1. S.G. Hübscher, Department of Pathology, University of Birmingham, Birmingham B15 2TT, U.K. The Spectrum of Findings in Protocol and Indicated Biopsies in Long-term Liver Allograft Recipients (with an emphasis on diagnostic challenges)

2. Histological findings in 1045 biopsies obtained > 1 year post-transplant, Liver Unit, QE Hospital, Birmingham Data from Liver Unit Database, Jan 2004 – Jan 2009 Reason for biopsy - 720 (69%) protocol, 325 (31%) clinically indicated

3. Histological findings in 1045 biopsies obtained > 1 year post-transplant, Liver Unit, QE Hospital, Birmingham Clinically Indicated versus Protocol Biopsies Data from Liver Unit Database, Jan 2004 – Jan 2009

4. Histological findings in late post-transplant biopsies Liver Unit, QE Hospital, Birmingham Data from Liver Unit Database, Jan 2004 – Jan 2009

5. Long-term Liver Allograft Pathology. How Frequently Does the Graft Remain Normal? CentreNo of biopsies Length of follow-up Normal histology Normal histology with: LFTs N LFTs raised Changing prevalence with time Pittsburgh (Pappo 1995) 65>5years34%64%25% KCH London (Slapak 1997) 116> 5years23%54%13% Valencia (Berenguer 2001) 254> 1 year44-58%--58% normal at 1 year 44% normal at 5 years Paris (Sebagh 2003) 143> 10years20%28%10%44 % normal at 5 years 20% normal at 10 years Several cases classified as normal had “mild non-specific changes”

6. Histological Findings in Protocol Biopsies from Patients with Normal LFTs InstitutionTime of biopsy No of biopsies Biopsies Abnormal Abormalities found Mayo Clinic (Abraham 2008) 3 months - 5 yrs 16527% Fatty liver disease (11%), recurrent disease (10%), rejection/central perivenulitis (7%), other (2%) (32% = “non-specific changes”)* Dallas (Khan, ILTS, Paris, July 2008) 1-20 yrs>4000< 5% had inflammation Acute rejection (2-3%), recurrent HCV (0.5-2%) London (Kings) (Bachina, ILTS, Paris, July 2008) > 10 yrs1392% Fibrosis (92%) – mod/severe in 30%, lymphocytic infiltration of variable severity (54%) Birmingham (Mells 2009, in press) 1-10 yrs237 76% Unexplained CH (33%), recurrent disease (23%), fatty liver disease (14%), other (5%) * Includes portal and lobular lymphocytic infiltration

7. Factors Influencing the Histology of Late Post-transplant Biopsies 1.Nature of original liver disease 2.Indication for liver biopsy (protocol or clinically indicated) 3.Duration of follow up 4.Type/amount of immunosuppression used 5.Diagnostic criteria/terminology

8. Main Pathological Changes in Biopsies >12 Months Post-transplant Rejection Less common than in early post-transplant period May have different histological features Worse outcome Recurrent disease General issues Assessment of biopsies from HCV-positive individuals De novo disease General issues De novo autoimmune hepatitis Other findings in late biopsies “ Idiopathic” chronic hepatitis Vascular/structural abnormalities

9. Main Pathological Changes in Biopsies >12 Months Post-transplant Rejection Less common than in early post-transplant period May have different histological features Worse outcome Recurrent disease General issues Assessment of biopsies from HCV-positive individuals De novo disease General issues De novo autoimmune hepatitis Other findings in late biopsies “ Idiopathic” chronic hepatitis Vascular/structural abnormalities

10. Acute Cellular Rejection – Typical Histological Features Portal inflammation (mixed population)Bile duct inflammation Venous endothelial inflammation

11. 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 as a manifestation of late rejection) More recent studies suggest that central perivenulitis may also be a feature of late cellular rejection (Banff Working Party, Hepatology 2006; 44: 489-501) Central perivenulitis can occur without typical portal changes. Isolated CP seen in: –28% of adult patients (protocol biopsy) > 3years post-transplant ( Krasinskas 2008) –22% of paediatric patients > 3 months post-transplant (Abraham 2008)

12. Liver Allograft Rejection - “Isolated Central Perivenulitis” Liver biopsy, 6 months post-transplant. Worsening LFTs (AST 2xN) Other possible causes of zone 3 necroinflammation Autoimmune hepatitis - recurrent AIH -de novo AIH Viral hepatitis (recurrent or acquired) - HBV - HCV

