1. Current problems in kidney transplantation: Clinical point of view
Stefan Schaub
Transplantation Immunology and Nephrology
University Hospital Basel, Switzerland
Many problems…….

2. 50% 50% Allograft loss Recipient death Allograft with functioning
failure
50%
50%
Age!!
Cardiovascular
Infection
Malignancy
I would like to start with a study from El-Zoghby, who investigated 153 graft losses from a population of 1317 transplants from Based on the clinical course and allograft biopsy results they determine why the allograft failed. As you can see on the slide, the leading cause for allograft failure could be identified in 90% of cases. The second important information on this slide is that allograft rejection is still the leading cause for allograft loss even with current immunosuppression. The question is, can we identify these pathological processes earlier allowing for a timely intervention to prevent deterioration of allograft function and loss.

3. Why do renal allograft fail?
Unknown
Acute rejection
10%
12%
Medical, surgical
16%
„Chronic“ rejection
24%
I would like to start with a study from El-Zoghby, who investigated 153 graft losses from a population of 1317 transplants from Based on the clinical course and allograft biopsy results they determine why the allograft failed. As you can see on the slide, the leading cause for allograft failure could be identified in 90% of cases. The second important information on this slide is that allograft rejection is still the leading cause for allograft loss even with current immunosuppression. The question is, can we identify these pathological processes earlier allowing for a timely intervention to prevent deterioration of allograft function and loss.
(Recurrent) GN
22%
IF-TA: other,
specified causes
CNI-toxicity 8% 1%
PyVAN 7%
Adapted from El-Zoghby. AJT, 2008

4. How to prevent acute / chronic rejection?
1) Avoid transplantation in high risk constellations
(e.g. preformed donor-specific memory)
2) Screening for early / subclinical rejection
I would like to start with a study from El-Zoghby, who investigated 153 graft losses from a population of 1317 transplants from Based on the clinical course and allograft biopsy results they determine why the allograft failed. As you can see on the slide, the leading cause for allograft failure could be identified in 90% of cases. The second important information on this slide is that allograft rejection is still the leading cause for allograft loss even with current immunosuppression. The question is, can we identify these pathological processes earlier allowing for a timely intervention to prevent deterioration of allograft function and loss.

5. How to prevent acute / chronic rejection?
1) Avoid transplantation in high risk constellations
(e.g. preformed donor-specific memory)
2) Screening for early / subclinical rejection
I would like to start with a study from El-Zoghby, who investigated 153 graft losses from a population of 1317 transplants from Based on the clinical course and allograft biopsy results they determine why the allograft failed. As you can see on the slide, the leading cause for allograft failure could be identified in 90% of cases. The second important information on this slide is that allograft rejection is still the leading cause for allograft loss even with current immunosuppression. The question is, can we identify these pathological processes earlier allowing for a timely intervention to prevent deterioration of allograft function and loss.

6. HLA-antibodies as a surrogate for memory
Pregnancy
Transfusion
Transplant Tn
Naive B-cell
IgM positiv
Naive T-cell Bn
Durch Transplantate, SS und Bluttransfusionen kann sich das menschliche Immunsystem gegen fremde HLA-Moleküle sensibilisieren. Meistens umfasst diese HLA-spezifische Immunantwort sowohl die zelluläre und die humorale Immunantwort. Da HLA-Antikörper viel einfacher zu detektieren und messen sind als HLA-spezifische T-Zellen, fungieren HLA-Antikörper als genereller Surrogatmarker für eine Sensibilisierung. Somit ist auch klar, dass hinter jedem IgG HLA-Antikörper auch memory T-Zellen mit der gleichen Spezifität stecken.
Activated T-cell Ta
Plasma cell
IgG positiv Tm PC
Memory T-cell
IgG HLA-Ab 6 6

