Risk Factors for Hepatocellular Carcinoma in Hepatitis C Patients

Journal of Viral Hepatitis 

Risk Factors for Hepatocellular Carcinoma in Hepatitis C Patients With Normal Alanine Aminotransferase Treated With Pegylated Interferon and Ribavirin 

N. Harada, N. Hiramatsu, T. Oze, N. Morishita, R. Yamada, H. Hikita, M. Miyazaki, T. Yakushijin, T. Miyagi, Y. Yoshida, T. Tatsumi, T. Kanto, A. Kasahara, M. Oshita, E. Mita, H. Hagiwara, Y. Inui, K. Katayama, S. Tamura, H. Yoshihara, Y. Imai, A. Inoue, N. Hayashi, T. Takehara

"All patients who developed HCC had HCV genotype 1 and a high viral load. The first reason is that the patient group with genotype 1 and high viral load is refractory to antiviral therapy. Non-SVR patients are liable to develop HCC. However, three of the 11 patients in this group had SVR. As some researchers have hypothesized, HCV genotype 1 may pose a higher carcinogenic hazard than other genotypes"

Abstract and Introduction
Abstract

Pegylated interferon (Peg-IFN) plus ribavirin combination therapy is effective in patients with hepatitis C virus (HCV) infection and normal alanine aminotransferase levels (NALT). However, it remains unclear whether the risk of hepatocellular carcinoma (HCC) incidence is actually reduced in virological responders. In this study, HCC incidence was examined for 809 patients with NALT (ALT ≤ 40 IU/mL) treated with Peg-IFN alpha-2b and ribavirin for a mean observation period of 36.2 ± 16.5 months. The risk factors for HCC incidence were analysed by Kaplan–Meier method and Cox proportional hazards model. On multivariate analysis among NALT patients, the risk of HCC incidence was significantly reduced in patients with sustained virological response (SVR) or relapse compared with those showing nonresponse (NR) (SVR vs NR, hazard ratio (HR): 0.16, P = 0.009, relapse vs NR, HR: 0.11, P = 0.037). Other risk factors were older age (≥65 years vs <60 years, HR: 6.0, P = 0.032, 60–64 vs <60 years, HR: 3.2, P = 0.212) and male gender (HR: 3.9, P = 0.031). Among 176 patients with PNALT (ALT ≤ 30 IU/mL), only one patient developed HCC and no significant risk factors associated with HCC development were found. In conclusion, antiviral therapy for NALT patients with HCV infection can lower the HCC incidence in responders, particularly for aged and male patients. The indication of antiviral therapy for PNALT (ALT ≤ 30 IU/mL) patients should be carefully determined.
Introduction

The goal of treatment for patients with hepatitis C virus (HCV) infection is not only elimination of the virus, but also prevention of hepatocellular carcinoma (HCC) and thus of death due to liver disease. Therefore, the indication of antiviral therapy should be based on whether or not antiviral therapy can suppress the incidence of HCC.

In the era of interferon (IFN) monotherapy, patients with chronic HCV infection and normal alanine transaminase (NALT) were not considered as candidates for antiviral therapy because SVR rates were very low (6–15%) and ALT flare-up occurred in some cases (47–62%) on IFN therapy.[1–3] Recently, however, the antiviral efficacy of pegylated interferon (Peg-IFN) plus ribavirin combination therapy for patients with chronic HCV infection has led to equivalent antiviral effects for patients with NALT levels, compared with those with elevated ALT levels (EALT).[4,5] Moreover, ALT flare-up after Peg-IFN plus ribavirin combination therapy for patients with NALT has been reported to be rare.[4] Thus, patients with chronic HCV infection and NALT have come to be considered as candidates for Peg-IFN plus ribavirin combination therapy. What remains unknown is whether or not HCC incidence can be reduced in patients with HCV infection and NALT responding to antiviral therapy. In this study, this is what we tried to elucidate.

Another issue is the risk factors for HCC incidence in patients with HCV infection and NALT. For example, among chronic hepatitis patients with NALT, it is still controversial whether or not the viral load of HCV is related to HCC incidence, although high levels of HBV DNA have been revealed to be a risk factor for HCC incidence in patients with hepatitis B virus (HBV) infection.[6] We therefore also examined patients with chronic HCV infection and NALT, who are at high risk of HCC.