13. Central Perivenulitis in Liver Allograft Rejection – Clinical Correlations Present later than cases with pure portal rejection Often associated with raised transaminase levels Less responsive to immunosuppression Increased risk of developing “adverse outcomes”: –Further episodes of acute rejection –Chronic (ductopenic) rejection –De novo AIH –Centrilobular fibrosis (Krasinskas 2001,Gouw 2002, Neil 2002,Sebagh 2002, Lovell 2004, Hassoun 2004, Junge 2005, Riva 2006, Sundaram 2006, Krasinskas 2008, Abraham 2008, Demirhan 2008)

14. Main Pathological Changes in Biopsies >12 Months Post-transplant Rejection Less common than in early post-transplant period May have different histological features Worse outcome Recurrent disease General issues Assessment of biopsies from HCV-positive individuals De novo disease General issues De novo autoimmune hepatitis Other findings in late biopsies “ Idiopathic” chronic hepatitis Vascular/structural abnormalities

15. Disease Recurrence in the Liver Allograft DISEASEFREQUENCY HEPATITIS B< 10% (up to 85% in earlier studies) HEPATITIS C> 80% PBC30-50% PSC20-30% AUTOIMMUNE HEPATITIS20-30% ALCOHOL 10 - 30% NASH (“cryptogenic” cirrhosis)20-40% Recurrent disease = commonest cause of late graft dysfunction

16. Disease Recurrence in Liver Allografts -Diagnostic Problems (1) Recurrent disease and other transplant complications (A)Histological Similarities Hepatitis Cv Acute rejection PBC/PSCvChronic rejection PSCvIschaemic biliary complications (B)Other Interactions Higher incidence of rejection (acute and chronic) in: –patients transplanted for autoimmune liver disease –recurrent HCV Changes seen in late biopsies often reflect more than one pathological process Clinical picture often complex Histology may help to identify the dominant cause of graft damage

17. Disease Recurrence in Liver Allografts -Diagnostic Problems (2) Effects of immunosuppression LESS aggressive disease- immune - mediated disease (e.g. AIH, PBC) MORE aggressive disease-viral hepatitis (atypical patterns)(e.g. HBV,HCV)

18. Hepatitis C in the Liver Allograft Differences Compared with HCV in the Native Liver More aggressive disease –More severe inflammatory activity (more rapid progression to fibrosis and cirrhosis) –Cholestatic features (fibrosing cholestatic hepatitis) Hepatitis C and rejection Hepatitis C with “autoimmune features” (? de novo AIH)

19. Recurrent Hepatitis C prominent lobular inflammation with zone 3 necrosis Are these changes related to HCV alone? or HCV + another graft complication - rejection with central perivenulitis - de novo AIH

20. Aggressive Recurrent HCV Male, age 52. 21 months post-LT for HCV Antiviral therapy recently stopped because of nephric abscess Presented with acutely deranged LFTs (AST 650) Became HCV-RNA positive

21. 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) In most cases the time of occurrence and pattern of inflammation enable a reasonably confident diagnosis to be made 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

22. 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. Poor correlation with HCV-RNA levels (Errico-Grigioni 2008, Sadamori 2009). High tissue expression helpful in confirming diagnosis of HCV hepatitis in 16 patients with inconclusive histopathological diagnosis (Grassi 2006). Problems with reproducibility, tissue processing (2/3 studies used frozen sections). Immunostaining for C4d (Schmeding, 2006, Lorho, 2006,Jain, 2006) Higher frequency of C4d positivity in acute rejection (45-80%) than in recurrent HCV infection (0-12%) BUT specificity for rejection not confirmed in other studies (e.g Bellamy 2007), nor has utility in cases with overlapping features of HCV and rejection.

