- Email: [email protected]
379 COMPARISSON OF HEPATITIS B CCCDNA BETWEEN PARAFFIN-EMBEDDED AND CRYO-PRESERVED LIVER BIOPSIES OF CHRONIC HEPATITIS B PATIENTS
S144
Poster Session − Thursday, April 23
Table 1 Category
All F=0 F=1 F=2 F=3 F=4
HBV
HCV
p
No. of cases
FS (kPa)
No. of cases
FS (kPa)
63 1 14 31 15 2
8.13±3.86 7.4 6.52±1.76 7.05±2.05 9.94±4.06 20.45±9.26
191 4 26 97 45 19
8.45±4.96 5.27±0.83 5.41±1.92 7.18±2.63 9.75±4.63 16.68±8.10
– small sample 0.0519 (NS) 0.8153 (NS) 0.5789 (NS) small sample
The mean value of LS the 120 inactive HBsAg carriers was 5.69±2.25kPa (2.7−20.1 kPa), statistically significant higher than in 177 patients with not known history of chronic liver disease (4.83±1.26kPa, p = 0.0002). In 11 (9.17%) cases, the LS was higher than 8 kPa, theese patients needing further evaluation. Conclusion: In our study, for the same degree of fibrosis, the LS is not statistically significant diffrent in patients with chronic B hepatitis than in patients with chronic C hepatitis. Inactive HBsAg carriers have higher LS values than normal individuals. Those with high values of LS need to be further evaluated, including by means of LB.
378 COMPARATIVE MORPHOMETRIC ASSESSMENT OF FIBROSIS IN CHRONIC HEPATITIS B (CHB) VERSUS CHRONIC HEPATITIS C (CHC) LIVER BIOPSIES: IMPLICATION FOR FIBROSIS ASSESSMENT BY NON INVASIVE METHODS N. Sturm1 , A. Cheveau2 , P. Arvers3 , V. Leroy2 , J.P. Zarski2 . 1 D´epartement d’Anatomie et de Cytologie Pathologiques, 2 D´epartement d’H´epatoGastroenterologie, CHU A Michallon, 3 D´epartement des Facteurs Humains, CRSSA, Grenoble, France E-mail: [email protected] Background and Aims: Accurate assessment of liver fibrosis is essential in the management of chronic viral hepatitis. While percutaneous biopsy remains the gold standard to assess liver histology, non invasive methods have been recently developed to evaluate fibrosis. They have been essentially validated in CHC and less in CHB. Preliminary studies even suggest that they were less performant in CHB fibrosis assessment than in CHC. The aim of this study was to evaluate amount and pattern of fibrosis in CHB, as compared to CHC. Patients and Methods: 120 liver biopsies (60 CHB and 60 CHC) from naive sex-aetiology and Metavir fibrosis stage-matched patients were included (10 biopsies per stage from F0 to F4). Semi-quantitative evaluation of Metavir activity, steatosis (%) and perisinusoidal fibrosis (absent/present) by a senior liver pathologist, and quantitative morphometric evaluation of total fibrosis ratio and perisinusoidal fibrosis ratio (red sirius stained-sections, station Nikon, Lucia G software) by an independant operator were performed without knowledge of aetiology. Results: Quality of biopsy (length, number of portal tracts, number of optical fields), activity and steatosis were not different between the two groups. Concordance between fibrosis ratio and fibrosis stage was good (r = 0.85), better in CHB (r = 0.90) than in CHC (r = 0.81). A significant increase in fibrosis ratio was only observed in both groups for progression from F2 to F3 and F3 to F4. Mean total fibrosis ratio was higher in CHC than in CHB for F0 (2.27% vs 1.17%, p = 0.005), F1 (3.25% vs 2.30%, NS) and F2 (3.53% vs 2.59%, p = 0.06), without difference for severe fibrosis and cirrhosis. Semi-quantitative and morphometric approaches demonstrated concordant higher perisinusoidal fibrosis in CHC than in CHB for F0 (p = 0.001), F1 (p = 0.08) and F2 (p = 0.004). Conclusions: Our study shows differential amount of fibrosis in CHB and CHC in F2, mainly due to higher perisinusoidal fibrosis in CHC. Our results also probably explain difficulty for non invasive methods to assess liver fibrosis in early stages of the disease, especially F1 and F2 which are differentiated on architectural histological criteria. They could support
