Low oxygen tension enhances hepatitis C virus replication.

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

Sorafenib: from literature to clinical practice.

Sorafenib is considered the standard systemic therapy for hepatocellular carcinoma (HCC), in patients with well-preserved liver function (Child-Pugh A class) and advanced-stage HCC (BCLC-C) or in patients with HCC progressing after locoregional therapies, with a high grade of recommendation. The approval of sorafenib for this indication was grounded on the efficacy and the safety results reported by two international randomized, controlled trials, the SHARP and the Asia-Pacific studies. In addition, the efficacy and the safety of sorafenib in clinical practice are addressed by several field-practice experiences, including the multinational GIDEON study and the SOFIA study. Finally, further research on sorafenib is ongoing to optimize the use of this molecule. This review aims to provide an overview of the most relevant clinical data on the efficacy and the safety of sorafenib in patients with HCC.

Safety, Efficacy and Distribution of Doxorubicin Loaded Radiopaque Beads in Chemoembolization in Intermediate Stage Hepatocellular Carcinoma (HCC) with Correlation with Local Response.

PURPOSE: The primary objectives of this study were to evaluate safety, and efficacy of Transarterial Chemoembolization (TACE) using doxorubicin-loaded radiopaque microspheres (DC Bead LUMI™) for the treatment of early and intermediate stage Hepatocellular Carcinoma (HCC) not amenable for curative treatments. Distribution of the microspheres was correlated with results post embolization. MATERIALS AND METHODS: This was a prospective, single arm, open label study. The primary outcome measures were distribution of the radiopaque microspheres as showed by computerized tomography (CT) and local response measured by modified Response Evaluation Criteria (mRECIST) after Magnetic Resonance Imaging (MRI). Secondary measures were Time to Progression (TTP) and Overall Survival (OS). RESULTS: Fifty patients were enrolled over 36 months. Median age was 69.0 years; mean sum of target lesions diameters was 78.6 ± 36.8 mm. There were no Grade 4 or 5 adverse events (AEs). At 6 months Complete Response (CR) (18%), Partial Response (PR) (62%), Objective Response OR (80%) and Stable Disease (SD) (20%) were recorded. Before embolization, Diffusion Weighted Imaging (DWI) showed high signal (restricted diffusion). Post procedure, patients with dense deposition (< 5 mm distance of microsphere aggregations) showed 100% absence of enhancement and no restriction in 30.6%. Median TTP was 8.3 months. TTP for patients with CR was 13.3 months and 7.2 and 5.6 for PR and SD, respectively. At 6 and 36 months, survival was 94% and 34%, respectively. CONCLUSION: DC Bead LUMI™ is well tolerated and effective in early and intermediate stage HCC with maximal necrosis obtained in dense deposition in the target.

High levels of HCV core+1 antibodies in HCV patients with hepatocellular carcinoma.

The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.

Long-term safety of anti-TNF treatment in patients with rheumatic diseases and chronic or resolved hepatitis B virus infection.

OBJECTIVES: The aim of this prospective study was to examine the safety of anti-tumour necrosis factor (TNF) therapy in patients with rheumatic disease and hepatitis B virus (HBV) infection. METHODS: 14 patients with chronic HBV infection, 19 HBV-vaccinated patients and 19 patients with resolved HBV infection were included in the study. All HBV-infected patients received combination therapy with oral antivirals and anti-TNF agents. During treatment the levels of hepatitis B surface antibodies (anti-HBs) in HBV-vaccinated patients and of serum HBV DNA in patients with chronic or resolved HBV infection were monitored. RESULTS: No viral reactivation was observed in patients with resolved HBV infection while anti-HBs titres decreased during anti-TNF treatment in vaccinated patients, similarly to patients treated with methotrexate alone. None of the HBV-infected patients developed liver decompensation or a significant increase in alanine aminotransferase levels. One patient (7%) treated with lamivudine and etanercept showed viral reactivation due to the emergence of a lamivudine-resistant mutant strain. CONCLUSIONS: Anti-TNF agents represent a safe option for patients with chronic HBV infection when combined with antiviral therapy, as well as in patients previously exposed to HBV receiving no HBV prophylaxis. Resistant HBV strains may arise in patients with chronic hepatitis B, necessitating the initial use of anti-HBV agents with a low risk of resistance.

