Beclin 1 mRNA strongly correlates with Bcl-XLmRNA expression in human hepatocellular carcinoma.

Beclin 1 physically associates with Bcl-x(L) and is considered as a haploinsufficient tumor suppressor. As the role of Beclin 1 in hepatocellular carcinoma (HCC) is unknown, we determined Beclin 1 mRNA expression in 27 pairs of tumoral/nontumoral (T/NT) liver samples. The Beclin 1 mRNA T/NT ratio was less than 0.5 in 2 tumors and more than 2 in 1 tumor, and was positively correlated with the Bcl-X(L) mRNA T/NT ratio (P < 0.001), but not with the proliferating cell nuclear antigen mRNA T/NT ratio. Coregulation of Beclin 1 and Bcl-X(L) expression in HCC may suggest cooperation in the regulation of apoptosis.

Validation and comparison of indexes for fibrosis and cirrhosis prediction in chronic hepatitis C patients: proposal for a pragmatic approach classification without liver biopsies.

Noninvasive indexes have been developed to predict fibrosis staging. The aim of this study was to assess the diagnostic accuracy of these indexes in comparison with liver histology in hepatitis C virus (HCV)-infected patients. A total of 235 consecutive patients with HCV infection from the Fibropaca multicentre independent study were included in this paper. FibroTest (FT), aspartate aminotransferase to platelet ratio index (APRI) and Forns score were assessed in the cohort and compared with liver histology performed on the same day. The main end point was the area under characteristic curves (AUCs) for the diagnosis of significant fibrosis (F2-F4) and cirrhosis (F4) by the METAVIR classification. Mean age was 46 (+/-11) years, 55% were males, 42% (n = 99) had significant fibrosis (F2-F4) and 7% (n = 16) had cirrhosis (F4). For the diagnosis of significant fibrosis, respective AUCs of FT, APRI and Forns score were 0.81 (95% confidence interval: 0.76-0.86), 0.71 (0.67-0.79) and 0.76 (0.70-0.82); for cirrhosis prognosis, AUCs of FT and APRI were 0.82 (0.77-0.87) and 0.81 (0.76-0.86) (AUCs not significantly different). Using each index independently, all patients were classified by FT, 214 (91%) patients were classified by APRI and 129 (55%) by Forns score. There were significantly more cases of discordances between APRI and liver biopsy than between FT or Forns score and liver biopsy (P < 0.05). Performing all scores (FT, Forns and APRI) without liver biopsy allowed fibrosis to be well evaluated in 191 patients (81.3%), including patients with FT failure. Liver biopsy remained mandatory to evaluate fibrosis in 44 patients (18.7%). Our study shows that performing all the tests and liver biopsy improves the diagnostic accuracy for liver fibrosis in chronic hepatitis C patients without patent comorbidities. The combination of all tests with liver biopsy allowed 225/235 (96%) patients to be correctly classified. The combination of all tests without liver biopsy allowed 191/235 (81.3%) patients to be correctly classified; liver biopsy remained mandatory in some patients (18.7%).

Human pancreatic mucinous cystadenoma is characterized by distinct mucin, cytokeratin and CD10 expression compared with intraductal papillary-mucinous adenoma.

AIMS: To examine cytokeratin, epithelial glycoprotein (mucin) and glycoprotein CD10 expression in benign mucinous cystdenomas (MCAs) in comparison with intraductal papillary mucinous adenomas (IPMAs). METHODS AND RESULTS: Thirty MCAs of the pancreas were analysed for immunohistochemical expression of cytokeratin (CK) 7, CK20, MUC1, MUC2, MUC5AC and CD10 and were compared with 16 IPMAs. CK7 was expressed in all neoplasms. CK20 was significantly more frequent in MCAs compared with IPMAs (56.66% versus 18.75%, P = 0.027). MUC1 was more frequent in MCAs (40% versus 12.5%, P = 0.0915), whereas MUC5AC was significantly less frequent in MCAs (33.33% versus 100%). MUC2 was expressed in goblet cells of seven MCAs. In MCAs, CD10 was observed both in epithelial cells and in the ovarian-type stromal cells (24/30). Epithelial expression of CD10 was significantly lower in IPMAs (66.66% versus 6.25%, p = 0.0001). CONCLUSIONS: MCA is characterized by a significantly greater frequency of expression of CK20 and CD10 when compared with IPMA, which preferentially expresses MUC5AC.

Interferon-alpha suppresses liver cell proliferation in patients with chronic hepatitis C virus infection.

