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Transcriptional profiling of primary sclerosing cholangitis identifies novel epithelial associated gene expression
364A
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
767
768
OBSTETRIC CHOLESTASIS W I T H ELEVATED 'y GLUTAMYLTRANSPEPTIDASE (GGT): INCIDENCE, PRESENTATION AND TREATMENT. P Milkiewicz, Liver Unit, Queen Elizabeth Hospital, Birmingham
ISOLATED CRYPTOGENIC DUCTULAR HYPERPLASIA (MINIMAL CHANGE CHOLANGIOPATHY). Aurelio Sonzogni, Ospedali Riuniti Ber-
United Kingdom; R Gallagher, Dept of Clinical Chemistry, Selly Oak Hospital, Birmingham United Kingdom; E Eggington, M Kilby, J Weaver, Women's Hospital, Birmingham United Kingdom; E Elias, Liver Unit, Queen Elizabeth Hospital, Birmingham United Kingdom Background. Obstetric cholestasis (OC) , thought to be a response to high hormonal levels of pregnancy in genetically predisposed individuals, causes pruritus which may be severe in the mother and may lead to foetal distress and stillbirth. Heterozygosity for mutations of the MDR3 gene has been suggested to play a role in the pathogenesis of OC when it is associated with elevated GGT levels(Lancet 1999;353:210). We have recently demonstrated missense mutation of MDR3 in association with OC which caused disruption of MDR3 translocaton to the cell membrane (Hum Mol Genet 2000;9:1209). Ursodeoxycholate (URSO) may facilitate the insertion of transporters into the canalicular membrane in cholestasis, as recently shown by us (Hepatol 1999;30:A1204 ) and others (Hepatol 2001;33;1206 ). The aim of the study is to i) assess the incidence of OC manifesting with elevated GGT; to compare (ii) the natural history and (iii) the effect of URSO in both groups of OC patients (pts). Methods. 81 pts with OC, treated in our institutions were analyzed. OC was diagnosed in pts with pruritus and elevated serum bile acids (SBA). 57 consented volunteer patients (70%) were treated with URSO. Results. GGT was elevated in 21 pts (30%). An elevated GGT at presentation was associated with significantly higher serum levels of AST (110+-25 vs. 66+-8 u/l; P<0.05); bilirubin (BIL) (25-+6 vs. 12+-1 umol/1; P<0.005) and SBA (67+- 13 vs. 43+-5 umol/l; P<0.05). OC first presented after 32+-2 week of pregnancy in the elevated GGT group and 31-+2 with normal GGT (P=NS). In pts not treated with URSO a comparison of LFT's at the time of diagnosis and at delivery showed no improvement of AST, ALT or BIL and SBA increased significantly. Pts from both groups responded to URSO with significant improvement of their AST, ALT and BIL but SBA fell significantly only in the normal GGT group. No side effect of URSO was observed. Conclusions. OC with elevated GGT ( i ) occurs in less than one third of U.K. pts.; (ii) is associated with significantly more impaired LFT's than in those with normal GGT, but is not associated with a different gestational age at onset. (ill)Treatment with URSO appears safe and significantly improves LFT's in patients with OC, excepting SBA in the high GGT group.
