It was shown that hepatitis C antibodies were present in one third to one half of alcoholics with alcoholic liver disease in. Spain and Italy (Brillanti et al., 1989; ...
Alcohol & Alcoholism Vol. 35, No. 3, pp. 286–295, 2000
EFFECT OF ALCOHOL CONSUMPTION ON THE PROGRESSION OF HEPATITIS C VIRUS INFECTION AND RISK OF HEPATOCELLULAR CARCINOMA IN JAPANESE PATIENTS KHALEQUE NEWAZ KHAN and HIROSHI YATSUHASHI* Institute for Clinical Research, Nagasaki Chuo National Hospital, WHO Collaborating Center for Reference and Research on Viral Hepatitis, 2-1001-1 Kubara, Omura City, Nagasaki 856, Japan (Received 3 August 1999; in revised form 7 December 1999; accepted 4 January 2000) Abstract — Chronic hepatitis C virus (HCV) infection is associated with a spectrum of liver diseases and a proportion of chronic cases progress through cirrhosis to hepatocellular carcinoma (HCC). The viral and host factors that are important in the clinical and histological progression of HCV infection are unclear. We investigated the effect of moderate (80 g/day) alcohol intake on the histological and clinical progression of HCV infection and their associated risk of hepatic cancer in a group of Japanese patients. A number of other variables were assessed to evaluate their impact on disease progression. We recruited 120 patients with HCV infection and categorized them into four groups, based on alcohol consumption pattern. All clinical and biochemical profiles were collected from recorded files. Liver biopsies were analysed for the degree of fibrosis, presence of cirrhosis and histological activity of necroinflammation. Hepatic tumours were detected by the follow-up imaging analysis. There was no difference in the age, length of exposure to HCV infection and HCV RNA serum levels in the alcohol and alcohol-free groups. The histological grading of necroinflammation, serum levels of alanine aminotransferase and HCV RNA did not have any correlation with each other in the alcohol and alcohol-free group. There was a 1.5–2.5-fold greater risk of liver cirrhosis and hepatocellular carcinoma in the alcohol intake group compared to the alcohol-free group. Kruskal–Wallis analysis among four groups demonstrated a significant transition to fibrosis (P < 0.05) for alcoholics with HCV infection. The increased risk of liver cancer in the alcohol group is independent of size and growth of tumours. The clinical manifestations of gastro-oesophageal variceal bleeding, ascites, and encephalopathy were also higher in the alcohol intake group. Alcohol consumption is an important risk factor in the histological and clinical progression of HCV infection and has no relation with HCV replication. Chronic HCV carriers should avoid excessive alcohol intake to reduce the acceleration of liver disease and risk of liver cancer.
INTRODUCTION
1993). Chronic hepatitis due to HCV in non-alcoholics tends to be somewhat less aggressive than other forms of chronic liver disease, but between 20% and 60% of cases nevertheless progress to cirrhosis (Di Bisceglie et al., 1991; Bach et al., 1992; Scheuer et al., 1992), and it is reasonable to presume that high alcohol intake will exacerbate this process. Cooksley (1996) reported that although HCV and alcohol produce different histological appearances in the pre-cirrhosis stage, they both progress to cirrhosis slowly and only a minority of people develop cirrhosis despite the combination of HCV and heavy alcoholism. However, the majority of evidence suggests that these insults are probably additive for the progression of fibrosis caused by an interaction of alcohol and HCV in the pathogenesis of chronic liver disease (McFarlane, 1993; Poynard et al., 1997). Alcohol intake has also been implicated as an independent risk factor in the progression of HCV infection, although its overall effect on both the histological and clinical progression of liver disease in patients chronically infected with HCV is still uncertain, and the published reports on this topic are very limited, except one from the United States (Wiley et al., 1998). In the present retrospective study, we examined the effect of moderate and heavy alcohol intake on the histological and clinical status of patients who were infected with HCV and their associated risk of liver cancer in a group of Japanese patients. Unlike previous studies, we also included patients with alcoholic liver disease without HCV infection as a positive control. Our study also indicates that patients who are infected with HCV and drink alcohol have a higher frequency of cirrhosis and liver cancer, and progress more frequently to clinically apparent liver disease irrespective of the viral load and severity of necroinflammation.
