D. SerónNephrology DepartmentHospital Vall d’HebronBarcelona

Interpretation of sequential protocol biopsies

in terms of

prognosis and clinical implications

1 2 3 4 5 years

300

250

200

150

100

50

0

BiopsySCr mol/l

Lesions are too advanced

The biopsy was done too late

There is nothing we can do

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

Characteristics of early routine renal allograft biopsies

Protocol biopsies done at 1-4 weeksQuantification of interstitial infiltrates

with a morphometric technique in HE stained biopsies

Diagnosis N cel/mm2 interstitium__________________________________________ATN in native kidney 9 451 101Stable function 4 1290 179Post-transplant ATN 7 1335 182Acute rejection 5 2269 215*__________________________________________

Burdick JF et al, Transplantation 1984; 38: 679

Protocol biopsies

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

CAN in (2y) protocol biopsies predicts renal function deterioration

Graft function deterioration: SCr >20% at 2-4yCADI: interstitial inflammation & fibrosis + glomerular sclerosis +mesangial matrix increase + vascular intimal proliferation + tubular atrophy

Stable Deteriorated_________________________________________CADI 1.79 1.89 5.67 2.94 <0.0001_________________________________________

N = 94 patients

Isoniemi H, Transplantation 1994

Chronic lesions at 6m and graft survival

0 1 2 3years

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

IF/TA is an independent predictor of graft survival

Serón D, Kidney Int 1997: 51:310

0 1 2 3 years

100

80

60

40

20

0

4 5 6 7

CAN=41

Normal=53

p=0.024

N=94 patients

% graft survival

RR 95% CI

_____________________________SCr 1.026 (1.005-1.0047)

(mol/l)CAN 5.98 (1.15-31.25)(yes vs. no) _____________________________

Sirius red derived VIntFib and time to graft failure

Grimm PC et al, J Am Soc Nephrol 2003

CAN, transplant vasculopathy and survival 3 m protocol Bx, n=282

0

20

40

60

80

100

0 20 40 60 80 100 120 meses

p < 0.001

% deatt censored graft survival

Normal

IF/TA (cv-score 1

Serón D, Transplantation 2000; 69: 1849

Univariate Multivariate

Variable RR 95%CI RR 95%CI

__________________________________SCr (mol/l) 1.009 (1.001-1.016) -

Prot (g/l) 1.002 (1.001-1.004) -

IF/TA 4.64 (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

SCR + IF/TA and graft survival95 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

Predicting decline in allograft functionBiopsy 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

SCR, CAN and graft survivalProtocol Bx < 6m; n=435

Moreso F et al Am J Transplant 2006; 6: 747

.25

.5

.75

1

0 50 100 150 200 months

Normal=186

SCR=74

IF/TA=110

IF/TA+SCR=65

Function and structure are independent predictors of outcome

Moreso F et al. AJT 2006; 6: 747

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

Surrogate Category Accuracy Sensitivity Specificity

______________________________________________________________

Acute rejection yes 72% 30% 80%

3-month SCr >1.8 mg/dl 73% 58% 76%

Protocol biopsy IF/TA 67% 65% 67%

Protocol biopsy IF/TA + cv-score 1 81% 21% 92%

Protocol biopsy IF/TA + SCR 78% 31% 86%

______________________________________________________________

Seron D & Moreso F. Kidney Int 200; 72:690

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

Variable Follow up Obs %Failed OR CI AUC_____________________________________________________________________________SCr at 1y 2y 74480 7.2 2.22 2.13-2.31 0.627SCr at 1y 7y 35255 45.2 2.4 2.31-2.50 0.624

_____________________________________________________________________________

Kaplan B et al. AJT 2003; 3: 1560

While renal function is a strong risk factorand highly correlated with graft failure, theutility of renal function as a predictive toolfor graft loss is limited

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 yn=430 early protocol BX

Unpublished observation

Histology is not only a predictive variable but a surrogate variable

SRL and CsA withdrawal

Randomization 3m: n = 430

SRL+CsA, n = 215 SRL, n = 215

SRL> 5 ng/mL

CsA150 - 400

ng/mL

Steroids+ +

N = 525

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

Benefit Risk

Questions

How much contributes one protocol biopsy to predict outcome?

