Banff 2013Summary of Liver Sessions

Antibody-Mediated Rejection - Histological FeaturesABO-compatible grafts with preformed anti-donor antibodies

(Demetris 1992, Watson 2006, Bellamy 2007, Kozlowski 2011, Lunz 2011, Musat 2011, Ali 2012, Paterno 2012)

Time Histological Features Comment

0-7 days Centrilobular ballooning/cholestasis/necrosis

Resembles preservation/reperfusion injury (PRI more frequent in XM+ cases - Ruiz 2012)

1-4 weeks Portal/periportal oedema Ductular reactionNeutrophil-rich infiltratePortal venulitis (mainly neutrophils)

Portal haemorrhage (more severe cases)Periportal necrosis (more severe cases)

Some features resemble biliary obstruction

Other possible antibody-mediated lesions:• Sinusoidal neutrophils• Lobular inflammation and necrosis

(including central perivenulitis)• Acute & chronic rejection

> 4 weeks Ischaemic bile duct necrosisVascular thrombosis

Centrilobular sinusoidal fibrosis

Present in more severe cases (failed allografts)

Similar change seen in studies of ABO-I grafts treated with aggressive immunosuppression(Haga 2004, Morioaka 2004, Usuda 2005, Haga 2006, Egawa 2008)

Immunostaining for C4d – Patterns of DepositionPortal Venules/Capillaries

• Most frequent pattern described

• Generally considered to be most specific

Antibody-Mediated Rejection - Role of C4d Immunostaining

Functional significance of different methods & patterns of staining uncertain

• Most studies use an immunoperoxidase method on formalin-fixed paraffin-embedded tissues and focus on staining in portal capillaries

• Recent studies have suggested that these staining patterns lack specificity for AMR (Koslowski 2011 & 2012)

– AMR should be diagnosed using immunofluorescence in frozen sections

– Linear/granular sinusoidal pattern correlates with presence of DSAs (and histological features) and therefore considered specific for AMR

Linear pattern Granular pattern

( from Kozlowski . Liver Transpl 2012; 18: 641-648)

Desley Neil, Birmingham, UKParticipants in general survey

39 centres17 North America16 Europe (+UK)3 Aus/Japan3 unknown

SUMMARY• Majority think AMR occurs• Majority use IP on FFPE• Work required to standardise method

• Identify positive liver cases• Send unstained sections to all labs• Assess reproducibility

• Work required to validate• HLA information

• Short term• On FFPE –any staining should be considered

positive / suspicious• High pH antigen retrieval appears more sensitive

Liver Antibody-Mediated Rejection Survey, Redux

• Close look at the HLA typing and antibody/crossmatch individual responses…– Perform HLA Typing?

• 25% Not Sure

– Crossmatch for Liver Tx? • 25% Not Sure

– If you crossmatch, what method? • 40% Not Sure

– HLA Antibody Screening? • 21% Not Sure

– Which method is used? • 31% Not Sure

From Fuggle and Martin Transplantation 2008

LuminexBead

PE labeled anti-human immunoglobin antibodyTypically pan IgG

HLA Antibody

HLA Antigen

Algorithmic recognition of AMR in human liver allografts:

Importance of clinical, serological, and histopathological observations including C4d staining pattern

Tomasz Kozlowski, MDAssociate Professor of Surgery

Department of Surgery

University of North Carolina at Chapel Hill

Banff Conference 2013

Summary of Ab study

• We observed two scenarios among recipients with positive crossmatch

1. Minority of Pre-Tx Pos. Crossmatch pts (20%) maintained positive crossmatch and developed early severe AMR

2. Majority spontaneously converted their Pos. Crossmatch into Neg. and maintained good long term liver function when on routine immunosuppression.

