This document summarizes a session from the 12th Banff Conference on Allograft Pathology. It discusses various topics relating to antibody-mediated rejection (AMR) in heart transplants, including: - The natural history of C4d deposition and progression to clinical AMR over 5 years in a large cohort. - An update on the revised International Society for Heart and Lung Transplantation grading system for AMR. - Practical issues and pitfalls in the pathological diagnosis of AMR using immunohistochemistry. - The clinician's perspective on pathological diagnosis of AMR and how donor-specific antibodies can complement diagnosis. - Monitoring donor-specific antibodies and how they relate

1. Summary Heart Session
E Rene Rodriguez, M.D. and Carmela D. Tan, M.D.
12th Banff Conference on Allograft Pathology
Comandatuba-Bahia, Brazil
August 23, 2013

2. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

3. Background - Clinical AMR
Tan et al, Am J Transplant. 2009 Sep;9(9):2075-84

4. Natural History of C4d deposition in Heart
Allografts

5. Results
C4d+
C3d-
48
C3d-
40
C3d+
8
C3d+
16
Index biopsies
Follow-up
biopsies
Average time for
conversion:
13.5 (1.1-31.9)
months
8/48 (17%) of patients
progressed to
C4d+C3d+

6. C4d C4d
converters
C4dC3d P-value
Average
(months)
13.2 7.38 12.6 NS
Range
(months)
0.2-61.1 0.7-17.4 0.2-37.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
24.0
26.0
28.0
30.0
32.0
34.0
36.0
38.0
40.0
42.0
44.0
46.0
48.0
0 5 10 15 20 25 30 35 40
Time to first positive biopsy
C4d C4d converters C4dC3d
Monthsposttransplant

7. Assessment of mortality odds ratio
Backward Logistic Regression Reduced Model Odds Ratio Estimates
Odds 95% Wald
Effect Ratio Confidence Limits P-Value
C4d+ vs. Control 4.690 1.959 11.227 .0005
(40 vs.232)
C4d+ converters vs. Control 5.824 1.221 27.775 .027
(8 vs. 232)
C4d+C3d+ vs. Control 6.166 1.844 20.615 .003
(16 vs.232)
The odds ratio for mortality in the three patient groups were
significant compared against the control group.

8. Cox Proportional Hazard Survival Curves
Control
C4d
C4d
Converter
C4dC3d

9. Assessment of Odds ratios for CAV
Exact Odds Ratios
Odds 95% Confidence
Parameter Ratio Limits p-Value
C4d+ vs. Control 2.611 0.228 19.006 0.5141
C4d+ converters vs. Control 5.324* 0 47.623 1.0000
C4d+C3d+ vs. Control 2.438* 0 20.529 1.0000
*Indicates a median unbiased estimate
There were no significant differences in CAV odds ratios as a function of
patient groupings.

10. Conclusions I
• C4d+ C3d+ capillary –> AMR with DSA and Dysfunction
• Majority of C4d+ only episodes are single occurrences
(60%) and subclinical (85%).
• Some patients with C4d staining alone (8 of 48, 17%) will
develop AMR (Convert to C4d+ C3d+) on follow-up.
Control C4d+
C4d+
Converters
C4d+C3d+ P-Value
Patient Gender
Male 176 33 7 8 0.08
Female 56 7 1 8
232 40 8 16

11. Conclusions II
• There is no increased risk for CAV associated with
complement deposition (C4d+ only or C4d+ C3d+).
• Cardiovascular mortality is increased in patients with
complement deposition relative to controls.

12. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

13. 2011 GRADING SCHEME now accepted by
ISHLT as the new Working Formulation for AMR
2013
• pAMR 0: Negative for pathologic AMR: both histological and
immunopathological studies are negative
• pAMR 1 (H+): Histopathological AMR alone: histopathological findings
present and immunopathological findings absent
• pAMR 1 (I+): Immunopathologic AMR alone: Immunopathological
findings present and histological findings absent
• pAMR 2: Pathologic AMR: both histological and immunopathological findings
present
• pAMR 3: Severe pathologic AMR: Rare cases of severe AMR with
histopathological findings of IS hemorrhage, capillary fragmentation, mixed
inflammation, endothelial cells pyknosis, karyorrhexis, marked edema

14. • Since this grading has been in use for two years as of this Banff
meeting it will be imperative to evaluate its accuracy in order to refine
or revise criteria.
• Usefulness of C4d and CD68 by IHC and how it correlates with DSA
and dysfunction
• Great goal for Banff 2015

15. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

16. •Many examples of Histologic pAMR H+
•Technical challenges of C4d by IHC

17. For EMB:
Assess Reproducibility of interpretation amongst centers
Quality Assurance / Control (technical (virtual and non-
virtual))
Refining and improving criteria ISHLT & Banff synergy

18. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

19. Clinical Cardiologists Need
• Consistency on interpretation
• Guidance on interpretation and correlation of DSA
and Bx with clinical AMR
• Validation through clicical trials
• Studies that correlate with outcomes
• AHA Scientific Statement on AMR in the process of
being published
• Expectation is to validate

20. Clinical Cardiologists Need
• Consistency on interpretation
• Guidance on interpretation and correlation of DSA
and Bx with clinical AMR
• Validation through clicical trials
• Studies that correlate with outcomes
• AHA Scientific Statement on AMR in the process of
being published
• Expectation is to validate

21. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

22. Wisdom on interpreting DSA in cardiac
AMR
• Whether and which kind of DSA should be considered in diagnosis
AMR
• Discussion of value of C1q assay (pre- post- transplant)
• Kinds of non HLA DSA in heart transplant discussed with new
information from Cedars Sinai – AT1 receptor, MICA
• The must be a STRONG recommendation somewhere about
utilization of monitoring DSA. This will help hospital administrators
and 3rd party payers to make up their minds and pay for useful tests.

23. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

24. •For the pediatric cardiologist
•Highly sensitized patients, common
•Monitoring DSA already being done. But…
•How often? How long?
•Better defined role of non HLA antibodies in the
pediatric patients
•Definitions of dysfunction not standardized, which
makes difficult to utilize ISHLT working formulation for
AMR
•How to address AMR without dysfunction if just a
pathologic diagnosis

25. 3:30 - 4:00 The natural history of C4d vs. clinical AMR in a large cohort over 5 years
Carmela D. Tan, Cleveland Clinic
4:00 - 4:30 Update on the revised ISHLT AMR grading system
Gerald Berry, Stanford University
4:30 - 5:00 Practical approach, diagnostic issues and pitfalls in the pathologic diagnosis of AMR by IHC
Martin Goddard – Papworth Hospital, UK
5:00 - 5:30 Pathologic diagnosis of AMR – The clinician’s perspective
Monica Colvin-Adams, University of Minnesota
6:00 - 6:30 How is DSA monitored and, how will it complement pAMR diagnosis
Dolly B. Tyan, Stanford University
6:30 - 7:00 AMR in the pediatric heart recipients
Janet Scheel, Johns Hopkins University
7:00 – 7:30 Future directions and challenges in the diagnosis and management of AMR
E Rene Rodriguez, Cleveland Clinic

26. Antibodies in Heart Allograft Rejection
• Antibody mediated rejection -> Capillary deposition -> Complement
activation and deposition
• Antibody dependent cellular cytotoxicity -> Mediated by Cells (NK
cells, eosinophils, neutrophils)

27. Antibodies in Heart Allograft Rejection
• Antibody mediated rejection -> Capillary deposition -> Complement
activation and deposition
• Antibody dependent cellular cytotoxicity -> Mediated by Cells (NK
cells, eosinophils, neutrophils)
Acute process – Devastating if not treated – May recur

28. Antibodies in Heart Allograft Rejection
• Antibody mediated rejection -> Capillary deposition -> Complement
activation and deposition
• Antibody dependent cellular cytotoxicity -> Mediated by Cells (NK
cells, eosinophils, neutrophils)
Acute process – Devastating if not treated – May recur
Slow (smoldering / chronic) process – Eventually devastating –
Not amenable to effective treatment of prevention

29. Endothelial lining in the heart
-Coronary arteries
Epicardial
Intramural
-Coronary arterioles
-Capillaries
-Venules
-Veins
-Endocardium
Atrial
Valvular
Ventricular

30. Endothelial lining in the heart
-Coronary arteries
Epicardial
Intramural
-Coronary arterioles
-Capillaries
-Venules
-Veins
-Endocardium
Atrial
Valvular
Ventricular
AMR

31. Endothelial lining in the heart
-Coronary arteries
Epicardial
Intramural
-Coronary arterioles
-Capillaries
-Venules
-Veins
-Endocardium
Atrial
Valvular
Ventricular
AMR

32. Endothelial lining in the heart
-Coronary arteries
Epicardial
Intramural
-Coronary arterioles
-Capillaries
-Venules
-Veins
-Endocardium
Atrial
Valvular
Ventricular
AMR

33. Endothelial lining in the heart
-Coronary arteries
Epicardial
Intramural
-Coronary arterioles
-Capillaries
-Venules
-Veins
-Endocardium
Atrial
Valvular
Ventricular
AMR
Can we really assume
that these are two
related processes?
They may share one
thing..
Antibody
But… The antibodies
can produce allograft
injury (CAV) by a
different pathogenetic
mechanism