SUMMARY: Interferon (IFN) therapy has been shown to reduce the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C, including virological nonresponders (NR). Whether IFN suppresses liver cell proliferation, i.e. the relevant risk factor for HCC, is unknown. The aim of the study was to evaluate the effect of IFN therapy on liver cell proliferation in chronic hepatitis C. The proliferating cell nuclear antigen-labelling index (PCNA-LI) was assessed prior to and at the end of therapy in the liver of 29 patients with chronic hepatitis C who received 3 MU IFN-alpha2b thrice weekly for 24-48 weeks. Overall, the median value of PCNA-LI was significantly reduced from 2.6% to 1.1% at the end of therapy (P < 0.0001). At baseline, PCNA-LI median values were similar in the 15 virological responders compared with the 14 NRs (2.3%vs 3.4%, P = 0.121) and at the end of therapy, median changes of PCNA-LI (-1.4%vs-1.1%, P = 0.089) were also similar although there was a higher decline of the proliferation index in responders with respect to NRs at the end of therapy (0.7%vs 1.6%, P = 0.004). In the two groups, the rate of fibrosis score reduction was also similar (7%vs 20%, P = 0.326). In contrast, the histological activity index was more often reduced in responders than in NRs both at the >or=2 and >or=4 points reduction level (80%vs 36%, P = 0.02 and 53%vs 14%, P = 0.03, respectively). The study showed a significant suppression of liver cell proliferation in IFN-treated patients with chronic hepatitis C. Although the strongest IFN effect was observed in virological responders, a reduction of proliferative activity was also seen in virological NRs.

Safety and efficacy of peginterferon plus ribavirin in patients with chronic hepatitis C and bridging fibrosis or cirrhosis.

The combination of pegylated interferon and ribavirin is the most effective therapy in patients with chronic hepatitis C. We evaluated this combination in unselected patients with bridging fibrosis or cirrhosis. Eighty patients were treated with peginterferon alpha-2b plus ribavirin. Hepatitis C virus serum RNA was monitored. Tolerance and safety were evaluated by the rate of treatment's discontinuation for any reason, and occurrence of serious clinical adverse events, respectively. Sustained virologic response (SVR) rate was 36.3% overall, and was observed in every group of patients except those who had previously failed to respond to the combination of interferon and ribavirin. No serious clinical adverse event occurred. Treatment was withdrawn in 18.7% of patients. Variables associated with discontinuation of treatment were low prothrombin index [OR: 1.16 (1.05;1.27)] and low body mass index [OR: 1.47 (1.12;1.92)]. Initial blood count abnormalities were not associated with cessation of treatment. Furthermore, early virologic response at week 8 and week 12 of treatment had similar predictive value for SVR. Combination therapy with peginterferon plus ribavirin seems effective in this group of patients, except in those who had previously failed to respond to the combination of interferon and ribavirin. This therapy is safe with appropriate monitoring, but tolerance seems worse in patients with the most advanced liver disease.

Expression of hypoxia-inducible factors is correlated with the presence of a fibrotic focus and angiogenesis in pancreatic ductal adenocarcinomas.

AIMS: To study the expression of hypoxia-regulated markers in pancreatic ductal adenocarcinomas (PA) in relationship to the presence of a fibrotic focus, angiogenesis quantification and clinical outcome. METHODS AND RESULTS: The expression of hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) was immunohistochemically detected in 50 PA and correlated with tumour characteristics, microvascular density (MVD) and survival. HIF-1alpha was expressed within tumour cells in 68%, HIF-2alpha in 46%, CA9 in 78% and VEGF in 52% of the cases. Stromal expression was also noted for HIF-2alpha and CA9 in, respectively, 42% and 48% of the cases. Tumour CA9 expression was associated with that of VEGF (P=0.004) and that of stromal HIF-2alpha (P=0.013), with the presence of a fibrotic focus (P=0.046) and with an increased MVD (P=0.034). Tumour VEGF expression correlated with the presence of a fibrotic focus (P=0.039) and a greater MVD (P=0.047). Both the presence of a fibrotic focus (P=0.0002) and high tumour CA9 expression (P=0.029) were associated with reduced overall survival. CONCLUSION: The strong association of the presence of a fibrotic focus with CA9 expression and lower survival demonstrates that hypoxia-driven angiogenesis plays an important role in the progression of PA.

In vitro chronic hepatic disease characterization with a multiparametric ultrasonic approach.