Hepatitis C virus (HCV) infection is a quiescent disease with patients rarely having clinical symptoms until cirrhosis develops. Following the discovery of an assay for HCV (Kuo et al., 1989), it was demonstrated that chronic hepatitis C has a prevalence of at least 1% worldwide (Alter, 1995). Mortality associated with chronic hepatitis C results mainly from the development of liver fibrosis and the subsequent occurrence of cirrhosis, with complications such as hepatocellular carcinoma (HCC) (Tong et al., 1995). Ten to thirty per cent of patients with HCV will develop cirrhosis over a 20–30 year period, and 1–4% will develop HCC (Di Bisceglie et al., 1991; Seeff et al., 1992; Takahashi et al., 1993). Host and viral factors that are important in enhancing the progression to cirrhosis are not entirely clear (Tassopoulos, 1996). Proposed contributory factors include viral genotype, circulating viral load, duration of infection, mode of infection, iron overload, and alcohol consumption (Di Bisceglie et al., 1991; Seeff et al., 1992; Takahashi et al., 1993; Tong et al., 1995; Tassopoulos, 1996). It was shown that hepatitis C antibodies were present in one third to one half of alcoholics with alcoholic liver disease in Spain and Italy (Brillanti et al., 1989; Bruix et al., 1989; Esteban et al., 1989; Pares et al., 1990) and similar figures were obtained from studies in the USA, Europe and Japan (Ishii et al., 1990; Mendelhall et al., 1991). In contrast, relatively low frequencies of HCV antibodies in alcoholic liver disease were also reported (Locarnini and Dudley, 1991; Herion et al.,
*Author to whom correspondence should be addressed. 286
© 2000 Medical Council on Alcoholism
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PATIENTS AND METHODS Patients One hundred and twenty patients with HCV chronic liver diseases were recruited for our current study. The medical records and liver biopsies of patients who were seen at the liver unit of Nagasaki Chuo National Hospital over the last 4 years were reviewed retrospectively. All patients were interviewed by use of a standardized questionnaire to obtain information about the type of alcoholic drink, the amount of alcohol consumed per day, duration of alcohol consumption, mode of HCV infection, and dates of transfusions during a subsequent visit to our hospital. Only cases in which there had been an established drinking pattern for more than 5 years were considered under past exposure. Although the exact duration of alcohol intake was not always recorded in the patient’s medical records, it was noted that this period exceeded 10 years in about two-thirds of the subjects. Therefore, we confirmed the consistency of the recorded alcohol history by subsequent assessment. Informed consent was obtained from all patients detailing the consequence of the study and our approved protocol was granted by the Nagasaki Chuo National Hospital Ethical Committee. Alcohol consumption was estimated by one attending physician (K.N.K.) and quantified in g/day. For this study, excessive alcohol intake consisted of moderate alcohol consumption of 80 g/day for >5 years during the time the patient was infected with HCV. The duration of exposure to HCV was estimated as being from the first blood transfusion before 1990 to the time of liver biopsy. No patient with a source of infection by intravenous drug use was found. We divided our patients into four groups depending on the amount of alcohol intake. Group A (14 patients), alcoholic liver disease with an alcohol intake of >80 g/day without any HCV infection; Group B (40 patients), HCV infection only and no regular alcohol intake with the possibility of only chance or social drinking; Group C (42 patients), HCV plus alcohol intake of 80 g/day. Groups A, C and D consisted of only male patients and Group B initially consisted of 50 patients with 10
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female patients. We excluded the 10 female patients from Group B and finally recruited 120 male patients. The clinical profiles and biochemical data of all these patients are shown in Table 1. Patients were considered to have alcoholic liver disease when they fulfilled the following criteria: (1) all viral and immunological markers are negative, (2) aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio greater than 1, (3) histological evidence of Mallory’s hyaline, polymorphonucleocytes surrounding hepatocytes, and/or significant central vein fibrosis. Patients were considered to have clinically manifested liver disease if they had a gastrooesophageal varix with or without episodes of variceal bleeding, ascites, encephalopathy, or developed a hepatocellular carcinoma. The severity of varices was based on the endoscopic formation (F) of varix according to the recommendation of the Japanese Endoscopic Society. F1 varix denotes straight and narrow varix; F2 indicates tortuous varix; F3 denotes large and engorged varix. Diagnosis of HCV infection HCV liver disease was