gamo, Bergamo Italy; Guido Colloredo, Seriate Hosp, Seriate Italy; Luigi Roffi, Monza Hosp, Monza Italy; Piero Del Poggio, Cernnseo sul Naviglio Hosp, Milan Italy; Marco Andreoletti, Cernusco sul Naviglio Hosp, Milano Italy; Bruno Brugnetti, Ponte S Pietro Hosp, Bergamo Italy; Luca Fabris, Mario Strazzabosco, Ospedali Riuniti Bergamo, Bergamo Italy Proliferation of duct ceils is a common finding during routine examination of liver biopsies. Occasionally,marginal ductular proliferation is the only histopathological change. On a retrospective review of 1235 percutaneous liver biopsies performed for suspected chronic liver diseases during 4 years in five northern Italian centers (1035 for viral disease, 157 for other causes), and after careful exclusion of drug intake and of known conditions associated to biIiary tract diseases, we have identified 43 cases (16 among virus-free and 27 among virus+ve subjects) in which the peculiar histological feature was a cryptogenic marginal ductular reaction (minimal change cholangiopathy, MCC) without significant fibrosis and/or necroinflammatory lesions and/or paucity of interlobular bile ducts. The mean age was 37 yrs. ( range 18-64) (26/17; M/F), At the time of the biopsy, mean duration of LFTs changes was 6.9 years (range 1-17). All liver specimens (containing at least six portal fields) were stained with H&E, d-PAS, Perls staining, tricrome staning, cytokeratin 19 (bfliary phenotype marker), Epithelial Membrane Antigen (EMA, a marker of mature, differentiated cholangiocytes), Neural Cell Adhesion Molecule (NCAM, a marker of immature atypical ductules) and Ki-67 (a marker of cell proliferation). Grading and staging were classified according to Ishak and al. (J. Hepatol 1995, 22:696). Ductular structures were classified according to Crawford (Hepatology, 1998; 28:323) as interlobular (portal) bile ducts (BD1), marginal (periportal) ductules (BD2) and solitary bile ducts, divided into cross-section profiles (BD3a), and longitudinal cuboidal strings (BD3b). Morphometrical analysis showed that, in comparison to published data for normal liver biopsies, in MCC samples typical marginal ductules (BD2) were clearly more represented (1.69+/-0.84 ductules per portal space vs 0.4+/-0.7 in Hepatology, 1998). Immunohistochemistry demonstrated that reactive ductules were well differentiated (EMA+ve and NCAM-ve) and without significant proliferative activity (Ki-67-ve). For all the morphological parameters considered there was no difference between HCV positive and negative patients. Furthermore, in four HCV-RNApositive patients, that achieved a sustained virological response to interferon, MCC was still present and unchanged more than one year after the end of treatment, suggesting the absence of pathogenetic relationships with the virus. In conclusion, MCC is a newly documented condition characterized by hyperplastic marginal ductular structures with mature phenotype in the absence of portal fibrosis/inflammation. It is present in young adults with mildly elevated liver function tests, irrespective of the viral status and is likely non progressive.
769
770
PROGRESSION OF PRIMARY SCLEROSING CHOLANGITIS IN CHILDREN. Luis A Batres, Div GI & Nutrition/The Children's Hospital of Philadel-
TRANSCRIPTIONAL PROFILING OF PRIMARY SCLEROSING CHOLANGITIS IDENTIFIES NOVEL EPITHELIAL ASSOCIATED GENE EXPRESSION. Nicholas A Shackel, Centenary Institute, Sydney Australia; Peter
phia, Philadelphia, PA; Pierre A Rnsso, Eduardo D Ruchelli, Dept of Pathology/ The Children's Hospital of Philadelphia, Philadelphia, PA; David A Piccoli, Div GI & Nutrition/The Children's Hospital of Philadelphia, Philadelphia, PA; Mark Mathews, Dept of Pathology/The Children's Hospital of Philadelphia, Philadelphia, PA; Emil Chuang, Div GI & Nutrition/The Children's Hospital of Philadelphia, Philadelphia, PA Background: Primary selerosing cholangitis (PSC) is rare in the pediatric population, and has few distinguishing clinical and pathological characteristics. Little information on the progression of the disease exists, and follow-up information is rare. The purpose of this study is to report our experience over the past 20 years emphasizing initial and progressive histologic features and clinical follow-up. Methods: We retrospectively reviewed the data of 20 subjects between 1981 and 2001, diagnosed at our institution with primary sclerosing cholangitis. Results: There were 20 subjects diagnosed to have PSC (6 females), between the ages of 0 to 15 years of age at presentation. A total of 72 liver specimens were reviewed, 15 subjects had two or more specimens. Eight children (40%) were diagnosed to have inflammatory bowel disease during the course of their illness. Nine subjects had positive pANCA. All subjects had biliary tract imaging, 14 subjects had an endoscopic retrograde cholangiopancreatography (ERCP), 4 had magnetic resonance cholangiography and 2 subjects had a percutaneous transhepatic cholangiogram. Eighteen subjects had classic radiographic changes. The remaining two subjects were subsequently diagnosed with PSC by biliary imaging or at the time of liver transplantation after initially normal radiographic studies. Initial biopsies in ten patients revealed advanced disease (bridging fibrosis or cirrhosis). Of the 10 subjects presenting with an earlier histologic stage, 4 progressed, 3 remained stable, and 3 subjects did not have follow up biopsies. Seven subjects underwent liver transplantation. Disease recurred in the allograft in three patients (average of 1.6 years after transplantation). Conclusions: Our review suggests that PSC in children is an insidious disease, frequently only detected at a relatively advanced histological stage, and which appears to be progressive in patients in whom it is detected earlier. There appears to be a high recurrence rate after transplantation.
