- Email: [email protected]
Hepatocellular proliferation in HCV-related chronic hepatitis
April 1995
pHARMACOKINETIC EVALUATION OF HEPATOCELLULAR FUNCTION IN CRITICAL ILLNESS. P.J. Fabri, C. Sodellas, W.R. Sower, Jr., M. Blankenship, Depts. of Surgery and Biochemistry, Univ. of So. FL, J.A. Haley VAH, Tampa, FL 33612. While it is widely recognized that liver dysfunction is a component of multiple organ system failure, there is currently no mechanism available to identify subclinical organ dysfunction. Similarly, once evidence of hepatocellular dysfunction presents, there is no clinically useful way to evaluate serial changes in bepatocellular function (HF). We developed a panel of pharmacokinetio probes to prospectively evaluate HF in normal volunteers, hospitalized controls, and critically ill SICU patients. HF was assessed by measurement of acetaminophen elimination, acetaminophen sulfate and glucuronide appearance, theophylline half-llfe, lidocaine clearance, MEGX appearance, acetaminophen mercapturate and cystinate appearance in urine, and galactose clearance (effective hepatic blood flow). Acetaminophen, metabolites, and theophylline were assayed by HPLC; galactose, lidocaine, and MEGX by fluorescence. Acetaminophen elimination and metabolite appearance measure conjugation and cytochrome P450 activity. Lidocaine elimination and MEGX appearance quantify blood flow limited HF and cytochrome P450 activity. Theophylline levels at 24 hours appear to be an excellent global indicator of intrinsic hepatocellular dysfunction. Normal individuals demonstrate variability in ell parameters. Hospitalized preoperative patients demonstrate mild impairment of HF which does not appear to worsen after clean, major surgical procedures. Patients with existing liver dysfunction demonstrate relative preservation of HF with mild deterioration postoperatively. critically ill patients demonstrate a wide variability in all parameters, suggesting the need to measure multiple hepatic components when evaluating HF. Kd(A) Ka(S) Ka(G) EHBF T24hr Normal s N=17 Mean -0. 002 0. 028 0. 027 1837 0.79 SEM 0.0002 0.004 0.005 159 0.07 SICU N=33 Mean -0.001 0.006 0.006 2244 2.90 SEM 0. 0001 0.001 0.001 232 0.27 A~A-Pre N=5 Mean -0. 002 0. 014 0. 017 1376 1.71 SEM 0.0003 0.002 0.003 222 0.48 AAA-Post N=5 Mean -0.002 0.008 0.009 1881 1.21 SEM 0.0003 0.002 0.002 575 0.34 PCS-Pre N=I7 Mean -0. 002 0.016 0.015 1166 2.25 SEM 0.0002 0.003 0.003 i01 0.35 PCS-Post N=I7 Mean -0. 002 0. 011 0. 010 1405 2.41 SEM 0.0003 0.002 0.003 169 0.33
• IMBALANCE BETWEEN CELL PROLIFERATION AND PROGRAMMED CELL DEATH "APOPTOSIS" IN HEPATOCELLULAR CARCINOMA Fang JWS, Lau VKT, Wu PC.1 , CL Lai2, Lau JYN. Section of Hepat~biliary Diseas% University of Florida, Gainesville, FL and Department of Pathology and Medicine ~, University of Hung Kong, Hung Kong. Background The cell turnover in normal humans is tightly regulated by the coordination between cell proliferation and "programmed cell death" (apoptosis). Hypothesis Cell proliferation exceeds apoptosis in hepatocellular carcinoma (HCC). Aim To determine cell proliferation, apoptosis and the expression of oscogene products known to regulate apoptosis (953, bcl-2) in HCC. Methods 150 Chinese patients with HCC were studied (M:F 128:22, age 14-88 yes, histological evidence of cirrhosis in 71/91 specimens with adequate non-tumorous tissue, HBsAg+ 86.3%, survival 1-212 weeks). Formalin-fixed, paraffin-embedded liver sections were employed for the detection of proliferation markers [immunohistochemistry, PCNA, Ki67 (with citrate buffer-microwave retrieval)] and apoptosis (terminal d___UTPnick_end labeling, TUNEL). Expression of related oncogene products (953, bd-2) and