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Liver fibrosis in adult thalassemia patients assessed by transient elastography
A6
Abstracts / Digestive and Liver Disease 40 (2008) A1–A40
ALT (p = 0.005), and diabetes (p = 0.01) were associated with fibrosis. In conclusion our data indicate that also patients with normal ALT should undergo liver biopsy particularly in the presence of diabetes, for an early diagnosis of fibrosis and prevention of progression of diseases.
pattern of disease, and for more selective use of liver biopsy before considering antiviral therapy. doi:10.1016/j.dld.2007.12.032
doi:10.1016/j.dld.2007.12.031
LIVER FIBROSIS IN ADULT THALASSEMIA PATIENTS ASSESSED BY TRANSIENT ELASTOGRAPHY
TRANSIENT ELASTOGRAPHY SEPARATES INACTIVE HBV CARRIERS FROM HBEAG MINUS DISEASE
M. Fraquelli a , E. Cassinerio b , E. Volpato b , C. Rigamonti c , M.D. Cappellini b , D. Conte a , M. Colombo c
S. Maimone a , V. Calvaruso a , M. Pleguezuelo a , G. Squadrito b , G. Raimondo b , G.M. Amaddeo b , G. a Dusheiko a
Centre for Hepatology, Royal Free and University College School of Medicine, London, United Kingdom b Clinical and Molecular Hepatology, Messina University, Italy Background and aim. Transient elastography (TE) is a novel, non-invasive technique of evaluating hepatic fibrosis. Few data are available in chronic hepatitis B. Liver biopsy is frequently required in HBeAg-negative disease to determine the stage of fibrosis. However, although most patients with inactive hepatitis B do not have severe fibrosis, it can be difficult to distinguish cohorts with negative HBeAg who may have varying degrees of fibrosis due to later seroconversion or intermittent fluctuations in disease. Patients and Methods. Two hundred and twenty HBsAg positive individuals were consecutively enrolled between January and October 2007. One hundred twenty-five of them (Group A) had an inactive HBsAg carrier state (as defined by longitudinally assessed negative HBeAg, positive anti-HBe, persistently normal aminotransferase (AT) levels, HBV DNA below 105 copies/ml) and 95 (Group B) were HBeAg negative, anti-HBe positive patients with persistently or intermittent elevation of AT and/or HBV DNA above 105 copies/ml. Liver biopsy was available in 39 Group B patients: 10 of whom had no fibrosis, 15 had mild fibrosis, 5 had moderate fibrosis, 3 severe and 3 had histological cirrhosis. Statistical analysis have been done by Student’s t-test; χ2 -test; binary logistic regression multivariate analysis. Results. Mean stiffness was 4.8 ± 1.2 kPa in the Group A vs. 8.5 ± 6 kPa in Group B (p < 0.001); we found also statistically significant differences between AST/ratio ALT/ratio, HBV DNA in A vs. B respectively (p < 0.001). In the multivariate analysis the only variable independently associated with the stage of fibrosis was the Stiffness (OR 1.5; 95% CI 1.02–2.2; p = 0.038). Conclusion. This study shows that mean hepatic stiffness determined by transient elastography is significantly lower in patients with inactive hepatitis B compared to those with HBeAg negative disease. For individual patients, the procedure is a useful adjunct to diagnosis, to offset the limitations of AT and HBV DNA measurements, to confirm a clinical
a
I Division of Gastroenterology, Policlinico Foundation IRCCS, University of Milan, Italy b Hereditary Anemia Center, Dept Internal Medicine, Policlinico Foundation IRCCS, University of Milan, Italy c II Division of Gastroenterology, Policlinico Foundation IRCCS, University of Milan, Italy Background and aim. Transient elastography (TE) is a non-invasive and reproducible technique that measures liver stiffness (LSM). It has been demonstrated to be a reliable tool for staging hepatic fibrosis in patients with chronic liver disease (CLD). However, its role in patients with -thalassemia has not been extensively investigated. Methods. From January to September 2007, 71 consecutive adults patients with -thalassemia referred to a single Italian Thalassemia Care Center in Italy were enrolled in the study. Twenty-nine patients (Group I: 7 M and 23 F, aged 31 ± S.D. 7.1 years) had -thalassemia major and 42 intermediate (Group II: 18 M and 24 F; aged 39 ± SD 12 years). Sixteen patients (55%) in Group I and nine (21%) in Group II were HCV–RNA positive. All patients were examined by TE (FibroScan® ; Echosens, Paris, France) and only the examinations with at least 10 validated measurements and a success rate of at least 60% were considered adequate. According to a previous study in CLD patients the considered TE cut