Jacquelin O'Leary Lecture Novartis Sofitel

1. Donor-Specific Antibodies
in Liver Transplantation
Jacqueline G. O'Leary, MD MPH
Medical Director, Hepatology Research
Baylor University Medical Center

2. DSA is not Dichotomous
Level N No DSA Class I Class II Class I & II p-value
Negative 1117 96% 61% 83% 14% <.0001
T-cell+ 22 1% 12% 0 12%
B-cell+ 32 2% 4% 10% 2%
T-cell+ B-cell+ 67 1% 23% 6% 71%
 DSA testing ≠ Pregnancy Testing
 DSA testing has improved
 DSA testing is still imperfect
 Denatured antigens
 MFI is at best semi-quantitative
 Effects of DSA are spectral

3. Liver’s Relative Resistance Ab
 Liver is relatively resistant to AMR:
 Soluble class I HLA antigens
 Kupffer cells clearance
 Immune complexes
 Activated complement
 Size – 100x microvasculature vs. kidney
 Regeneration
 Decreased clotting
 Low Platelets
 Coagulopathy
 Dual blood supply
 Hypocomplementemia
O’Leary JG, et al. AJT 2014

4. Many Faces of DSA in LT
 Acute AMR
 Acute rejection
 Chronic rejection
 Plasma cell hepatitis
 Biliary strictures
 “Idiopathic” fibrosis
 Fibrosis progression – HCV+
 Renal AMR in SLKT
Prevalence
1-2%
10% Year 1
8% At risk
O’Leary JG, et al. AJT, 2014

5. In the Beginning…
 Early reports of LT did not show preformed DSA
increased graft loss/rejection.
 Graft failure rates were high
 Technology and medications were limited
Gordon RD, et al. Surgery 1986
The Liver Is Different.

6. Early Graft Loss
Takaya S, et al. Transplant Proceedings 1991;
Ogura K., et al. Transplant Proceedings 1992;
Ogura K, et al. Clin Transplantation 1994;
 Retrospective evaluations since the early 90s have
shown graft failure in pts w/ + crossmatch.
1-Month Graft Loss
Year 1991 1992
Takaya/Starzl
(n=600)
Ogura/Terasaki
(n=290)
Technology Crossmatch Crossmatch
Positive 29% 47%
Negative 16% 12%
Stratification by strength of crossmatch

7. “Rejection”
Takaya S, et al. Transplant Proceedings 1991
 “Rejection was the most common cause of
primary liver allograft failure” w/ + crossmatch.
+12.4%
- 4.2%
Graft loss from rejection
0 1 9
Months

9. Hyperacute Rejection?
 3 cases of SLKT.
 Hyperacute rejection of the kidney
 Precipitous liver allograft loss
 Kindey = “canary” heralding liver AMR
 Histology = diffuse coagulative necrosis with
IgM and C1q + staining = hyperacute rejection

10. “Rejection”
Manez R, et al. Hepatology 1995
Manez R, et al. Transplant Proceedings 1993
 Rejection leading to early graft loss:
 High-titer anti-donor IgG antibodies
 Crossmatch remained +
 Falling complement
 Increased CIC
 Refractory thrombocytopenia
 Pathology = “Rejection”

11. Graft Loss from DSA
197 transplants over 3 years 10% crossmatch +.
15/19 had repeat - crossmatch 2 weeks post-LT.
 3 pts “AMR” (DSA, allograft dysfunction, C4d)
 3-7 distinct HLA DSA
 MFI 4,982-14,174
 Outcome:
 2 died (40 and 116 days)
 One re-transplanted--13 months
Kozlowski T, et al. LT 2011:17; 357
 Many preformed DSA “resolve”
 Those that do not may lead to devastating AMR

12. 1-Year Graft Loss & Rejection?
Castillo-Rama M, et al. Liver Transplantation 2008
 896 LT for preformed HLA Ab:
 rejection in patients with Class II +/- I DSA.
Class 1 -Year Graft Loss
I 25%
II 29%
I & II 32%
None 17%
Problem: No multivariate analysis
A, B, DR

13. Acute & Chronic Rejection
 Evaluated 43 LT –indication liver biopsy—C4d & DSA.
 Antibody characteristics:
 MFI >500 = +
 22 pts class II, 1 class I, 4 both
Musat AI, et al. AJT 2011:11; 500
DSA can play a role in
Acute and Chronic Rejection
DSA/ C4d Other P-value
Number 17 26
Acute
Rejection 88% 50% 0.02
SRR 41% 19% 0.03
N DSA/ C4d
ACR +
Ductopenia 6 100%