Patients with AMR had more advanced changes on H&E histologythat co-existed with C4d linear deposits in liver sinusoids

when compared to patients only with ischemia-reperfusion injury and without AMR

Summary of H&E histology findings

Donor-Specific Antibodies in Liver Transplantation-

BUMC Experience

Jacqueline G. O'Leary, MD MPH

Medical Director, Inpatient Liver & Transplant UnitBaylor University Medical Center, Dallas TX

SurvivalP

atie

nt S

urvi

val

Preformed Class I Preformed Class II

P < 0.001No AntibodyClass I DSA >5000 P = 0.018

No AntibodyClass 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 <0.001Hepatitis C viremia* 2.13 <0.001Donor age > 50 1.51 0.012Cytomegalovirus infection 1.54 0.015Recipient age > 60 1.58 0.028Alcohol, NASH or cryptogenic cirrhosis 1.6 0.045

*Censored at aviremia

Preformed DSA - HCV Fibrosis Progression

P = 0.012No AntibodyClass I DSA >5000

Years

Sta

ge 2

-4 F

ibro

sis/

HC

V r

ela

ted

dea

th

HR p-valuePreformed Class I MFI >5000 1.45 0.04Recipient AA 2.15 <0.001Induction 1.75 <0.001CMV 1.65 <0.001Rejection 1.43 0.003Recipient Age >50 0.74 0.014Donor AA 0.61 0.003

* Patients are not treated for HCV until ≥Stage 2 Fibrosis

P = 0.006No AntibodyClass II DSA >5000

Years

Sta

ge 2

-4 F

ibro

sis/

HC

V r

ela

ted

dea

th

HR p-valuePreformed Class II MFI >5000 1.74 0.005Recipient AA 2.03 <0.001Induction 1.73 <0.001CMV 1.6 0.002Rejection 1.4 0.006Donor > 50 years old 1.33 0.03MELD >15 at transplant 1.32 0.02Donor AA 0.56 <0.001

Class I Class II

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) Histopathological evidence of diffuse microvascular injury

1 3 5 7 9 13 16 23 29 360

1000

2000

3000

4000

5000

6000

7000

0

1

2

3

4

5

6

AST

Bilirubin

↓

RejectionSteroid recycle

↓

RejectionSteroid recycle

↓↓

PODPlts 127 50 54 61 80 182 68 73 188 87Tx plts 2 in OR

↓

Pre-op MFI with dilutions

“Preservation Injury”

“Lobular congestion, dropout”

neat 1:3 1:9 1:27 1:810

5000

10000

15000

20000

25000

mean ...

MFI

Figure 2: Patient 1 was a 56 year-old female (with a gynecologic history significant for one still-birth) with a MELD of 12, who underwent a primary LT for autoimmune hepatitis with a 37 year-old male donor after 12.2 hours of cold ischemia in 1990. She had saturation of single antigen beads of numerous DSAs at 1:81 dilution (A) against class I pre-transplant, (B) 7 distinct DSAs (MFI reported at 1:3 dilution) in total, and (C) a clinical course characterized by early post-operative platelet consumption and recurrent rejection. (D) The post reperfusion biopsy (top panels) showed endothelial reactivity and diffuse C4d positivity followed by evidence of a combined AMR and ACR on day 16 (bottom panels) characterized by diffuse microvascular endothelial cell injury.

C)

A) B) IgG MFI

(1:3)IgG

subclassC1q MFI

(1:3)

A2 19,711 1+2 12,423

A26 21,581 1 15,854

B52 22,124 1+3 14,498

B62 23,209 1+2+3 13,458

Cw3 18,844 1+2 1,939

DR13* 8,310 1 0

DR52* 9,429 1 0

* Not plotted

Acute AMR Courtesy Jackie O’Leary

Indication Bx

Variable inflammation + C4d

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

Pe

rcen

t Gra

ft 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 IgG w/o IgG3

Multiple IgG w/IgG3 p=0.009

79%

50%

71%

B

Conclusions Class I & II DSA are different. MFI DSA to result in pathology liver. Preformed class I:

Vast majority resolution High titer class I that persists post-LT AMR Associated with HCV fibrosis progression

Preformed class II: Resolution depends on MFI Increases the risk of early rejection

Liver allograft Renal AMR

Associated with HCV fibrosis progression. Associated with chronic rejection.

Acute AMR occurs in LT, but is rare. Characteristic histologic features that can be recognized

Study Design

Stable liver recipients

>3 years after Tx

2 yearsStable liver function

(Tolerant)

Immunosuppression withdrawal6-9 months

12 month follow-up

Withdrawal FailureRejection

(Non-Tolerant)

Liver Biopsy Liver Biopsy

Stable liver function

(Tolerant)

Liver Biopsy

Time since transplantation (years)

