Summary by Dr. Rene Rodriguez of the heart sessions from the 2011 Eleventh Banff Conference on Allograft Pathology, June -10, 2011 in Paris, France.

1. Heart Sessions Summary 11 th Banff Conference on Allograft Pathology June 6 -10, 2011 Enghien les Bains, France

3. 2001 Banff – 6 th Banff Conference √ -First time a Heart Symposium was formally convened √ - ISHLT WF had withstood the test of time (11 years or worldwide use) √ Grade 2 benchmark used in trials and to trigger therapy √ -Criteria for kids not tailored for their particular problems (in particular “humoral” rejection) -Criteria for “humoral” rejection needed

4. 2003 Aberdeen – 7th Banff Conference √ -Non HLA antigens important (but frequency??) -Role of chemokine expression as markers -Role of coagulation/fibrinolysis in vasculopathy (not yet reproduce as of 2011) √ -Expression profiling of Cardiac Allograft Vasculopathy identified molecules in CAV and not present in Atherosclerosis Hopkins/Yale √ -Problems with reproducibility in the UK needed to be considered for an international classification

6. 2005 Edmonton – 8th Banff conference -Expression profiling – Allomap Test (Panacea!)(think again… useful tool to rule out inflammation in the heart) 1:00-1:30 The new working formulation of ISHLT. - Susan Stewart* 1:30 - 2:00 Definition antibody mediated rejection at ISHLT. - E. Rene Rodriguez* 2:00 - 2:30 The future role of pathologists in cardiac transplantation, can we meet the clinician's needs. - Randall Starling* 2:30 - 3:00 Refreshment Break (Empire Ballroom Foyer) 3:00 - 3:30 The complement system in antibody mediated rejection. - Wink Baldwin* 3:30 - 4:00 The complement regulatory proteins. - Gonzalo Gonzalez-Stawinski*

7. 2007 La Coruña – 9 th Banff conference - Review of the updated ISHLT WF 2004 √ - What about kids? - Patient with severe allograft dysfunction, no evidence of CR or AMR on biopsy what can pathology (anatomic, clinical, molecular) offer ? (Low frequency number of patient but still unresolved) √ -What’s new on non-HLA antigens? What are they? What is the Prevalence? Clinical Significance?

8. 2007 – 9 th Banff conference √ - Are there any new molecular tools that work on real time? - Can the diagnostic criteria of AMR be standardized to be at least as good ( reproducible ) the criteria for CR? ? - Is there are cause effect relationship between Antibody-Complement and CAV? ? - Since AMR can be devastating in an acute setting and may be associated with CAV, can we modulate it with pharmacologic therapy?

10. Banff 2007 going into Banff 2009 -> Goal: Standardize C4d & C3d Immunoperoxidase staining for discussion at Banff 2009. Done using Digital Pathology

12. Mayo < 24 h C4d as stained by all centers

13. Tissue Array Cleveland C3d

14. Centers in the C4d C3d IHC Reproducibility Study Center Cases Rcd C4d Rcd C3d 1 Cleveland 2 1 1 2 Rigshospitalet 1 1 1 3 Pompidou 1 1 1 4 Harefield 2 1 1 5 Columbia 2 1 1 6 Mayo 1 1 0 7 Padova 1 1 1 8 U Maryland 2 0 0 9 Birmingham UK 1 1 1 10 Brigham & Women's 0 1 0 11 U Alberta 0 1 0 12 Stanford 0 1 0 13 U la Coruña 0 1 0 14 Montefiore 0 1 1 15 U Minnesota 0 1 1 16 Herzzentrum 0 1 1 17 Sentara 0 1 0 18 UPMC 0 1 0 19 U Colorado 0 1 0 20 U Arizona 0 1 1 21 CC Immunology 0 1 0 22 U Chicago 0 1 23 UCLA 0 1 24 Cedars Sinai 0 1 13 23 11

15. Is IF equivalent to IHC?

16. http://www.e-heart.org

17. http://www.e-heart.org

18. Miller DV et al Arch Pathol Lab Med 2010: 134; 1679-1684 e, Diffuse strong staining by IF (3+); f, Diffuse strong staining by IP (3+)

20. 2009 – Banff – Consensus Serology and Reproducibility (I) Milestone – ISHLT and Banff working together (Board of Directors (Lori West)). Consensus was achieved on most of the goals for this session Serologic aspects in the diagnosis of AMR: Collaborative work on evaluating patients with suspected or proven AMR (i.e. immunologists, pathologists, cardiologists) Guidelines for minimum required action for establishing serologic diagnosis of DSA . Evaluate DSA over time Interpretation of tests for complement deposition: Agreement on what structures should be interpreted in the assessment of AMR by light microscopy and by immunofluorescence / immunohistochemistry (i.e. capillary endothelial cells only). Advocate further use of Digital Pathology

21. 2009 – Banff – Consensus Serology and Reproducibility (II) Recommendations for the technical aspects: Obviate the need of numerous immunostains that do not have sensitivity or specificity Use of C4d and reproduce the usefulness of a combination of C4d & C3d to add specificity to the diagnosis of AMR. Evaluate capillaries only. With standardization of criteria for diagnosing clinical AMR it will be then possible to start evaluating “in a standardized” manner subclinical AMR and the impact of this on allograft longevity or epicardial coronary disease (allograft vasculopathy). AMR is a late phenomenon in most cases

29. C4d positive biopsy without dysfunction: Accommodation vs subclinical rejection? Carmela D. Tan, M.D. Cleveland Clinic 08 June 2010 11 th Banff Conference on Allograft Pathology Enghien-les-Bains, France

31. Conclusions : Redefining late rejection Late rejection occurs in 5-10% of patients with long term survival Late rejection frequently exhibits features of pAMR, presence of DSA and may be associated with progression of CAV Late rejection may be the witness of an ongoing graft injury during the process of AMR Dr. Jean-Paul Duong Van Huyen

32. Outstanding issues Late protocol biopsies? Criteria for mixed rejection? Relevance of new entities: MI+ C4d- DSA+? Risk stratification/therapeutic intervention?

33. Autoantibodies are formed early after transplantation. De-novo DSA occur 1-8 years After transplantation Smith et al, AJT, 2011. 0 5 10 15 20 25 30 0 1 2 3 4 5 6 Yrs Post transplant % Antibody positive Vimentin IgM non-HLA HLA Dr. John Smith 86 67 43 23 40 70 56 23 21 35 72 56 38 23 35

34. De novo HLA DSA are strongly associated with development of AMR, particularly if directed against donor HLA-DR or –DQ. Demonstrates the importance of post transplant monitoring for antibody development. Antibodies directed against non-HLA determinants may also be associated with AMR. Pre-transplant non-HLA abs may be predictive of AMR, especially in the cardiomyopathy patients. It may explain patients who are C4d+ve in the absence of DSA-HLA. Definition of DSA-HLA by titre and/or C’ fixing may be useful for stratifying risk. Conclusions Dr. John Smith

35. In summary, we have identified themes that needed to be: -Reassured -> (ISHLT WF CR Withstood test of time) -Reproduced -> (Markers in tissue) -Synchronized -> Serologic approach and AMR pathology -Standardized -> (C4d C3d reading by IF and IHC) -Used Digital pathology for rapid evaluation -ISHLT reacted to these initiatives at Banff NOT DONE i.e. Challenges for future work Pediatric focused issues with AMR Cause effect of anything and CAV What triggers AMR as a late (de novo) phenomenon?