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

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 year6236 5 year graft survival80%35% Escofet et al. Transplantation 2003;75:344-6

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

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 ReferenceNumber of patientsTime of biopsySubclinical AR (%)Borderline (%) Rush et al, 95251 month2044 3 months1628 6 months128 Seron et al, 97983 months412 Rush et al, 98351 month4311 3 months2721 6 months1529 Jain et al, 00783 months111 Jain et al, 00501 week48 Nankivell et al, 011023 months2949 Shapiro et al, 01281 week2521 Gloor et al, 021143 months311 Roberts et al, 041151 week1312 1 month816

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-MMF1 CsA(S)-azathioprine21.8 CsA(N)-azathioprine22.1 CsA(N)-MMF11 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 scoreChronic score Tacrolimus0.61 ± 1.011.57 ± 1.97 CsA1.26 ± 1.451.51 ± 1.59 p=0.0115p=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 Roberts et al ERA/EDTA 2005 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

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 rejection10 (13%) 8 treated immediately1 of the 2 untreated patients developed CR in <3 days Borderline changes 9 (12%) 4 treated immediately3 of the 5 untreated patients developed CR in <3 days Roberts et al Transplantation 2004;77:1194-98 The incidence of early SCR is less than it appears

15. 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 The incidence of early SCR is less than it appears

16. 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 How common is subclinical antibody-mediated rejection?

17. Koo et al. Transplantation 2004;78:398-403 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

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. 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 Does early SCR contribute to chronic graft injury?

21. 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 Does early SCR contribute to chronic graft injury?

22. 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 Does treatment of SCR improve graft outcome?

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. Assumptions: 1.Early histological evidence of chronic damage predicts chronic graft failure. 2.Intervention in patients with good renal function but CAN on biopsy can prevent/limit progression. Problems: 1.Sampling error: subcapsular scar, vessel disease 2.Stable chronic damage Protocol biopsies and the early diagnosis of CAN

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 months2 years CAN25%50% grade I710 grade II15 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 biopsy54 (35%)100% CAN in 2nd but not 1st39 (25%)81.6% CAN in 1st but not 2nd19 (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:baseline1 year3 years 1.3 ± 1.13.3 ± 1.84.1 ± 2.2 CADI score 1 year 4 Graft failure 3 years04.6%16.7% CADI score predicts graft survival even if normal renal function Yilmaz et al. J Am Soc Nephrol 2003;14:773-9

28. 51 consecutive cadaveric transplant recipients Protocol biopsies at 0, 3, 6, 12 months 3 months6 months12 months24 months CAN4%12%49% Cr Cl56 ± 2 mL/min 56 ± 2 mL/min Baboolal et al. Kidney Int 2002;61:686-96 Temporal relationship between histological CAN & graft function

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. 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 Temporal relationship between histological CAN & graft function

31. Does type of CAN predict chronic graft failure? 3 month protocol biopsies in 280 patients All stable renal function, sCr <300 micromol/L. Number10 year graft survival No CAN17495% CAN (cv0)8782% CAN (cv1+)2141% 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? Sund et al. Nephrol Dial Transplant 2004;19:2838-45 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

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: 1.Progression of CAN - need at least 2 biopsies 2.Type of CAN - cv lesions most significant 3.Severity of CAN - quantitative image analysis better than Banff scoring 4.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.