23. Hepatitis C versus Acute Rejection - Other Approaches Immunostaining for Mcm-2 (Unitt Liver Transpl 2009; 15: 306-312) Mcm-2 expression also helpful in 11 HCV-infected patients with uncertain diagnosis 7 eventually diagnosed as HCV, 4 as superimposed corticosteroid responsive ACR

24. Hepatitis C with “Autoimmune Features” ( de novo AIH) Following Interferon Therapy (Cholongitas 2006,Kontorinis 2006,Berardi 2007,Merli 2009) 14 cases – all had features compatible with de novo AIH 12/14 were HCV-RNA negative 10/14 responded to treatment with immunosuppression Unrelated to Interferon Therapy (Khettry 2007, Fiel 2008) 47 patients - plasma cell rich portal and lobular infiltrates (“plasma cell hepatitis”) Central perivenulitis in 43/47 Worse outcome than cases of “typical” recurrent HCV –Progression to cirrhosis, retransplantation or death 75% had increased serum immunoglobulins and/or autoantibodies (Khettry 2007) 82% had suboptimal immunosuppression, auto-antibodies in 61% (low titre) –probably a form of rejection (Fiel 2008)

25. Main Pathological Changes in Biopsies >12 Months Post-transplant Rejection Less common than in early post-transplant period May have different histological features Worse outcome Recurrent disease General issues Assessment of biopsies from HCV-positive individuals De novo disease General issues De novo autoimmune hepatitis Other findings in late biopsies “ Idiopathic” chronic hepatitis Vascular/structural abnormalities

26. De Novo Disease in the Liver Allograft DISEASEDE NOVO OCCURRENCE Hepatitis BYES Hepatitis CYES PBCNO PSC? (ischaemic cholangitis resembles PSC)* Autoimmune HepatitisYES AlcoholPossible NASH YES (several risk factors, including recurrent HCV) * One possible case, 5 years post-LT (McPartland Path Int 2009; 59: 312-316)

27. ‘De Novo’ Autoimmune Hepatitis in the Liver Allograft (Kerkar 1998, Jones 1999, Gupta 2001, Heneghan 2001, Salcedo 2002, D’Antiga 2002, Miyagawa- Hayashino 2004, Mieli-Vergani 2004, Aguilera 2004 & 2005, Riva 2006, Avitzur 2007, Rodriguez- Mahou 2007, Vernick 2007, Salcedo 2009). 1.Classical biochemical, serological and histological features of AIH may develop in patients transplanted for other diseases 2.Higher prevalence in paediatric population (5-10%), compared with adults (1-2%) ? Immunosuppressive drugs interfering with normal T cell maturation 3.Most cases respond to increased immunosuppression. Occasional cases have progressed to graft failure 4.Areas of overlap between de novo AIH and rejection - antibodies to graft antigens may indicate an alloimmune response - de novo AIH could represent a form of late cellular rejection 5.Areas of overlap/interaction between de novo AIH and hepatitis C

28. Chronic Hepatitis in the Liver Allograft Features favouring an autoimmune aetiology Portal inflammation with numerous plasma cells Prominent interface hepatitis Lobular inflammation (plasma cell rich) with zone 3 necrosis Lobular changes in de novo AIH more prominent than in AIH in the native liver (Salcedo 2002, Aguilera 2004)

29. Main Pathological Changes in Biopsies >12 Months Post-transplant Rejection Less common than in early post-transplant period May have different histological features Worse outcome Recurrent disease General issues Assessment of biopsies from HCV-positive individuals De novo disease General issues De novo autoimmune hepatitis Other findings in late biopsies “ Idiopathic” chronic hepatitis Vascular/structural abnormalities

30. Main Pathological Changes in Biopsies >12 Months Post-transplant “Idiopathic” chronic hepatitis Common(est) histological diagnosis in late post-transplant biopsies In cases where viral and autoimmune causes have been excluded, could this be a form of rejection? (late rejection may have hepatitic features) A potentially important cause of graft fibrosis

31. Chronic Hepatitis in the Liver Allograft Portal Inflammatory Changes

32. Chronic Hepatitis in the Liver Allograft Lobular Inflammatory Changes

33. Chronic Hepatitis in the Liver Allograft -Periportal Fibrosis Mild (fibrous portal expansion) Moderate (bridging fibrosis) Severe (cirrhosis)

34. Chronic Hepatitis in the Liver Allograft – Centrilobular Fibrosis

35. Chronic Hepatitis in Late Post-Transplant Biopsies Is this a form of late cellular rejection? In the adult population, excluding recurrent disease as a cause for chronic hepatitis is difficult –Most of the diseases for which transplantation carried out in adults have the potential to recur, with features of chronic hepatitis HBV, HCV, AIH, PBC, PSC –Histological features of “non-specific” chronic hepatitis may precede other diagnostic abnormalities of recurrent disease (AIH and PBC)