biliary cell markers (AE1/AE3 and cytokeratin 19) in HCC were also assessed by immunohistocbemistry. Expression of proliferation, oncogene, and biliary markers was assessed semiquantitatively (0=0%, l=1-24%, 2=25-49%, 3=50-74%, 4__>75% "+" cells) and T U N E L labeling was quantitated in four random low power fields. Results Cell Proliferation 98% and 95% of HCC had PCNA (median 2+) and Ki67 (median 2+) detected respectively and there was an excellent correlation between PCNA and Ki67 (r=0.365, p<0.001). There was also a good correlation between the expression of proliferation markers and the proportion Of mitotic cells (vs PCNA, r=0.242, p<0.001). No correlation existed between the expression of proliferation markers and survival, Apoptosis T U N E L labeling was detected in only a small number of tumour cells (no labeling in 11%, median 1/1000 cell labeled, range: 0-70/1000 cells). There was no correlation between the clinical parameters (sex, age, cirrhosis, and survival) with either cell proliferation or apoptosis. There was also no difference in T U N E L labeling between HCC with and withouiL biliary markers. Oncogene Product Expression p53 was detected in 53% of the patients (median 1 +, range: 0-4+) and bcl-2 was detected in only 11% (range: 0-1+). The expression of p53 was associated with an increased expression of proliferation markers (PCNA and Ki67, r=ll.254 and 0.396 respectively, p<0.001 for both cases) but not with T U N E L labeling, bcl-2 expression did not correlate with either TUNEL labeling or survival. Conclusions (1) Cell proliferation in HCC is unmatched with apoptosis, accounting for the rapid growth of this tumour. (2) The lack of apoptosis in H C C is unrelated to bcl-2 over-expression which is uncommonly expressed in HCC, suggesting that ofher factors (e.g. mutant p53, a lack of c-fas or c-myc expression) may be important in either not inducing or inhibiting apoptosis in HCC.
AASLD
A1063
PREVALENCE OF HCV INFECTION AFFER HEART TRANSPLANT S. Fagiuoli, A. Jazzar, N. DeMaria, H. Farukl, S. Deal, T. Hassanein, A. Colantoni, S. Sisson, J. Tripp, J.S. Gavaler, DH Van Thiel, DKC Cooper, N. Zuhdi. Oklahoma Transplant Institute, Baptist Medical Center of Oklahoma, Oklahoma City, Oklahoma; Central Blood Bank of Pittsburgh, Pittsburgh, Pennsylvania Heart transplantation (OHTx) is a well-accepted treatment for individuals with end stage heart disease. Patient graft survival rates are more than 80% and 70% at 1 and 3 years respectively. Despite the high rate of prior cardiac surgery in these patients, there are only a few data that examine the rate of HCV infection in OHTx recipients before and after OHTx. To resolve this information deficit, the prevalence of HCV infection was assessed by means of H CV-RNA in a large series of consecutive OHTx recipients studied between I989 and I994. 100 consecutive patients with a mean age of 50 years, ranged 15 to 68, who underwent successful OHTx were studied. Those who died within the first postoperative week were excluded from the analysis. The mean follow-up was 27 months with a range of 4 and 46 months. A serologic panel consisting of anti-HAV lgG and IgM, HBsAg, HBeAg, anti-HBs, anti-HBc IgG and lgM, HBV-DNA, anti-HCV (ELISA II), HCV-RNA by RT-PCR and anti-HDV was performed on serum samples obtained pre- and at 1, 6 and 12 months post-OHTx. Sera from all of the donors were tested for the same viral markers. Anti-HCV positive and anti-HBc positive donors were excluded from use. Liver injury parameters (AST, ALT, Alkaline Phosphatase, Gamma-Glutamyl Transpeptidase and Total Bilirubin) were assessed in each recipient on a monthly basis following OHTx. Actual patient survival rate was 98% at 