14. Pathology
 Pathology described in Crossmatch +
 Preservation Injury
 Vanishing Bile Duct Syndrome
 Chronic Rejection

15. Chronic Rejection
 Risk chronic rejection in Crossmatch +
 26.3% vs. 4.9%
 39 LT patients w/ CR vs. matched comparators.
Chronic Rejection
(N=39)
Comparator
(N=39)
P-value
Any DSA ever 92% 61% P = 0.003
Preformed 60% 41% NS
De novo 62% 38% P = 0.047
DSA post-OLT 79% 56% P = 0.047
Any DSA ever Class I 10% 5% P = 0.008
Class II 38% 33%
Class I & II 44% 23%
none 8% 39%
Demetris AJ, et al. Transplant Proceedings 1987
Takaya S, et al. Transplant Proceedings 1999
O’Leary JG, et al. AJT 2011:11;1868

16. Post-OLT Total MFI
Post-OLT high MFI Class II DSA.
ClassII
ClassI
O’Leary JG, et al. AJT 2011:11;1868

17. IgG subclass DSA?
 Does IgG subclass matter?
 Multiple IgG subclasses more common in pts w/ graft dysfunction.
 IgG3 is present in pts w/ most rapid graft failure.
 Multivariable analysis:
 IgG3 HR graft loss = 3.35
Kaneku H, O’Leary JG, et al. LT 2012
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10
Years from detection of IgG subclass DSA
Single IgG
Multiple IgG
w/o IgG3
Multiple IgG w/IgG3
p=0.009
79%
50%
71%
B
1) Surrogate “robust”
immune attack
2) Intrinsic about IgG3
3) IgG3 test more
specific

18. Biliary Strictures?
 risk of bile duct complications:
 37% vs. 10%
 Risk factors for anastomotic strictures:
162 LT
 ABO-incompatible: 13% vs. 2%
 Class II DSA: 23% vs. 8%
Takaya S, et al. Transplant Proceedings 1999
Iacob S, et al. Liver International 2012

19. BUMC Experience
 Large Project:
1/00 to 5/09
 Analyze all 1270 pts
(4.2% missing data)
 Pre-LT and 1-year
post-LT sample.
 Blinded--Terasaki
Foundation
Laboratory
Age 52
Model for End-Stage Liver Disease 16
Cold ischemia time 7.7
Male Sex 65%
Race African-American 8%
Caucasian 73%
Liver Disease HCV 47%
PSC/PBC/AIH 15%
Alcohol 14%
NASH/CC 12%
Hepatocellular Carcinoma 24%
Donor age 42
Rejection 46%
Induction 42%
CNI* 3 months Tacrolimus 65%
Cyclosporine 30%
Steroids* 54%
Mycophenolate* 51%

20. Antibody Characteristics
 1270 patients:
Class I DSA MFI ≥5000
Preformed 134 (10.5%)
De Novo 3 (0.2%)
Class II DSA MFI ≥5000
Preformed 100 (7.9%)
De Novo 82 (7.5%)

21. Pre Post
20,000
15,000
10,000
5,000
MFI
MFI % persistent P-value
Preformed
Class I
1,000-4,999 0% 0.02
5,000-9,999 5%
>10,000 5%
Class I Persistence
O’Leary JG, et al. Liver Transplantation, 2013

22. Early Graft Loss
 60 cases of “Idiopathic” Graft loss <90 days
 Pre-transplant sample tested DSA
 Path was re-reviewed by Dr. Demetris
 Post-perfusion
 Indication
 Explant / Autopsy
 Acute AMR Dx:
1) DSA in serum
2) Diffuse C4d
3) Exclusion of other causes of a similar type of injury
4) Microvascular injury
O’Leary JG, et al. Liver Transplantation, In Press

23. Case 1
Class IgG MFI
(1:3)
IgG
subclass
C1q
(1:3)
I 6,365 Neg 0
I 23,091 1+2+3 16,307
I 4,373 Neg 0
I 23,127 1+2+3 17,309
II 9,531 3 0
II 24,883 1+3 16,389
II 23,158 1+3 9,512
II 17,068 1+3 10,692
0
5000
10000
15000
20000
25000
neat 1:3 1:9 1:27 1:81
MFI
mean class I
mean class II
0
5
10
15
20
25
30
0
500
1000
1500
2000
2500
1 2 3 4 5 6 10 14 18
Bilirubin
AST
POD
Plts 55 45 51 12 23 20 11
Tx plts 3 1 2
Read as
“biliary obstruction”
↓