7. Luminex Multiplex technology
bead A1 A2 A3
A25
A24
A11 B7 B8
B27
B62
B52
B51
Color-coded beads
Flow cytometer
Data

8. Clinical relevance of HLA-DSA
detected by Luminex
Author
Year N
DSA+
AMR
Graft survival
Patel
2007 60 20 ↑ =
Gupta
2008
121 16 ↓
Berg Loonen 34 13
Aubert
2009
114 11
Amico
334 67
Wahrmann
338 39
Vlad
325 27
Lefaucheur
2010
402 76
Willicombe
2011
480 45
Caro-Oleas
2012
892 50
Otten
837
290
Durch Transplantate, SS und Bluttransfusionen kann sich das menschliche Immunsystem gegen fremde HLA-Moleküle sensibilisieren. Meistens umfasst diese HLA-spezifische Immunantwort sowohl die zelluläre und die humorale Immunantwort. Da HLA-Antikörper viel einfacher zu detektieren und messen sind als HLA-spezifische T-Zellen, fungieren HLA-Antikörper als genereller Surrogatmarker für eine Sensibilisierung. Somit ist auch klar, dass hinter jedem IgG HLA-Antikörper auch memory T-Zellen mit der gleichen Spezifität stecken. 8 8

9. Complex biology… 1. Magnitude and durability of the humoral memory
T-cell
2. Binding strength of HLA-DSA to the target epitope
3. Capacity of HLA-DSA to activate complement
5. Protective factors and ‚absorptive capacity‘
of endothelial cells
1. Magnitude and durability
of the humoral memory
response
4. Density of
HLA-molecule
expression
B-cell
Plasma cell
Complement
anti-HLA-antibodies
Donor
HLA
Amico P. Curr Opin Organ Transplant 2009 9 9

10. Organ allocation HLA-antibodies No HLA-antibodies
Try to transplant around DSA
- Acceptable mismatch program
- Living donor exchange program
Proceed with transplant
Transplantation around DSA
not achievable
- Adapt immunosuppression!!

11. How to prevent acute / chronic rejection?
1) Avoid transplantation in high risk constellations
(e.g. preformed donor-specific memory)
2) Screening for early / subclinical rejection
I would like to start with a study from El-Zoghby, who investigated 153 graft losses from a population of 1317 transplants from Based on the clinical course and allograft biopsy results they determine why the allograft failed. As you can see on the slide, the leading cause for allograft failure could be identified in 90% of cases. The second important information on this slide is that allograft rejection is still the leading cause for allograft loss even with current immunosuppression. The question is, can we identify these pathological processes earlier allowing for a timely intervention to prevent deterioration of allograft function and loss.

12. Subclinical allograft pathologies
„Clinical“ pathologies
Serum creatinine threshold
Rejection (AMR, TCMR)
CNI-toxicity
Polyomavirus nephropathy
Nickerson P. JASN 1998
Rush D. AJT, 2007
Loupy A. AJT, 2009
„Subclinical“
pathologies
Nankivell B. NEJM, 2003
However, there are many pathologies that develop and evolve while serum creatinine still remains stable. These pathologies have been coined „subclinical“ and can only be detected by so-called protocol or surveillance biopsies. The most important pathologies are subclinical TMR and AMR. In the last 15 years several groups around the world found that subclinical rejection leads to chronic irreversible allograft damage, which will slowly diminish allograft function and reduce long-term allograft survival. Opponents of surveillance biopsies often make the argument that subclinical rejection has a prevalence of only 10-15% within the first year post-transplant using tacrolimus-based triple immunosuppression and thus it is not justified to perform surveillance biopsies in all patients. However, we have to keep in mind that there are currently many efforts to minimize immunosuppression which will likely again increase the incidence of subclinical rejection.
Schaub S. AJT, 2010

13. Clinical relevance of subclinical “TCMR”
The Mayo group went even a step further and investigated whether subclinical tubulointerstitial inflammation with at most meeting only borderline rejection criteria by the current BANFF classification represent a risk for allograft function decline. None of the investigated patients had clinical or subclinical rejejction within the first year post-transplant. Based on the 1 year surveillance biopsy, they defined three groups: Normal, IF alone, IF plus I. Indeed, compared to patients with normal protocol biopsies and patients with only IF/TA at 1 year, patients demonstrating IF/TA and tubulointerstitial inflammation experienced functional decline over the next 3 years. Thus, the authors concluded that patients with IF/TA and tubulointerstitial inflammation in one year surveillance biopsies predict subsequent functional decline. This well performed study further supports the concept that subclinical rejection processes are clinically relevant.
If we add up all subclinical pathologies, at least a third of all patients are affected within the first year.
Interstitial fibrosis with inflammation at one year
predicts decline of allograft function
Park WD. JASN, 2010

