Pathology of Recurrent Hepatitis C Diagnostic Problems and Differential Diagnosis: Are there Predictors of Graft Outcome? Stefan Hübscher, School of Cancer Sciences, University of Birmingham and Department of Cellular Pathology, Queen Elizabeth Hospital, Birmingham. U.K.

Hepatitis C in the Liver Allograft HCV cirrhosis commonest indication for liver transplantation Re-infection is universal –begins within few hours of reperfusion –HCV-RNA >> pre-transplant levels Most cases (70-90%) result in graft inflammation Many progress to cirrhosis –20-30% by 5 years, up to 50% at 10 years 10-30% develop graft failure (Roche 2010, Ponziani 2011) – most common indication for late retransplantation

Hepatitis C in the Liver Allograft Histological Features Typical Histological Features - as seen in the non-transplanted liver Atypical Histological Features - modified by immunosuppression - interaction with other complications Many post-transplant biopsies have features reflecting more than one process Liver biopsy may help to identify main cause of graft damage

Hepatitis C in the Liver Allograft Histological Features Typical Histological Features - as seen in the non-transplanted liver Atypical Histological Features - modified by immunosuppression - interaction with other complications

Hepatitis C in Liver Allografts - Evolution of Histological Changes Phase (time post-transplant) HistologyComments Early infection (0-2 months) Mild non-specific changes Lobular disarray Acidophil bodies Lack of inflammation Steatosis Established infection (2-6 months) Acute hepatitisLobular inflammation (usually mild) Progressive damage (> 6 months) Chronic hepatitisPortal/periportal inflammation Fibrosis/cirrhosis

Hepatitis C in Liver Allografts - Evolution of Histological Changes Phase (time post-transplant) HistologyComments Early infection (0-2 months) Mild non-specific changes Lobular disarray Acidophil bodies Lack of inflammation Steatosis Established infection (2-6 months) Acute hepatitisLobular inflammation (usually mild) Progressive damage (> 6 months) Chronic hepatitisPortal/periportal inflammation Fibrosis/cirrhosis

Recurrent HCV –5 weeks post-OLT

Early Recurrent HCV lobular disarray, variation in cell size

Hepatitis C in Liver Allografts - Evolution of Histological Changes Phase (time post-transplant) HistologyComments Early infection (0-2 months) Mild non-specific changes Lobular disarray Acidophil bodies Lack of inflammation Steatosis Established infection (2-6 months) Acute hepatitisLobular inflammation (usually mild) Progressive damage (> 6 months) Chronic hepatitisPortal/periportal inflammation Fibrosis/cirrhosis

Recurrent HCV – 3 months post-OLT spotty lobular inflammation (mild)

Hepatitis C in Liver Allografts - Evolution of Histological Changes Phase (time post-transplant) HistologyComments Early infection (0-2 months) Mild non-specific changes Lobular disarray Acidophil bodies Lack of inflammation Steatosis Established infection (2-6 months) Acute hepatitisLobular inflammation (usually mild) Progressive damage (> 6 months) Chronic hepatitisPortal/periportal inflammation Fibrosis/cirrhosis

Recurrent Hepatitis C – 6 months post-OLT Chronic Hepatitis

Chronic Hepatitis C in the Liver Allograft Role of Protocol Liver Biopsies Most centres still obtain protocol annual review biopsies from HCV-positive patients e.g. 65% in recent survey of 35 LT centres (vs 25% for non- HCV patients) (Mells & Neuberger 2008) Histological abnormalities frequently present in HCV-positive patients with normal LFTs (Berenguer 2001, Sebagh 2003)

Chronic Hepatitis C in the Liver Allograft Role of Protocol Liver Biopsies Mainly used to assess disease severity (especially fibrosis) Implications for prognosis (e.g. presence/severity of fibrosis at 1 year predicts subsequent progression to fibrosis/cirrhosis/graft failure) May influence treatment (anti-viral therapy to prevent fibrosis progression) Scoring systems (e.g. METAVIR, Ishak) should be used with caution and only in patients with “pure” HCV infection. Non-invasive methods (serum markers, transient elastography) increasingly used to assess fibrosis in post-transplant HCV Mainly useful to identify mild/early fibrosis versus advanced fibrosis/cirrhosis May also help to predict fibrosis progression

