The European experience with protocol biopsies

1. Oxford Pathology The European experience with protocol biopsies Ian Roberts Oxford, UK

2. Should protocol biopsies be part of routine patient monitoring? What is the evidence from the European experience of protocol biopsies? How does this differ from the American, Asian and Australian experience? How relevant is published data from elsewhere in the world to European practice?

3. Should protocol biopsies be part of routine patient monitoring? Protocol biopsies = allograft biopsies performed at pre-determined time points, irrespective of graft dysfunction. Functions: graft selection, donor disease, baseline for post-transplant pathology, subclinical rejection, early diagnosis and quantification of chronic allograft nephropathy. Risk morbidity associated with allograft biopsy Benefit information that can be acted on to improve graft outcome

4. Impact of disease in the donor kidney: implantation biopsies Biopsies of donor kidneys: 210 transplants, 129 no GS, 81 showed 1-60% GS No GS >20% GS GFR at 1 year 62 36 5 year graft survival 80% 35% Escofet et al. Transplantation 2003;75:344-6

5. Impact of disease in the donor kidney: implantation biopsies Baseline for interpretation of later biopsy changes. Interpretation of sub-optimal function. Choice of immunosuppressive regimen.

6. What level of risk is associated with protocol biopsies? Retrospective audit of 2127 protocol biopsies audited for complications No deaths One graft loss 3 episodes of haemorrhage requiring intervention. 3 further patients requiring transfusion. 2 episodes of peritonitis Furness et al. Transplantation 2003;76:969-73

7. What determines the potential benefit of protocol biopsies? For protocol biopsies performed to diagnose subclinical rejection, the likelihood of benefit to an individual patient is a product of: Frequency of subclinical rejection at the time of protocol biopsy in the patient population X The increased graft longevity to be gained from treating the subclinical rejection

8. Incidence of subclinical rejection Reference Number of patients Time of biopsy Subclinical AR (%) Borderline (%) Rush et al, 95 25 1 month 20 44 3 months 16 28 6 months 12 8 Seron et al, 97 98 3 months 4 12 Rush et al, 98 35 1 month 43 11 3 months 27 21 6 months 15 29 Jain et al, 00 78 3 months 1 11 Jain et al, 00 50 1 week 4 8 Nankivell et al, 01 102 3 months 29 49 Shapiro et al, 01 28 1 week 25 21 Gloor et al, 02 114 3 months 3 11 Roberts et al, 04 115 1 week 13 12 1 month 8 16

9. Impact of immunosuppression on subclinical rejection 119 SPK recipients Subclinical rejection 60.% at 1 month, 45.7% at 3 months No significant impact of immunosuppression on subclinical rejection at 1 month. Protocol biopsies 3-12 months: odds ratio Tacrolimus-MMF 1 CsA(S)-azathioprine 21.8 CsA(N)-azathioprine 22.1 CsA(N)-MMF 11 Nankivell et al Transplantation 2004;78: 242-49

10. Impact of immunosuppression on subclinical rejection Case-control study 98 patients Tacrolimus-MMF-prednisolone vs CsA-MMF-prednisolone 4 months protocol biopsies scored using Banff 97: Acute score Chronic score Tacrolimus 0.61 ± 1.01 1.57 ± 1.97 CsA 1.26 ± 1.45 1.51 ± 1.59 p=0.0115 p=ns Relative risk of acute inflammation with Tacrolimus 0.3 (CI 0.11-0.84) Moreso et al Transplantation 2004;78: 1064-8

11. Impact of immunosuppression on subclinical rejection Comparison of subclinical rejection in two eras: Group 1 (1992-5) received CsA, azathioprine and prednisolone Group 2 (2001-3) stratified at transplantation according to immunological ris: High risk: basiliximab induction, tacrolimus, MMF & pred Medium risk: CsA, MMF & pred Low risk: CsA, azathioprine & pred Roberts et al ERA/EDTA 2005

12. Impact of immunosuppression on subclinical rejection Roberts et al ERA/EDTA 2005

13. The incidence of early SCR is less than it appears SCR = histological changes meeting recognised criteria for acute rejection in patients with good, stable graft function. but this also applies to pre-clinical and post-clinical rejections. True SCR = immune-mediated graft injury, not associated with acute graft dysfunction if untreated.