off for diagnosing different stages of hepatic fibrosis were: >7.9 kPa for F ≥ 2; >10.3 for ≥F3 and >11.9 for F = 4. Twelve patients (all in Group I) also had undergone liver biopsy. Necroinflammation and fibrosis were scored by METAVIR classification; liver iron concentration (LIC, mg/g of liver dry weight) was measured on fresh tissue cores by atomic absorption spectrometry. Twenty-five patients underwent liver iron determination by T2* magnetic resonance imaging (MRI) assessment. Results. In patients who underwent liver biopsy, LSM increased proportionally to the METAVIR stage and a significant positive correlation was observed between LSM and fibrosis stage (r = 0.57, p = 0.039). Patients in Group I had significantly higher mean LSM values (10.6 ± SD 9.3 kPa) and serum ferritin (SF) (1367 ± SD 1169 ng/ml) than those in Group II (6.4 ± SD 3.3 kPa and 778 ± SD 693 ng/ml, respectively) (p < 0.01). In Group I a significant positive correlation was observed between LSM and SF (r = 0.47, p = 0.008), ALT (r = 0.87, p = 0.0001) and GGT levels (r = 0.80, p = 0.0001). In Group II LSM values were
Abstracts / Digestive and Liver Disease 40 (2008) A1–A40
correlated with iron overload measured by T2* MRI (r = 0.61, p = 0.001) and GGT levels (r = 0.51; p = 0.001). Among patients with -thalassemia major LSM values did not differ significantly between HCV–RNA positive (13.0 ± SD 12.5 kPa) and negative patients (7.7 ± SD 3.3 kPa) (p = NS) as well as the proportion of patients with significant (F ≥ 2) or severe fibrosis (F ≥ 3) did not differ significantly according to HCV viremic status. Conclusion. TE is a reliable non-invasive technique to stage liver fibrosis in patients with -thalassemia major. In these patients with concomitant HCV infection a significant or severe fibrosis was observed in about one third of the cases. Apart from fibrosis also serum necroinflammatory activity, GGT levels and SF levels may influence LSM values. Interestingly, we observed a significant positive correlation in patients with -thalassemia intermediate between T2* MRI values and the degree of hepatic fibrosis assessed by LSM. doi:10.1016/j.dld.2007.12.033 PHES STANDARDIZATION IN ITALY AND ITS COMPARISON TO EEG FINDINGS F. Campagna a , S. Olianas b , D. Mapelli b , P. Iannizzi a , M. Penzo a , P. Angeli a , A. Gatta a , P. Amodio a a
Dpt. of Clinical and Experimental Medicine, Italy b Dpt. of General Psychology, Cirmanmec, University of Padua, Italy The Psychometric Hepatic Encephalopathy Score (PHES) is used to detect minimal hepatic encephalopathy. This battery was standardized only for German and Spanish populations and, thus, needs to be standardized also for Italian population. This study aimed at standardizing the PHES in the Italian population and comparing Italian PHES standardization with the German and Spanish ones. The standardization sample for the PHES battery comprised 228 healthy subjects (108 males, 120 female). The PHES was also administered to 100 cirrhotic patients (71 males, 29 females) without diseases influencing mental function that underwent EEG spectral analysis to compare the Italian and the German and Spanish standardizations. The time and the errors of the line tracing test of the PHES (LTT) were found to be related, therefore error-weighted time was used to express LTT performance. The main predictor for all tests was age. Education level and working activity were significant predictors at univariate analysis, in contrast gender did not have any role. Multivariate analysis showed that age and education level were significant predictors; therefore, age–education adjusted z scores for each test were calculated and rounded to integers, as usual [1]. In the cirrhotic patients PHES battery showed 10% subjects with low PHES scores. The German and the Spanish standardizations provided higher prevalence of low values than the Italian one (32% and 17% vs. 10%, respectively). PHES and electroencephalographic spectral analysis showed low agreement: 31% of patients were positive to EEG spectral analysis
A7
and, of these, only 2% had PHES’ alterations. At any rate, the Italian standardization was correlated to MDF more than the German and the Spanish ones (r = 0.38, r = 0.31, r = 0.33, respectively, p < 0.01 for all of them). In conclusion, (i) the evaluation of LTT in the PHES battery deserves caution, (ii) PHES battery require adequate standardization in each country before its use, (iii) EEG and PHES findings are only roughly related each other. Reference [1] Romero-Gomez M. Hepatology 2007;45:879–85.
