Antibody-Mediated Rejection - Histological Features
ABO-compatible grafts with preformed anti-donor antibodies
(Demetris 1...
Immunostaining for C4d – Patterns of Deposition
Portal Venules/Capillaries
• Most frequent pattern described
• Generally c...
Antibody-Mediated Rejection - Role of C4d Immunostaining
Functional significance of different methods & patterns of staini...
39 centres
17 North
America
16 Europe
(+UK)
3Aus/Japan
3 unknown
Liver Antibody-Mediated Rejection
Survey, Redux
• Close look at the HLA typing and
antibody/crossmatch individual response...
From Fuggle and Martin
Transplantation 2008
Luminex
Bead
PE labeled anti-human
immunoglobin antibody
Typically pan IgG
HLA...
Algorithmic recognition of AMR in
human liver allografts:
Importance of clinical, serological, and histopathological
obser...
Summary of Ab study
• We observed two scenarios among recipients with
positive crossmatch
1. Minority of Pre-Tx Pos. Cross...
Patients with AMR had more advanced changes on H&E histology
that co-existed with C4d linear deposits in liver sinusoids
w...
Donor-Specific Antibodies
in Liver Transplantation-
BUMC Experience
Jacqueline G. O'Leary, MD MPH
Medical Director, Inpati...
Survival
PatientSurvival
Preformed Class I Preformed Class II
P < 0.001No Antibody
Class I DSA >5000 P = 0.018
No Antibody...
Preformed DSA - HCV Fibrosis
Progression
P = 0.012No Antibody
Class I DSA >5000
Years
Stage2-4Fibrosis/
HCVrelateddeath
HR...
Early Graft Loss
 60 cases of “Idiopathic” Graft loss <90 days
 Pre-transplant sample tested DSA
 Path was re-reviewed ...
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
↓
Re...
Indication Bx
 Variable inflammation
 + C4d
IgG subclass DSA?
 Does IgG subclass matter?
 Multiple IgG subclasses more common in pts w/ graft dysfunction.
 IgG3 is...
Conclusions
 Class I & II DSA are different.
 MFI DSA to result in pathology liver.
 Preformed class I:
 Vast majority...
Study Design
Stable liver
recipients
>3 years after Tx
2 yearsStable liver
function
(Tolerant)
Immunosuppression
withdrawa...
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...
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
...
No differences in anti-HLA antibodies between
TOL and Non-TOL recipients
Benitez C, Hepatology 2013
Presence of Abs at
Bas...
Conclusions
 In clinical liver transplantation operational tolerance is
more prevalent than originally estimated.
 Multi...
Fibrosis in pediatric LT
patterns & possible mechanisms
Annette S.H. Gouw
Dept. of Pathology and Medical Biology
Universit...
Incidence (3)
• Mean incidence recent studies 54% unaltered
• More emphasis on sinusoidal and centrilobular
fibrosis (men...
Patterns: histology
Venturi C et al, Am J of Transpl 2012; 12: 2986–2996
Histologic features and staging definitions of the liver allograft fi...
summarizing
Late stage fibrosis in pediatric LT:
• Heterogenous in pattern and clinical
context
• Alloimmune reaction play...
Pattern of Fibrosis in Long-
Surviving Liver Allografts
Use of Novel Grading Systems
and Functional Consequences
Oyedele A...
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 transplantat...
Late biopsy (>30 POD) from 30 adult and 84
pediatric patients (mostly for routine check-up)
anti HLA-DR DSA
5000 MFI cut-o...
SUMMARY
• In late allograft biopsy, presence of anti-class II
antibody was not uncommon and may be
associated with graft f...
Liver Tx Tolerance: Histology, DSA, C4d
Overview of ITN Withdrawal Trials:
DSA, C4D Score, Allograft Histology
Sandy Feng,...
Liver Tx Tolerance: Histology, DSA, C4d
ITN029ST: 12 of 20 participants were operationally tolerant
12 of 20
subjects
off ...
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 Tolerant
0
1
2
3
4
5
6
7 IgG2 or IgG4 Positive DSA
IgG2 and IgG4 Nega...
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 ...
Liver Tx Tolerance: Histology, DSA, C4d
Conclusions
 Inflammation and fibrosis do not appear to progress in the majority
...
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
Banff 2013 liver session summary
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Banff 2013 liver session summary

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

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Banff 2013 liver session summary

  1. 1. 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)
  2. 2. Immunostaining for C4d – Patterns of Deposition Portal Venules/Capillaries • Most frequent pattern described • Generally considered to be most specific
  3. 3. 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)
  4. 4. 39 centres 17 North America 16 Europe (+UK) 3Aus/Japan 3 unknown
  5. 5. 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
  6. 6. From Fuggle and Martin Transplantation 2008 Luminex Bead PE labeled anti-human immunoglobin antibody Typically pan IgG HLA Antibody HLA Antigen
  7. 7. 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
  8. 8. 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.
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. Indication Bx  Variable inflammation  + C4d
  16. 16. 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
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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)
  22. 22. 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.
  23. 23. Fibrosis in pediatric LT patterns & possible mechanisms Annette S.H. Gouw Dept. of Pathology and Medical Biology University Medical Center Groningen Groningen, the Netherlands
  24. 24. 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
  25. 25. Patterns: histology
  26. 26. 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)
  27. 27. 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
  28. 28. 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
  29. 29. Immunologic Injury as a Probable Pathophysiologic mechanism? CD68
  30. 30. 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
  31. 31. 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
  32. 32. 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
  33. 33. 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
  34. 34. 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
  35. 35. Liver Tx Tolerance: Histology, DSA, C4d ITN029: Pt 3 (No DSA) Baseline 5+ years laterinflammation fibrosis C4d 0 0
  36. 36. Liver Tx Tolerance: Histology, DSA, C4d Baseline 5+ years later ITN029: Pt 3 (No DSA)
  37. 37. 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
  38. 38. 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
  39. 39. 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.

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