14. Day 7 protocol biopsies Acute rejection 10 (13%) 8 treated immediately 1 of the 2 untreated patients developed CR in <3 days Borderline changes 9 (12%) 4 treated immediately 3 of the 5 untreated patients developed CR in <3 days The incidence of early SCR is less than it appears Roberts et al Transplantation 2004;77:1194-98

15. The incidence of early SCR is less than it appears Day 28 protocol biopsy 10/13 patients who had subclinical borderline changes had been treated for acute rejection in the previous 3 weeks Roberts et al. Transplantation 2004;77:1194-98 Protocol biopsies 10 days after treatment of clinical acute rejection. Over 25% of patients with full clinical response to anti-rejection therapy have persistent histological signs of rejection. Mazzucchi et al. Transplantation 1999;67:430-34

16. How common is subclinical antibody-mediated rejection? Day 7 protocol biopsies in 37 living donor recipients C4d positivity in 11 (30%) 9/11 showed clinical rejection 6/11 showed histological rejection 2/37 (6%) subclinical C4d positivity Sund et al. Transplantation 2003;75:1204-13

17. How common is subclinical antibody-mediated rejection? 48 day 7 protocol biopsies (Cad/LRD 41/7) C4d positivity in 6 (13%) 5/6 showed clinical and histological rejection all 5 with rejection had post-transplant DRA One patient C4d positive, no histological rejection, no circulating alloantibodies, stable graft function (ie. doesn’t meet Banff criteria for humoral rejection) - never treated for rejection, sCr 104 micromol/L at 3 months Koo et al. Transplantation 2004;78:398-403

18. How common is subclinical antibody-mediated rejection? Multicentre study, 551 protocol biopsies Diffuse C4d positivity in 2% Focal C4d positivity in 2.4% Inter-centre variation reflecting variable numbers of pre-sensitised patients. Subclinical C4d positivity had no significant impact on graft outcome. Mengel et al. Am J Transplant 2005;5:1050-56

19. How common is subclinical antibody-mediated rejection? Does subclinical C4d positivity equate to subclinical antibody-mediated rejection? C4d positivity not due to antibody deposition (false positive). Accommodation?

20. Does early SCR contribute to chronic graft injury? 120 recipients of kidney-pancreas grafts Protocol biopsies at 1 & 2 weeks, 1, 3, 6 & 12 months, annually thereafter. 70% of patients suffered clinical or SCR/borderline changes in first 3 months. Patients with acute SCR in first 3 months showed higher grades of CAN in subsequent biopsies than did patients with no SCR (2.48 ±0.25 vs 0.32 ±0.09). But: 3 month risk of SCR was increased by a previous episode of severe rejection (hazard ratio 2.46), no data on treatment of SCR. Nankivell et al. N Engl J Med 2003;349:2326-33

21. Does early SCR contribute to chronic graft injury? Day 28 protocol biopsies: 19 patients with subclinical rejection or borderline changes. 4 treated immediately, 3 for clinical rejection within one week. 12 never subsequently received treatment for rejection: 1 late graft failure at 6 years post-transplantation 2 patients died with functioning grafts (sCr 159 & 168) 9 had functioning grafts at 6 years median sCr 119 umol/l (range 90-185) Roberts et al Transplantation 2004;77:1194-98

22. Does treatment of SCR improve graft outcome? 72 patients randomised to protocol biopsies 1, 2, 3, 6 and 12 months vs 6 and 12 months only. In the biopsy group, 80% of 1 month biopsies showed either acute rejection or borderline changes. Sub-clinical rejection treated. Early biopsy group had less chronic damage in 6 month biopsies and lower serum creatinine at 2 years than controls. Rush et al. J Am Soc Nephrol 1998;9:2129-34

23. Protocol biopsies and the early diagnosis of CAN Rationale: Renal scarring on histology is an earlier/more sensitive marker of chronic damage than is serum creatinine/GFR - by the time renal function deteriorates, it’s too late.

24. Protocol biopsies and the early diagnosis of CAN Assumptions: Early histological evidence of chronic damage predicts chronic graft failure. Intervention in patients with good renal function but CAN on biopsy can prevent/limit progression. Problems: Sampling error: subcapsular scar, vessel disease Stable chronic damage

25. How common is early CAN? 41 patients (10 HLA identical LRD, 31 cadaveric donor kidneys). Protocol biopsies at 3 months and 2 years. No CAN in recipients of HLA identical kidneys. Cadaveric kidneys: 3 months 2 years CAN 25% 50% grade I 7 10 grade II 1 5 CAN correlates with donor age Legendre et al. Transplantation 1998;65:1506-09

26. Is sampling error a problem in CAN diagnosis? 310 protocol biopsies from 155 patients. Protocol biopsies at 4 & 14 months, follow-up 5-12 years. graft survival No CAN in either biopsy 54 (35%) 100% CAN in 2nd but not 1st 39 (25%) 81.6% CAN in 1st but not 2nd 19 (12%) 82.6% CAN in both 43 (28%) 69.4% Assuming CAN does not regress, 25% of biopsies are misclassified, greater than increase in CAN from 4-14 months. Seron et al. Kidney Int 2002;61:727-33