Primary end point

% n

YES 12.5 3

NO 87.5 21

≤ 5.7

Primary end point

% n

YES 38 19

NO 62 31

5.7-10.6 >10.6

Primary end point

% n

YES 79.2 19

NO 20.8 5

Age at transplantation

Primary end point

% n

YES 0 0

NO 100 14

Primary end point

% n

YES 30 3

NO 70 7

≤ 49.6 >49.6

INFLUENCE OF TIME AND AGE ON THE DEVELOPMENT OF OPERATIONAL TOLERANCE

Benitez C, Hepatology 2013

Baseline 1 y 3 y0.0

0.5

1.0

1.5

2.0

2.5

Portal inflamation

Sco

re

Baseline1 y3 y

Baseline 1 y 3 y-0.5

0.0

0.5

1.0

1.5

2.0

2.5

Interface Hepatitis

Sco

re

Baseline1 y3 y

Baseline 1 y 3 y-0.5

0.0

0.5

1.0

1.5

2.0

2.5

Bile Duct Lesions

Sco

re

Baseline1 y3 y

In tolerant patients drug withdrawal is associated with transient (spontaneously resolving) mild

liver inflammation

* **

Benitez, C. et al. Hepatology 2013

Mean ± SD

No differences in anti-HLA antibodies between TOL and Non-TOL recipients

Benitez C, Hepatology 2013

Presence of Abs at Baseline

Detection of de novo Abs at the dnd of

study

TOL Non-TOL TOL Non-TOL

Anti-class I 11% 14% - -

Anti-class II 31% 20% 1/15 5/23

Elisa class I and IIComplement binding by CDC +/- DTT

Specificities confirmed by single-Ag beads in Luminex platform

(performed by Frans Claas, Leiden, NL)

Conclusions

In clinical liver transplantation operational tolerance is more prevalent than originally estimated.

Multiple pathways are likely to operate in combination to maintain tolerance.

The graft itself likely plays a central role in inducing and/or maintaining tolerance

In patients without chronic HCV cohort and RCT studies are in preparation.

In patients with chronic HCV no clinical studies are planned.

Fibrosis in pediatric LTpatterns & possible mechanisms

Annette S.H. Gouw

Dept. of Pathology and Medical Biology

University Medical Center Groningen

Groningen, the Netherlands

Incidence (3)

• Mean incidence recent studies 54% unaltered• More emphasis on sinusoidal and centrilobular

fibrosis (mentioned in 8/11 studies)• Heterogenous studies

– Varying Context of Studies– Protocol – indication Bx– With/without inflammation – Normal – abnormal LFTs– Several grading systems– Variable reporting of type of fibrosis

Patterns: histology

Venturi C et al, Am J of Transpl 2012; 12: 2986–2996

Histologic features and staging definitions of the liver allograft fibrosis semiquantitative scoring system (LAFSc)

summarizing

Late stage fibrosis in pediatric LT:• Heterogenous in pattern and clinical

context• Alloimmune reaction plays a major role• Silent fibrosis: insidious, progressive• Possible mechanism(s): early events

complemented by continuous damage

Pattern of Fibrosis in Long-Surviving Liver Allografts

Use of Novel Grading Systems and Functional Consequences

Oyedele A. AdeyiUniversity Health Network & The University of Toronto

Toronto, ON, Canadaoyedele.adeyi@uhn.ca

20 August, 2013

Immunologic Injury as a Probable Pathophysiologic mechanism?

CD68

Prevalence of Donor-specific anti-HLA Class II Abs more than 5 years after transplantation

in pediatric liver transplantation (n = 79)

Luminex bead assay

(>1000 MFI)

Not detected

Class I only

Class II only

Class I and II

DSA 62%* 3% 38%** 0%

*Included cases without donor HLA typing** Most (87%) were anti-DR Abs

Late biopsy (>30 POD) from 30 adult and 84 pediatric patients (mostly for routine check-up)

anti HLA-DR DSA5000 MFI cut-off

High MFI (n = 25)

Low MFI(n = 79) P value

Mean age 7.9 16.5 0.07Mean years after LT 8.2 7.6 0.8

Mean ALT (IU/L) 49 52 0.9Mean T-Bil (mg/dL) 0.9 1.7 0.2

≥ F2 fibrosis 52% 27% 0.03ACR% 32% 8% 0.004

C4d (any) 56% 27% 0.01

C4d (diffuse or strong) 12% 1% 0.04

SUMMARY

• In late allograft biopsy, presence of anti-class II antibody was not uncommon and may be associated with graft fibrosis

