1. D. Serón Nephrology Department Hospital Vall d’Hebron Barcelona Interpretation of sequential protocol biopsies in terms of prognosis and clinical implications

2. 12345years 300 250 200 150 100 50 0 Biopsy SCr  mol/l

4. Lesions are too advanced The biopsy was done too late There is nothing we can do

5. The assumption that renal allograft histology should be perfectly normal during quiescence has not been adequately investigated Burdick JF et al, Transplantation 1984; 38: 679

6. Characteristics of early routine renal allograft biopsies Protocol biopsies done at 1-4 weeks Quantification of interstitial infiltrates with a morphometric technique in HE stained biopsies DiagnosisNcel/mm 2 interstitium __________________________________________ ATN in native kidney9451  101 Stable function41290  179 Post-transplant ATN71335  182 Acute rejection52269  215* __________________________________________ Burdick JF et al, Transplantation 1984; 38: 679

7. Protocol biopsies Are lesions observed in protocol biopsies relevant from the clinical point of view?

8. CAN in (2y) protocol biopsies predicts renal function deterioration Graft function deterioration:  SCr >20% at 2-4y CADI: interstitial inflammation & fibrosis + glomerular sclerosis +mesangial matrix increase + vascular intimal proliferation + tubular atrophy StableDeteriorated _________________________________________ CADI1.79  1.895.67 2.94 <0.0001 _________________________________________ N = 94 patients Isoniemi H, Transplantation 1994

9. Chronic lesions at 6m and graft survival 01 23 years 100 80 60 40 20 0 CGD<6 (n=54) CGD>6 (n=35) p=0.0009 Dimény E, Clin Transplantation 1995; 58(11): 1195 N = 89 patients %graft survival

10. IF/TA is an independent predictor of graft survival Serón D, Kidney Int 1997: 51:310 0 123years 100 80 60 40 20 0 4567 CAN=41 Normal=53 p=0.024 N=94 patients % graft survival RR95% CI _____________________________ SCr 1.026 (1.005-1.0047) (  mol/l) CAN 5.98(1.15-31.25) (yes vs. no) _____________________________

11. Sirius red derived V IntFib and time to graft failure Grimm PC et al, J Am Soc Nephrol 2003

12. CAN, transplant vasculopathy and survival 3 m protocol Bx, n=282 0 20 40 60 80 100 020406080100120 meses p < 0.001 % deatt censored graft survival Normal IF/TA (cv-score  1 Serón D, Transplantation 2000; 69: 1849 UnivariateMultivariate VariableRR 95%CI RR 95%CI __________________________________ SCr (  mol/l)1.009 (1.001-1.016)- Prot (g/l)1.002 (1.001-1.004)- IF/TA4.64 (1.44-14.95)4.53(1.39-14.82) IF/TA (cv-score)13.61(3.73-49.62)9.45 (2.32-38.41) IF/TA

13. SCR + IF/TA and graft survival 95 pediatric recipients from a living donor Shishido et al, JASN 2003; 14: 1046 IF/TA without SCR IF/TA with SCR Normal 1 year protocol Bx

14. Predicting decline in allograft function Biopsy at 1 year (living 69%), Tx 1998-2001, n=292 Primary endpoint: death censored graft loss or > 50%  GFR beyond 1y Cosio FG et al, Am J Transplant 2005

15. SCR, CAN and graft survival Protocol Bx < 6m; n=435 Moreso F et al Am J Transplant 2006; 6: 747.25.5.75 1 050100150200 months Normal=186 SCR=74 IF/TA=110 IF/TA+SCR=65

16. Function and structure are independent predictors of outcome Moreso F et al. AJT 2006; 6: 747

17. Predictive value of clinical variables and different histological patterns on 7 y death censored graft survival n=361 pts, protocol Bx before 6 m, follow up > 7y SurrogateCategoryAccuracySensitivitySpecificity ______________________________________________________________ Acute rejectionyes72%30%80% 3-month SCr>1.8 mg/dl73%58%76% Protocol biopsyIF/TA67%65%67% Protocol biopsyIF/TA + cv-score  181%21%92% Protocol biopsy IF/TA + SCR78%31%86% ______________________________________________________________ Seron D & Moreso F. Kidney Int 200; 72:690

18. Poor predictive value of serum creatinine for renal allograft loss 1st RT > 17y, 1988-1999, at least 2y follow up SCr > 1.8 mg/dl VariableFollow upObs%FailedORCI AUC _____________________________________________________________________________ SCr at 1y 2y744807.22.222.13-2.310.627 SCr at 1y7y3525545.22.42.31-2.500.624 _____________________________________________________________________________ Kaplan B et al. AJT 2003; 3: 1560 While renal function is a strong risk factor and highly correlated with graft failure, the utility of renal function as a predictive tool for graft loss is limited