Two sequential protocol biopsies improve the predictive value of histology

Questions

How much contributes a protocol biopsy to predict outcome?

Two Sequential protocol biopsies improve the predictive value of histology?

Inclusion criteria

Protocol Bx < 6m

GFR (MDRD4) > 30 ml/min/1.73 m2

Proteinuria < 1g/day

Stable function

> 5 years of follow up

Protocol Bx > 12-24 m

EARLY Prot Bx LATE Prot Bx

Patients and biopsiesjune 88-december 2003

Bx < 6m 458 Bx 12-24m 250

Bx < 6m 430with tissue

Bx 12-24m 231with tissue

PREDICTIVE VALUE OF

ONE BIOPSY

n=430

Statistical approach

Cox proportional hazard model

a.) Predictive clinical variables

b.) Predictive clinical and histological variables

Characteristics of patientsn=430

Donor age 37±17Donor sex (%male) 70%Recipient age 46±14Recipient sex (%male) 63%PRA (%) 7.5±19HLA DR mm 0.63±0.58CIT (h) 22±6Retransplantation 64/430 (17.5%)VHC 16%DGF 17%Acute rejection 19%Graft loss 146 (33.2%) Death censored graft loss 104 (24.2%)

Time of biopsy (months) 4.3±1.7

GFR ml/min/1.73m2 53±14Proteinuria g/d 0.30±0.21

Histological data at the time of biopsyn=430

______________________________N glomeruli 13±8N arteries 5±4g 0.15±0.48i 0.58±0.68t 0.38±0.61v 0.01±0.11ah 0.16±0.45Acute score 1.13±1.31cg 0.13±0.34ci 0.46±0.64ct 0.45±0.62cv 0.20±0.54mm 0.25±0.45Chronic score1.24±1.65______________________________

Clinical variables and death censored graft survival

Variable Univariate Multivariate

RR (95% CI) P RR (95% CI) p

Donor age 1.013 (1.002-1.025) 0.027 1.02 (1.006-1.034) 0.004

Recipient age 0.98 (0.97-0.99) 0.015 0.96 (0.95-0.98) 0.000

PRA (%) 1.014(1.007-1.021) 0.000 1.011 (1.003-1.020) 0.008

GFR (ml/min) 0.97 (0.96-0.99) 0.000 0.98 (0.96-0.99) 0.004

HCV pos 2.29 (1.49-3.52) 0.000 1.56 (0.94-2.58) ns

Proteinuria mg/d 1.001 (1.000-1.002) 0.041 1.001 (1.000-1.001) ns

SCR - IF/TA

No SCR - IF/TA

SCR - no IF/TA

No SCR - no IF/TA

,5

,6

,7

,8

,9

1

Cum

. S

urv

ival

0 50 100 150 200 250

Time (months)

P = 0.037

SCR - IF/TA

no SCR - IF/TA

Histological diagnosis and graft survival

Clinical variables and death censored graft survival

Variable Univariate Multivariate

RR (95% CI) P RR (95% CI) p

Donor age 1.013 (1.002-1.025) 0.027 1.02 (1.007-1.034) 0.003

Recipient age 0.98 (0.97-0.99) 0.015 0.97 (0.95-0.98) 0.001

PRA (%) 1.014(1.007-1.021) 0.000 1.011 (1.003-1.020) 0.008

GFR (ml/min) 0.97 (0.96-0.99) 0.000 0.98 (0.96-0.99) 0.009

HCV (pos) 2.29 (1.49-3.52) 0.000 1.62 (0.99-2.67) ns

Proteinuria mg/d 1.001 (1.000-1.002) 0.041 1.001 (1.000-1.001) ns

SCR-IF/TA 1.92 (1.18-3.12) 0.009 1.75 (1.06-2.89) 0.029

Is it worth to include histology in multivariate models to predict graft

survival?