Although, high resolution, real-time ultrasonic (US) imaging is routinely available, image interpretation is based on grey-level and texture and quantitative evaluation is limited. Other potentially useful diagnostic information from US echoes may include modifications in tissue acoustic parameters (speed, attenuation and backscattering) resulting from disease development. Changes in acoustical parameters can be detected using time-of-flight and spectral analysis techniques. The objective of this study is to explore the potential of three parameters together (attenuation coefficient, US speed and integrated backscatter coefficient-IBC) to discriminate healthy and fibrosis subgroups in liver tissue. Echoes from 21 fresh in vitro samples of human liver and from a plane reflector were obtained using a 20-MHz central frequency transducer (6-30 MHz bandpass). The scan plane was parallel to the reflector placed beneath the liver. A 30 x 20 matrix of A-scans was obtained, with a 200-microm step. The samples were classified according to the Metavir scale in five different degrees of fibrosis. US speed, attenuation and IBC were estimated from standard methods described in the literature. Statistical tests were applied to the results of each parameter individually and indicated that it was not possible to identify all the fibrosis groups. Then a discriminant analysis was performed for the three parameters together resulting in a reasonable separation of fibrotic groups. Although the number of tissue samples is limited, this study opens the possibility of enhancing the discriminant capability of ultrasonic parameters of liver tissue disease when they are combined together.

Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression.

Tumour-associated angiogenesis is partly regulated by the hypoxia-inducible factor (HIF) pathway. Endocrine tumours are highly vascularised and the molecular mechanisms of their angiogenesis are not fully delineated. The aim of this study is to evaluate angiogenesis and expression of HIF-related molecules in a series of patients with pancreatic endocrine tumours (PETs). The expression of vascular endothelial growth factor (VEGF), HIF-1alpha, HIF-2alpha and carbonic anhydrase 9 (CA9) was examined by immunohistochemistry in 45 patients with PETs and compared to microvascular density (MVD), endothelial proliferation, tumour stage and survival. Microvascular density was very high in PETs and associated with a low endothelial index of proliferation. Microvascular density was significantly higher in benign PETs than in PETs of uncertain prognosis, well-differentiated and poorly differentiated carcinomas (mean values: 535, 436, 252 and 45 vessels mm(-2), respectively, P < 0.0001). Well-differentiated tumours had high cytoplasmic VEGF and HIF-1alpha expression. Poorly differentiated carcinomas were associated with nuclear HIF-1alpha and membranous CA9 expression. Low MVD (P = 0.0001) and membranous CA9 expression (P = 0.0004) were associated with a poorer survival. Contrary to other types of cancer, PETs are highly vascularised, but poorly angiogenic tumours. As they progress, VEGF expression is lost and MVD significantly decreases. The regulation of HIF signalling appears to be specific in pancreatic endocrine tumours.

Angiogenesis and extracellular matrix remodelling in bronchioloalveolar carcinomas: distinctive patterns in mucinous and non-mucinous tumours.

AIM: Bronchioloalveolar carcinomas (BACs) are rare primitive lung adenocarcinomas growing along the alveolar septum without stromal, vascular or pleural invasion. We report an immunohistochemical study of their vascular microenvironment. METHODS AND RESULTS: In three mucinous BACs (M-BAC) and three non-mucinous BACs (NM-BAC) we examined the following parameters in comparison with the normal lung: (i) constituents of the alveolar extracellular matrix; (ii) qualitative and quantitative changes of alveolar capillaries; and (iii) expression of vascular endothelial growth factor (VEGF) by tumour cells. In M-BAC, the alveolar matrix was unchanged compared with the normal parenchyma. Capillaries expressed normal alveolar endothelial markers and their average surface was calculated, as in normal lung, as 8%. VEGF was negative in tumour cells. In NM-BAC, the alveolar wall was thickened by deposits of fibronectin and type III collagen containing myofibroblasts and the basement membrane was disrupted. Capillaries did not retain alveolar endothelial markers and their surface was calculated as 19%. Tumour cells expressed high levels of VEGF. CONCLUSIONS: In contrast to NM-BAC, M-BAC do not modify the alveolar structure and seem to exploit the normal alveolar vascular bed to grow, without inducing neoangiogenesis. A better understanding of the mechanisms of growth of lung cancers may have implications for future anti-angiogenic therapeutic strategies.

Liver fibrosis is not associated with steatosis but with necroinflammation in French patients with chronic hepatitis C.