14. Natural history of de novo DSA and AMR
Hourmant. JASN 2005
Moreso. Transplant 2012
Wiebe. AJT 2012
Liefeldt. AJT 2012
Event driving early inflammatory events: poor matching, poor adherence, IS minimization
Wiebe C. AJT 2012;12: 1157–1167

15. Screening for subclinical TCMR/AMR Non-invasive rejection biomarkers
Surveillance biopsies
In which patients?
When?
How often?
My hope for the future is that we can tailor surveillance biopsies to the individual needs of the patients. I think, that non-invasive rejection biomarkers are the key element for such an individualized diagnostic surveillance. I hope we will get there in the next 5-10 years……
Thank you for your attention.
Non-invasive rejection biomarkers
to tailor surveillance allograft biopsy frequency to the individual needs of every patient.

16. De novo DSA as a non-invasive biomarker
for subclinical AMR
Not useful <1 year post-transplant (low prevalence)
Annually beyond the 1st year. Restricted to patients at risk?
Detection of de novo DSA should be followed by a biopsy
Treatment options for chronic active AMR are very limited
Prevention of development of de novo DSA is important:
- Screen for and treat subclinical TCMR
- Do not minimize IS in patients with repeated TCMR
- Reinforce drug adherence and improve DR/DQ-matching

17. Urinary CXCL10 chemokine as a biomarker
for subclinical TCMR
CXCL10
Several studies have demonstrated that the urinary CXCL10 chemokine is a promising biomarker for tubulo-interstitial inflammation. Indeed, urinary CXCL10 predicts acute clinical allograft inflammation as rejection and polyomavirus-BK nephropathy. Furthermore, it correlated with the extent of subclinical tubulitis in two independent patient cohorts. A key limitation of all these studies is that the CXCL10 chemokine has only been evaluated in highly selected patient groups. As a consequence, the frequency of the investigated pathologies did not reflect real life.
Jackson JA, AJT 2011
Ho J, Transplantation 2011
Schaub S, AJT 2009
Hu H. Transplantation 2009
Matz M, KI 2006
Hauser IA, JASN 2005
Hu H, AJT 2004 17 17

18. Demographic data – surveillance biopsies (n=362)
Acute score zero
(n=206)
Interstitial infiltrates only
(n=37)
Tubulitis t1
+ any i/v/g/ptc
(n=86)
Tubulitis t2-3
(n=21)
Isolated vascular compartment inflammation
(n=12)
P-level
Acute Scores
- i
- t
- v
- g
- ptc
1.2±0.4
1.2±0.6 1
0.1±0.3
0.2±0.4
0.2±0.5
2.0±0.7
2.2±0.4
0.3±0.5
0.6±0.5
<0.0001
eGFR
47 (39-58)
51 (45-59)
47 (37-58)
43 (31-57)
48 (36-58)
0.57
Proteinuria
- Prot/creat
- a1m/creat
13 (8-21)
4 ( )
13 (10-24)
6 ( )
14 (9-24)
5 ( )
12 (8-19)
6 ( )
12 (8-15)
5 ( )
0.40
0.21
On this slide you can see a short version of the demographic data of the 362 surveillance biopsies. Of all surveillance biopsies acute and chronic Banff scores were assessed and biopsies were assigned to five histological groups according to the acute scores: First group is the “acute score zero group” with per definition no acute Banff scores. (i.e. t0 i0 g0 v0 ptc0). Second group is the interstitial infiltrates only group with per definition only an i score (i.e. t0 i1-3 g0 v0 ptc0). Third group is the tubulitis t1 group plus any other inflammation (i.e. t1 i0-3 g0-3 v0-3 ptc0-3). Forth group ist the tubulitis t2-3 group plus any other inflammation (i.e. t2-3 i0-3 g0-3 v0-3 ptc0-3) and the last group is the isolated vascular compartment inflammation group with a t score of zero and any i scores and per definition any vascular compartment scores (a glomerulitis, endothelialitis or peritubular capillaritis) (i.e. t0 i0-3 g0-3 v0-3 ptc0-3).
All acute Banff scores (t, i, v, g, ptc) were significantly different across the groups. Concomittant BKV-viremia was observed more often in the tubulitis t1 and tubulitis t2-3 group. However, eGFR and proteinuria did not differ among the groups (p≥0.21).
Hirt-Minkowski P. AJT 2012