lesser performance for non invasive tests in CHB and necessity to interpret data with caution. 379 COMPARISSON OF HEPATITIS B CCCDNA BETWEEN PARAFFIN-EMBEDDED AND CRYO-PRESERVED LIVER BIOPSIES OF CHRONIC HEPATITIS B PATIENTS B. Takkenberg1 , H. Zaaijer2 , S. Menting2 , C. Weegink1 , V. Terpstra3 , M. Dijkgraaf4 , M. Cornelissen5 , P. Jansen1 , H. Reesink1 , M. Beld6 . 1 AMC Liver Center; Department of Gastroenterology and Hepatology, 2 Laboratory of Clinical Virology, Department of Medical Microbiology, 3 Department of Pathology, 4 Department of Clinical Epidemiology and Biostatistics, 5 Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center; University of Amsterdam, 6 Royal Tropical Institute, KIT Biomedical Research, Amsterdam, The Netherlands E-mail: [email protected] Background and Aim: To detect and analyze cccDNA in formalin-fixed, paraffin embedded (FFPE) liver tissue and compare results with cryopreserved (CP) liver biopsies, individual plasma ALT levels and Histology Activity Index (HAI). Material and Methods: cccDNA was quantified by selective primers that target the gap region between the two Direct Repeat Regions (DR1 and DR2). To quantify cells, a commercially available b`eta actin assay was used. Real-time PCR was performed in the Roche Lightcycler® 480. From 56 FFPE biopsies and 36 cryo preserved biopsies of HBeAg positive and HBeAg negative CHB patients, 60 mm was cut in slides of 10 mm each and processed for DNA quantification. Results: cccDNA was detectable in 40/56 FFPE biopsies and 36/36 CP biopsies. Mean number of hepatocytes was significantly higher in CP biopsies compared to FFPE biopsies (1.0×105 cells vs 1.0×103 cells; SD 2×103 ; p = 0.002). No difference was found in cccDNA levels/hepatocyte between FFPE and CP biopsies (1.0 copies/hepatocyte vs 0.8 copies/hepatocyte; p = 0.53). Median levels of cccDNA/hepatocyte in both FFPE and CP biopsies were significantly higher in biopsies from HBeAg positive patients than in biopsies from HBeAg negative patients (3.5 and 1.8 copies/hepatocyte vs 0.12 and 0.08 copies/hepatocyte p < 0.0001). Overall there was a significant correlation between the level of cccDNA/hepatocyte and HBV viral load (cop/mL) (correlation coefficient (R) = 0.6; p < 0.0001), but no correlation between cccDNA levels/hepatocyte and HAI-score (p = 0.10). In HBeAg negative CHB patients, median cccDNA levels/hepatocyte was significantly higher in patients with active hepatitis (HAI > 3) than in patients with inactive hepatitis (HAI 3) (p = 0.009 in FFPE and p = 0.006 in CP biopsies). Conclusions: In both FFPE and CP biopsies a strong correlation between cccDNA level and HBV viral load was observed. However, detection of cccDNA in FFPE biopsies was approximately 100 times less efficient than in CP biopsies. Only in HBeAg negative CHB patients, cccDNA copies/hepatocyte was significantly higher in CHB patients with HAI > 3. 380 SERUM HBsAg CONCENTRATION: RELATIONSHIP TO INTRAHEPATIC AND SERUM MARKERS OF HBV REPLICATION A. Thompson1,2 , T. Nguyen1,2 , E. Gane3 , W. Abbott3 , G. Lau4 , P. Desmond1 , S. Bowden2 , S. Locarnini2 . 1 Department of Gastroenterology, St Vincent’s Hospital, Fitzroy, 2 R&MD, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia; 3 Auckland City Hospital, Auckland, New Zealand; 4 Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong S.A.R. E-mail: [email protected] Background: HBsAg seroconversion is the ultimate goal of therapy for patients with chronic hepatitis B (CHB). Data from clinical trials suggests a role for quantitative HBsAg titres in guiding interferon-based therapies. However, HBsAg titres have not been well-characterized during the natural history of CHB and the relationship of serum HBsAg titre to serum viral