Patients and Methods

This retrospective, multicentre study was conducted by Osaka University Hospital and institutions participating in the Osaka Liver Forum. Of 4640 patients with chronic HCV infection who had been treated by Peg-IFN plus ribavirin combination therapy between December 2004 and December 2009, this study enrolled 809 patients with NALT (ALT ≤ 40 IU/mL) at the start of the therapy who had not suffered from HCC. Among them, 431 patients showed persistent normal alanine aminotransferase (PNALT), defined as an ALT value of ≤40 IU/mL (PNALT40) or ≤30 IU/mL (PNALT30), on two to three occasions separated by at least a month over a period of 6 months.[7,8] PNALT30–40 was defined as the observation of ALT values of 31–40 IU/mL on at least one occasion among PNALT40 patients. This study excluded patients who developed HCC within 12 months from the start of Peg-IFN plus ribavirin combination therapy, patients with co-infection with hepatitis B or human immunodeficiency virus, patients with drug-induced or alcoholic liver disorder and patients with autoimmune hepatitis. The protocol was performed after obtaining informed consent from each patient before treatment in accordance with the ethical guidelines of the 1975 Declaration of Helsinki amended in 2002.

Treatment Protocol

All patients received Peg-IFN alpha-2b (PEGINTRON; Merck & Co. Inc. Whitehouse Station, NJ, USA) plus ribavirin (REBETOL; Merck & Co). The serum HCV RNA levels were qualitatively analysed using the COBAS AMPLICOR HCV test, version 2.0 (lower limit of detection 50 IU/mL; Roche Diagnostics, Branchburg, NJ, USA), and the COBAS AMPLICOR HCV MONITOR test, version 2.0 (detection range 6–5000 K IU/mL; Roche Diagnostics). Among the patients with HCV genotype 1, as a rule, the treatment duration was 48 weeks. However, patients with detectable HCV RNA (≥50 IU/mL) at week 12 and undetectable HCV RNA (<50 IU/mL) at week 24 were treated for 72 weeks. Patients with HCV genotype 2 were treated for 24 weeks.
Definition of Virological Response

A sustained virological response (SVR) was defined as HCV RNA being undetectable at the end of treatment and at 24 weeks after completion of treatment. A relapse was defined as undetectable HCV RNA at the end of treatment, but detectable HCV RNA at 24 weeks after completion of treatment. A nonresponse (NR) was defined as detectable HCV RNA at the end of treatment.
Histological Evaluation

Liver biopsy was performed immediately before initiating the Peg-IFN plus ribavirin combination therapy. Liver biopsy specimens were scored using the METAVIR system, and the grade of activity and stage of fibrosis were evaluated.[9]
Surveillance of HCC

Ultrasonography or computed tomography (CT) was carried out before Peg-IFN plus ribavirin combination therapy and every 3–6 months during the follow-up period. New space-occupying lesions detected or suspected at the time of ultrasonography were further examined by CT or hepatic angiography. HCC was diagnosed by the presence of typical hypervascular characteristics on angiography, in addition to the CT findings. If no typical image of HCC was observed, fine-needle aspiration biopsy was carried out with the patient's consent, or the patient was carefully followed until a diagnosis was possible with a definite observation by CT or angiography.
End Point

The observation period started from the date of the start of Peg-IFN plus ribavirin combination therapy. Patients who developed HCC and patients who were retreated after completion of Peg-IFN plus ribavirin combination therapy were defined as censored cases at that point in time.
Statistical Analysis

Baseline data for various demographic, biochemical and virological characteristics of the patients were expressed as mean ± SD. The variables of age, sex, body mass index, HCV genotype, grading and staging of liver histology, platelet count, serum ALT level and virological response to the combination therapy were examined as correlates of HCC development. The Kaplan–Meier method was used to assess the cumulative incidence of HCC, and the groups were compared using the log-rank test. The Cox proportional hazards model was used to identify the independent factors associated with developing HCC. A value of P < 0.05 (two-tailed) was considered to indicate significance. Statistical software IBM SPSS for Windows, version 19.0.0 (IBM, Armonk, NY, USA), was used for this analysis.