confirmed in each patient by the detection of anti-HCV antibody in serum tested by second generation radioimmunoblot assay or serum HCV RNA by the the polymerase chain reaction. All patients with hepatitis B surface antigen positivity and immunological disorders of chronic liver disease were excluded from our study. Patients who had not received interferon therapy before liver biopsy were included in this study. HCV RNA was measured in serum by the signal amplification technique employing branched deoxyribonucleic acid (bDNA) in a sandwich hybridization assay (Quantiplex version 1.0, Chiron Corp., USA) (Lau et al., 1993). Serum HCV RNA titres were expressed as mega equivalent copies of viral genome per millilitre of serum (Meq/ml). Histology All patients had a liver biopsy perfomed within the last 4 years as part of their standard medical evaluation. Liver biopsy specimens were collected by either blind biopsy or during peritoneoscopy and were fixed in 10% neutral formalin. Sections were cut at 3–4 µm thickness and stained with
Table 1. Clinical profiles of patients with hepatitis C virus (HCV) liver diseases and alcohol consumption Patient groups Parameter No. of patients Sex (M/F) Age (years) Level of alcohol intake (g/day) AST (U/l) (11–39) ALT (U/l) (5–35) MCV (U/l) (80–94) PLT (×104/µl) (15–38) γ-GTP (U/l) (0–50) LAP (U/l) (40–100) Albumin (g/dl) (3.8–4.9) TB (U/l) (0.2–1.0)
A
B
C
D
14 14/0 61.6 ± 11.7 119.3 ± 47.2 54.6 ± 20.0 39.2 ± 23.3 96.6 ± 3.3 16.1 ± 8.7 111.2 ± 98.4 113.7 ± 42.9 3.8 ± 0.4 0.9 ± 0.3
40 40/0 59.5 ± 6.4 0 75.3 ± 45.9 125.6 ± 82.9 95.6 ± 1.9 12.3 ± 4.5 57.8 ± 54.3 90.6 ± 29.5 3.9 ± 0.3 0.7 ± 0.2
42 42/0 59.6 ± 7.5 65.4 ± 15.2 70.6 ± 46.5 103.1 ± 70.6 96.5 ± 6.6 11.4 ± 6.5 74.3 ± 85.6 95.8 ± 26.7 3.6 ± 0.5 1.1 ± 0.6
24 24/0 60.6 ± 9.2 115.2 ± 43.6 93.4 ± 64.4 113.9 ± 72.1 95.6 ± 8.4 13.0 ± 6.9 86.8 ± 80.9 106.7 ± 38.3 3.6 ± 0.5 1.0 ± 0.6
All results are expressed as mean ± SD. Group A, alcoholic liver disease; Group B, HCV only; Group C, HCV+ 80 g/day of alcohol. Abbreviations used: AST, aspartate aminotransferase; ALT, alanine aminotransferase; MCV, mean corpuscular volume; PLT, platelet; γ-GTP, gamma-glutamyl transpeptidase; LAP, leucine aminopeptidase; TB, total bilirubin.
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haematoxylin–eosin and Masson trichrome or silver stain for reticulin fibres (Yano et al., 1996). Tissue specimens were studied independently by a senior pathologist of our Institution (H.Y.). For each biopsy specimen, grading of necroinflammation and staging of fibrosis were based on recommendations by Desmet et al. (1994) and Scheuer (1991). The disease activity was assessed by a final grading of necroinflammation as described before (Yano et al., 1996). Detection of HCC HCCs were detected in our recruited patients during followup screening by abdominal sonography and finally confirmed by computed tomographic scan, histology and/or angiography. The risks of HCC among the four groups of patients and their occurrence in different stages of fibrosis were analysed. Size and doubling time of all detected tumours were calculated from computer-preserved data files of the patients and based on the formula reported earlier (Collins, 1956; Schwartz, 1961). Statistical analysis All results are expressed as means ± SD. Student’s t-test and Kruskal–Wallis analysis were obtained for assessing significance of values between non-paired groups and overall transition of fibrosis among four groups. The correlation efficacy in non-paired groups was assessed by Spearman regression analysis.χ2-test was also undertaken to analyse the risk of liver cirrhosis, HCC, and appearance of clinical signs of liver disease. P < 0.05 was considered as statistically significant. RESULTS Demographic and biochemical data There was no difference in the age of all male patients in the alcohol and alcohol-free groups (Table 1). The median alcohol intake for Groups A and D was similar and was considered indicative of heavy alcohol consumption. The median alcohol intake for Group C was 65 g/day and this was considered as a group of moderate alcohol consumption. The AST/ALT ratio was >1 for Group A, but was 80 g/day) could not induce an increased disease activity. A possible alternative explanation, that the increase in circulating endotoxins leading to activation of Kupffer cells causes ethanol-induced liver damage (Thurman et al., 1998), needs to be evaluated. The enhanced degree of fibrosis, the 1.5–2.5-fold increased risk of liver cirrhosis and the clinical presentation of liver disease complicated by HCV and alcohol intake, as observed in our current study, concur with the results published recently (Wiley et al., 1998). In a large population-based study from Italy, concomitant HCV infection increased the incidence of cirrhosis 10-fold in chronic alcoholics, compared with alcoholics who were not infected with HCV (Bellentani et al., 1994). A 2-fold greater frequency of cirrhosis was also noted in a French study in HCV-infected patients who drank (Roudot-Thoraval et al., 1997). Also, the prognosis of alcoholic liver disease was worse in patients who were HCV Abpositive (Mendelhall et al., 1991). In fact, stage 4 fibrosis was