30 days, 94% at 1 year and 92% at 2 years. None of the recipients were HBsAg positive prior to transplantation. Three were HBsAb positive (only 2 were anti-HBc IgG positive). One was both HCV-RNA positive and anti-HCV positive prior to transplantation. No cases of HBV infection were detected during the follow-up period. In contrast, seven recipients became HCVRNA positive by RT-PCR within 6 months of the transplant procedure. Three more became anti-HCV positive (EL1SA II) but continued to he HCV-RNA negative by RT PCR. None of these 3 anti-HCV(+)/HCV-RNA(-) recipients manifested any biochemical or clinical evidence of liver dysfunction during their follow-up. In contrast, all of the HCV-RNA(+) recipients manifested biochemical liver dysfunction during their follow-up. AI1 4 had liver biopsies consistent with CAH. Two progressed to cirrhosis and died of end stage liver disease at 26 and 48 months following OHTx. Based upon this experience, it can be concluded that: I) The prevalence of HCV infection after successful OHTx is 7%; 2) Post-OHTx HCV infection leads to chronic liver disease, which is more aggressive than that observed in a non-transplant setting; and 3) HBV serologic and HCV-RNA surveillance as well as liver biopsy data are essential in order to detect, stage and manage cases of hepatitis occurring in OHTx recipients.
HEPATOCELLULAR PROLIFERATION IN HCV-RELATED CHRONIC HEPATITIS. F.Farinati, R.Cardin, A. D'Errico*, W.Grigioni*, C.Marafin, N.De Maria, A . C e c c h e t t o ^, R . N a c c a r a t o . Depts. G a s t r o e n t e r o l o g y & Pathology ^ , Padova University, Dept. P a t h o l o g y B o l o g n a U n i v e r s i t y * , Italy. HCV-related liver damage is l i n k e d to an increased risk of h e p a t o c e l l u l a r c a r c i n o m a (HCC), the underlying m e c h a n i s m s b e i n g s t i l l u n c l e a r . We evaluated extent and pattern of hepatocyte p r o l i f e r a t i o n in 56 p t s w i t h c h r o n i c h e p a t i t i s Cat histology) of different etiology (HCV [34], H C V + a l c o h o l [5], HBV [i0], alcohol abuse [4], hemochromatosis [3]). Proliferation was assayed using the Ki67 mcnoclonal antibody MIBI (AMAC Inc., Westbrook, USA), as the % of positive hepatocytesin both periportal and centrilobular area, a m i n i m u m of i000 b e i n g c o u n t e d . T h e r a t e w a s c o r r e l a t e d w i t h age, g e n d e r , e t i o l o g y , K n o d e l l index, inflammation score, liver iron (atomic absorbtion), glutathione (HPLC) a n d f r e e r a d i c a l s production (colorimetric assay of malondialdehyde). Results: MIBI+ cells in t h e c e n t r i l o b u l a r a r e a w e r e m o r e f r e q u e n t l y d e t e c t e d in H C V positive pts ( p < 0 . 0 5 ) . In the periportal area, HCV+ patients had significantly more MIBI+ cells than HBsAg+, a l c o h o l a b u s e o r h e m o c h r o m a t o s i s pts (median 3.5% v e r s u s 2%, 1% a n d 1% r e s p e c t i v e l y , p<0.015). A correlation was found between number of M I B I + h e p a t o c y t e s in t h e p e r i p o r t a l a r e a a n d I. Knodell index (p<0.02), 2. inflammation score ( p < 0 . 0 1 ) , 3. A L T l e v e l s ( p < 0 . 0 0 0 1 ) and, i n v e r s e l y , 4. liver iron (p<0.05). ALT levels were not significantly different in the 5 groups. In summary, hepatocyte p r o l i f e r a t i o n is i n c r e a s e d in pts with H C V - r e l a t e d d a m a g e a n d s h i f t s t o w a r d the p e r i c e n t r a l area. These data suggest that HCV infection is correlated with increased and a b n o r m a l p r o l i f e r a t i o n , w h i c h m i g h t be i n v o l v e d in t h e i n c r e a s e d H C C r i s k of H C V - p o s i t i v e pts.