Hepatitis C in the Liver Allograft Differences Compared with HCV in the Native Liver 1.More aggressive disease –More severe inflammatory activity –more rapid progression to fibrosis and cirrhosis (20- 30% cirrhotic by 5 years, up to 50% by 10 years) 2.Atypical features –Cholestatic features (“fibrosing cholestatic hepatitis”) –Autoimmune features ( de novo AIH / “plasma cell hepatitis”) 3.Interaction with other graft complications –Rejection (acute and chronic) –Non-alcoholic fatty liver disease

Hepatitis C in the Liver Allograft Differences Compared with HCV in the Native Liver 1.More aggressive disease –More severe inflammatory activity –more rapid progression to fibrosis and cirrhosis (20- 30% cirrhotic by 5 years, up to 50% by 10 years) 2.Atypical features –Cholestatic features (“fibrosing cholestatic hepatitis”) –Autoimmune features ( de novo AIH / “plasma cell hepatitis”) 3.Interaction with other graft complications –Rejection (acute and chronic) –Non-alcoholic fatty liver disease

Recurrent Hepatitis C – Liver Biopsy 12 months post-transplant prominent lobular inflammation with zone 3 necrosis (central perivenulitis) Are these changes related to HCV alone? or Is this HCV + another graft complication? - Late acute rejection -De novo AIH

Are these changes related to HCV alone? or Is this HCV + another graft complication? - Late acute rejection -De novo AIH Histological distinction often difficult -Clinical context (e.g. recent changes in immunosuppression and/or anti- viral therapy) -Results of other investigations (e.g. HCV-RNA levels, auto-antibodies) Recurrent Hepatitis C – Liver Biopsy 12 months post-transplant prominent lobular inflammation with zone 3 necrosis (central perivenulitis)

Aggressive Recurrent HCV Male, age months post-LT for HCV Antiviral therapy recently stopped because of nephric abscess Presented with acutely deranged LFTs (AST 650) Became HCV-RNA positive

Hepatitis C in the Liver Allograft Differences Compared with HCV in the Native Liver 1.More aggressive disease –More severe inflammatory activity –more rapid progression to fibrosis and cirrhosis (20- 30% cirrhotic by 5 years, up to 50% by 10 years) 2.Atypical features –Cholestatic features (“fibrosing cholestatic hepatitis”) –Autoimmune features ( de novo AIH / “plasma cell hepatitis”) 3.Interaction with other graft complications –Rejection (acute and chronic) –Non-alcoholic fatty liver disease

Hepatitis C with Cholestatic Features (“Fibrosing Cholestatic Hepatitis”) Pathogenesis Most cases present within first 12 months post-transplant (peak 2-6 months) High viral RNA levels (serum and intra-hepatic) Impaired HCV-specific immune response High viral load in hepatocytes may result in: Direct cytopathic injury to hepatocytes (ballooning, cholestasis) Replicative senescence in hepatocytes –Stimulus for progenitor cell activation & ductular reaction –Hepatic stellate cell activation Fibrosis

Hepatitis C with Cholestatic Features (“Fibrosing Cholestatic Hepatitis”) Prevalence & Clinical Features Prevalence Reported frequency varies (2-13%) May reflect different diagnostic criteria Higher frequency in HIV co-infected patients (up to 20% - Antoni 2011) Clinical Features Jaundice and cholestatic liver biochemistry –Other causes of cholestasis should be excluded (e.g. biliary obstruction, drug toxicity) Severe cases progress rapidly to graft failure

Hepatitis C with Cholestatic Features (“Fibrosing Cholestatic Hepatitis”) Histological Features Early Features Hepatocyte ballooning and cholestasis, mainly perivenular Lobular disarray, little inflammation Perisinusoidal fibrosis (? related to hepatic stellate cell activation) Later Changes Portal expansion with marginal ductular reaction Progressive fibrosis (periportal and bridging) Severe cases progressing rapidly to graft failure may be associated with multi-acinar necrosis and collapse