Poster Session − Thursday, April 23
Table 1 Category
All F=0 F=1 F=2 F=3 F=4
HBV
HCV
p
No. of cases
FS (kPa)
No. of cases
FS (kPa)
63 1 14 31 15 2
8.13±3.86 7.4 6.52±1.76 7.05±2.05 9.94±4.06 20.45±9.26
191 4 26 97 45 19
8.45±4.96 5.27±0.83 5.41±1.92 7.18±2.63 9.75±4.63 16.68±8.10
– small sample 0.0519 (NS) 0.8153 (NS) 0.5789 (NS) small sample
The mean value of LS the 120 inactive HBsAg carriers was 5.69±2.25kPa (2.7−20.1 kPa), statistically significant higher than in 177 patients with not known history of chronic liver disease (4.83±1.26kPa, p = 0.0002). In 11 (9.17%) cases, the LS was higher than 8 kPa, theese patients needing further evaluation. Conclusion: In our study, for the same degree of fibrosis, the LS is not statistically significant diffrent in patients with chronic B hepatitis than in patients with chronic C hepatitis. Inactive HBsAg carriers have higher LS values than normal individuals. Those with high values of LS need to be further evaluated, including by means of LB.
378 COMPARATIVE MORPHOMETRIC ASSESSMENT OF FIBROSIS IN CHRONIC HEPATITIS B (CHB) VERSUS CHRONIC HEPATITIS C (CHC) LIVER BIOPSIES: IMPLICATION FOR FIBROSIS ASSESSMENT BY NON INVASIVE METHODS N. Sturm1 , A. Cheveau2 , P. Arvers3 , V. Leroy2 , J.P. Zarski2 . 1 D´epartement d’Anatomie et de Cytologie Pathologiques, 2 D´epartement d’H´epatoGastroenterologie, CHU A Michallon, 3 D´epartement des Facteurs Humains, CRSSA, Grenoble, France E-mail: [email protected] Background and Aims: Accurate assessment of liver fibrosis is essential in the management of chronic viral hepatitis. While percutaneous biopsy remains the gold standard to assess liver histology, non invasive methods have been recently developed to evaluate fibrosis. They have been essentially validated in CHC and less in CHB. Preliminary studies even suggest that they were less performant in CHB fibrosis assessment than in CHC. The aim of this study was to evaluate amount and pattern of fibrosis in CHB, as compared to CHC. Patients and Methods: 120 liver biopsies (60 CHB and 60 CHC) from naive sex-aetiology and Metavir fibrosis stage-matched patients were included (10 biopsies per stage from F0 to F4). Semi-quantitative evaluation of Metavir activity, steatosis (%) and perisinusoidal fibrosis (absent/present) by a senior liver pathologist, and quantitative morphometric evaluation of total fibrosis ratio and perisinusoidal fibrosis ratio (red sirius stained-sections, station Nikon, Lucia G software) by an independant operator were performed without knowledge of aetiology. Results: Quality of biopsy (length, number of portal tracts, number of optical fields), activity and steatosis were not different between the two groups. Concordance between fibrosis ratio and fibrosis stage was good (r = 0.85), better in CHB (r = 0.90) than in CHC (r = 0.81). A significant increase in fibrosis ratio was only observed in both groups for progression from F2 to F3 and F3 to F4. Mean total fibrosis ratio was higher in CHC than in CHB for F0 (2.27% vs 1.17%, p = 0.005), F1 (3.25% vs 2.30%, NS) and F2 (3.53% vs 2.59%, p = 0.06), without difference for severe fibrosis and cirrhosis. Semi-quantitative and morphometric approaches demonstrated concordant higher perisinusoidal fibrosis in CHC than in CHB for F0 (p = 0.001), F1 (p = 0.08) and F2 (p = 0.004). Conclusions: Our study shows differential amount of fibrosis in CHB and CHC in F2, mainly due to higher perisinusoidal fibrosis in CHC. Our results also probably explain difficulty for non invasive methods to assess liver fibrosis in early stages of the disease, especially F1 and F2 which are differentiated on architectural histological criteria. They could support