McGuinness, Cathy Abbott, Mark Gorrell, Geoff McCaughan, Royal Prince Alfred Hospital, Sydney NSW Australia Background: Primary sclerosing cholangitis (PSC) is a classical autoimmune biliary epithelial disease. Although PSC is morphologically well defined, the molecular events in PSC pathobiology remain mostly uncharacterised, eDNA array analysis, through its unparalleled simultaneous examination of multiple gene expression, was used to achieve new insights into PSC pathogenesis. Methods: cDNA array analysis used two commercial arrays of 856 genes to profile and charaeterise intrahepatic gene expression in PSC cirrhosis (n=4) compared to non-diseased liver tissue (n=8), primary biliary cirrhosis (PBC n= 6), hepatitis C cirrhosis (HCV n=6) and autoimmune hepatitis (AIH n= 4). Intrahepatic gene expression was linked to the pathogenic processes of inflammation, fibrosis, proliferation and apoptosis. Selected gene expression was confirmed by quantitative real-time RT-PCR. Results: Gene profiling showed that PSC clustered with primary biliary cirrhosis and overall was characterised by 10 to 15% greater gene upregulation than PBC, HCV or AIH cirrhosis compared to non-diseased tissue. Comparison of PSC cirrhosis with PBC associated gene expression demonstrated that many epithelial associated genes not previously implicated in liver disease, such as the growth associated coltrectum cell-derived growth factor (amphiregulin) and bullous pemphigoid antigen 1, were uniquely expressed in PSC cirrhosis. Inflammation in PSC was characterised by a mixed Thl and Th2 immune response and expression of IL-8, CXCR4 and a number of endothelial associated inflammatory mediators such as endothelial-monocyte activating polypeptide II (EMAP-II) and endothelial leucocyte adhesion molecule I (ELAM-I). Fibrogenesis was characterised by increased expression of genes such as connective tissue growth factor (CTGF) which was also observed in PBC, HCV and AIH cirrhosis. An active proliferative response was observed in PSC with increased cell cycle associated kinases (CDK7, CCNB1, CCNB3, CDKN3) and insulin growth factor axis associated gene expression (IGF IA, 1GFR II, IGF II, IGFBP 1,2,3,5,6). Apoptosis gene expression in PSC contrasted with PBC and was characterised by multiple pro-apoptotic (caspases 2,6,8, Bak, Bik) and anti-apoptotie gene expression (Bcl-2, Bcl-2A1, inhibitor of apoptosis 1,2,3). Conclusion: PSC cirrhosis was charaeterised by a pro-inflammatory, pro-proliferative and mixedapoptosis associated gene profile. Epithelial associated gene expression suggests that there are unique pathogenic pathways in PSC cirrhosis. It is intruiging that there was increased expression of genes such as coloreetum cell-derived growth factor that may link the pathobiology of PSC with ulcerative colitis.
