The liver session summary from the 12th Banff Conference on Allograft Pathology held in Comandatuba-Bahia, Brazil, August 19-23, 2013.

2. Antibody-Mediated Rejection - Histological Features
ABO-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 reaction
Neutrophil-rich infiltrate
Portal 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 necrosis
Vascular 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)

3. Immunostaining for C4d – Patterns of Deposition
Portal Venules/Capillaries
• Most frequent pattern described
• Generally considered to be most specific

4. 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)

5. 39 centres
17 North
America
16 Europe
(+UK)
3Aus/Japan
3 unknown

7. 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

8. From Fuggle and Martin
Transplantation 2008
Luminex
Bead
PE labeled anti-human
immunoglobin antibody
Typically pan IgG
HLA Antibody
HLA Antigen

9. Algorithmic recognition of AMR in
human liver allografts:
Importance of clinical, serological, and histopathological
observations including C4d staining pattern
Tomasz Kozlowski, MD
Associate Professor of Surgery
Department of Surgery
University of North Carolina
at Chapel Hill
Banff Conference 2013

10. 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.

11. Patients with AMR had more advanced changes on H&E histology
that 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

12. Donor-Specific Antibodies
in Liver Transplantation-
BUMC Experience
Jacqueline G. O'Leary, MD MPH
Medical Director, Inpatient Liver & Transplant Unit
Baylor University Medical Center, Dallas TX

13. 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

14. Preformed DSA - HCV Fibrosis
Progression
P = 0.012No Antibody
Class I DSA >5000
Years
Stage2-4Fibrosis/
HCVrelateddeath
HR p-value
Preformed Class I MFI >5000 1.45 0.04
Recipient AA 2.15 <0.001
Induction 1.75 <0.001
CMV 1.65 <0.001
Rejection 1.43 0.003
Recipient Age >50 0.74 0.014
Donor AA 0.61 0.003
* Patients are not treated for HCV until ≥Stage 2 Fibrosis
P = 0.006
No Antibody
Class II DSA >5000
Years
Stage2-4Fibrosis/
HCVrelateddeath
HR p-value
Preformed Class II MFI >5000 1.74 0.005
Recipient AA 2.03 <0.001
Induction 1.73 <0.001
CMV 1.6 0.002
Rejection 1.4 0.006
Donor > 50 years old 1.33 0.03
MELD >15 at transplant 1.32 0.02
Donor AA 0.56 <0.001
Class I Class II

15. 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

16. 0
1
2
3
4
5
6
0
1000
2000
3000
4000
5000
6000
7000
1 3 5 7 9 13 16 23 29 36
Bilirubin
AST
↓
Rejection
Steroid recycle
↓
Rejection
Steroid recycle
↓
↓
POD
Plts 127 50 54 61 80 182 68 73 188 87
Tx plts 2 in OR
↓
Pre-op MFI with dilutions
“Preservation
Injury”
“Lobular congestion,
dropout”
0
5000
10000
15000
20000
25000
neat 1:3 1:9 1:27 1:81
MFI
mean…
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
subclass
C1q 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

18. Indication Bx
 Variable inflammation
 + C4d

19. 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

20. 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

21. Study Design
Stable liver
recipients
>3 years after Tx
2 yearsStable liver
function
(Tolerant)
Immunosuppression
withdrawal
6-9 months
12 month follow-up
Withdrawal Failure
Rejection
(Non-Tolerant)
Liver Biopsy Liver Biopsy
Stable liver
function
(Tolerant)
Liver Biopsy

22. 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

23. B ase lin e 1 y 3 y
0.0
0.5
1.0
1.5
2.0
2.5
P ortal inflam atio n
Score
Baseline
1 y
3 y
B ase lin e 1 y 3 y
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
Interface H epatitis
Score
Baseline
1 y
3 y
B ase lin e 1 y 3 y
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
B ile D u ct L esio n s
Score
Baseline
1 y
3 y
In tolerant patients drug withdrawal is associated
with transient (spontaneously resolving) mild
liver inflammation
* **
Benitez, C. et al. Hepatology 2013
Mean SD

24. 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 II
Complement binding by CDC +/- DTT
Specificities confirmed by single-Ag beads in Luminex platform
(performed by Frans Claas, Leiden, NL)

25. 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.

26. Fibrosis in pediatric LT
patterns & possible mechanisms
Annette S.H. Gouw
Dept. of Pathology and Medical Biology
University Medical Center Groningen
Groningen, the Netherlands

27. 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

28. Patterns: histology

29. 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)

30. 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

31. Pattern of Fibrosis in Long-
Surviving Liver Allografts
Use of Novel Grading Systems
and Functional Consequences
Oyedele A. Adeyi
University Health Network & The University of Toronto
Toronto, ON, Canada
oyedele.adeyi@uhn.ca
20 August, 2013

33. Immunologic Injury as a Probable
Pathophysiologic mechanism?
CD68

35. 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

36. Late biopsy (>30 POD) from 30 adult and 84
pediatric patients (mostly for routine check-up)
anti HLA-DR DSA
5000 MFI cut-off
High MFI
(n = 25)
Low MFI
(n = 79)
P value
Mean age 7.9 16.5 0.07
Mean years after LT 8.2 7.6 0.8
Mean ALT (IU/L) 49 52 0.9
Mean T-Bil (mg/dL) 0.9 1.7 0.2
≥ F2 fibrosis 52% 27% 0.03
ACR% 32% 8% 0.004
C4d (any) 56% 27% 0.01
C4d (diffuse or strong) 12% 1% 0.04

37. 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

38. Liver Tx Tolerance: Histology, DSA, C4d
Overview of ITN Withdrawal Trials:
DSA, C4D Score, Allograft Histology
Sandy Feng, MD,PhD
Professor of Surgery
University of California San Francisco
Banff Conference
Comandatuba, Brazil
August 20, 2013

39. 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 months
Feng et al.,
JAMA 2012

40. Liver Tx Tolerance: Histology, DSA, C4d
ITN029: Pt 3 (No DSA)
Baseline 5+ years laterinflammation
fibrosis
C4d
0 0

41. Liver Tx Tolerance: Histology, DSA, C4d
Baseline 5+ years later
ITN029: Pt 3 (No DSA)

42. Liver Tx Tolerance: Histology, DSA, C4d
Non-Tolerant Tolerant
0
1
2
3
4
5
6
7 IgG2 or IgG4 Positive DSA
IgG2 and IgG4 Negative DSA
#ofSubjects
IgG subtypes at Baseline: Tolerant versus Non-Tolerant

43. Liver Tx Tolerance: Histology, DSA, C4d
IgG1 IgG2 IgG3 IgG4
BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y
DQ
IgG1 IgG2 IgG3 IgG4
BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y BL 3Y 5Y
DR
1
2
5
9
11
12
17
Positive
Negative
Not
Present
Longitudinal Analysis of IgG Subgroups: Tolerant Subjects

44. 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.