BACKGROUND: In chronic hepatitis C, it has been suggested that steatosis could accelerate progression of fibrosis. However, results of the few published studies are controversial. AIM: To determine the characteristics (epidemiological, biological, and histological) associated with steatosis and its relationship with liver lesions (grade of necroinflammation and stage of fibrosis) in patients with chronic hepatitis C. METHODS: From November 2000 to July 2001, untreated consecutive adults with chronic hepatitis C admitted for liver biopsy were included in this study. On the day of liver biopsy, a questionnaire for risk factors was completed prospectively, and a blood sample was obtained for laboratory analysis. RESULTS: Our study included 290 patients (143 men, 147 women). Mean body mass index (BMI) was 24 (3.8) kg/m(2). Proportions of patients with genotypes 1 and 3 were, respectively, 48% and 18%. A total of 135 patients (46.6%) had steatosis. Liver steatosis, in multivariate analysis, was associated with hepatitis C virus genotype 3, higher grade of necroinflammation, and higher BMI. There was no significant association between stage of fibrosis and liver steatosis. In multivariate analysis, high stage of fibrosis was associated with male sex, age over 50 years, high BMI, and high grade of necroinflammation. CONCLUSION: In our population of patients with chronic hepatitis C, steatosis does not seem to be an important determinant of liver fibrosis. High grade of necroinflammation is associated with a high stage of fibrosis.

Idiopathic non-cirrhotic intrahepatic portal hypertension in the West: a re-evaluation in 28 patients.

BACKGROUND: Non-cirrhotic portal hypertension of unknown cause is a poorly understood condition attributed to obstructive portal venopathy. AIM: To reassess the manifestations, course, and causes, with special attention to thrombosis. METHODS: Analysis of a cohort of 28 patients. RESULTS: Gastrointestinal bleeding occurred in 11 patients. Liver failure developed at the time of concurrent disease in eight patients, including all four patients who died. Portal vein thrombosis developed in 13 patients. A prothrombotic disorder was found in 12 of 23 fully investigated patients. Hepatoportal sclerosis was observed in 11 patients (with associated perisinusoidal fibrosis and/or nodular regenerative hyperplasia in six); periportal fibrosis, perisinusoidal fibrosis, nodular regenerative hyperplasia, or a combination thereof were observed in other patients. A morphometric evaluation showed an increased number of portal vessels in patients with hepatoportal sclerosis. There was no relation between pathological results and haemodynamic findings or prothrombotic disorders. CONCLUSIONS: Outcome was related to associated conditions. Overlap in pathological, haemodynamic, and causal features suggests a single entity, with prothrombotic disorders as major causal factors, and injury to sinusoids as well as to portal venules as the primary mechanism. Activated coagulation could mediate vascular injury in the absence of thrombosis. Anticoagulation should be considered.

Hypervariable region 1 quasispecies in hepatitis C virus genotypes 1b and 3 infected patients with normal and abnormal alanine aminotransferase levels.

The role of hepatitis C virus (HCV) heterogeneity in the severity of chronic hepatitis C infection remains unclear. Our aim was to study the hypervariable region 1 (HVR1) heterogeneity in patients with chronic hepatitis C infected with genotype 1b or 3 and with normal or abnormal alanine aminotransferase (ALT). HVR1 quasispecies were assessed by single strand conformational polymorphism (SSCP) in 67 patients with chronic hepatitis C, including 35 with persistently normal ALT and 32 with abnormal ALT. Sixty-two patients underwent a liver biopsy. Among the 67 patients, 40 were infected with genotype 1b and 27 with genotype 3. In univariate analysis, low heterogeneity (

Integrin up-regulation in chronic liver disease: relationship with inflammation and fibrosis in chronic hepatitis C.

In 94 patients with chronic hepatitis C, the pattern of integrin expression was correlated with firstly, the histological activity index, necro-inflammatory grade, and stage of fibrosis; secondly, the expression of inflammatory markers including ICAM-1; and thirdly, the extent and intensity of laminin deposition in the perisinusoidal matrix. Immunohistochemical results were evaluated according to a semi-quantitative scoring system or by image analysis. Increased beta1 expression was observed in 88.2% of cases. The expression of alpha1 and alpha5 was increased in 55% and 58.5% of cases, respectively. alpha6 chain was detected in 78.7% of cases. There were no statistically significant differences in integrin expression level according to Knodell's score, inflammatory grade, or stage of fibrosis. ICAM-1 expression was higher in patients with high scores for beta1 expression, but the differences were not statistically significant. There were significantly more patients with high scores for beta1 expression among those with continuous perisinusoidal deposition of laminin. Moreover, a close statistical correlation was observed between alpha6 induction and perisinusoidal laminin deposition (p<0.001). The results suggest that integrin up-regulation in chronic hepatitis C is more closely related to the fibrotic process than to the inflammatory lesions. This reinforces the idea that integrin induction in chronic liver disease is part of a coordinated process involved in the progression of liver fibrosis.

The influence of human immunodeficiency virus coinfection on chronic hepatitis C in injection drug users: a long-term retrospective cohort study.