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
767
768
OBSTETRIC CHOLESTASIS W I T H ELEVATED 'y GLUTAMYLTRANSPEPTIDASE (GGT): INCIDENCE, PRESENTATION AND TREATMENT. P Milkiewicz, Liver Unit, Queen Elizabeth Hospital, Birmingham
ISOLATED CRYPTOGENIC DUCTULAR HYPERPLASIA (MINIMAL CHANGE CHOLANGIOPATHY). Aurelio Sonzogni, Ospedali Riuniti Ber-
United Kingdom; R Gallagher, Dept of Clinical Chemistry, Selly Oak Hospital, Birmingham United Kingdom; E Eggington, M Kilby, J Weaver, Women's Hospital, Birmingham United Kingdom; E Elias, Liver Unit, Queen Elizabeth Hospital, Birmingham United Kingdom Background. Obstetric cholestasis (OC) , thought to be a response to high hormonal levels of pregnancy in genetically predisposed individuals, causes pruritus which may be severe in the mother and may lead to foetal distress and stillbirth. Heterozygosity for mutations of the MDR3 gene has been suggested to play a role in the pathogenesis of OC when it is associated with elevated GGT levels(Lancet 1999;353:210). We have recently demonstrated missense mutation of MDR3 in association with OC which caused disruption of MDR3 translocaton to the cell membrane (Hum Mol Genet 2000;9:1209). Ursodeoxycholate (URSO) may facilitate the insertion of transporters into the canalicular membrane in cholestasis, as recently shown by us (Hepatol 1999;30:A1204 ) and others (Hepatol 2001;33;1206 ). The aim of the study is to i) assess the incidence of OC manifesting with elevated GGT; to compare (ii) the natural history and (iii) the effect of URSO in both groups of OC patients (pts). Methods. 81 pts with OC, treated in our institutions were analyzed. OC was diagnosed in pts with pruritus and elevated serum bile acids (SBA). 57 consented volunteer patients (70%) were treated with URSO. Results. GGT was elevated in 21 pts (30%). An elevated GGT at presentation was associated with significantly higher serum levels of AST (110+-25 vs. 66+-8 u/l; P<0.05); bilirubin (BIL) (25-+6 vs. 12+-1 umol/1; P<0.005) and SBA (67+- 13 vs. 43+-5 umol/l; P<0.05). OC first presented after 32+-2 week of pregnancy in the elevated GGT group and 31-+2 with normal GGT (P=NS). In pts not treated with URSO a comparison of LFT's at the time of diagnosis and at delivery showed no improvement of AST, ALT or BIL and SBA increased significantly. Pts from both groups responded to URSO with significant improvement of their AST, ALT and BIL but SBA fell significantly only in the normal GGT group. No side effect of URSO was observed. Conclusions. OC with elevated GGT ( i ) occurs in less than one third of U.K. pts.; (ii) is associated with significantly more impaired LFT's than in those with normal GGT, but is not associated with a different gestational age at onset. (ill)Treatment with URSO appears safe and significantly improves LFT's in patients with OC, excepting SBA in the high GGT group.