19. GFR AUC = 0.679 (0.581 - 0.777); p=0.001 Banff score AUC = 0.685 (0.598 - 0.771); p=0.001 ROC AUC for graft failure at 5 y n=430 early protocol BX Unpublished observation

20. Histology is not only a predictive variable but a surrogate variable

21. SRL and CsA withdrawal Randomization 3m: n = 430 SRL+CsA, n = 215 SRL, n = 215 SRL > 5 ng/mL CsA 150 - 400 ng/mL Steroids ++ N = 525

22. Oberbauer R, Transpl Int 2005; 1: 22 A reduction in CADI score is associated with improved survival Mota A et al., AJT 2004; 4: 953

23. Benefit Risk

24. Questions How much contributes one protocol biopsy to predict outcome? Two sequential protocol biopsies improve the predictive value of histology

25. Questions How much contributes a protocol biopsy to predict outcome? Two Sequential protocol biopsies improve the predictive value of histology?

26. Inclusion criteria Protocol Bx < 6 m GFR (MDRD4) > 30 ml/min/1.73 m2 Proteinuria < 1 g/day Stable function > 5 years of follow up Protocol Bx > 12-24 m EARLY Prot Bx LATE Prot Bx

27. Patients and biopsies june 88-december 2003 Bx < 6m458Bx 12-24m250 Bx < 6m 430 with tissue Bx 12-24m 231 with tissue

28. PREDICTIVE VALUE OF ONE BIOPSY n=430

29. Statistical approach Cox proportional hazard model a.) Predictive clinical variables b.) Predictive clinical and histological variables

30. Characteristics of patients n=430 Donor age37±17 Donor sex (%male)70% Recipient age46±14 Recipient sex (%male)63% PRA (%)7.5±19 HLA DR mm0.63±0.58 CIT (h)22±6 Retransplantation64/430 (17.5%) VHC16% DGF17% Acute rejection 19% Graft loss 146 (33.2%) Death censored graft loss104 (24.2%) Time of biopsy (months) 4.3±1.7 GFR ml/min/1.73m253±14 Proteinuria g/d0.30±0.21

31. Histological data at the time of biopsy n=430 ______________________________ N glomeruli13±8 N arteries5±4 g0.15±0.48 i0.58±0.68 t0.38±0.61 v0.01±0.11 ah0.16±0.45 Acute score1.13±1.31 cg0.13±0.34 ci0.46±0.64 ct0.45±0.62 cv0.20±0.54 mm0.25±0.45 Chronic score1.24±1.65 ______________________________

32. Clinical variables and death censored graft survival VariableUnivariateMultivariate RR (95% CI)P p Donor age1.013 (1.002-1.025)0.0271.02 (1.006-1.034)0.004 Recipient age0.98 (0.97-0.99)0.0150.96 (0.95-0.98)0.000 PRA (%)1.014(1.007-1.021)0.0001.011 (1.003-1.020)0.008 GFR (ml/min)0.97 (0.96-0.99)0.0000.98 (0.96-0.99)0.004 HCV pos2.29 (1.49-3.52)0.0001.56 (0.94-2.58)ns Proteinuria mg/d1.001 (1.000-1.002)0.0411.001 (1.000-1.001)ns

33. SCR - IF/TA No SCR - IF/TA SCR - no IF/TA No SCR - no IF/TA,5,6,7,8,9 1 Cum. Survival 050100150200250 Time (months) P = 0.037 SCR - IF/TA no SCR - IF/TA Histological diagnosis and graft survival

34. Clinical variables and death censored graft survival VariableUnivariateMultivariate RR (95% CI)P p Donor age1.013 (1.002-1.025)0.0271.02 (1.007-1.034)0.003 Recipient age0.98 (0.97-0.99)0.0150.97 (0.95-0.98)0.001 PRA (%)1.014(1.007-1.021)0.0001.011 (1.003-1.020)0.008 GFR (ml/min)0.97 (0.96-0.99)0.0000.98 (0.96-0.99)0.009 HCV (pos)2.29 (1.49-3.52)0.0001.62 (0.99-2.67)ns Proteinuria mg/d1.001 (1.000-1.002)0.0411.001 (1.000-1.001)ns SCR-IF/TA1.92 (1.18-3.12)0.0091.75 (1.06-2.89)0.029

