Meeting summary by Dr. A.J. Demetris of the liver section of 2011 Eleventh Banff Conference on Allograft Pathology, June -10, 2011 in Paris, France.

1. Liver Session Summary

3. Adult Long-TermPathologyM. Sebagh

4. LFTs and biopsy findings at 20 years PPV= 6/33 (18%) NPV = 55/58 (95%)

5. Conclusion Rate of histological abnormalities (90%) Rate of combined insults (31%) Onset of hepatic structural abnormalities Both global and individual histological progression

6. Conclusion Most of the main histological abnormalities might warrant treatment Chronic rejection (23%): increase in immunosuppression Viral chronic hepatitis (46%): introduction of an antiviral treatment IPTH (8.8%): increase in immunosuppression LFD (11%): altered immunosuppression Recurrent diseases (8%): altered immunosuppression Hepatic structural abnormalities (20%): ?

7. Conclusion Partial unreliability of LFTs Partial unreliability of non-invasive markers (TE and FT-AT) despite of adequate samples - for fibrosis but discriminative ability for significant fibrosis (> F2) - for combined disorders Impact on immunosuppressive and antiviral therapies

8. Histological Findings in Paediatric Allograft Biopsies > 1 year Post-Transplant( Protocol Biopsies, > 50% have normal LFTs) Birmingham, Groningen - prevalence & severity of abnormal histology increase with time

10. Late Protocol Biopsies – Biopsy-directed Changes in Immunosuppression(Mells G, Mann C, Hubscher S, Neuberger J. Liver Transplantation 2009 ; 15 : 931-8) 235 protocol biopsies (> 1yr) from adult patients with normal LFTs 76 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)

11. Summary & Conclusions Histological abnormalities are commonly present in late-post transplant biopsies from paediatric liver allograft recipients. Many of changes seen (including graft fibrosis or cirrhosis) are present in children who appear to be clinically well with good graft function. In comparison with adults, children are more prone to develop late rejection biliary complications, de novo AIH, idiopathic chronic hepatitis and nodular changes. Late rejection, de novo AIH and idiopathic CH are probably part of a spectrum of late allo-immune graft injury Idiopathic chronic hepatitis is frequently sub-clinical, but is associated with the development of graft fibrosis and cirrhosis. Further studies are required to investigate the role of protocol liver biopsies in determining therapeutic strategies in children who appear to have good graft function using non-invasive investigations.

12. Liver transplantation & survival Overall survival according to year of transplant in Europe (ELTR)

13. Liver transplantation & survival Survival beyond year-1 in the UK 1994 - 2007 (n = 4483)

14. Mortality beyond year-1 in liver recipients since 1994 (n = 206)

15. ‘Immune senescence’

16. Immune senescence in ‘healthy’ liver recipients Paris, June 2011

17. ‘Routine’ histology

18. Study group Minimal inflammation & steatosis groups: more likely to be: older aetiology related to alcohol, fatty liver, metabolic/genetic or acute Less likely to have: aetiology related to autoimmune liver disease Paris, June 2011

19. Outcome of Kyoto Weaning Protocol (June, 1990- April, 2008) 675 pediatric LDLT 540 survived patients 340 Weaning never been attempted 200 weaning attempted -152 protocol IS weaning -48 non-elective IS off 50 Group-intolerance (24: rejection, 26: fibrosis) 84 Group-tolerance 66 under weaning Ohe H, et al. 2011 Transplantation

20. Operational Tolerance (OT) vs IS group

21. Fibrosis in the graft (Evaluated by Masson-Trichrome)

22. Summary 1) The frequency of both conventional and naïve Tregs was high in OT 2) Both Tregs exerted donor-specific suppressive activity in OT only 3) OT in this population was non-deletional 4) Strikingly, the number of naïve Tregs increased with time after cessation of IS, but not conventional Tregs OT : operational tolerance

23. Summary Not intragraft FOXP3mRNA level, but the increased number of FOXP3 protein positive CD4+ cells characterized OT These cells are accumulated in the portal area