gamo, Bergamo Italy; Guido Colloredo, Seriate Hosp, Seriate Italy; Luigi Roffi, Monza Hosp, Monza Italy; Piero Del Poggio, Cernnseo sul Naviglio Hosp, Milan Italy; Marco Andreoletti, Cernusco sul Naviglio Hosp, Milano Italy; Bruno Brugnetti, Ponte S Pietro Hosp, Bergamo Italy; Luca Fabris, Mario Strazzabosco, Ospedali Riuniti Bergamo, Bergamo Italy Proliferation of duct ceils is a common finding during routine examination of liver biopsies. Occasionally,marginal ductular proliferation is the only histopathological change. On a retrospective review of 1235 percutaneous liver biopsies performed for suspected chronic liver diseases during 4 years in five northern Italian centers (1035 for viral disease, 157 for other causes), and after careful exclusion of drug intake and of known conditions associated to biIiary tract diseases, we have identified 43 cases (16 among virus-free and 27 among virus+ve subjects) in which the peculiar histological feature was a cryptogenic marginal ductular reaction (minimal change cholangiopathy, MCC) without significant fibrosis and/or necroinflammatory lesions and/or paucity of interlobular bile ducts. The mean age was 37 yrs. ( range 18-64) (26/17; M/F), At the time of the biopsy, mean duration of LFTs changes was 6.9 years (range 1-17). All liver specimens (containing at least six portal fields) were stained with H&E, d-PAS, Perls staining, tricrome staning, cytokeratin 19 (bfliary phenotype marker), Epithelial Membrane Antigen (EMA, a marker of mature, differentiated cholangiocytes), Neural Cell Adhesion Molecule (NCAM, a marker of immature atypical ductules) and Ki-67 (a marker of cell proliferation). Grading and staging were classified according to Ishak and al. (J. Hepatol 1995, 22:696). Ductular structures were classified according to Crawford (Hepatology, 1998; 28:323) as interlobular (portal) bile ducts (BD1), marginal (periportal) ductules (BD2) and solitary bile ducts, divided into cross-section profiles (BD3a), and longitudinal cuboidal strings (BD3b). Morphometrical analysis showed that, in comparison to published data for normal liver biopsies, in MCC samples typical marginal ductules (BD2) were clearly more represented (1.69+/-0.84 ductules per portal space vs 0.4+/-0.7 in Hepatology, 1998). Immunohistochemistry demonstrated that reactive ductules were well differentiated (EMA+ve and NCAM-ve) and without significant proliferative activity (Ki-67-ve). For all the morphological parameters considered there was no difference between HCV positive and negative patients. Furthermore, in four HCV-RNApositive patients, that achieved a sustained virological response to interferon, MCC was still present and unchanged more than one year after the end of treatment, suggesting the absence of pathogenetic relationships with the virus. In conclusion, MCC is a newly documented condition characterized by hyperplastic marginal ductular structures with mature phenotype in the absence of portal fibrosis/inflammation. It is present in young adults with mildly elevated liver function tests, irrespective of the viral status and is likely non progressive.
769
770
PROGRESSION OF PRIMARY SCLEROSING CHOLANGITIS IN CHILDREN. Luis A Batres, Div GI & Nutrition/The Children's Hospital of Philadel-
TRANSCRIPTIONAL PROFILING OF PRIMARY SCLEROSING CHOLANGITIS IDENTIFIES NOVEL EPITHELIAL ASSOCIATED GENE EXPRESSION. Nicholas A Shackel, Centenary Institute, Sydney Australia; Peter
phia, Philadelphia, PA; Pierre A Rnsso, Eduardo D Ruchelli, Dept of Pathology/ The Children's Hospital of Philadelphia, Philadelphia, PA; David A Piccoli, Div GI & Nutrition/The Children's Hospital of Philadelphia, Philadelphia, PA; Mark Mathews, Dept of Pathology/The Children's Hospital of Philadelphia, Philadelphia, PA; Emil Chuang, Div GI & Nutrition/The Children's Hospital of Philadelphia, Philadelphia, PA Background: Primary selerosing cholangitis (PSC) is rare in the pediatric population, and has few distinguishing clinical and pathological characteristics. Little information on the progression of the disease exists, and follow-up information is rare. The purpose of this study is to report our experience over the past 20 years emphasizing initial and progressive histologic features and clinical follow-up. Methods: We retrospectively reviewed the data of 20 subjects between 1981 and 2001, diagnosed at our institution with primary sclerosing cholangitis. Results: There were 20 subjects diagnosed to have PSC (6 females), between the ages of 0 to 15 years of age at presentation. A total of 72 liver specimens were reviewed, 15 subjects had two or more specimens. Eight children (40%) were diagnosed to have inflammatory bowel disease during the course of their illness. Nine subjects had positive pANCA. All subjects had biliary tract imaging, 14 subjects had an endoscopic retrograde cholangiopancreatography (ERCP), 4 had magnetic resonance cholangiography and 2 subjects had a percutaneous transhepatic cholangiogram. Eighteen subjects had classic radiographic changes. The remaining two subjects were subsequently diagnosed with PSC by biliary imaging or at the time of liver transplantation after initially normal radiographic studies. Initial biopsies in ten patients revealed advanced disease (bridging fibrosis or cirrhosis). Of the 10 subjects presenting with an earlier histologic stage, 4 progressed, 3 remained stable, and 3 subjects did not have follow up biopsies. Seven subjects underwent liver transplantation. Disease recurred in the allograft in three patients (average of 1.6 years after transplantation). Conclusions: Our review suggests that PSC in children is an insidious disease, frequently only detected at a relatively advanced histological stage, and which appears to be progressive in patients in whom it is detected earlier. There appears to be a high recurrence rate after transplantation.