Results

Baseline Characteristics of Patients With Chronic HCV Infection and NALT

Baseline characteristics of the 809 HCV-infected patients with NALT are shown in  Table 1.  The mean age was 56.7 ± 11.0 years, and the ratio of men was 67%. A total of 550 patients (69%) were infected with HCV genotype 1. Liver biopsies were performed for 587 cases, and the ratio of patients with progression of liver fibrosis (METAVIR fibrosis score 3 or 4) was 7.8%. The rate of patients with SVR was 48.7% (263 of 550) in genotype 1 and 74.5% (184 of 247) in genotype 2. The mean observation period was 36.2 ± 16.5 months.

Baseline Characteristics of Patients Who Developed HCC

Eleven patients developed HCC during the observation period. The baseline clinical features before Peg-IFN plus ribavirin combination therapy and the features of the HCC that developed are shown in Table 2. HCC development was observed often in aged patients and male patients.

All who developed HCC were infected with HCV genotype 1 and had a high viral load.

Risk Factors Associated With Incidence of HCC Among Patients With Chronic HCV Infection and NALT

Patients with chronic HCV infection and NALT showed HCC incidences of 1.1% and 3.3% at 3 years and 5 years, respectively, in this study. Significant association with HCC development on univariate analysis was found for age, sex, platelet count and virological response to combination therapy, but not for the amount of HCV RNA (Table 3). Significant factors included older age, male gender and virological response, which were independent factors of HCC incidence in the Cox proportional hazards model (Table 3). The adjusted cumulative incidence of HCC at 3 years by the Cox proportional hazards model was 0.19% among patients <60 years of age, 0.59% among patients 60–64 years of age and 1.12% among patients ≥65 years of age. The cumulative incidence of HCC at 3 years was 0.96% in men and 0.25% in women. As shown in Fig. 1, patients with SVR or relapse had a significantly lower cumulative incidence of HCC than that of patients with NR (cumulative incidence of HCC at 3 years: SVR, 0.20%; relapse, 0.30%; NR, 1.89%).

Figure 1.Cumulative rate of HCC incidence by Cox proportional hazards model according to antiviral effect.

Baseline Characteristics and Risk Factors Associated With HCC Incidence Among Patients With Chronic HCV Infection and PNALT

In this study, patients who met the definition of PNALT40 were 431 of the 809 patients with NALT during the mean observation period of 35.5 ± 15.9 months. Baseline characteristics of the 431 HCV-infected patients with PNALT40 are shown in Table 1. Among the PNALT40 patients, three developed HCC during the observation period (Table 2, cases 4, 9 and 10). Significant association with HCC development by log-rank test was found for older age, male gender, lower platelet count and NR (Table 4). No significant risk factors were found in multivariate analyses of these four variables.

Baseline characteristics of the 176 HCV-infected patients with PNALT30 are shown in Table 1. There were more female PNALT30 patients, more HCV genotype 2 patients, lower BMI, lower activity and lower fibrosis compared with PNALT30–40 patients. Among them, one patient developed HCC during the mean observation period of 33.5 ± 15.9 months (Table 2, case 4). There were no significant risk factors associated with HCC development among these patients (Table 4).

Discussion

Continuous infection with HCV is a leading cause of liver fibrosis which may progress to cirrhosis and HCC.[10,11] HCV carriers with NALT levels are generally considered to have a low risk of carcinogenesis because of minimal liver inflammation and slow progression of liver fibrosis.[12,13] Thus, IFN therapy has not been used for NALT patients. Against this background, there has been no report of antiviral therapy for HCV carriers with NALT reducing HCC incidence. However, a simulation using the Markov model has suggested that Peg-IFN plus ribavirin combination therapy could decrease HCV-related morbidity and mortality in patients with NALT.[14] This needed to be evaluated with patients using Peg-IFN plus ribavirin combination therapy. In the present study, we examined the efficacy of antiviral therapy in patients with chronic HCV infection and NALT from the viewpoint of reducing the risk of HCC. This is the first report indicating that virological responders for antiviral therapy had a significantly lower cumulative incidence rate of HCC than those with NR in patients with chronic HCV infection and NALT.

In the present study, we examined whether successful virological response to Peg-IFN plus ribavirin combination therapy was associated with a low HCC incidence in NALT patients. Considering that reducing the risk of HCC incidence is the goal of antiviral therapy, this result indicates that patients with HCV infection and NALT can also be candidates for antiviral therapy. In particular, male patients ≥65 years of age should be treated earlier even if the ALT level is within the normal range because the factors of age ≥65 years and male gender were identified as independent risk factors for HCC incidence. Several studies have reported that patients ≥65 years of age or male patients generally had a higher risk of HCC incidence among CHC patients with EALT.[15–17] Our results suggest that older male patients with HCV infection should be treated even if they are NALT patients.