Hepatitis C with Cholestatic Features - Histological Features Differences Compared with Fibrosing Cholestatic Hepatitis B Cholestatic features frequently co-exist with more typical hepatitic changes Fibrosis less prominent (cholestatic hepatitis C) Clinico-pathological severity variable –Mild/early cases may respond to reduction in immunosuppression +/- anti-viral therapy –Overall mortality 50% (Narang 2010)

Cholestatic Hepatitis C – Lobular Changes Ballooning, lobular disarrayBilirubinostasis

Cholestatic Hepatitis C – Portal &Periportal Changes

keratin 7 Ductular reaction Also seen in: Biliary obstruction Acute rejection Leads to periportal fibrosis Cholestatic Hepatitis C – Portal &Periportal Changes

Cholestatic Hepatitis C – Fibrosis - Periportal and Perisinusoidal

Hepatitis C with “Autoimmune Features” ( de novo autoimmune hepatitis, “plasma cell hepatitis”) 1.Following Interferon Therapy (Cholongitas 2006,Kontorinis 2006,Berardi 2007,Merli 2009) 14 cases – all had biochemical, serological and histological features compatible with autoimmune hepatitis (“de novo AIH”) Prevalence 20% (9/44) in largest study (Berardi 2006) 12/14 were HCV-RNA negative 10/14 responded to treatment with immunosuppression

Chronic Hepatitis in the Liver Allograft Features favouring an autoimmune aetiology Portal inflammation with plasma cells Interface hepatitis Lobular inflammation (plasma cell rich) Confluent necrosis (Central perivenulitis)

Hepatitis C with “Autoimmune Features” ( de novo autoimmune hepatitis, “plasma cell hepatitis”) 2.Unrelated to Interferon Therapy (Khettry 2007, Fiel 2008) 47 patients (prevalence 10%) - plasma cell rich portal and lobular infiltrates (“plasma cell hepatitis”) Centrilobular necro-inflammatory changes (“central perivenulitis”) in 43/47 Worse outcome than cases of “typical” recurrent HCV Pathogenesis 75% had increased serum immunoglobulins and/or autoantibodies (Khettry 2007) In keeping with DNAIH 82% had suboptimal immunosuppression, auto-antibodies only in low titre (Fiel 2008) Probably a form of rejection 40% of patients with plasma cell hepatitis post-LT had plasma cell-rich infiltrates in native liver at transplantation (versus 18% in control group) (Ward 2009) Increasing evidence to suggest that DNAIH and late rejection are part of an overlapping spectrum of immune-mediated graft injury (pathogenesis and histological features)

Hepatitis C in the Liver Allograft Differences Compared with HCV in the Native Liver 1.More aggressive disease –More severe inflammatory activity –more rapid progression to fibrosis and cirrhosis (20- 30% cirrhotic by 5 years, up to 50% by 10 years) 2.Atypical features –Cholestatic features (“fibrosing cholestatic hepatitis”) –Autoimmune features ( de novo AIH / “plasma cell hepatitis”) 3.Interaction with other graft complications –Rejection (acute and chronic) –Non-alcoholic fatty liver disease

Hepatitis C and Rejection Rejection/immunosuppression = risk factors for severe/progressive HCV Immunosuppression allows increased viral replication Predisposes to more severe inflammation when immunosuppression reduced Higher incidence of acute and chronic rejection in HCV-positive patients HCV-associated changes may augment alloimmune-mediated lesions Common patterns of liver injury (e.g.portal inflammation, bile duct damage) Effects of anti-viral therapy (interferon) Stimulation of immune system Improved hepatocellular metabolism of immunosuppressive drugs after viral clearance from hepatocytes