doi:10.1016/j.dld.2007.12.034 REDOX-SENSITIVE GSK-3 INHIBITION AND LATE VEGF RELEASE MEDIATE HYPOXIADEPENDENT EPITHELIAL–MESENCHYMAL TRANSITION AND INCREASED INVASIVENESS IN HUMAN HEPG2 CELLS S. Cannito, E. Novo, A. Compagnone, L. Valfr`e di Bonzo, C. Busletta, E. Zamara, A. Bandino, C. Cravanzola, V. Bravoco, S. Colombatto, M. Parola Dip. Medicina e Oncologia Sperimentale, University of Turin, Turin, Italy Background/aims. Epithelial–mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many tumour cells. In this study poorly invasive HepG2 cells and other cell lines of epithelial origin of the GI tract (Panc1, HT-29) have been exposed to carefully controlled hypoxic conditions (3% O2 ) in order to investigate whether hypoxia “per se” may trigger EMT program and related signalling mechanisms. Methods. EMT, signal transduction pathways, MMP-2 and MMP-9, chemotaxis and invasion were analysed by morphological, cell and molecular biology techniques, gelatine zimography and migration/invasion assays, respectively. Results. Hypoxia induces EMT changes in all neoplastic cells within 72 h, including nuclear translocation of SNAIL, Ecadherin disassembly, expression of N-cadherin, vimentin, ␣-SMA, MMP-2 and MMP-9. Moreover, all cancer cell lines responded to hypoxia by increased cell migration and Matrigel invasion, without significant changes in viability. Hypoxic medium collected after 24 to 48 hrs was found by itself to increase migration and invasiveness of normoxic cells in a VEGF-sensitive way. However, phosphorylation and inactivation of GSK3 was detected long before nuclear translocation of SNAIL and -catenin, suggesting a very early, hypoxia – dependent, involvement of Wnt/-catenin signalling machinery and EMT was indeed reproduced in normoxia by either recombinant Wnt-3A or LiCl. Early inhibition of GSK3 in hypoxic cells was found to be independent on VEGF and mostly related to intracellular generation of reactive oxygen species (ROS) by NADPH-oxidase and mitochondria.
Abstracts / Digestive and Liver Disease 40 (2008) A1–A40
ALT (p = 0.005), and diabetes (p = 0.01) were associated with fibrosis. In conclusion our data indicate that also patients with normal ALT should undergo liver biopsy particularly in the presence of diabetes, for an early diagnosis of fibrosis and prevention of progression of diseases.
pattern of disease, and for more selective use of liver biopsy before considering antiviral therapy. doi:10.1016/j.dld.2007.12.032
doi:10.1016/j.dld.2007.12.031
LIVER FIBROSIS IN ADULT THALASSEMIA PATIENTS ASSESSED BY TRANSIENT ELASTOGRAPHY
TRANSIENT ELASTOGRAPHY SEPARATES INACTIVE HBV CARRIERS FROM HBEAG MINUS DISEASE
M. Fraquelli a , E. Cassinerio b , E. Volpato b , C. Rigamonti c , M.D. Cappellini b , D. Conte a , M. Colombo c
S. Maimone a , V. Calvaruso a , M. Pleguezuelo a , G. Squadrito b , G. Raimondo b , G.M. Amaddeo b , G. a Dusheiko a
Centre for Hepatology, Royal Free and University College School of Medicine, London, United Kingdom b Clinical and Molecular Hepatology, Messina University, Italy Background and aim. Transient elastography (TE) is a novel, non-invasive technique of evaluating hepatic fibrosis. Few data are available in chronic hepatitis B. Liver biopsy is frequently required in HBeAg-negative disease to determine the stage of fibrosis. However, although most patients with inactive hepatitis B do not have severe fibrosis, it can be difficult to distinguish cohorts with negative HBeAg who may have varying degrees of fibrosis due to later seroconversion or intermittent fluctuations in disease. Patients and Methods. Two hundred and twenty HBsAg positive individuals were consecutively enrolled between January and October 2007. One hundred twenty-five of them (Group A) had an inactive HBsAg carrier state (as defined by longitudinally assessed negative HBeAg, positive anti-HBe, persistently normal aminotransferase (AT) levels, HBV DNA below 105 copies/ml) and 95 (Group B) were HBeAg negative, anti-HBe positive patients with persistently or intermittent elevation of AT and/or HBV DNA above 105 copies/ml. Liver biopsy was available in 39 Group B patients: 10 of whom had no fibrosis, 15 had mild fibrosis, 5 had moderate fibrosis, 3 severe and 3 had histological cirrhosis. Statistical analysis have been done by Student’s t-test; χ2 -test; binary