Survivin expression in colorectal carcinomas: correlations with clinicopathological parameters and survival.

BACKGROUND: Survivin is a new member of the Inhibitor of apoptosis protein family that has a dual function as a mitotic regulator and apoptosis inhibitor. Survivin is prominently expressed in transformed cell lines and in many human cancers, including colorectal carcinoma. The aim of this study is to investigate the expression of survivin in colorectal carcinomas and its possible associations with clinicopathological parameters and patient survival. MATERIALS AND METHODS: Sections of formalin-fixed paraffin-embedded tissues from 77 colorectal carcinomas were immunohistochemistry stained for survivin. RESULTS: Survivin was mainly detected in the bottom of the glands of normal mucosa with mainly cytoplasmic localization. No survivin expression was found in infiltrating lymphocytes, fibroblasts, smooth muscle cells or neural tissue. Survivin staining was detected in 68/77 (88.3%) colorectal carcinomas. Survivin expression was found to be significantly associated with tumor differentiation (P = 0.02) but not with gender, age or Dukes stage. Survival did not differ according to survivin expression. CONCLUSION: Survivin was found in the majority of colorectal carcinomas, suggesting that its expression is an early event in colorectal carcinogenesis. Its expression is statistically significantly associated with tumor differentiation but not with patient survival.

Prevalence of glucose intolerance in patients with chronic hepatitis B or C: a prospective case-control study.

Despite several studies, the association of glucose intolerance with chronic hepatitis B (CHB) or C (CHC) virus infection remains controversial. We evaluated the prevalence of glucose intolerance by oral glucose tolerance test (OGTT) in patients with CHB or CHC in comparison with matched controls. In total, 189 consecutive outpatients with CHB or CHC and 189 subjects individually matched for age, sex and body mass index (BMI) were included. OGTT was performed in all cases, except in known diabetics, and glucose intolerance was defined as impaired glucose tolerance (IGT), OGTT-diabetes or known diabetes. Most patients with abnormal OGTT had normal fasting glucose (IGT: 69.8%, OGTT-diabetes: 54.5%). Compared with their own controls, CHB patients had a higher prevalence of IGT (13.6% vs 2.5%, P = 0.018) and family history of diabetes (34.6% vs 16.0%, P = 0.011), while CHC patents had higher prevalence of glucose intolerance (37.0% vs 15.7%, Rho = 0.001), mostly because of more frequent IGT (21.3% vs 6.5%, Rho = 0.003). After age and BMI adjustment, patients with CHC compared with those with CHB had significantly higher prevalence of glucose intolerance (37.0% vs 29.6%, P = 0.037). In conclusion, increased prevalence of glucose intolerance is documented by OGTT both in CHC and CHB patients compared with age, sex and BMI matched controls. Glucose intolerance is more frequent in CHC than CHB patients, regardless of known risk factors. An OGTT might be necessary at the baseline work-up of CHB or CHC patients, as a normal fasting glucose value does not exclude IGT or OGTT-diabetes.

Changes in the epidemiology of hepatitis B virus infection following the implementation of immunisation programmes in northeastern Greece.

