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
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, [email protected]
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