36. Chronic Hepatitis (Interface Hepatitis) in Paediatric Liver Transplant Patients Birmingham (Evans 2006) Montreal (Herzog 2008) Number of children158119 Biopsy indicationsProtocol biopsies at 1,5, 10 yearsClinically indicated biopsies Prevalence/time of presentation with chronic hepatitis 22% at 1year 43 % at 5 years, 64 % at 10 years 24% ( median 2 years) Progressive fibrosis (by 10 years) 37% -bridging fibrosis 15% - cirrhosis 35% -bridging fibrosis 35% - cirrhosis Other findings70-80% had auto-antibodies Only 6% diagnosed as de novo AIH (AST levels < 2x normal) No association with auto-antibodies 55% had features of chronic rejection ( Evans. Hepatology 2006; 43: 1109-1117, Herzog Liver Transpl 2008; 14: 946-955 )

37. Graft Histology in Long-Term Survivors of Paediatric Liver Transplantation Ekong, Liver Transplantation 2008; 14: 1582-1587 (Northwestern University, Chicago) 63 biopsies from children surviving > 3 years post-LT 39 normal LFTs, 24 clinically indicated 01234 Portal inflammation Batts &Ludwig 1995 19261620 Fibrosis stage METAVIR, Bedossa 1996 23017140 Aetiology uncertain. Only 6/63 had features suggestive of chronic rejection. Factors correlating with > stage 3 fibrosis –Time post-transplant > 6years –Inflammatory grade > 2 –NO correlation with abnormal LFTs

38. Unexplained chronic hepatitis in adults following liver transplantation (Syn WK, Nightingale P, Gunson B, Hubscher SG, Neuberger JM. Liver Transplantation 2007, 13: 984-9) 288 adults transplanted without features of disease recurrence (ALD, drug-induced liver failure) Chronic hepatitis present in 46/143 (32%) patients biopsied > 6 months post-transplant. Median time of diagnosis 15 months (6-72) No obvious correlation with AST levels 43% developed progressive fibrosis in follow-up biopsies – median 4 years (10% cirrhotic) Factors correlating with fibrosis progression –High titre autoantibodies (ANA > 1:1600) –Plasma cell rich infiltrate in index biopsy

39. Graft Monitoring Routine LFTs unreliable Role for protocol biopsies Autoantibody testing (particularly in children) Treatment (immunosuppression to prevent disease progression?) criteria for treatment monitoring therapeutic responses patients transplanted for hepatitis C Many patients have mild changes with no evidence of fibrosis Does mild (non-progressive) portal hepatitis represent a form of graft tolerance? Can liver biopsy help to identify patients in whom immunosuppression can be reduced or withdrawn? Chronic Hepatitis with “Autoimmune Features” Clinical Implications

40. Protocol Liver Allograft Biopsies Mells G, Neuberger JM. Transplantation 2008; 85: 1686-1692 Survey of 35 liver transplant centres (North America 19, Europe 13, Other 3) –23 (65%) protocol annual biopsies for HCV-positive patients –9 (25%) protocol biopsies for other patients Reasons for discontinuing protocol biopsies –Risk of potentially serious complications –Cost –Non-invasive alternatives (e.g. to assess fibrosis) –Problems with histological interpretation –Findings don’t influence clinical management Healy’s Law (From Knisely & Thompson 2008) “Don’t order a test if you lack the facts to know how to interpret the result” Healy B, N Engl J Med 1997; 336: 1448-9

41. Late Protocol Biopsies in the Liver Allograft – A Neglected Investigation? Mells G, Mann C, Hubscher S, Neuberger J. Liver Transplantation 2009, in press 235 biopsies > 1yr post –LT from 176 patients (ALD 49, AIH 20, PBC 107) All had normal LFTs at time of biopsy ALD (n=60) AIH ( n= 28) PBC (n=147) TOTAL (n=235) Normal18%29%26%24% Fatty liver42%14%4%14% Idiopathic CH28%18%38%33% Rec disease (AIH/PBC)N/A39%29%23% De novo AIH2%N/A3%2% Other10%01%3% Idiopathic Chronic Hepatitis Cases (n=78) Inflammatory grade – mild 75 (96%), moderate 3 (4%), severe 0 Fibrosis stage – none 29 (37%), mild 41 (53%), moderate 8 (10%), severe (0)