The objective of this study was to investigate changes in the epidemiology of hepatitis B virus infection in the general population and selected groups of immigrants in the region of northeastern Greece over the last decade in relation to the introduction of hepatitis B vaccination programmes. Two population-based seroprevalence surveys were carried out during the years 1992-1994 and 1998-2006. In total, 25,105 individuals were tested for the presence of hepatitis B virus markers: HBsAg, anti-HBs and anti-HBc. Childhood/adolescence immunisation programmes began early in 1994 in selected groups of immigrants and were complemented by the national vaccination programme in 1998. Between 1992-1994 and 1998-2006, the HBsAg carrier rate declined from 5.4% [95% CI: 4.5-5.9] in adults (20-60 years old) and 1.9% [95% CI: 1.6-2.4] in children/adolescents (5-19 years old) of indigenous residents to 3.4% [95% CI: 2.9-3.8] and 0.6% [95% CI: 0.2-1.4] respectively (p<0.05). In spite of a decrease compared with 1992-1994, the percentage of HBsAg carriers was still relatively high in 1998-2006 among the Muslim religious minority group (8.2% [95% CI: 8.0-8.7] in adults and 2% [95% CI: 1.7-2.4] in children/adolescents) and in immigrants from the former Soviet Union (4.3% [95% CI: 3.6-4.7] in adults and 1.1% [95% CI: 0.8-2.4] in children/adolescents) (p<0.05 for both selected groups versus general population). The decline of the prevalence of HBsAg in the general population and selected groups of immigrants in northeastern Greece over the last decade supports the effectiveness of the ongoing immunisation programme although the information on the actual number of cases of acute HBV infection is not available.

Five-Years Outcome Analysis of 142 Consecutive Hepatocellular Carcinoma Patients Treated with Doxorubicin Eluting Microspheres 30-60 µm: Results from a Single-Centre Prospective Phase II Trial.

PURPOSE: To assess prospectively long-term results of doxorubicin-loaded HepaSphere 30-60 µm in consecutive patients with hepatocellular carcinoma (HCC) not amenable to curative treatments. PATIENTS AND METHODS: Single-center study from June 2011 to December 2015 in 151 patients treated with 75 mg of doxorubicin per HepaSphere vial. Baseline: Barcelona Clinic Liver Cancer BCLC A/B was 49.3%/50.7%, and median diameter 6.1 cm (mean 6.7 ± 2.0). Liver function, local response (mRECIST), liver time to progression (LTTP), progression-free survival (PFS), overall survival (OS) and adverse events (AEs) were recorded. RESULTS: Final analysis included 142 patients with median follow-up of 46.8 months (range 4-72) without grade 4/5 AEs, and 30-day mortality was 0%. Mean number of scheduled treatments was 2.6 (range 1-3) and on demand 3 (range 1-8). Complete response for single tumor ≤ 5 cm was 75.0% and 66.7% for Child A and Child B, while for > 5 cm was 28.6% and 11.8%, respectively. OS was 31.0 months (mean 33.3 ± 15.2; range 8-69), notably for BCLC A 41 months (mean 41.1 ± 15.3; range 13-69) and for BCLC B 26.0 (mean 26.0 ± 10.5; range 8-51). OS at 1, 3 and 5 years: 95.8%, 75.7% and 21.4% for BCLC A, and 94.4%, 36.1% and 2.7% for BCLC B. Median LTTP for BCLC A was 11 months (mean 11.9 ± 4.7; range 3-24) and 7.5 for BCLC B (mean 7.9 ± 2.9). Local response was significant for OS and LTTP (p < 0.0001), while size and lesion number affected LPFS and OS (p < 0.001). CONCLUSIONS: HepaSphere 30-60 µm loaded with doxorubicin provides a safe and effective treatment option for patients with HCC.

Evaluation of alternative serum biomarkers to monitor the progression of chronic HBV and HCV infection.

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are among the most serious health conditions affecting about 600 million people worldwide leading to a number of severe liver diseases. Due to the lack of warning signs or mild symptoms during the early stage of the infection, a molecular signature associated with disease progression would be useful. Based on our recent paper where candidate biomarkers were determined through topological and modularity analysis of protein interaction networks (PINs), this study was focused on the evaluation of MIF, TNFRSF1A, FAS and TMSB4X as diagnostic biomarkers in chronic HBV and HCV infections. The aim was to establish a molecular profile, by combining those markers, that would discriminate the different stages during the progression of chronic hepatitis. One hundred and fifteen patients infected with HBV or HCV categorized into three groups: non-cirrhotic, cirrhotic and with HCC, and 20 healthy subjects were enrolled in this study. Serum levels of the aforementioned factors were measured by ELISA. TNFRSF1A serum levels appeared statistically significantly increased in all patient groups compared to control group with a p-value of <0.05. Furthermore, the combination of TNFRSF1A and TMSB4X serum levels successfully classified 63, 47% of patients indicating an association with HBV and HCV infections. Thus, variations of serum levels of TNFRSF1A and TMSB4X could be associated with the different stages of the disease and may be utilized for further research. On the other hand, we found no contribution of MIF and FAS serum levels for successful classification of patients.