logistic regression multivariate analysis. Results. Mean stiffness was 4.8 ± 1.2 kPa in the Group A vs. 8.5 ± 6 kPa in Group B (p < 0.001); we found also statistically significant differences between AST/ratio ALT/ratio, HBV DNA in A vs. B respectively (p < 0.001). In the multivariate analysis the only variable independently associated with the stage of fibrosis was the Stiffness (OR 1.5; 95% CI 1.02–2.2; p = 0.038). Conclusion. This study shows that mean hepatic stiffness determined by transient elastography is significantly lower in patients with inactive hepatitis B compared to those with HBeAg negative disease. For individual patients, the procedure is a useful adjunct to diagnosis, to offset the limitations of AT and HBV DNA measurements, to confirm a clinical
a
I Division of Gastroenterology, Policlinico Foundation IRCCS, University of Milan, Italy b Hereditary Anemia Center, Dept Internal Medicine, Policlinico Foundation IRCCS, University of Milan, Italy c II Division of Gastroenterology, Policlinico Foundation IRCCS, University of Milan, Italy Background and aim. Transient elastography (TE) is a non-invasive and reproducible technique that measures liver stiffness (LSM). It has been demonstrated to be a reliable tool for staging hepatic fibrosis in patients with chronic liver disease (CLD). However, its role in patients with -thalassemia has not been extensively investigated. Methods. From January to September 2007, 71 consecutive adults patients with -thalassemia referred to a single Italian Thalassemia Care Center in Italy were enrolled in the study. Twenty-nine patients (Group I: 7 M and 23 F, aged 31 ± S.D. 7.1 years) had -thalassemia major and 42 intermediate (Group II: 18 M and 24 F; aged 39 ± SD 12 years). Sixteen patients (55%) in Group I and nine (21%) in Group II were HCV–RNA positive. All patients were examined by TE (FibroScan® ; Echosens, Paris, France) and only the examinations with at least 10 validated measurements and a success rate of at least 60% were considered adequate. According to a previous study in CLD patients the considered TE cut off for diagnosing different stages of hepatic fibrosis were: >7.9 kPa for F ≥ 2; >10.3 for ≥F3 and >11.9 for F = 4. Twelve patients (all in Group I) also had undergone liver biopsy. Necroinflammation and fibrosis were scored by METAVIR classification; liver iron concentration (LIC, mg/g of liver dry weight) was measured on fresh tissue cores by atomic absorption spectrometry. Twenty-five patients underwent liver iron determination by T2* magnetic resonance imaging (MRI) assessment. Results. In patients who underwent liver biopsy, LSM increased proportionally to the METAVIR stage and a significant positive correlation was observed between LSM and fibrosis stage (r = 0.57, p = 0.039). Patients in Group I had significantly higher mean LSM values (10.6 ± SD 9.3 kPa) and serum ferritin (SF) (1367 ± SD 1169 ng/ml) than those in Group II (6.4 ± SD 3.3 kPa and 778 ± SD 693 ng/ml, respectively) (p < 0.01). In Group I a significant positive correlation was observed between LSM and SF (r = 0.47, p = 0.008), ALT (r = 0.87, p = 0.0001) and GGT levels (r = 0.80, p = 0.0001). In Group II LSM values were
Abstracts / Digestive and Liver Disease 40 (2008) A1–A40
correlated with iron overload measured by T2* MRI (r = 0.61, p = 0.001) and GGT levels (r = 0.51; p = 0.001). Among patients with -thalassemia major LSM values did not differ significantly between HCV–RNA positive (13.0 ± SD 12.5 kPa) and negative patients (7.7 ± SD 3.3 kPa) (p = NS) as well as the proportion of patients with significant (F ≥ 2) or severe fibrosis (F ≥ 3) did not differ significantly according to HCV viremic status. Conclusion. TE is a reliable non-invasive technique to stage liver fibrosis in patients with -thalassemia major. In these patients with concomitant HCV infection a significant or severe fibrosis was observed in about one third of the cases. Apart from fibrosis also serum necroinflammatory activity, GGT levels and SF levels may influence LSM values. Interestingly, we observed a significant positive correlation in patients with -thalassemia intermediate between T2* MRI values and the degree of hepatic fibrosis assessed by LSM. doi:10.1016/j.dld.2007.12.033 PHES STANDARDIZATION IN ITALY AND ITS COMPARISON TO EEG FINDINGS F. Campagna a , S. Olianas b , D. Mapelli b , P. Iannizzi a , M. Penzo a , P. Angeli a , A. Gatta a , P. Amodio a a