pHARMACOKINETIC EVALUATION OF HEPATOCELLULAR FUNCTION IN CRITICAL ILLNESS. P.J. Fabri, C. Sodellas, W.R. Sower, Jr., M. Blankenship, Depts. of Surgery and Biochemistry, Univ. of So. FL, J.A. Haley VAH, Tampa, FL 33612. While it is widely recognized that liver dysfunction is a component of multiple organ system failure, there is currently no mechanism available to identify subclinical organ dysfunction. Similarly, once evidence of hepatocellular dysfunction presents, there is no clinically useful way to evaluate serial changes in bepatocellular function (HF). We developed a panel of pharmacokinetio probes to prospectively evaluate HF in normal volunteers, hospitalized controls, and critically ill SICU patients. HF was assessed by measurement of acetaminophen elimination, acetaminophen sulfate and glucuronide appearance, theophylline half-llfe, lidocaine clearance, MEGX appearance, acetaminophen mercapturate and cystinate appearance in urine, and galactose clearance (effective hepatic blood flow). Acetaminophen, metabolites, and theophylline were assayed by HPLC; galactose, lidocaine, and MEGX by fluorescence. Acetaminophen elimination and metabolite appearance measure conjugation and cytochrome P450 activity. Lidocaine elimination and MEGX appearance quantify blood flow limited HF and cytochrome P450 activity. Theophylline levels at 24 hours appear to be an excellent global indicator of intrinsic hepatocellular dysfunction. Normal individuals demonstrate variability in ell parameters. Hospitalized preoperative patients demonstrate mild impairment of HF which does not appear to worsen after clean, major surgical procedures. Patients with existing liver dysfunction demonstrate relative preservation of HF with mild deterioration postoperatively. critically ill patients demonstrate a wide variability in all parameters, suggesting the need to measure multiple hepatic components when evaluating HF. Kd(A) Ka(S) Ka(G) EHBF T24hr Normal s N=17 Mean -0. 002 0. 028 0. 027 1837 0.79 SEM 0.0002 0.004 0.005 159 0.07 SICU N=33 Mean -0.001 0.006 0.006 2244 2.90 SEM 0. 0001 0.001 0.001 232 0.27 A~A-Pre N=5 Mean -0. 002 0. 014 0. 017 1376 1.71 SEM 0.0003 0.002 0.003 222 0.48 AAA-Post N=5 Mean -0.002 0.008 0.009 1881 1.21 SEM 0.0003 0.002 0.002 575 0.34 PCS-Pre N=I7 Mean -0. 002 0.016 0.015 1166 2.25 SEM 0.0002 0.003 0.003 i01 0.35 PCS-Post N=I7 Mean -0. 002 0. 011 0. 010 1405 2.41 SEM 0.0003 0.002 0.003 169 0.33
• IMBALANCE BETWEEN CELL PROLIFERATION AND PROGRAMMED CELL DEATH "APOPTOSIS" IN HEPATOCELLULAR CARCINOMA Fang JWS, Lau VKT, Wu PC.1 , CL Lai2, Lau JYN. Section of Hepat~biliary Diseas% University of Florida, Gainesville, FL and Department of Pathology and Medicine ~, University of Hung Kong, Hung Kong. Background The cell turnover in normal humans is tightly regulated by the coordination between cell proliferation and "programmed cell death" (apoptosis). Hypothesis Cell proliferation exceeds apoptosis in hepatocellular carcinoma (HCC). Aim To determine cell proliferation, apoptosis and the expression of oscogene products known to regulate apoptosis (953, bcl-2) in HCC. Methods 150 Chinese patients with HCC were studied (M:F 128:22, age 14-88 yes, histological evidence of cirrhosis in 71/91 specimens with adequate non-tumorous tissue, HBsAg+ 86.3%, survival 1-212 weeks). Formalin-fixed, paraffin-embedded liver sections were employed for the detection of proliferation markers [immunohistochemistry, PCNA, Ki67 (with citrate buffer-microwave retrieval)] and apoptosis (terminal d___UTPnick_end labeling, TUNEL). Expression of related oncogene products (953, bd-2) and biliary cell markers (AE1/AE3 and cytokeratin 19) in HCC were also assessed by immunohistocbemistry. Expression of proliferation, oncogene, and biliary markers was assessed semiquantitatively (0=0%, l=1-24%, 2=25-49%, 3=50-74%, 4__>75% "+" cells) and T U N E L labeling was quantitated in four random low power fields. Results Cell Proliferation 98% and 95% of HCC had PCNA (median 2+) and Ki67 (median 2+) detected respectively and there was an excellent correlation between PCNA and Ki67 (r=0.365, p<0.001). There was also a good correlation between the expression of proliferation markers and the proportion Of mitotic cells (vs PCNA, r=0.242, p<0.001). No correlation existed between the expression of proliferation