Liver transplantation in Greek patients: epidemiological data, morbidity, and mortality of 71 patients from a single center with 6 years of mean follow-up.

Liver transplantation remains an underdeveloped technique in Greece; currently there is no information on outcomes in Greek patients. In this study, data were provided on the outcomes of liver transplantation in 71 patients with a mean follow-up of 6 (0.1 to 16) years in our center. Mean age at transplantation was 46 +/- 13 years, while the main cause for transplantation was hepatitis B (16 patients, 23%) or C (six patients, 8%) virus. In the first posttransplantation year, three patients died, while 18 (25%) required at least one hospitalization with a median stay of 30 days. At the end of follow-up, 56 patients (79%) are alive. The leading cause of death was de novo malignancies (40%), appearing at a mean of 5.2 +/- 3.3 years. Late adverse effects of immunosuppressive therapy included hypertension (42%), hyperlipidemia (24%), chronic renal failure (21%), and diabetes mellitus (24%). With the exception of diabetes, all the above abnormalities were significantly associated with cyclosporine-based but not with tacrolimus-based immunosuppressive regimens. Relapse of primary disease in liver transplants occurred in 21 (29.6%) patients at a mean time of 1.5 +/- 1.4 years, of whom 67% were related to viral hepatitis. The quality of life (Karnofsky scale 1 to 6) was excellent in 64% of surviving patients, affordable in 21%, and poor in 15%. In conclusion, after 6 mean years, the majority of Greek liver transplant recipients conduct a normal life, although metabolic abnormalities are often observed. A national registry is needed to provide more solid evidence of outcomes.

Overlapping syndrome of autoimmune hepatitis and primary sclerosing cholangitis associated with pyoderma gangrenosum and ulcerative colitis.

We describe the case of a 18-year-old male patient who first presented with decompensated cirrhosis, fever and generalized lymphadenopathy. He had abnormal results for liver biochemical tests, with a hepatitic-like picture and high titre of antinuclear antibodies. According to the scoring system proposed by the International Autoimmune Hepatitis Group he had 'definite' autoimmune hepatitis and responded well to immunosuppressive treatment. One year later he developed pyoderma gangrenosum which was successfully treated with cyclosporine. Two years later he experienced bloody diarrhoea as a first presentation of ulcerative colitis. At that time both the cholestatic biochemical picture and the cholangiographic appearances of the biliary tree were consistent with primary sclerosing cholangitis. Despite the addition of azathioprine and ursodeoxycholic acid to his treatment regime he developed recurrent episodes of cholangitis and intractable pruritus for which he underwent successful liver transplantation.

Combined hepatocellular-cholangiocarcinoma presented with massive pulmonary embolism.

A 30-year-old HBsAg-positive woman was admitted to the hospital because of 6 days of progressive shortness of breath. She was in severe respiratory distress with circulatory collapse. She had an enlarged liver but no stigmata of chronic liver disease or signs of cirrhosis. She had rapidly developed respiratory arrest and was transferred to intensive care unit. Heart ultrasonography and Doppler scan showed right heart straining and high pulmonary artery pressure. Despite cardiovascular and respiratory support she died a few hours after admission. Autopsy revealed combined hepatocellular-cholangiocarcinoma infiltrating the entire liver, metastatic invasion of lung blood vessels and absence of right ventricular hypertrophy. The incidence of hepatocellular-cholangiocarcinoma, a variant of hepatocellular carcinoma, is roughly 2-3% and the presenting symptoms are abdominal pain, weight loss, jaundice, fever or decompensation of liver disease. Associated HBsAg positivity and cirrhosis are reported in 20-30% and 60% of patients, respectively. Metastases to lungs are relatively frequent but this is the first report of hepatocellular-cholangiocarcinoma presented with acute respiratory distress due to massive pulmonary embolism.