• Although not sensitive, C4d IHC-P may be useful for screening of possible antibody-associated fibrosis

Liver Tx Tolerance: Histology, DSA, C4d

Overview of ITN Withdrawal Trials:DSA, C4D Score, Allograft Histology

Sandy Feng, MD,PhDProfessor of Surgery

University of California San Francisco

Banff ConferenceComandatuba, Brazil

August 20, 2013

Liver Tx Tolerance: Histology, DSA, C4d

ITN029ST: 12 of 20 participants were operationally tolerant

12 of 20 subjects

off IS

57.0 months

77.7 monthsFeng et al., JAMA 2012

Liver Tx Tolerance: Histology, DSA, C4d

ITN029: Pt 3 (No DSA)

Baseline 5+ years laterinflammation

fibrosis

C4d

0 0

Liver Tx Tolerance: Histology, DSA, C4d

Baseline 5+ years later

ITN029: Pt 3 (No DSA)

Liver Tx Tolerance: Histology, DSA, C4d

Non-Tolerant Tolerant0

1

2

3

4

5

6

7 IgG2 or IgG4 Positive DSAIgG2 and IgG4 Negative DSA

# of

Sub

ject

s

IgG subtypes at Baseline: Tolerant versus Non-Tolerant

Liver Tx Tolerance: Histology, DSA, C4d

IgG1 IgG2 IgG3 IgG4BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y

DQIgG1 IgG2 IgG3 IgG4

BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y

DR

1

2

5

9

11

12

17

PositiveNegative Not Present

Longitudinal Analysis of IgG Subgroups: Tolerant Subjects

Liver Tx Tolerance: Histology, DSA, C4d

Conclusions

Inflammation and fibrosis do not appear to progress in the majority of operationally tolerant pediatric liver transplant recipients

C4d scores generally increase over 5 years of follow-up in operationally tolerant children

∆ No apparent correlation to inflammation / fibrosis or DSA

Immunosuppression withdrawal frequently precipitates the emergence of DSA against Class II.

∆ Adult and pediatric withdrawal experiences

∆ Antibodies emerge even in subjects who are “operationally tolerant”

In children, DSA of subtype IgG3 is rarely present.

Recommended HLA and Serological Testing in Liver Transplantation

• Criteria for establishing a definite diagnosis of AMR requires DSA testing, preferably obtained at the time of biopsy

• Allo-Ab monitoring can identify DSA only if donor HLA type is known.

• Minimum Recommendations: save donor & recipient sera and DNA or cells for future testing in the event that the clinicopathological profile of the patient warrants further testing.

• Ideal Recommendations: Complete donor and recipient HLA typing and pre-transplant crossmatch with DSA testing and repeat DSA testing with a preference, if possible, to determining the IgG subtype and functional activity (C1q assay, etc.), as needed, based on indication based on clinicopathological profile.

Tissue Sampling in Suspected AMR• Minimum Recommendations: At least one, 20 mm, needle

core, 16-gauge routinely FFPE processing.• Most centers biopsy triage routine includes preparation of

formalin-fixed, paraffin-embedded (FFPE) sections, which are less sensitive than frozen tissue for detection of C4d tissue deposits.

• Fresh-frozen (FZ) tissue samples have improved sensitivity to detect C4d deposits but have practical limitations. A 3 mm fragment may be fresh-frozen in OCT compound and the remainder of the core undergoes FFPE processing.

Working Draft Criteria for Acute AMR

1. Tissue injury in a pattern consistent with acute AMR:1. See text

2. Diffuse microvascular C4d positivity:1. FFPE: > 50% portal vein and capillaries positive

2. Or FZ: >50% linear sinusoidal positivity

3. Circulating DSA: 1. Positive single antigen bead DSA testing. See text.

4. Reasonable exclusion of other causes of graft dysfunction with a similar pattern of injury, see text

Working Draft of Criteria for Chronic AMR

• See text– Tissue injury in a pattern consistent with chronic AMR:

• Perivenular and/or subsinusoidal fibrosis• ? Stellate cell activation• ? Portal venopathy

– Diffuse microvascular C4d positivity:• ? Portal vs central vs sinusoidal

– Circulating DSA:• DSA testing with a preference, if possible, to determining the IgG

subtype and functional activity (C1q assay, etc.), as needed, based on indication based on clinicopathological profile

– Reasonable exclusion of other causes of a similar pattern of injury