1. C4d in pancreas transplant biopsies: the Leiden experience Ingeborg Bajema Ingeborg Bajema Ingeborg Bajema

2. Introduction Taking biopsies from pancreas transplants was uncommon until the early 1990s when a technique of percutaneous needle biopsy was introduced Each year, circa 3000 pancreas transplants are performed worldwide, 75% in the setting of a SPKT, mostly for patients with diabetes type 1 Some hospitals follow serum creatinine as a surrogate markers for pancreas rejection

3. Pancreas rejection: Drachenberg et al, Am J Transpl 2008 Working proposal for grading pancreas allograft rejection 1.Normal 2.Indeterminate 3.Cell-mediated rejection (grade I, II, III) 4.Antibody-mediated rejection 5.Chronic allograft rejection/graft sclerosis 6.Other histological diagnosis (e.g. CMV pancreatitis, PTLD)

4. Pancreas rejection: Drachenberg et al, Am J Transpl 2008 Working proposal for grading pancreas allograft rejection 1.Normal 2.Indeterminate 3.Cell-mediated rejection (grade I, II, III) 4.Antibody-mediated rejection 5.Chronic allograft rejection/graft sclerosis 6.Other histological diagnosis (e.g. CMV pancreatitis, PTLD) The role of a C4d staining in relation to AMR is only beginning to be described

5. Pancreas rejection: Drachenberg et al, Am J Transpl 2008 Working proposal for grading pancreas allograft rejection Antibody-mediated rejection: Complement deposition in vessels (i.e. capillary C4d deposition) which can be accompanied by monocyte/macrophage and neutrophil margination within interstitial capillaries Graft dysfunction Donor-specific antibodies (DSA) in serum

6. Melcher in 2006 were the first to report a patient with a SPKT who developed AMR with donor-specific HLA-DR allo- antibodies and had positive C4d staining in the pancreas one month after a SPKT. The renal transplant biopsy of this patient taken at day 10 was also positive for C4d. Overview of literature: Melcher, Am J Transplant, 2006 pancreas kidney

7. Carbajal et al a year later reported a patient with pancreas after kidney transplantation (PAK) who developed AMR of the pancreas with C4d positivity in the biopsy. The kidney, which was from a different donor, remained unaffected, but the pancreas graft was lost Overview of literature: Carbajal et al, NDT 2007

8. Gaber reported one case of a C4d positive surveillance biopsy taken 2 weeks after transplantation from a patient who was sensitized by two previous islet transplants and had detectable anti-HLA antibodies Overview of literature: Gaber, Arch Pathol Lab Med 2007

9. Pascual reported 13 patients with an acute rejection of the pancreas of which 2 were biopsy-proven, and these were positive for C4d staining. In one patient, two biopsies of the pancreas were taken. DSA were negative at the time of the first biopsy, which was C4d positive. Three months later, a second biopsy was taken which was C4d positive as well, and by that time DSA were also positive Overview of literature: Pascual, JASN 2008

10. Torrealba et al reported on the potential role of C4d in pancreas transplant biopsies with AMR in a group of 18 patients 27 biopsies from 18 patients Correlation between C4d+, development of DSA and graft dysfunction There is potential value of C4d staining in the diagnosis of AMR of pancreas allografts Overview of literature: Torrealba et al, Transplantation 2008

11. C4d+ can be encountered in various clinical settings: 2/4 patients with C4d+ had typical AMR In one patient, C4d+ was associated with recurrent diabetes Another patient (with circulating DSA), remained euglycemic after 12 months of follow-up and without treatment of AMR Overview of literature: comment by Munivenkatappa et al. to Torrealba et al, Transplantation 2009

12. …. there is perhaps a spectrum of clinical scenarios, tissue evidence of antibody activation, and serologic patterns that still need to be defned to better understand AMR as an entity in pancreas allograft. Overview of literature: reply from Torrealba et al. To comment by Munivenkatappa et al., Transplantation 2009

13. Whole pancreas transplantation at LUMC 109 pancreas transplants between 1991 – 2001 All SPKT Pancreas transplant biopsies were taken on clinical indication until 2001 19 biopsies from pancreas transplants in this study 26 pre-transplant biopsies

14. Pancreas transplant biopsies from LUMC 19 biopsies from 18 patients 17/19 biopsies taken because of clinically suspected rejection 10 renal biopsies taken at the same time H&E slides scored according to BANFF working proposal C4d immunostaining was performed on 4 μm paraffin sections C4d positivity: Diffuse: > 50% of interacinar capillaries positive staining Focal: 5 to 50% of interacinar capillaries positive staining Minimal: < 5% of interacinar capillaries positive staining Of 13/18 patients, DSA assessment was available

15. C4d staining patterns negativediffusely + focally +

16. Recipient characteristics Mean age: 38.9 years (SD: 8.3) Gender, % male: 67% Years of DM1 at Tx: 6.9 years % of women pregnant before Tx: 67% Biopsy taken at day (mean, range): 97 (12-481) DSA: negative in 6, positive in 7

17. Results 2 / 24 pre-transplant biopsies showed diffuse C4d staining pattern

18. R=rejection o=no rejection n =negative F=focal positive D=diffuse positive Rest (83%) > 5yr graft survival Results

19. R=rejection o=no rejection n =negative F=focal positive D=diffuse positive Rest (83%) > 5yr graft survival Results

20. R=rejection o=no rejection n =negative F=focal positive D=diffuse positive Rest (83%) > 5yr graft survival Results

21. R=rejection o=no rejection n =negative F=focal positive D=diffuse positive Rest (83%) > 5yr graft survival Results

22. R=rejection o=no rejection n =negative F=focal positive D=diffuse positive Rest (83%) > 5yr graft survival Results

23. C4d staining in simultaneous pancreas and kidney biopsies (case 8) focally +

24. A cooperation between LUMC and UMMC UMMC: 148 pancreas transplantations between 2000 and 2007 37 pancreas transplant biopsies, taken on clinical indication

25. Results

26. AMR and graft survival 27 patients with C4d and DSA results available were categorized into 3 groups: I: minimal or no C4d staining, no DSA (N=13) II: diffuse C4d staining, no DSA (N=2); focal C4d staining and DSA (N=2); minimal/no C4d staining and DSA (2) III: Diffuse C4d staining and DSA (N=9)

27. Results

28. Conclusion C4d+ in the pancreas is independent of Banff classification C4d staining patterns can differ per organ (kidney/pancreas) C4d positivity alone, has no effect on pancreas survival 1 patient  DSA, Banff and C4d positive  pancreas lost

29. Conclusions -Diffuse C4d positivity with concurrent DSA had poor graft survival, most likely related to AMR -Patients did well who had: -either DSA or C4d alone -or no DSA or C4d -The sole occurrence of either C4d or DSA poses no additional risk for poor outcome of the pancreas graft

30. Conclusions The results from our study emphasize the importance of taking into account a combination of C4d staining pattern, histological diagnosis and presence of DSA before a diagnosis of AMR of the pancreas is made

31. Acknowledgements LUMC, the Netherlands Department of Pathology Hanneke de Kort Jan A. Bruijn Emile de Heer Department of Nephrology Eelco de Koning Cees van Kooten Stefan Berger Department of ImmunoHematology and Blood Transfusion (IHB) Michael Eikmans Univ. of Maryland, School of Medicine, Baltimore, USA Department of Pathology Raghava Munivenkatappa Cinthia Drachenberg Benjamin Philosophe All doctors who contributed patients