McGuinness, Cathy Abbott, Mark Gorrell, Geoff McCaughan, Royal Prince Alfred Hospital, Sydney NSW Australia Background: Primary sclerosing cholangitis (PSC) is a classical autoimmune biliary epithelial disease. Although PSC is morphologically well defined, the molecular events in PSC pathobiology remain mostly uncharacterised, eDNA array analysis, through its unparalleled simultaneous examination of multiple gene expression, was used to achieve new insights into PSC pathogenesis. Methods: cDNA array analysis used two commercial arrays of 856 genes to profile and charaeterise intrahepatic gene expression in PSC cirrhosis (n=4) compared to non-diseased liver tissue (n=8), primary biliary cirrhosis (PBC n= 6), hepatitis C cirrhosis (HCV n=6) and autoimmune hepatitis (AIH n= 4). Intrahepatic gene expression was linked to the pathogenic processes of inflammation, fibrosis, proliferation and apoptosis. Selected gene expression was confirmed by quantitative real-time RT-PCR. Results: Gene profiling showed that PSC clustered with primary biliary cirrhosis and overall was characterised by 10 to 15% greater gene upregulation than PBC, HCV or AIH cirrhosis compared to non-diseased tissue. Comparison of PSC cirrhosis with PBC associated gene expression demonstrated that many epithelial associated genes not previously implicated in liver disease, such as the growth associated coltrectum cell-derived growth factor (amphiregulin) and bullous pemphigoid antigen 1, were uniquely expressed in PSC cirrhosis. Inflammation in PSC was characterised by a mixed Thl and Th2 immune response and expression of IL-8, CXCR4 and a number of endothelial associated inflammatory mediators such as endothelial-monocyte activating polypeptide II (EMAP-II) and endothelial leucocyte adhesion molecule I (ELAM-I). Fibrogenesis was characterised by increased expression of genes such as connective tissue growth factor (CTGF) which was also observed in PBC, HCV and AIH cirrhosis. An active proliferative response was observed in PSC with increased cell cycle associated kinases (CDK7, CCNB1, CCNB3, CDKN3) and insulin growth factor axis associated gene expression (IGF IA, 1GFR II, IGF II, IGFBP 1,2,3,5,6). Apoptosis gene expression in PSC contrasted with PBC and was characterised by multiple pro-apoptotic (caspases 2,6,8, Bak, Bik) and anti-apoptotie gene expression (Bcl-2, Bcl-2A1, inhibitor of apoptosis 1,2,3). Conclusion: PSC cirrhosis was charaeterised by a pro-inflammatory, pro-proliferative and mixedapoptosis associated gene profile. Epithelial associated gene expression suggests that there are unique pathogenic pathways in PSC cirrhosis. It is intruiging that there was increased expression of genes such as coloreetum cell-derived growth factor that may link the pathobiology of PSC with ulcerative colitis.