CT portography and post-lipiodol CT in the preinterventional work-up of primary and secondary liver tumors. A single center experience.

BACKGROUND/AIMS: The evaluation of the clinical use of CT portography (CTp) and post-lipiodol CT (CT post-lip) in terms of therapeutic implications in patients with liver malignancies, particularly hepatocellular carcinoma (HCC). METHODOLOGY: We prospectively evaluated 130 patients with CTp and CT post-lip: 109 with HCC and underlying cirrhosis (group I) and 21 with liver metastases considered for surgical resection (group II). All patients also underwent hepatic angiography (hA). Mean lesion size was 4.6 cm and 2.2 cm for group I and II respectively. Previous contrast-enhanced CT studies were available for comparison. RESULTS: Diagnostic CTp examinations resulted in only 84.4% of group I due to enhancement in homogeneities and in all patients from group II. In comparison with the referral CT, additional lesions were seen in 83.6% of the HCC group and in 66.6% of the metastatic group that implicated treatment alterations in 15.21% and in 23.8% of them, respectively. Hepatic angiography revealed hypervascularity in 91.3% of HCC lesions and in 33.3% of metastatic ones. CT post-lip images suitable for evaluation resulted in 104/130 patients (80%). At CT post-lip false negative results were observed in 33.73% patients with HCC and in 30.95% patients with liver metastases. Selective lipiodol retention was seen in only 50% of the biopsy proved satellites. CONCLUSIONS: CTp reveals additional lesions that have therapeutic implications in at least 15% of HCC patients and in 20% of patients with metastatic disease, and should be routinely included in the preinterventional work-up particularly for cases in which intraarterial or percutaneous treatment is scheduled. By contrast, CT post-lip seems to be of limited value unless it is evaluated in combination with CTp and angiography.

Treatment of chronic active hepatitis B (CAH B) with chloroquine: a preliminary report.

Corticosteroids, azathioprine and antiviral agents have a questionable effect on CAH B. Chloroquine, a lysosomotropic agent, was used to treat 7 patients with histologically confirmed CAH B. All were HBeAb positive. A working hypothesis considering cellular death in CAH B as the result of lysosomal enzyme liberation by activated Kupffer cells was the basis for treatment. In this model T lymphocytes have only an immunoregulatory role. Clinical and laboratory follow-up was done for 6-16 months (median 12 months). Serum chloroquine levels were recorded by a fluorimetric method. 150-450 mg of chloroquine base were administered according to bio-chemical disease activity. In all patients AST and ALT values returned to normal and there was a fall in serum delta GT and improvement of prothrombin time. an increase of globulins was noted. Inadvertent drug withdrawal resulted in aminotransferase increase in 3 patients with prompt restoration of normal values on readministration. One patient refused to continue the drug and died after two months. Variceal bleeding was the cause of death of a second patient. No side effects were noted. A repeat liver biopsy, a year later (4 patients) revealed inactive cirrhosis in all. Chloroquine administration is a safe treatment for patients with CAH B. Further studies are justified.

The role of tenofovir in preventing and treating hepatitis B virus (HBV) reactivation in immunosuppressed patients. A real life experience from a tertiary center.