Hepatitis C versus Rejection

Hepatitis C and Acute Rejection (Demetris Am J Surg Pathol 2004, Banff Working Party 2006) AR and recurrent HCV have overlapping histological features (portal inflammation, bile duct inflammation, venous endothelial inflammation) and may occur at the same time In most cases the pattern of inflammation enables the main cause of graft dysfunction to be identified Cases in which distinction between HCV and AR is difficult probably have a dual pathology –In most of these cases rejection changes are mild –HCV best considered as the primary diagnosis –No additional immunosuppression required Increased immunosuppression should be considered as a treatment option if –rejection changes moderate or severe –features suggest progression to (early) chronic rejection

Late Cellular Rejection - Different Histological Features (Snover 1988, Kemnitz 1989, Cakaloglu 1995, Pappo 1995) Portal inflammation predominantly mononuclear Inflammation of bile ducts and venular endothelium less conspicuous More prominent interface hepatitis and lobular inflammation Overall features resemble chronic hepatitis (e.g. viral or autoimmune)

“Idiopathic” chronic hepatitis Common(est) histological diagnosis in late post-transplant biopsies 10-50% of biopsies > 1yr have unexplained inflammation (mainly portal, usually mild) that could be classified as “idiopathic” CH (Shaikh & Demetris 2007) In cases where viral and autoimmune causes have been excluded: May be a form of rejection (late rejection with hepatitic features) A potentially important cause of graft fibrosis –50-70% of children with CH have bridging fibrosis/cirrhosis, 10 year post-transplant (Evans 2006, Herzog 2008) “Idiopathic” Chronic Hepatitis Cirrhosis 5 years post-transplant

Hepatitis C versus Acute Rejection - Other Approaches Immunostaining for HCV Antigens (Grassi 2006, Errico-Grigioni 2008, Sadamori 2009) Higher frequency of staining and greater proportion of positively staining hepatocytes in HCV than rejection. Immunostaining for C4d (Schmeding, 2006, Lorho, 2006,Jain, 2006, Bellamy 2007) Higher frequency of C4d positivity in acute rejection (45-80%) than in recurrent HCV infection (0-12%) Immunostaining for cell cycle entry protein mcm-2 (Unitt 2009) More mcm-2 positive portal lymphocytes in rejection than HCV Immunostaining for MxA protein – marker of type 1 IFN activation (MacQuillan 2010) More hepatocellular expression in HCV than rejection

Hepatitis C versus Acute Rejection - Other Approaches Applications & Limitations of Immunohistochemistry Most studies have looked at “typical” cases of acute rejection and HCV, which are clearly distinguishable by routine microscopy. Problems with reproducibility (HCV tissue detection) Problems with specificity (C4d immunostaining) May help to identify main cause of graft damage in some cases where routine histological findings inconclusive High tissue expression of HCV antigens confirmed diagnosis of HCV hepatitis in 16 patients with inconclusive histological diagnosis (Grassi 2006) Mcm-2 expression pointed to correct diagnosis in 11 HCV infected patients with uncertain diagnosis – 7 HCV, 4 superimposed ACR (Unitt 2009)

Recurrent Hepatitis C Are there predictors of graft outcome? Can one identify risk factors that predispose to more severe disease (rapid progression to fibrosis/cirrhosis/graft failure) ? May have implications for donor/ patient selection prior to transplantation and for post-transplant management (e.g. selecting patients for anti-viral therapy)

Viral FactorsHCV genotype (1b or 4) HCV-RNA levels (pre- and post-transplant) Recipient FactorsAge, female gender Non-white race Co-infection with HIV IL-28b polymorphism TGF beta-1 polymorphism Donor factorsAge White race HLA mis-matching Hepatic iron concentration Steatosis (controversial) Donation after cardiac death (DCD) (controversial) Transplant-related factors Perioperative  warm and cold ischaemic times, preservation/reperfusion injury Acute rejection  severity and number of episodes Immunosuppression  amount/type of immunosuppression given  rapid withdrawal of cortiocosteroids Co-infection with herpesviruses (CMV, HHV-6) Metabolic syndrome/NAFLD Biliary complications Recurrent HCV – Risk Factors for Severe/Progressive Disease

