy must be validated in larger prospective RCTs.

Conclusions

Several previous RCTs investigating the effects of IFN on survival and tumor recurrence after hepatic resection were inconclusive. However, in the current study, peg-IFN therapy following hepatic resection improved the survival rates of hepatectomized patients with HCV-related HCC. The results of this study suggest that peg-IFN therapy is effective as an adjuvant chemopreventive agent after hepatic resection in patients with HCV-related HCC.