PROGRESSION OF HCV INFECTION BY ALCOHOL
significantly higher in the HCV plus alcohol-intake patients, compared to patients with HCV infection only or alcoholic liver disease only. It is important to note that the histological activity of necroinflammation was significantly lower in patients with alcoholic liver disease without HCV infection, even when they ingested a toxic concentration of ethanol, when compared with the other groups of patients. However, their degree of fibrosis was significantly higher and similar to patients infected only with HCV. In contrast, the patients with concomitant HCV infection and alcohol ingestion displayed a significantly higher degree of fibrosis and consequently increased risk of cirrhosis. Since the median age of our patient population was similar, our findings further confirm the role of alcohol in inducing fibrosis. A relation between fibrosis and age, and fibrosis and past alcohol consumption has already been described (Deny et al., 1994; Mochida et al., 1996; Poynard et al., 1997; Roudot-Thoraval et al., 1997; Khan et al., 1998; Ostapowicz et al., 1998). Although we could not detect other parenteral risk factors, except blood transfusion, in a small group of patients with HCV infection, our study supports other observations indicating that progression of liver disease is not dependent on the mode of infection (Strasser et al., 1991; Kao et al., 1994). Again, the progression to cirrhosis was found to be independent of the length of exposure to HCV infection. However, high-alcohol-consuming patients with HCV infection developed cirrhosis a little faster, although not significantly so. The non-homogeneous distribution of our patients might affect the results as reported recently (Wiley et al., 1998). In addition to the increased risk of liver cirrhosis, we also reported that about one-half of our recruited patients developed HCC in the clinical course of liver disease. Among these, more than 70% of HCC belonged to the HCV-infected patients who drank either moderate or heavy amounts of alcohol. This means a 1.5–2.5-fold increased risk of HCC was noticed in HCV-infected patients who consumed alcohol. The other clinical complications related to decompensated liver disease, such as gastro-oesophageal varices and their bleeding episodes, endoscopic formation of varices, ascites and encephalopathy, were also markedly higher in HCV-infected patients who consumed alcohol excessively. The increased risk of HCC as a complication of alcohol ingestion was mostly observed in patients with stage 4 liver cirrhosis and was similar to the report by Donato et al. (1997). However, a recent study suggested that the degree of fibrosis was not a significant risk factor for the development of HCC in patients infected with HCV (Kasahara et al., 1998). In the natural course of HCV-infected liver disease, repeated regeneration due to persistent liver injury by HCV may cause hepatocyte DNA to become susceptible to mutagenesis, resulting in gene instability (Shiratori, 1996). Therefore, ethanol-induced enzymatic activation for the conversion of procarcinogens into carcinogens and consequent induction of hepatic neoplasm (Farinati et al., 1985; Lieber et al., 1986; Garro and Lieber, 1990) might be an additional factor for the increased risk of HCC in our study. We did not find any difference of tumour sizes between HCV-infected patients who drank and those who did not. When the doubling times of all these tumours were analysed in all tumour-bearing patients to assess the effect of alcohol on tumour cell proliferation, we did not
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find any group difference, in contradiction to the results of Matsuhashi et al. (1996). It has been reported that p53 gene mutations are associated with tumour progression as a late event in hepatocarcinogenesis, and that heavy drinking correlates with p53 gene mutations in squamous cell carcinoma of the head and neck (Field et al., 1994; Hayashi et al., 1995). As for HCC, it may be speculated that specific p53 mutations are not associated with HCC patients who are alcohol misusers. More epidemiological and molecular studies are necessary to confirm the effect of alcohol on tumour growth of HCC. In conclusion, our results further strengthen the evidence that alcohol consumption can be an additional insult for the progression of fibrosis and risk of cirrhosis in HCV liver disease. It is independent of HCV replication and severity of disease activity. Chronic HCV carriers should avoid excessive alcohol consumption if they are to reduce the progression of fibrosis, incidence of liver cirrhosis, clinical manifestations complicating decompensated liver disease and also to reduce the risk of HCC.
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