lesser performance for non invasive tests in CHB and necessity to interpret data with caution. 379 COMPARISSON OF HEPATITIS B CCCDNA BETWEEN PARAFFIN-EMBEDDED AND CRYO-PRESERVED LIVER BIOPSIES OF CHRONIC HEPATITIS B PATIENTS B. Takkenberg1 , H. Zaaijer2 , S. Menting2 , C. Weegink1 , V. Terpstra3 , M. Dijkgraaf4 , M. Cornelissen5 , P. Jansen1 , H. Reesink1 , M. Beld6 . 1 AMC Liver Center; Department of Gastroenterology and Hepatology, 2 Laboratory of Clinical Virology, Department of Medical Microbiology, 3 Department of Pathology, 4 Department of Clinical Epidemiology and Biostatistics, 5 Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center; University of Amsterdam, 6 Royal Tropical Institute, KIT Biomedical Research, Amsterdam, The Netherlands E-mail: [email protected] Background and Aim: To detect and analyze cccDNA in formalin-fixed, paraffin embedded (FFPE) liver tissue and compare results with cryopreserved (CP) liver biopsies, individual plasma ALT levels and Histology Activity Index (HAI). Material and Methods: cccDNA was quantified by selective primers that target the gap region between the two Direct Repeat Regions (DR1 and DR2). To quantify cells, a commercially available b`eta actin assay was used. Real-time PCR was performed in the Roche Lightcycler® 480. From 56 FFPE biopsies and 36 cryo preserved biopsies of HBeAg positive and HBeAg negative CHB patients, 60 mm was cut in slides of 10 mm each and processed for DNA quantification. Results: cccDNA was detectable in 40/56 FFPE biopsies and 36/36 CP biopsies. Mean number of hepatocytes was significantly higher in CP biopsies compared to FFPE biopsies (1.0×105 cells vs 1.0×103 cells; SD 2×103 ; p = 0.002). No difference was found in cccDNA levels/hepatocyte between FFPE and CP biopsies (1.0 copies/hepatocyte vs 0.8 copies/hepatocyte; p = 0.53). Median levels of cccDNA/hepatocyte in both FFPE and CP biopsies were significantly higher in biopsies from HBeAg positive patients than in biopsies from HBeAg negative patients (3.5 and 1.8 copies/hepatocyte vs 0.12 and 0.08 copies/hepatocyte p < 0.0001). Overall there was a significant correlation between the level of cccDNA/hepatocyte and HBV viral load (cop/mL) (correlation coefficient (R) = 0.6; p < 0.0001), but no correlation between cccDNA levels/hepatocyte and HAI-score (p = 0.10). In HBeAg negative CHB patients, median cccDNA levels/hepatocyte was significantly higher in patients with active hepatitis (HAI > 3) than in patients with inactive hepatitis (HAI 3) (p = 0.009 in FFPE and p = 0.006 in CP biopsies). Conclusions: In both FFPE and CP biopsies a strong correlation between cccDNA level and HBV viral load was observed. However, detection of cccDNA in FFPE biopsies was approximately 100 times less efficient than in CP biopsies. Only in HBeAg negative CHB patients, cccDNA copies/hepatocyte was significantly higher in CHB patients with HAI > 3. 380 SERUM HBsAg CONCENTRATION: RELATIONSHIP TO INTRAHEPATIC AND SERUM MARKERS OF HBV REPLICATION A. Thompson1,2 , T. Nguyen1,2 , E. Gane3 , W. Abbott3 , G. Lau4 , P. Desmond1 , S. Bowden2 , S. Locarnini2 . 1 Department of Gastroenterology, St Vincent’s Hospital, Fitzroy, 2 R&MD, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia; 3 Auckland City Hospital, Auckland, New Zealand; 4 Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong S.A.R. E-mail: [email protected] Background: HBsAg seroconversion is the ultimate goal of therapy for patients with chronic hepatitis B (CHB). Data from clinical trials suggests a role for quantitative HBsAg titres in guiding interferon-based therapies. However, HBsAg titres have not been well-characterized during the natural history of CHB and the relationship of serum HBsAg titre to serum viral