35. Is it worth to include histology in multivariate models to predict graft survival?

36. The contribution of histology to predict death-censored graft failure Donor age Recipient age, PRA, GFR Clinical variables Clinical + histological variables Donor age Recipient age PRA GFR Histology Model 1 Model 2

37. The contribution of histology to predict death-censored graft failure Donor age Recipient age, PRA, GFR Clinical variables Clinical + histological variables Donor age Recipient age PRA GFR Histology Model 1 Model 2

38. The contribution of histology to predict death-censored graft failure Donor ageyears Recipient ageyears PRA% GFRml/min/1.73m 2 Histologyyes/no

39. First classification of acute rejction

41. The contribution of histology to predict death-censored graft failure Donor ageyears Recipient ageyears PARA% GFRml/min Histologyyes/no risk

42. Beta coefficient of Cox regression model to calculate risk scores Variableβ coefficient β coefficient without histologywith histology ___________________________________________________ Donor age (year)+0.020(+2.0)+0.020 (+2.0) Patient age (year)-0.035(-3.5)-0.035 (-3.5) GFR (ml/min)-0.024(-2.4)-0.022 (-2.2) PARA (%)+0.011(+1.1)+0.011 (+1.1) SCR&IF/TAn.a.+0.559 (+55.9) ____________________________________________________ H(t)=H 0 (t) x exp (β 1 x 1 + β 2 x 2 + β 3 x 3 +…+ β k x k )

43. Beta coefficient of Cox regression model to calculate risk scores Risk score without histology =(2*Donor age)+ (-3.5*patient age)+ (-2.4*GFR)+ (1.1*PRA) Risk score with histology =(2*Donor age)+ (-3.5*patient age)+ (-2.2*GFR)+(1.1*PRA)+(55.9*SCR&IF/TA)

44. Q1Q1 Q2Q2 Q3Q3 Q4Q4 Classification of patients according to risk scores Risk score

45. Q1Q2Q3Q4 Q19951O Q269262 Q3098116 Q4001887 With histology Without histology p<0.0001

46. Changes in quartile classification due to inclusion of histology in the statistical model: 15%

47. Death censored graft failure using quartiles of risk scores Without histologyWith histology months

48. Validation Modelling sample Testing sample

49. TWO BIOPSIES <6m and 12-24m N=231

50. Two sequential biopsies N=231 6m12-24m _____________________________________________ Time of biopsy (M) 4.3±1.716.5±6.0 GFR ml/min/1.73m253±1452±15ns Proteinuria g/d0.30±0.210.37±0.490.01 ______________________________________________

51. Acute scoreChronic score P=0.0001 P=0.003

52. progression Reliability of IF/TA diagnosis I II III Protocol Bx regression 2 nd without 2 nd withIF/TA _____________________________________ 1 st without IF/TA54 (34.8%)39 (25.2%) 1 st with IF/TA 19 (12.2%)43 (27.7%) _____________________________________ N Serón D et al. KI 2002; 61:727

53. Error associated with the diagnosis of IF/TA in sequential protocol biopsies Progression to IF/TA25.2% Regression of IF/TA12.2% 25% Sampling + intraobserver error Serón D et al. KI 2002; 61:727

54. Two sequential Bx 1st Bx 1st diagnosis 2ndBx 2nd diagnosis Integrated diagnosis Prediction of graft survival

55. Interpretation of sequential protocol biopsies in terms of prognosis and clinical implications Normal 2ndSCR 2ndIF/TA 2ndSCR+IFTA 2nd Normal 1st5353416 SCR 1st154127 IF/TA 1st161269 SCR+IF/TA 1st91158 IF/TA IF/TA + SCR SCR normal

56. Two sequential biopsies ( integrated diagnosis) (n = 231),5,6,7,8,9 1 Cum. Survival 050100150200250300 Time IF/TA - SCR IF/TA - no SCR No IF/TA - SCR No IF/TA - no SCR p=0.04

57. SCR-IF/TA No SCR-IF/TA SCR-noIF/TA NoSCR-noIF/TA,5,6,7,8,9 1 050100150200250300 Time P = 0.12,5,6,7,8,9 1 050100150200250300 Time P = 0.29 Early and late biopsies (n=231) Early Late

58. Comments Histology contributes to better define patients at risk for graft failure Two sequential biosies done 1 year apart increase the predictive value of histology on graft failure

59. Acknowledgements F Moreso D Hernandez M Hueso C Fernandez Gamiz M Gomà JM Cruzado O Bestard JM Grinyo M Carrera