24. Indication Bx
 Variable inflammation
 + C4d

25. Early Graft Loss
 AMR - caused or contributed to:
 20% idiopathic graft loss if DSA +
 Preformed DSA with bead
saturation at 1:27
 80% DSA caused or contributed to
graft loss
AMR No AMR
Highest Class I 20,000 3,000
Highest Class II 12,000 2,000
MFISUM 96,000 3,500

26. Early Graft Loss
 AMR - caused or contributed to:
 20% idiopathic graft loss if DSA +
 Preformed DSA with bead
saturation at 1:27
 80% DSA caused or contributed
to graft loss
AMR No AMR
Highest Class I 20,000 3,000
Highest Class II 12,000 2,000
MFISUM 96,000 3,500
Antibody Soup

27. Pre Post
25,000
20,000
15,000
10,000
5,000
MFI
%
persistent P-value
Preformed
Class II
1,000-4,999 1.5% <0.001
5,000-10,000 23%
>10,000 33%
Class II Persistence
O’Leary JG, et al. Liver Transplantation, 2013
MFI

28. Preformed Class II - Risk of
Acute Rejection
All other patients
Class II DSA >5000
%Rejection
P = 0.046
Months
HR p-value
Preformed Class II MFI ≥5000 1.58 0.004
Non-compliance 1.31 0.12
Autoimmune Etiology 0.99 0.9
Recipient age >50 0.72 <0.001
Recipient African-American 1.37 0.04
Steroids week 4 1.54 <0.001
Mycophenolate week 4 1.23 0.04

29. Induction Therapy
%Rejection
Months
P < 0.001
Class II, Ø
Ø, Ø
Ø, Induction
Class II, Induction
1) Induction rejection
2) No ∆ in survival

30. Survival
PatientSurvival
Preformed Class I Preformed Class II
P < 0.001No Antibody
Class I DSA >5000 P = 0.018
No Antibody
Class II DSA >5000
Years Years
HR p-value
Preformed Class I or II MFI ≥5000 vs. no MFI ≥1000 1.51 0.015
Recipient African-American 2.48 <0.001
Hepatitis C viremia* 2.13 <0.001
Donor age > 50 1.51 0.012
Cytomegalovirus infection 1.54 0.015
Recipient age > 60 1.58 0.028
Alcohol, NASH or cryptogenic cirrhosis 1.6 0.045
*Censored at aviremia

31. Multivariable Modeling
De Novo DSA Patient Survival
HR P-value
De Novo Class II DSA (>5000) 1.99 0.005
HCV viremia 1.68 0.002
Recient Age >60 1.76 0.008
Donor age >50 1.52 0.01
African-American Race 1.77 0.02
Kaneku H, et al. AJT 2013
Incidence de novo DSA
8% in first year

32. Multivariable Modeling
De Novo DSA Formation
 Independent predictors of de novo Class II >5000
DSA
HR P-value
Cyclosporine 2.5 0.004
Low CNI levels 2.66 0.02
MELD >15 0.47 0.02
Recipient age >60 0.26 0.03
Kaneku H, et al. AJT 2013

33. Fibrosis?
Miyagawa-Hayashino A, et al Liver Transplantation 2012
 79 pediatric LT—fibrosis progression:
 Stage 3/4: 88% vs. 17%

34. HCV & Fibrosis Progression
 1/00 to 5/09
 507 HCV viremic patients.
>80% compliance w/ protocol liver bx at year 1, 2, 5.
 HCV fibrosis progression is immune mediated.
 Hypothesized: Preformed DSA Accelerated fibrosis
in HCV viremic pts.
O’Leary JG, et al Liver Transplantation ePub