24. Summary 1) In the presence of reintroduction of minimal maintenance IS, most patients with fibrosis exhibited improvement or no progression of fibrosis 2) In single case, fibrosis rapidly progressed in the absence of IS, but such a case was not observed in patients given minimal maintenance IS 3) Thus, fibrosis in IS free patients or patients during weaning process may be at least in part antigen-dependent

25. ITN030: Tapering Status of HCV Recipients

26. ITN030: Tapering Status of Non-HCV Recipients

27. 12 of 20 Participants Met the Primary Endpoint:Off Immunosuppression for 30.0 – 50.7 Months

28. Clinical Factors Associated with Tolerance (1)

29. Clinical Factors Associated with Tolerance (2)

30. Histologic Factors Associated with Tolerance (1)H and E Findings

31. Histologic Factors Associated with Tolerance (2)Total C4d Score P = 0.019 C4d / CD31

32. Correlation of Baseline Total C4d Score and DSA Strength 20 16 12 Total C4d Score 8 Pearson Coefficient = 0.752 P = <0.001 4 Non - Tolerant Tolerant 0 15K 20K 35K 40K 0 5K 10K 25K 30K DSA Strength (MFI)

33. ALT and GGT Profiles of Tolerant Participants:Group A

34. Fibrosis Over Time

35. Alloantibody Evolution During Withdrawal:Five Sensitized and Successful Participants 5 of 10 sensitized participants achieved the 1º endpoint. 4 of 9 participants with DSA achieved the 1º endpoint.

36. 497 Patients were screened 395 Patients were excluded 160 had been transplanted for < 3 years 58 had medical disorders incompatible withthesafeconductofthestudy and/orinterpretationoftheresults. 35 declined to participate 30 were included in another clinical trial 29 had history of autoimmune liver disease 19 had abnormal liver function tests exceeding pre-specified criteria 19 exhibited abnormalities in basal liver biopsy exceeding pre-specified criteria 18 could not be closely followed-up 14 had history of rejection in the previous 12 months 9 had no side effects of IS drugs 4 were already receiving no immunosuppressive drugs 102 Patients were enrolled 4 Patients withdrawn from study during dose reduction classified as Non-TOLERANT in the ITT analysis 57 Patients REJECTED 41 Patients were TOLERANT

37. Patientdemographics

38. Search for the immunological signature of operational tolerance in liver transplantation (clinicaltrials.gov NCT00647283) Multivariableanalysis

39. Intra-graft expression differences between tolerant and non-tolerant grafts Non-TOL TOL FDR <25%

41. The most significant molecular differences between tolerant and non-tolerant recipients are related to genes involved in the regulation of iron metabolism.

42. Hepcidin and hepatocyte iron deposition are likely to be involved in the attenuation of liver-directed alloimmune responses.

43. Other mechanisms must also be implicated in the maintenance of the tolerant state (CD26, ADORA3, ADDSL1)

45. Higher fibrosis stages related to increased Ig, mast cell, T cell transcripts and decreased liver transcripts High fibrosis stage (p=0.008) and high plasma cell transcripts (p=0.070) were both related to late time post transplant. Values represent Spearman's correlation coefficient, p<0.05 Correlation coefficients >+/- 0.50 are in bold. NS, non significant.

46. Conclusions -1 Increased expression of IFNG induced transcripts correlates with Hepatitis C activity and abnormal LFTs. High fibrosis stages and cirrhosis correlate with increased plasma cell and mast cell transcripts. It is yet to be determined whether transcripts can predict progression to fibrosis. (only three progressors present in this dataset)

47. Conclusions -2 As expected, increased expression of IFNG-induced transcripts and T cell transcripts was shared across recurrent HCV, ACR, cirrhosis, and hepatocellular carcinoma. However, NK cell transcripts were selectively increased in recurrent HCV or HCV-related cirrhosis, but not in ACR, dysplasia or hepatocellular carcinoma. In addition, liver transcripts were strikingly lower in ACR and HCC than in hepatitis C and cirrhosis. These preliminary data suggest that transcript measurements (i.e. high NK transcripts without significant loss of liver transcripts) may help to distinguish recurrent hepatitis C from ACR.