Hepatitis C In Liver Allografts Histological Factors Predictive For Severe/Progressive Disease (rapid progression to advanced fibrosis/cirrhosis) 1.Steatosis 2.Necro-inflammatory activity 3.Hepatocyte apoptosis/replicative senescence 4.Hepatic stellate cell activation 5.Early fibrosis

Hepatitis C In Liver Allografts Histological Factors Predictive For Severe/Progressive Disease (rapid progression to advanced fibrosis/cirrhosis) 1.Steatosis Presence/severity during 1 st month (Pelletier 2000) Presence at 1 year (Ghabril 2011) May reflect viral factors (genotype 3) or host factors (NAFLD) Risk factor for poor response to anti-viral therapy (Testino 2011)

Hepatitis C In Liver Allografts Histological Factors Predictive For Severe/Progressive Disease (rapid progression to advanced fibrosis/cirrhosis) 2.Necro-inflammatory activity Severity at time of first presentation with HCV hepatitis (during 1 st 6 months) (Sreekumar 2000, Guido 2003) Severity in protocol biopsies at 4-12 months also predictive (Gane 1996, Prieto 1999, Firpi 2004)

Hepatitis C In Liver Allografts Histological Factors Predictive For Severe/Progressive Disease (rapid progression to advanced fibrosis/cirrhosis) 3.Hepatocyte apoptosis/replicative senescence Amount of hepatocyte apoptosis in stage F0 biopsies (Meriden 2010) Hepatocellular expression of senescence-associated beta galacosidase in biopsies during 1 st year (Trak-Smyra 2004) Hepatocellular expression of cell cycle entry protein Mcm-2 in early biopsies (median 3 months) (Marshall 2005) Initial cell cycle entry Subsequent cell cycle arrest

Hepatitis C In Liver Allografts Histological Factors Predictive For Severe/Progressive Disease (rapid progression to advanced fibrosis/cirrhosis) 4.Hepatic stellate cell activation MarkerPredictive value/other comments Smooth muscle actin (Guido 1997, Carpino 2005, Gawrieh 2005, Russo 2005) Numbers of SMA-positive cells in protocol biopsies (3-6 months) predicts development of fibrosis at 1-2 years GFA P (Carotti 2008) GFAP – marker of early HSC activation % of GFAP-positive cells in early biopsies correlates with rate of fibrosis progression Vimentin (Meriden 2010) Numbers of vimentin positive cells in stage F0 biopsies predicts rapid progression to F3/F4 fibrosis Mainly portal/periportal, associated with ductular reaction (CK 19 positive cells) “Slow fibroser” 4 month biopsy - immunostaining for SMA (from Russo 2005) “Rapid fibroser”

Hepatitis C In Liver Allografts Histological Factors Predictive For Severe/Progressive Disease (rapid progression to advanced fibrosis/cirrhosis) 5.Early fibrosis Fibrosis stage (Ishak 2-3/6) at 1 year predicts progression to cirrhosis at 5 years (Firpi 2004) Fibrosis stage (Ishak > 2) at 1 year predicts progression to graft failure (Gallegos-Orozco 2009) Digital image analysis to measure collagen proportionate area (CPA) in Sirius red stained sections (Manousou 2011) CPA at one year strongly predictive for subsequent hepatic decompensation (better than Ishak stage or HVPG)

Pathology of Recurrent Hepatitis C – Summary & Conclusions 1.Liver biopsy continues to play an important role in the management of post-transplant HCV. 2.In most cases with typical features, biopsy mainly used to assess disease severity (fibrosis stage). 3.Cases with atypical features (e.g cholestatic HCV, HCV with “autoimmune features”) are more challenging and require careful clinico- pathological correlation. Implications for prognosis and treatment. 4.Hepatitis C and rejection frequently co-exist. Liver biopsy helps to identify the predominant cause of graft damage. 5.Some features seen in early biopsies (protocol or clinically-indicated) appear to be predictive of rapidly progressive graft injury. These may also have therapeutic implications (e.g. early use of anti viral therapy)

Monday 28 April 2008