markers and survival, Apoptosis T U N E L labeling was detected in only a small number of tumour cells (no labeling in 11%, median 1/1000 cell labeled, range: 0-70/1000 cells). There was no correlation between the clinical parameters (sex, age, cirrhosis, and survival) with either cell proliferation or apoptosis. There was also no difference in T U N E L labeling between HCC with and withouiL biliary markers. Oncogene Product Expression p53 was detected in 53% of the patients (median 1 +, range: 0-4+) and bcl-2 was detected in only 11% (range: 0-1+). The expression of p53 was associated with an increased expression of proliferation markers (PCNA and Ki67, r=ll.254 and 0.396 respectively, p<0.001 for both cases) but not with T U N E L labeling, bcl-2 expression did not correlate with either TUNEL labeling or survival. Conclusions (1) Cell proliferation in HCC is unmatched with apoptosis, accounting for the rapid growth of this tumour. (2) The lack of apoptosis in H C C is unrelated to bcl-2 over-expression which is uncommonly expressed in HCC, suggesting that ofher factors (e.g. mutant p53, a lack of c-fas or c-myc expression) may be important in either not inducing or inhibiting apoptosis in HCC.
AASLD
A1063
PREVALENCE OF HCV INFECTION AFFER HEART TRANSPLANT S. Fagiuoli, A. Jazzar, N. DeMaria, H. Farukl, S. Deal, T. Hassanein, A. Colantoni, S. Sisson, J. Tripp, J.S. Gavaler, DH Van Thiel, DKC Cooper, N. Zuhdi. Oklahoma Transplant Institute, Baptist Medical Center of Oklahoma, Oklahoma City, Oklahoma; Central Blood Bank of Pittsburgh, Pittsburgh, Pennsylvania Heart transplantation (OHTx) is a well-accepted treatment for individuals with end stage heart disease. Patient graft survival rates are more than 80% and 70% at 1 and 3 years respectively. Despite the high rate of prior cardiac surgery in these patients, there are only a few data that examine the rate of HCV infection in OHTx recipients before and after OHTx. To resolve this information deficit, the prevalence of HCV infection was assessed by means of H CV-RNA in a large series of consecutive OHTx recipients studied between I989 and I994. 100 consecutive patients with a mean age of 50 years, ranged 15 to 68, who underwent successful OHTx were studied. Those who died within the first postoperative week were excluded from the analysis. The mean follow-up was 27 months with a range of 4 and 46 months. A serologic panel consisting of anti-HAV lgG and IgM, HBsAg, HBeAg, anti-HBs, anti-HBc IgG and lgM, HBV-DNA, anti-HCV (ELISA II), HCV-RNA by RT-PCR and anti-HDV was performed on serum samples obtained pre- and at 1, 6 and 12 months post-OHTx. Sera from all of the donors were tested for the same viral markers. Anti-HCV positive and anti-HBc positive donors were excluded from use. Liver injury parameters (AST, ALT, Alkaline Phosphatase, Gamma-Glutamyl Transpeptidase and Total Bilirubin) were assessed in each recipient on a monthly basis following OHTx. Actual patient survival rate was 98% at 30 days, 94% at 1 year and 92% at 2 years. None of the recipients were HBsAg positive prior to transplantation. Three were HBsAb positive (only 2 were anti-HBc IgG positive). One was both HCV-RNA positive and anti-HCV positive prior to transplantation. No cases of HBV infection were detected during the follow-up period. In contrast, seven recipients became HCVRNA positive by RT-PCR within 6 months of the transplant procedure. Three more became anti-HCV positive (EL1SA II) but continued to he HCV-RNA negative by RT PCR. None of these 3 anti-HCV(+)/HCV-RNA(-) recipients manifested any biochemical or clinical evidence of liver dysfunction during their follow-up. In contrast, all of the HCV-RNA(+) recipients manifested biochemical liver dysfunction during their follow-up. AI1 4 had liver biopsies consistent with CAH. Two progressed to cirrhosis and died of end stage liver disease at 26 and 48 months following OHTx. Based upon this experience, it can be concluded that: I) The prevalence of HCV infection after successful OHTx is 7%; 2) Post-OHTx HCV infection leads to chronic liver disease, which is more aggressive than that observed in a non-transplant setting; and 3) HBV serologic and HCV-RNA surveillance as well as liver biopsy data are essential in order to detect, stage and manage cases of hepatitis occurring in OHTx recipients.