INTRODUCTION/AIM: Patients who present HBV reactivation during immunosuppressive treatment are prone to develop life threatening decompensation of the liver function, therefore prophylaxis and treatment are strongly recommended. So far there are no data regarding the role of tenofovir in this context. Therefore, the aim of our study was to describe our "real life" experience with the use of tenofovir (TDF) in patients who underwent immunosuppressive treatment. RESULTS: 38 patients with immunosuppression received antiviral treatment with tenofovir (25 patients as prophylaxis and 13 patients as treatment of HBV reactivation). In all 25 patients in whom prophylactic treatment with tenofovir was administered no HBV flare occurred during immunosuppression and the levels of serum HBV-DNA became or remained undetectable during the follow up period (mean follow up 17.2 months, range 6-54). One patient experienced HBsAg seroconversion. In the 13 patients who exhibited HBV reactivation TDF treatment resulted in complete biochemical and virological response within 6 months except two patients with high pretreatment HBV-DNA levels who became HBV-DNA negative at 9 months. No exacerbation of liver disease or liver related death has been observed. One patient who presented with decompensated cirrhosis during HBV reactivation returned into a compensated state after treatment. No side effects of tenofovir have been documented. CONCLUSION: Tenofovir seems to be highly effective and safe in the prophylaxis and rescue treatment of HBV reactivation in patients who receive immunosuppression therapy.

Severe hepatitis C virus-related cryoglobulinaemic sensory-motor polyneuropathy treated with pegylated interferon-a2b and ribavirin: clinical, laboratory and neurophysiological study.

BACKGROUND/AIMS: Severe involvement of central and/or peripheral nervous system is a rare complication of hepatitis C virus (HCV)-related cryoglobulinaemia. METHOD: Four patients with HCV-related type II/III cryoglobulinaemia (three males with genotype 1, one female with genotype 3) who presented with severe sensory-motor polyneuropathy, one with central nervous system involvement as well, were treated with pegylated IFNa-2b 1.5 microg/kg/week and ribavirin 10.6 mg/kg/daily for 48 weeks. Neurological evaluation involved detailed clinical motor and sensory scores/scales and neurophysiological studies before and after treatment. RESULTS/CONCLUSION: Three out of four patients had undetectable serum HCV-RNA, normal levels of aminotransferases and substantially lower or undetectable levels of cryoglobulins at the end of treatment and at 24 weeks follow-up period. Treatment was well tolerated and all patients exhibited significant improvement of neuropathy based on solid clinical and laboratory criteria that was associated with the virological response.

Hepatic expression of the proliferative marker Ki-67 and p53 protein in HBV or HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma.

To evaluate hepatic expression of the nuclear proliferative marker Ki-67 and the p53 oncoprotein in hepatitis B virus (HBV)/HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma (HCC). We studied needle liver biopsies from 107 patients with cirrhosis and no HCC (52 HBV, 55 HCV) who had been assessed for protocol studies, and 57 cirrhotic patients with HCC (40 HBV, 17 HCV). We evaluated small and large cell dysplastic changes along with the expression of Ki-67 and p53 by immunohistochemistry. The labelling index (LI) was defined as the proportion (%) of positive-stained nuclei of the 500 measured. Large and small cell dysplastic changes were observed in 12 and 9% of specimens respectively. Only small cell changes were associated with Ki-67 expression. Ki-67 LI was 5.50 +/- 5.7 in cirrhosis (13.90 +/- 3.84 in those with small cell dysplastic changes vs 4.64 +/- 4.98 in those without, P < 0.01), 10.2 +/- 5.95 in cirrhosis with HCC (P < 0.05) and 18.56 +/- 10 in HCC (P < 0.01). Neither the presence of small cell dysplastic changes nor the expression of Ki-67 was related to severity or aetiology of cirrhosis. Expression of p53 was observed in 30% of the non-tumorous and in 53% of the neoplastic tissue obtained from patients with HCC, with no differences between HCV and HBV. Ki-67 and p53 expression was associated with the tumour grade (P < 0.001). Our observations clearly demonstrate the association between the proliferation activity and the morphological changes in the cirrhotic liver from the non-dysplastic to dysplastic lesion to HCC. They also support the hypothesis that p53 alterations are a rather late event in carcinogenesis and related to HCC grade. And finally, they suggest that the final steps of hepatocarcinogenesis are common and independent of the aetiology of the chronic viral infection.