35. Preformed DSA – Fibrosis Progression
Years Years
Stage2-4Fibrosis
Fibrosis Progression HR P-value
Preformed Class I MFI 5000 1.44 0.04
Recipient African-American 1.89 0.002
Induction 1.74 <0.001
Donor African-American 0.60 0.003
Cytomegalovirus infection 1.49 0.007
Rejection 1.26 0.05
Donor > 50 years old 1.14 0.30
MELD >15 at transplant 1.27 0.06
Recipient > 50 years old 0.74 0.58
P = 0.02
No Antibody
Class I DSA >5000
Fibrosis ≥ Stage 2 HR P-value
Preformed Class II MFI 5000 1.86 <0.001
Recipient African-American 1.87 0.002
Induction 1.73 <0.001
Donor African-American 0.53 <0.001
Cytomegalovirus infection 1.51 0.006
Rejection 1.22 0.10
Donor > 50 years old 1.19 0.19
MELD >15 at transplant 1.35 0.02
Recipient > 50 years old 0.73 0.01
P = 0.006
No Antibody
Class II DSA >5000

36. Preformed DSA - Patient Survival
PatientSurvival
Years
HR P-value
Preformed Class I MFI 5000 1.63 0.03
Sustained Virologic Response 0.21 <0.001
Recipient African-American 2.85 <0.001
Cytomegalovirus 1.60 0.02
sk
381 370 350 336 324 300
59 49 44 42 39 37
0
1
0 1 2 3 4 5
Years
0.0
0.2
0.4
0.6
0.8
1.0
PatientSurvival
1
0
Logrank p= 0.014
0 1 2 3 4 5
Years
0.0
0.2
0.4
0.6
0.8
1.0
PatientSurvival
1
0
Logrank p= 0.014
P = 0.02
No Antibody
Class I DSA >5000
HR P-value
Preformed Class II MFI 5000 1.72 0.03
Sustained Virologic Response 0.22 <0.001
Recipient African-American 2.62 <0.001
Cytomegalovirus 1.74 0.003
Induction 1.44 0.04
At Risk
381 370 350 336 324 300
46 38 34 33 32 31
0
1
0 1 2 3 4 5
Years
0.0
0.2
0.4
0.6
0.8
1.0
PatientSurvival
1
0
Logrank p= 0.043
0 1 2 3 4 5
Years
0.0
0.2
0.4
0.6
0.8
1.0
PatientSurvival
1
0
Logrank p= 0.043
P = 0.043
No Antibody
Class II DSA >5000

37. Dogma: “Liver protects kidney”
Saidman SL, Transplant Immunology 1994
17%
Liver “protects” kidney
from hyperacute rejection from class I

38.  86 patients:
Preformed De Novo
 In those with DSA:
DSA MFI >2000 MFI >2000
Class I 10 (11.6%) 0
Class II 10 (11.6%) 8 (9%)
Class I & II 10 (11.6%) 1 (1%)
None 56 (65%) 56 (65%)
No sample 0 21 (25%)
Median MFI IQR Median MFI IQR
Class I 18,550 5,000-23,000 10,000 NA
Class II 19,400 9,100-25,100 10,000 9,000-23,000
Preformed De Novo
SLKT
Saidman SL, et al. Transplant Immunology 1994
Olausson M, et al. AJT 2007
Dar W, et al. AJT 2011
Reichman TW, et al. AJT 2009
O’Leary JG, et al, AJT 2011

39. Preformed Class I DSA MFI>2000
Months Months
Kidney Liver
%Rejection
P = NS
None
Class I DSAP = NS
None
Class I DSA
 No effect of Preformed Class I DSA on:
 Patient survival
 Liver allograft survival
 Renal allograft survival
 Renal function

40. Preformed Class II DSA MFI>2000
Months Months
ACR AMR
%Rejection
P = NS None
Class II DSA
P = 0.006None
Class II DSA
Renal

41. Preformed Class II DSA Survival
Years
Patient
%Survival
P = 0.02
None
Class II DSA
HR p-value
Class II DSA 2.2 0.043
Steroids 1 month 0.03 0.004
Recipient Age >50 6.4 <0.001

42. Conclusions
 3 main reasons why DSA has been under-
appreciated in LT:
 DSA is not dichotomous.
 Liver is relatively resistant to AMR.
 Many faces of DSA in LT.
 Acute AMR occurs in LT but is rare.
 Highest risk = Class I DSA MFI >15,000 at 1:27
dilution.
 Preformed and de novo DSA can impair graft
outcomes.

43. Acknowledgements
 Baylor University Medical Center
 Göran Klintmalm, MD PhD
 Linda Jennings, PhD
 Terasaki Foundation Laboratory
 Hugo Kaneku, MD
 Paul Terasaki, PhD
 University of Pittsburgh Medical Center
 Anthony J. Demetris, MD
 Emory University
 Howie Gebel, PhD
 Allan Kirk, MD