27. Does early CAN predict chronic graft failure? 621 protocol biopsies in two multicentre MMF trials Protocol biopsies at baseline, 1 & 3 years. 1 year: sCr (>1.5 mg/100ml) in 20%, CADI score (>2) in 60% Mean CADI score: baseline 1 year 3 years 1.3 ± 1.1 3.3 ± 1.8 4.1 ± 2.2 CADI score 1 year <2 2-3.9 >4 Graft failure 3 years 0 4.6% 16.7% CADI score predicts graft survival even if normal renal function Yilmaz et al. J Am Soc Nephrol 2003;14:773-9

28. Temporal relationship between histological CAN & graft function 51 consecutive cadaveric transplant recipients Protocol biopsies at 0, 3, 6, 12 months 3 months 6 months 12 months 24 months CAN 4% 12% 49% Cr Cl 56 ± 2 mL/min 56 ± 2 mL/min Baboolal et al. Kidney Int 2002;61:686-96

29. Does early CAN predict chronic graft failure? 57 living donor transplants Protocol biopsy at day 0 (n=57), 1 week (n=43) & 1 year (n=33) Poor correlation between arteriosclerosis in day 0 and 1 week biopsies (sampling error). Glomerulosclerosis, interstitial fibrosis and tubular atrophy, but not arteriosclerosis, increased from baseline to 1 year. Extent of chronic tubulointerstitial damage in 1 year biopsies, but not baseline biopsies, correlated with sCr at 1 & 3 years. Best predictor of renal function at 3 years was sCr at 1 year. Sund et al. Nephrol Dial Transplant 1999;14:2445-54

30. Temporal relationship between histological CAN & graft function 258 patients Protocol biopsies at 6, 12 and 26 weeks In multivariate analysis, CAN in biopsy 3 (n=70) predicted by: CAN in biopsy 2 Lower GFR at biopsy 2 Nephrocalcinosis Acute rejection Renal functional decline preceded morphological changes of CAN Schwarz et al 2005;67:341-48

31. Does type of CAN predict chronic graft failure? 3 month protocol biopsies in 280 patients All stable renal function, sCr <300 micromol/L. Number 10 year graft survival No CAN 174 95% CAN (cv0) 87 82% CAN (cv1+) 21 41% Seron et al. Transplantation 2000;69:1849-55

32. Can we do better than traditional histology? Computerised image analysis with Sirius Red stain Protocol biopsies in 68 patients at 6 months Volume of interstitial fibrosis correlated better with time to graft failure (r=0.64) than did Banff chronic and acute sum scores (r=0.28 & 0.35). Multivariate model including all of the above was highly correlated with time to graft failure (r=0.7, p=<0.0001). Grimm et al. J Am Soc Nephrol 2003;14:1662-8

33. Can we do better than traditional histology? • TGF-beta • Protocol biopsies in 40 patients at day 0, 1 wk, 6 mnths • IF for active TGF-beta & semiquantitative scanning laser confocal microscopy. • active TGF-beta from day 0 to 1 wk to 6 mnths (p<0.0001) DGF  TGF-beta, but no association with acute rejection or IS drug (CsA vs Tac). No correlation between TGF-beta expression at any timepoint and isotope GFR at 12 months. Jain et al. Transplant Int 2002;15:630-4

34. Can we do better than traditional histology? Other techniques with potential: computerised morphometry combined with IH for: type III collagen tenascin smooth muscle actin

35. Can we do better than traditional histology? Computerised image analysis (Sirius red) vs Banff scoring Protocol biopsies in 33 patients at day 0, 1 year Sirius red (Vint) correlates with Banff chronic score r=0.439, p=0.0003 1 year biopsies: Banff chronic score correlated with 8-10 year sCr, p=0.01, and with late graft loss, p=0.0445 Sirius red (Vint) showed similar trend but did not reach statistical significance Sund et al. Nephrol Dial Transplant 2004;19:2838-45

36. Can we do better than traditional histology? Other techniques with potential: quantification of mast cells Roberts et al. J Clin Pathol 2000;53:858-62

37. Can we do better than traditional histology? Other techniques with potential: quantification of mast cells Roberts et al. J Clin Pathol 2000;53:858-62

38. CAN as a predictor of graft outcome CAN alone is not a useful label. What matters is: Progression of CAN - need at least 2 biopsies Type of CAN - cv lesions most significant Severity of CAN - quantitative image analysis better than Banff scoring Immune activity

39. Does intervention in subclinical CAN make a difference? Assumption 2: Intervention in patients with good, stable renal function but CAN on biopsy can prevent/limit progression. Evidence? Would you change immunosuppression in a patient with good stable graft function on the basis of some minor chronic changes in the biopsy?

40. Should protocol biopsies be part of routine patient management? Baseline biopsies: yes - useful in the interpretation of subsequent pathology. Post-transplantation: maybe, but must be clear on why they are being performed and have a protocol for acting on the results of the biopsies. Subclinical rejection: overall benefit depends on the frequency of SCR in the unit. The benefit of treating SCR remains unproven. Early CAN: a good early surrogate marker for graft survival in clinical trials. Relevance to management of individual patients unclear.