Dpt. of Clinical and Experimental Medicine, Italy b Dpt. of General Psychology, Cirmanmec, University of Padua, Italy The Psychometric Hepatic Encephalopathy Score (PHES) is used to detect minimal hepatic encephalopathy. This battery was standardized only for German and Spanish populations and, thus, needs to be standardized also for Italian population. This study aimed at standardizing the PHES in the Italian population and comparing Italian PHES standardization with the German and Spanish ones. The standardization sample for the PHES battery comprised 228 healthy subjects (108 males, 120 female). The PHES was also administered to 100 cirrhotic patients (71 males, 29 females) without diseases influencing mental function that underwent EEG spectral analysis to compare the Italian and the German and Spanish standardizations. The time and the errors of the line tracing test of the PHES (LTT) were found to be related, therefore error-weighted time was used to express LTT performance. The main predictor for all tests was age. Education level and working activity were significant predictors at univariate analysis, in contrast gender did not have any role. Multivariate analysis showed that age and education level were significant predictors; therefore, age–education adjusted z scores for each test were calculated and rounded to integers, as usual [1]. In the cirrhotic patients PHES battery showed 10% subjects with low PHES scores. The German and the Spanish standardizations provided higher prevalence of low values than the Italian one (32% and 17% vs. 10%, respectively). PHES and electroencephalographic spectral analysis showed low agreement: 31% of patients were positive to EEG spectral analysis
A7
and, of these, only 2% had PHES’ alterations. At any rate, the Italian standardization was correlated to MDF more than the German and the Spanish ones (r = 0.38, r = 0.31, r = 0.33, respectively, p < 0.01 for all of them). In conclusion, (i) the evaluation of LTT in the PHES battery deserves caution, (ii) PHES battery require adequate standardization in each country before its use, (iii) EEG and PHES findings are only roughly related each other. Reference [1] Romero-Gomez M. Hepatology 2007;45:879–85.
doi:10.1016/j.dld.2007.12.034 REDOX-SENSITIVE GSK-3 INHIBITION AND LATE VEGF RELEASE MEDIATE HYPOXIADEPENDENT EPITHELIAL–MESENCHYMAL TRANSITION AND INCREASED INVASIVENESS IN HUMAN HEPG2 CELLS S. Cannito, E. Novo, A. Compagnone, L. Valfr`e di Bonzo, C. Busletta, E. Zamara, A. Bandino, C. Cravanzola, V. Bravoco, S. Colombatto, M. Parola Dip. Medicina e Oncologia Sperimentale, University of Turin, Turin, Italy Background/aims. Epithelial–mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many tumour cells. In this study poorly invasive HepG2 cells and other cell lines of epithelial origin of the GI tract (Panc1, HT-29) have been exposed to carefully controlled hypoxic conditions (3% O2 ) in order to investigate whether hypoxia “per se” may trigger EMT program and related signalling mechanisms. Methods. EMT, signal transduction pathways, MMP-2 and MMP-9, chemotaxis and invasion were analysed by morphological, cell and molecular biology techniques, gelatine zimography and migration/invasion assays, respectively. Results. Hypoxia induces EMT changes in all neoplastic cells within 72 h, including nuclear translocation of SNAIL, Ecadherin disassembly, expression of N-cadherin, vimentin, ␣-SMA, MMP-2 and MMP-9. Moreover, all cancer cell lines responded to hypoxia by increased cell migration and Matrigel invasion, without significant changes in viability. Hypoxic medium collected after 24 to 48 hrs was found by itself to increase migration and invasiveness of normoxic cells in a VEGF-sensitive way. However, phosphorylation and inactivation of GSK3 was detected long before nuclear translocation of SNAIL and -catenin, suggesting a very early, hypoxia – dependent, involvement of Wnt/-catenin signalling machinery and EMT was indeed reproduced in normoxia by either recombinant Wnt-3A or LiCl. Early inhibition of GSK3 in hypoxic cells was found to be independent on VEGF and mostly related to intracellular generation of reactive oxygen species (ROS) by NADPH-oxidase and mitochondria.