All patients who developed HCC had HCV genotype 1 and a high viral load. The first reason is that the patient group with genotype 1 and high viral load is refractory to antiviral therapy. Non-SVR patients are liable to develop HCC. However, three of the 11 patients in this group had SVR. As some researchers have hypothesized, HCV genotype 1 may pose a higher carcinogenic hazard than other genotypes.[18,19]

In Japanese treatment guidelines for patients with chronic HCV infection and NALT levels, HCV carriers with NALT (≤40 IU/mL) at the initial visit are classified into four groups according to their ALT levels (≤30 IU/mL or ≥31 IU/mL) and platelet counts (≥15 × 104 per mm3 or <15 × 104 per mm3). In the univariate analysis according to those categories, low platelet counts (<15 × 104 per mm3) were shown to be the only risk factor for HCC incidence, irrespective of the ALT levels.[20] In the present study, risk factors for HCC incidence were examined among NALT patients by multivariate analysis. As a result, no significant relationship was found between platelet counts or progression of liver fibrosis and HCC incidence. In fact, older patients who developed HCC in this study had relatively high platelet counts and minimal liver fibrosis. Previous studies have shown that older patients with HCV infection even without severe liver fibrosis are at risk of developing HCC.[21] Although the reason for this is not known, mechanisms such as those associated with telomeres may be in part responsible.[22,23] Older patients require extra attention with respect to HCC surveillance, regardless of the progression of liver fibrosis.

In the present study, 431 patients with PNALT40, identified by an ALT value of ≤40 IU/mL on two to three occasions separated by at least a month over a period of 6 months, were examined for HCC incidence. Univariate analysis showed that older age (≥65 years), male gender, lower platelet counts (<12 × 104 per μL) and SVR to combination therapy were significant risk factors for HCC incidence, which were very similar to those for NALT patients; the difference was only that being in relapse was not a significant risk factor for HCC incidence in patients with PNALT40. A longer observation period may reveal a reduction in HCC incidence among relapse patients with PNALT40.

If the definition of PNALT were an ALT value of ≤30 IU/mL on two to three occasions separated by at least a month over a period of 6 months, then 176 of our patients would fit this classification. There were no significant risk factors for HCC incidence among patients with PNALT30, and only one patient with relapse developed HCC during the observation period. No patients with SVR and NR developed HCC. Therefore, little or no liver inflammation among patients with HCV infection and PNALT30 might indicate a fairly low risk of HCC incidence; that is, it may suggest that patients with PNALT30 can be followed up without antiviral therapy from the viewpoint of reducing the risk of HCC incidence. On the other hand, significant risk factors, including male gender and viral response, were found among patients with PNALT30–40 from the univariate analysis. Considering the high risk of HCC incidence, this suggests that patients with HCV infection and PNALT30–40 should be candidates for antiviral therapy.

The limitation of this study was that the HCC incidence was not compared between the treatment and nontreatment groups. HCC incidence was significantly lower in patients with SVR or relapse compared with those with NR as a control group among patients with Peg-IFN plus ribavirin combination therapy. This does not show a clear suppressive effect of antiviral therapy for HCC incidence in NALT patients with HCV infection. However, patients with NR treated with antiviral therapy are reported to have about the same HCC incidence as patients without antiviral therapy.[24] Moreover, Peg-IFN plus ribavirin combination therapy in NALT patients is not effective if a successful virological response does not lead to the reduction in HCC incidence. Our findings that patients with SVR or relapse had significantly lower HCC incidence compared with those with NR suggest that patients with HCV infection and NALT should be candidates for Peg-IFN plus ribavirin combination therapy. A prospective randomized study should be performed to verify the suppressive effect of antiviral therapy for HCC.

In conclusion, among patients with chronic HCV infection and NALT, the cumulative incidence of HCC was significantly lower in patients with SVR and relapse than those with NR to Peg-IFN plus ribavirin combination therapy in the same manner as any EALT patient with HCV infection. Therefore, patients with chronic HCV infection and NALT should be considered as candidates for antiviral therapy from the viewpoint of reducing HCC incidence, especially aged patients (≥65 years) and/or male patients. However, the indication of antiviral therapy for PNALT (ALT ≤ 30 IU/mL) should be carefully examined.

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