19. Urinary CXCL10 – subclinical pathologies
≥24
p=0.01 22 20 18
p=0.30 16 14
CXCL10/creat
[ng/mmol] 12 10 8 6 4 2
Urinary CXCL10 chemokine measurements were performed by an established ELISA. In order to correct for different urine dilutions, excretion of urinary CXCL10 chemokine is given in relation to urine creatinine (ng protein/mmol creatinine). On this slide you can see the results of the urinary CXCL10/creatinine ratios according to the five different histological groups within the surveillance biopsies. Median CXCL10/creatinine ratio was lowest in the acute score zero group (0.65ng/mmol) with a stepwise increase to the interstitial infiltrates only (1.19ng/mmol; p=0.004), the tubulitis t1 (1.89ng/mmol; p<0.0001), and the tubulitis t2-3 group (5.5ng/mmol; p<0.0001) . The isolated vascular compartment inflammation group had a higher median urinary CXCL10/creatinine ratio (1.74ng/mmol) than the acute score zero group, but this did not reach statistical significance (p=0.07) .
Acute Banff
score zero
(n=206)
Interstitial
infiltrates
only
(n=37)
Tubulitis t1
+ any i/v/g/ptc
(n=86)
Tubulitis t2-3
+ any i/v/g/ptc
(n=21)
Isolated
vascular
compartment
inflammation
(n=12)
Urinary CXCL10 correlates with the extent of
subclinical tubulo-interstitial inflammation
Hirt-Minkowski P. AJT 2012 19 19

20. Urinary CXCL10 as a non-invasive biomarker
Urinary CXCL10 correlated with the extent of clinical and subclinical tubulointersitital inflammation.
Moderate sensitivity (61-63%) and specificity (72-80%)
- Problem 1: tubulitis t1 (=borderline changes)
 clinical relevance of tubulitis t1?
- Problem 2: Urinary CXCL10 does not reflect
vascular compartment inflammation
In order to bring non-invasive rejection biomarkers for guidance of surveillance biopsies to clinics, three important steps are required.
First, the biomarkers have to be developed in subclinical rejection processes with adequate control groups.
If a promising biomarker has then been identified, it has to be validated in unselected and consecutive patients to determine the diagnostic performance in real life. Finally, if this step is passed a prospective interventional study should be performed to investigate whether the biomarker-guided diagnostic algorhythm improves outcomes. I becomes obvious that development of a non-invasive rejection biomarker is very laborious and requires a lot of time. Unfortunately, most proposed rejection biomarkers were not developed in a subclinical setting and were never tested in Step 2 and 3. I think this is the reason, why currently proposed non-invasive rejection biomarkers are not routinely used in clinics. 20 20

21. Current problems in kidney transplantation
Summary
Current problems in kidney transplantation
To adapt the immunosuppression to the individual needs of every patient
- Surveillance biopsies
- Non-invasive biomarker to guide performance of
surveillance biopsies
To accept the facts, that…
- allograft recipients are getting older…
- organ donors are getting older…
- the deceased donor pool will not match the demand
of the ever increasing waiting list…

22. Acknowledgement Transplant Immunology and Nephrology
Transplantation and Nephrology
Winnipeg, Canada
Gideon Hönger
Patrizia Amico
Patricia Hirt-Minkowski
Felix Burkhalter
Michael Dickenmann
Jürg Steiger
Denise Bielmann
Doris Lutz
Claudia Petit
Peter Nickerson
David Rush
Julie Ho
Institute of Pathology
Helmut Hopfer
Michael Mihatsch