In this study we analyzed the influence of human immunodeficiency virus (HIV) infection on the course of chronic hepatitis C through multivariate analysis including age, alcohol consumption, immune status, and hepatitis C virus (HCV)-related virologic factors. Eighty HIV-positive and 80 HIV-negative injection drug users included between 1980 and 1995 were matched according to age, gender, and duration of HCV infection and followed-up during 52 months. The progression to cirrhosis was the primary outcome measure. The impact of HIV on HCV-RNA load, histologic activity index, response to interferon therapy, and liver-related death was also considered. In HIV-positive patients, chronic hepatitis C was characterized by higher serum HCV-RNA levels (P =.012), higher total Knodell score (P =.011), and poorer sustained response to interferon therapy (P =.009). High serum HCV-RNA level was associated with low CD4-lymphocyte count (P =.001). Necroinflamatory score was higher in HIV-positive patients (P =.023) independently of the CD4-lymphocyte count, whereas increased fibrosis was related to decreased CD4-lymphocyte count (P =.011). The progression to cirrhosis was accelerated in HIV-positive patients with low CD4 cell count (RR = 4.06, P =.024) and in interferon-untreated patients (RR = 4.76, P =.001), independently of age at HCV infection (P =.001). Cirrhosis caused death in 5 HIV-positive patients. The risk of death related to cirrhosis was increased in heavy drinkers (RR = 10.8, P =.001) and in HIV-positive patients with CD4 cell count less than 200/mm(3) (RR = 11.9, P =.007). In this retrospective cohort study, HIV coinfection worsened the outcome of chronic hepatitis C, increasing both serum HCV-RNA level and liver damage and decreasing sustained response to interferon therapy. Age and alcohol were cofactors associated with cirrhosis and mortality. Interferon therapy had a protective effect against HCV-related cirrhosis no matter what the patient's HIV status was.

Primary liver carcinoma in genetic hemochromatosis reveals a broad histologic spectrum.

Hepatocellular carcinoma (HCC) is a well-known complication of genetic hemochromatosis (GH). However, the frequency of primary liver carcinoma (PLC) with biliary differentiation, such as cholangiocarcinoma (CC) and combined hepatocholangiocarcinoma (CHCC), in GH remains unclear We analyzed the histologic type of 20 PLCs occurring in the background of GH; all patients were homozygotic for the C282Y mutation. Ten were depleted of iron by successive phlebotomies, while the remaining 10 were untreated. Histologically, 13 cases were classified as HCC, 3 as CC, and 4 as CHCC. Immunohistochemical detection of Hep Par 1, cytokeratin 19 (CK19), and MUC1 supported this classification; PLC with biliary differentiation was immunoreactive for MUC1 in 86% (6/7) of cases and for CK19 in 100% (7/7) of cases. The nontumoral liver exhibited no cirrhosis or extensive fibrosis in 6 cases. Von Meyenburg complexes were present in 11 cases and intraparenchymal bile duct adenomas in 3. These data suggest that PLCs in patients with GH present a wide histologic spectrum, with tumors showing frequent biliary differentiation; may arise on a nonfibrotic or a cirrhotic liver; and often are associated with Von Meyenburg complexes and to a lesser extent with bile duct adenomas.

Quantitation of hepatic hepatitis C virus RNA in patients with chronic hepatitis C. Relationship with severity of disease, viral genotype and response to treatment.

BACKGROUND/AIMS: To determine the correlation between hepatic hepatitis C virus (HCV) RNA and histological lesions, viral genotype or response to alpha interferon therapy. METHODS: Forty-three patients with chronic hepatitis C (14 sustained responders (SR) and 29 non-sustained responders (NSR)) were studied. A liver tissue sample was obtained before and 1 year after treatment. Quantitation of hepatic HCV-RNA was performed by competitive PCR. RESULTS: Before treatment, HCV-RNA was detectable in all liver samples. There was no association between hepatic HCV-RNA and the severity of liver lesions. There was a significant association between old age and hepatic HCV-RNA (P = 0.03). There was an association, at the limit of significance, between genotype 1 and high hepatic HCV-RNA amounts (15 x 106 and 4.1 x 10(6) copies/g, P = 0.05). Pre-treatment hepatic HCV-RNA amounts were lower in SRs than in others (0.65 x 10(6) and 13.2 x 10(6) copies/g, P = 0.0002). After treatment, no liver HCV-RNA was detectable in the SRs while in the NSRs, the HCV-RNA amounts were unchanged. CONCLUSIONS: The amount of hepatic HCV-RNA is correlated to genotype and response to interferon therapy but not to histologic lesions. Hepatic HCV-RNA clearance is observed in SRs, suggesting viral eradication.