HEPATOCELLULAR PROLIFERATION IN HCV-RELATED CHRONIC HEPATITIS. F.Farinati, R.Cardin, A. D'Errico*, W.Grigioni*, C.Marafin, N.De Maria, A . C e c c h e t t o ^, R . N a c c a r a t o . Depts. G a s t r o e n t e r o l o g y & Pathology ^ , Padova University, Dept. P a t h o l o g y B o l o g n a U n i v e r s i t y * , Italy. HCV-related liver damage is l i n k e d to an increased risk of h e p a t o c e l l u l a r c a r c i n o m a (HCC), the underlying m e c h a n i s m s b e i n g s t i l l u n c l e a r . We evaluated extent and pattern of hepatocyte p r o l i f e r a t i o n in 56 p t s w i t h c h r o n i c h e p a t i t i s Cat histology) of different etiology (HCV [34], H C V + a l c o h o l [5], HBV [i0], alcohol abuse [4], hemochromatosis [3]). Proliferation was assayed using the Ki67 mcnoclonal antibody MIBI (AMAC Inc., Westbrook, USA), as the % of positive hepatocytesin both periportal and centrilobular area, a m i n i m u m of i000 b e i n g c o u n t e d . T h e r a t e w a s c o r r e l a t e d w i t h age, g e n d e r , e t i o l o g y , K n o d e l l index, inflammation score, liver iron (atomic absorbtion), glutathione (HPLC) a n d f r e e r a d i c a l s production (colorimetric assay of malondialdehyde). Results: MIBI+ cells in t h e c e n t r i l o b u l a r a r e a w e r e m o r e f r e q u e n t l y d e t e c t e d in H C V positive pts ( p < 0 . 0 5 ) . In the periportal area, HCV+ patients had significantly more MIBI+ cells than HBsAg+, a l c o h o l a b u s e o r h e m o c h r o m a t o s i s pts (median 3.5% v e r s u s 2%, 1% a n d 1% r e s p e c t i v e l y , p<0.015). A correlation was found between number of M I B I + h e p a t o c y t e s in t h e p e r i p o r t a l a r e a a n d I. Knodell index (p<0.02), 2. inflammation score ( p < 0 . 0 1 ) , 3. A L T l e v e l s ( p < 0 . 0 0 0 1 ) and, i n v e r s e l y , 4. liver iron (p<0.05). ALT levels were not significantly different in the 5 groups. In summary, hepatocyte p r o l i f e r a t i o n is i n c r e a s e d in pts with H C V - r e l a t e d d a m a g e a n d s h i f t s t o w a r d the p e r i c e n t r a l area. These data suggest that HCV infection is correlated with increased and a b n o r m a l p r o l i f e r a t i o n , w h i c h m i g h t be i n v o l v e d in t h e i n c r e a s e d H C C r i s k of H C V - p o s i t i v e pts.