42. Late Protocol Biopsies in the Liver Allograft – A Neglected Investigation? Mells G, Mann C, Hubscher S, Neuberger J. Liver Transplantation 2009, in press 76/235 cases had change in immunosuppression after biopsy 11 increased (active inflammation in protocol biopsy) 58 reduced (lack of inflammation in protocol biopsy) 7 switched to CNI-sparing regime (active inflammation and renal impairment)

43. Idiopathic Post-Transplantation Hepatitis Following LDLT Miyagawa-Hiyashino Transpl Int 2009: 22: 303-312 IPTH in 42/944 (4.4%) liver allograft recipients (NO protocol biopsies) Higher prevalence in children (6.5%) than adults (1.3%) Associated Factors Previous acute rejection (82%) Positive auto-antibodies (68%) –no differences between patients with low ( 1: 160) titres Outcome (serial biopsies in 29 patients) 8 - progressive fibrosis (5 developed cirrhosis & retransplanted) 21 - improvement in fibrosis (possibly due to increase in immunosuppression after diagnosis of IPTH)

44. Unexplained Chronic Hepatitis or Periportal Fibrosis in Late Post-Transplant Biopsies Are there other causes?

45. Chronic Hepatitis E Virus Infection in Liver Allograft Recipients Haagsma 2008, Kamar 2008, Gerolami 2008 11 transplant patients (5 liver, 4 kidney, 2 kidney/pancreas) All 11 patients developed biochemical and histological features of chronic hepatitis, with persistently elevated serum HEV-RNA levels Outcome variable: –2 patients retransplanted with severe fibrosis/cirrhosis (Haagsma 2008) –Other 9 patients clinically well with persistently abnormal LFTs (raised AST/ALT). 7/9 have METAVIR fibrosis stage < 2

46. Graft Fibrosis after Paediatric Liver Transplantation: Ten Years of Follow-up Scheenstra Hepatology 2009; 49: 880-886 (Groningen) 39/55 (69%) children biopsied at 10 years had fibrosis –17 mild, 11 bridging, 16 cirrhosis Associated with abnormal LFTs (ALP, GGT) Risks Factors –Young age of recipient –High donor/recipient age ratio –Cold ischaemia time –Reduced size graft Factors NOT associated with fibrosis –Rejection –Chronic hepatitis (only 2 patients had unexplained CH)

47. Main Pathological Changes in Biopsies >12 Months Post-transplant Rejection –Less common than in early post-transplant period –May have different histological features Recurrent disease –General issues –Assessment of biopsies from HCV-positive individuals De novo disease –General issues –De novo autoimmune hepatitis Other findings in late biopsies –“Idiopathic” chronic hepatitis –Vascular/structural abnormalities

48. Nodular Regenerative Hyperplasia Male age 46 – protocol biopsy 12 months post-transplant for Wilson’s disease

50. Perisinusoidal Fibrosis

51. Frequency2% - 82% Possible Causes (impaired sinusoidal blood flow) –Vascular problems (portal/hepatic venous insufficiency) –Drug toxicity (azathioprine) –Immune mediated (rejection related damage to sinusoidal/vascular sinusoidal endothelium) Clinical Consequences –6/26 cases reported from KCH London required retransplantation (Slapak Hepatology 1997; 25: 195-202) –7/14 cases from Mayo Clinic symptomatic with features of portal hypertension (ascites -7, varices- 4) (Devarbhavi Liver Transpl. 2007;13:1552-6.) –Others noted as incidental finding Nodular changes in late post-transplant biopsies (Pappo 1995, Slapak 1997, Sebagh 2003, Devarbhavi 2007)

52. Late Post-Transplant Biopsies – Future Studies 1.HCV with features of rejection or autoimmune hepatitis 2.Biopsies with (mild) unexplained inflammatory changes Terminology (chronic hepatitis vs other terms) Characterising the nature of the inflammatory infiltrate (alloimmune or tolerogenic, fibrogenic cytokine profiles) In cases thought to be alloimmune (with fibrosis) - clinical trial of immunosuppression vs no immunosuppression. 3.Nodular changes morphological spectrum and natural history ? more common in reduced-sized grafts

53. Banff 2009 – The Year of the Hat