The contribution of histology to predict death-censored graft failure

Donor age Recipient age, PRA, GFR

Clinical variables

Clinical + histologicalvariables

Donor ageRecipient agePRAGFRHistology

Model 1 Model 2

The contribution of histology to predict death-censored graft failure

Donor age Recipient age, PRA, GFR

Clinical variables

Clinical + histologicalvariables

Donor ageRecipient agePRAGFRHistology

Model 1 Model 2

The contribution of histology to predict death-censored graft failure

Donor age yearsRecipient age yearsPRA %GFR ml/min/1.73m2

Histology yes/no

First classification of acute rejction

The contribution of histology to predict death-censored graft failure

Donor age yearsRecipient age yearsPARA %GFR ml/minHistology yes/no

risk

Beta coefficient of Cox regression model to calculate risk scores

Variable β coefficient β coefficientwithout histology with 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/TA n.a. +0.559 (+55.9) ____________________________________________________

H(t)=H0(t) x exp (β1x1+ β2x2+ β3x3+…+ βkxk)

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)

Q1 Q2Q3 Q4

Classification of patients according to risk scores

Risk score

Q1 Q2 Q3 Q4

Q1 99 5 1 O

Q2 6 92 6 2

Q3 0 9 81 16

Q4 0 0 18 87

With histologyW

ith

ou

t h

isto

log

y

p<0.0001

Changes in quartile classification due to inclusion of histology in the

statistical model:

15%

0

,2

,4

,6

,8

1

Cum

. S

urvi

val

0 50 100 150 200 250

Time

Q4

Q3

Q2

Q1

Death censored graft failure using quartiles of risk scores

Without histology

0

,2

,4

,6

,8

1

Cum

. S

urvi

val

0 50 100 150 200 250

Time

Q4

Q3

Q2

Q1

With histology

months months

Validation

Modelling sample

Testing sample

TWO BIOPSIES<6m and 12-24m

N=231

Two sequential biopsies N=231

6m 12-24m_____________________________________________Time of biopsy (M) 4.3±1.7 16.5±6.0

GFR ml/min/1.73m2 53±14 52±15 ns

Proteinuria g/d 0.30±0.21 0.37±0.49 0.01______________________________________________

Acute score Chronic score

P=0.0001P=0.003

progression

Reliability of IF/TA diagnosis

I II III

Protocol Bx

regression

2nd without 2nd with IF/TA IF/TA

_____________________________________

1st without IF/TA 54 (34.8%) 39 (25.2%)

1st with IF/TA 19 (12.2%) 43 (27.7%)

_____________________________________

N

Serón D et al. KI 2002; 61:727

Error associated with the diagnosis of IF/TA in sequential protocol biopsies

Progression to IF/TA 25.2%Regression of IF/TA 12.2%

25%

Sampling + intraobserver error

Serón D et al. KI 2002; 61:727

Two sequential Bx

1st Bx1st diagnosis

2ndBx2nd diagnosis

Integrated diagnosis

Prediction of graft survival

Interpretation of sequential protocol biopsies

in terms of prognosis and clinical implications Normal 2nd SCR 2nd IF/TA 2nd SCR+IFTA

2nd

Normal 1st 53 5 34 16

SCR 1st 15 4 12 7

IF/TA 1st 16 1 26 9

SCR+IF/TA 1st 9 1 15 8

IF/TA

IF/TA + SCR

SCR

normal

Two sequential biopsies (integrated diagnosis)

(n = 231)

,5

,6

,7

,8

,9

1

Cum

. Surv

ival

0 50 100 150 200 250 300

Time

IF/TA - SCR

IF/TA - no SCR

No IF/TA - SCR

No IF/TA - no SCR

p=0.04

SCR-IF/TANo SCR-IF/TASCR-noIF/TANoSCR-noIF/TA

,5

,6

,7

,8

,9

1

0 50 100 150 200 250 300

Time

P = 0.12

,5

,6

,7

,8

,9

1

0 50 100 150 200 250 300

Time

P = 0.29

Early and late biopsies(n=231)

Early Late

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

Acknowledgements

F MoresoD Hernandez

M HuesoC Fernandez Gamiz

M GomàJM CruzadoO BestardJM GrinyoM Carrera