48. K Tinckam Tx Review 2009: 80

49. C4d staining decreased with the distance away from the afferent blood supply Portal Tract PV Sinusoids CV HA BD Portal Capillaries Hepatic Artery - 16% Portal Vein - 24% Portal Capillary - 23% Sinusoids – 4% Central Vein – 2% Percentage of early (<3 weeks post-Tx) biopsies irregardless of crossmatch status with either Focal or Diffuse C4d in each anatomical compartment. A similar C4d staining pattern was also seen in late (>3 weeks) biopsies. Lunz 2011

50. Correlation of C4d staining early (<3 weeks) after transplant with histopathological findings Total C4d score exhibited a positive correlation with components of the Rejection Activity Index (RAI) score. C4d+ biopsies also showed an insignificant trend (p=0.26) toward having a higher incidence of acute cellular rejection (63% vs. 55%).

51. Total C4d Score Increases with Crossmatch Score P=0.02 Lunz et al in preparation 2011

52. C4d Stain Pattern in Liver Negative Stromal Nonspecific injury AMR Endothelial Endothelial AMR AMR

53. Summary: Use of C4d Grading of AMR Severe/potentially irreversible AMR is suspected Very high postoperative isoagglutinin titer Use of both CDC and AHG-CDC positive donor Stromal ± endothelial C4d staining Mild/potentially reversible AMR is suspected High postoperative isoagglutinin titer Use of CDC or AHG-CDC positive donor Endothelial only C4d staining Detection of de novo DSA rejection End of the Slides

54. Ductopenic rejection 71 y/o man OLT for A1AT deficiency cirrhosis. POD 22: ACR, resistant to steroids and ATG. POD 43: ductopenia. High DSA class I and II. Diffuse portal C4d. C4d

55. Summary Based on detection of DSA in conjunction with C4d deposition: Humoral alloreactivity -accompanies at least 50% of ACR/ does not require treatment beyond the usual -appears significantly associated with -steroid and even thymoglobulin resistant rejection -ductopenic rejection -identifies a group of patients that are at risk of losing the allograft due to unrelenting, rapidly progressing cholestasis and ductopenia especially early post-transplantation (1.5 % of liver transplants) DSA may serve as a donor-specific marker of immune reactivity to the graft, In assessing the risk of rejection and need for augmented IS and to decide who may tolerate weaning and potentially elimination of immunosuppression

56. Conclusions Modern techniques of detection of DSA and C4d deposition provide further support to earlier observations that humoral alloreactivity -is frequently intertwined with cellular mechanisms of acute and chronic rejection and -may on its own destroy the liver allograft Prospective studies on consecutive patients with protocol biopsies / C4d staining and determination of DSA complement fixing and DSA subclasses are needed to better gauge the contribution of humoral alloreactivity to liver graft damage

57. 17 individual endothelial genes are increased in ABMR Also not in our strict definition of ENDAT list, but increased in ABMR: CDH13 Duffy blood group SOX7 THBD MALL Welch t test FDR 0.05 Red arrows indicate genes that are known to be involved in endothelial cell activation Sis et al. AJT 2009;9:2312-23

58. Differentially expressed transcripts between TG vs. non TG(p<0.05 FDR=0.05)

59. NK cells and macrophages in antibody mediated peritubular capillaritis p=0.03 A. B. p=0.09 C4d+ ABMR p=0.006 C4d- ABMR TCMR Mean number of positive cells in five peritubular capillaries CD56+ CD68+ CD3+ CD3 CD68 CD56 C. D. E. Hidalgo et al. AJT 2010; 10: 1812–1822

60. Consensus Document Table 3. BASELINE OR PRE-WEANING BIOPSY FINDINGS CONDUCIVE TO MINIMIZATON OF IS *Does not include patients with underlying AIH, HCV, PBC or PSC (see text).

61. Consensus Document Table 4. FOLLOW-UP BIOPSY FINDINGS THAT MERIT CONCERN AND CONSIDERATION OF CLOSE FOLLOW-UP DURING OR AFTER WEANING *Does not include patients with underlying AIH, HCV, PBC or PSC (see text).