Jacquelin O'Leary Lecture Novartis Sofitel

Donor-Specific Antibodies

in Liver Transplantation

Jacqueline G. O'Leary, MD MPH

Medical Director, Hepatology Research

Baylor University Medical Center

DSA is not Dichotomous

Level N No DSA Class I Class II Class I & II p-valueNegative 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

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

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

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.

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

“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

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

“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”

Graft Loss from DSA197 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

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 analysisA, B, DR

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%

Pathology Pathology described in Crossmatch +

Preservation Injury

Vanishing Bile Duct Syndrome

Chronic Rejection

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

Post-OLT Total MFI

Post-OLT high MFI Class II DSA.

Cla

ss I

I

Cla

ss I

O’Leary JG, et al. AJT 2011:11;1868

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 = 3.35Kaneku H, O’Leary JG, et al. LT 2012

0

10

20

30

40

50

60

70

80

90

100

Pe

rcen

t G

raft S

urv

iva

l

0 1 2 3 4 5 6 7 8 9 10

Years from detection of IgG subclass DSA

Single IgG

Multiple IgGw/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

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

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%

Antibody Characteristics

1270 patients:

Class I DSA MFI ≥5000Preformed 134 (10.5%)De Novo 3 (0.2%)

Class II DSA MFI ≥5000Preformed 100 (7.9%)De Novo 82 (7.5%)

Pre Post

20,000

15,000

10,000

5,000

MF

I

MFI % persistent P-valuePreformed

Class I1,000-4,999 0% 0.02

5,000-9,999 5%

>10,000 5%

Class I Persistence

O’Leary JG, et al. Liver Transplantation, 2013

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

Case 1Class 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 10,6920

5000

10000

15000

20000

25000

neat 1:3 1:9 1:27 1:81

MF

I

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

Biliru

binA

ST

POD

Plts 55 45 51 12 23 20 11

Tx plts 3 1 2

Read as

“biliary obstruction”

↓

Indication Bx

Variable inflammation

+ C4d

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

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

Pre Post

25,000

20,000

15,000

10,000

5,000

MFI

%

persistent P-valuePreformed

Class II1,000-4,999 1.5% <0.001

5,000-10,000 23%

>10,000 33%

Class II Persistence

O’Leary JG, et al. Liver Transplantation, 2013

MF

I

Preformed Class II - Risk of

Acute RejectionAll other patients

Class II DSA >5000

% R

eje

ction

P = 0.046

Months

HR p-value

Preformed Class II MFI ≥5000 1.58 0.004Non-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

Induction Therapy%

Reje

cti

on

Months

P < 0.001

Class II, Ø

Ø, Ø

Ø, Induction

Class II, Induction

1) Induction rejection

2) No ∆ in survival

SurvivalP

atient

Surv

ival

Preformed Class I Preformed Class II

P < 0.001No Antibody

Class I DSA >5000 P = 0.018No Antibody

Class II DSA >5000

Years Years HR p-value

Preformed Class I or II MFI ≥5000 vs. no MFI ≥1000 1.51 0.015Recipient 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

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

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

Fibrosis?

Miyagawa-Hayashino A, et al Liver Transplantation 2012

79 pediatric LT—fibrosis progression:

Stage 3/4: 88% vs. 17%

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

Preformed DSA – Fibrosis Progression

Years Years

Sta

ge 2

-4 F

ibro

sis

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 = 0.02No 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 = 0.006No Antibody

Class II DSA >5000

Preformed DSA - Patient SurvivalP

atie

nt S

urv

iva

l

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

At Risk381 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

Patie

nt S

urv

ival

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

Patie

nt S

urv

ival

1

0

Logrank p= 0.014P = 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 Risk381 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

Patie

nt S

urv

ival

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

Patie

nt S

urv

ival

1

0

Logrank p= 0.043P = 0.043

No Antibody

Class II DSA >5000

Dogma: “Liver protects kidney”

Saidman SL, Transplant Immunology 1994

17%

Liver “protects” kidney

from hyperacute rejection from class I

86 patients: Preformed De Novo

In those with DSA:

DSA MFI >2000 MFI >2000 Class I 10 (11.6%) 0Class 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 IQRClass 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

Preformed Class I DSA MFI>2000

Months Months

Kidney Liver

% R

eje

cti

on P = NS

None

Class I DSAP = NSNone

Class I DSA

No effect of Preformed Class I DSA on: Patient survival

Liver allograft survival

Renal allograft survival

Renal function

Preformed Class II DSA MFI>2000

Months Months

ACR AMR

% R

eje

cti

on

P = NS None

Class II DSAP = 0.006None

Class II DSA

Renal

Preformed Class II DSA Survival

Years

Patient

% S

urv

ival

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

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.

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