1. Summary / Report Heart Session Tenth Banff Conference on Allograft Pathology Banff, Canada 2009 E Rene Rodriguez Adriana Zeevi Annalisa Angelini and Lori West

2. Milestone: 1. In April 2009 at the ISHLT Scientific sessions in Paris there was a mandate from the members of the Pathology and Basic Science Council to the Chair (E R Rodriguez) and incoming Chair (S Ensminger) of the council to update the current Working formulation for the diagnosis of rejection in heart transplantation. 2. After consultation with Dr. Solez, the option of working with the Banff allograft pathology group was presented to the Board of Directors during the Council Reports Session of the ISHLT Scientific Sessions in Paris. Despite some early misgivings, the Board of Directors did not object to this initiative to synergize, in order to begin work on a new revision of the Working Formulation. The Chair of the Pathology and Basic Science Council was transferred to Dr. Ensminger and the incoming Chair Dr. A Angelini. The Board of Directors gave the charge to the past chair, chair and incoming chair to move forward with the revision of the Working Formulation With the clear mandate from the council membership to standardize the criteria for the diagnosis of Antibody Mediated Rejection in a manner that is reproducible and practical for everyday use in heart transplant centers around the world.

3. 3. The Chair and Co-Chair for the Banff Heart Session were approved by the Banff Conference organizers. ( ER Rodriguez and Adriana Zeevi) 4. The President of ISHLT (J Kirklin) sent his regrets that he would not be able to attend as his schedule had a conflict with another trip. He requested that member of the Board of Directors of ISHLT would be present at Banff. Dr. Lori West accepted to be the Board of Directors representative at the conference. 5. The goals of the Banff Heart Session were publicized in May 2009 through the Heart Failure Council and the Pathology Council, as well as the President of the ISHLT in the form of emails web link a PDF document stating these goals. In addition this was also publicized electronically through the Society for Cardiovascular Pathology and also through the Banff Conference on Allograft Pathology website

4. Attendance to the Banff Heart Session had > than 60 attendees including cardiologists, immunologists and pathologists The goals of the session were explained and followed by presentations including: 1. Gene expression studies that reflect the microvascular compartment in heart biopsies 2. Current understanding of the complement system and its regulators 3. Current armamentarium of tools to characterize assessment of sensitized patients, temporal appearance of DSA and follow up DSA 4. Clinical controversies and needs for improvement of the information provided to clinicians by pathologists and immunologists 5. Two surveys of current practice in the diagnosis of AMR in Europe and North America were presented. 6. Clinical experience on the diagnosis of AMR in heart transplant from Birmingham UK, Boston and Cleveland were presented.

5. Presence of donor specific HLA antibodies is associated with large scale gene expression changes mainly related to inflammatory processes in cardiac allograft biopsies HLA antibodies are associated with increased expression of: – Ifnγ regulated transcripts – T cells, macrophage and endothelial transcripts DSA is associated with changes in gene expression, myocardial capillary inflammation, and decreased cardiac graft function, even in biopsies with no C4d or C3d deposition Current immunohistologic criteria for ABMR need to be improved to increase its sensitivity. Conclusions Edmonton, Banu Sis et. al.

6. Summary C4d staining relatively uncommon Both DSA and highest grade of C4d correlates with death Correlation between C4d and DSA –? improved by C3d Neither C4d or DSA in isolation is sensitive at a single time point – C4d - comes and goes, precede inflam and symptoms – Repeat DSAs (- to + in 4/7) Birmingham, UK Desley Neil

7. Sound Criteria with Data for Definition of AAbMR 1. Cardiac Dysfunction 2. C4d/C3d; marker of alloantibody; marker of endothelial activation/injury 3. DSA C4d/C3d differential staining does not identify dysfunction C3d delays/disappears > C4d Histological Criteria in present classification poor: C4d+ Cases Two types of cardiac AHR ? with inflammation and dysfunction ?severe without inflammation but with dysfunction Accommodation? without inflammation without dysfunction StableUnstable Macrophage +++ Macrophage -++ Neal Smith, Boston

8. Correlation of C4d and C3d with DSA, allograft dysfunction and mortality Tan CD et al, Am J Transplant Epub 2009 Jul 16 Tan et al, Cleveland

9. Tan CD et al, Am J Transplant Epub 2009 Jul 16 Group 1Group 2 C4d+/C3d+ Diffuse DSA + Allograft Dysfunction C4d+ diffuse/C3d – DSA - No Allograft dysfunction Tan et al, Cleveland

10. In summary, A panel of C4d and C3d is more useful than C4d alone in the evaluation of AMR in heart transplants. Presence of C4d and C3d correlates with DSA and cardiac allograft dysfunction. Incidence of AMR in this cohort: 5% AMR can occur months to years after transplantation. Regulators of complement activation CD55 and CD59 may provide a protective mechanism from complement-mediated damage to the allograft. Tan et al Cleveland Tan CD et al, Am J Transplant Epub 2009 Jul 16

11. C4d methodology and interpretation in cardiac allograft biopsies - the European perspective Margaret Burke (Harefield, UK) Desley Neil (Birmingham, UK) Annalisa Angelini (Padua, IT) 10 th Banff Conference on Allograft Pathology August 2009 Survey # 1 – European Centers

12. Key points from questionnaire Of 35 European centres canvassed, 29 (85%) responded, of whom 24 (69%) did C4d staining, 21 centres (60%) using paraffin material C3d staining is done by only a few centres Selection of additional antibodies in an “AMR panel” to supplement any positive C4d staining of paraffin (or frozen) sections is variable Interpretation and scoring of C4d staining (structures stained, pattern, intensity) varies between centres Access to local immunology expertise “patchy” A small number of pathologists appear to “work in a vacuum” [do not do C4d staining/no clinical input at time of bx or clinical feedback afterwards] Survey # 1 – European Centers

13. Survey of pathologists at US and Canadian heart transplantation centers Queried about use and comfort with the ISHLT 2004 criteria & AMR-related practices Survey # 2 – Northamerican Centers Johns Hopkins University SOM Lauren Kucirka Joseph Maleszewski Dorry Segev 94 Survey Respondents Chi Lai Dylan Miller Charles Steenbergen Carmela Tan John Veinot

14. 94 Respondents –78% of transplant centers –82% of all transplants in 2008 Represents a good cross-section of US and Canadian centers Survey # 2 Northamerican Centers

15. 90% of centers reported evaluating for AMR. Centers that did not evaluate tended to perform fewer biopsies per year but were otherwise similar to other centers. However details on how this is done were not presented at this meeting ( comment added by ERR ) Survey # 2 - Northamerican Centers

16. “It would be good to know what the standard is for screening for AMR. It seems that by the time we are seeing positive IF (done only by request based on clinical suspicion), the clinical picture is so dire that the patients do not do well.” “Criteria on when to automatically test for humoral rejection would be useful. We occasionally see staining of only a few capillaries or blood vessels with C4d. We comment on it, but it would be nice to have a standardized way of grading / handling C4d staining.” “I have tried to assess humoral rejection and have found it impossible to interpret.” Survey # 2 - Northamerican Centers

17. “The aspect of biopsy grading that needs further standardization is AMR. When you talk to colleagues at other institutions, everyone is doing something different - different indications, different techniques, different interpretation. The clinical side needs to be addressed as well. If we have positive C4d staining and the patient is fine, no one knows what to do.” Survey # 2 - Northamerican Centers

18. HOW DO WE MAKE THE PATHOLOGIC DIAGNOSIS OF AMR? Recommendations for the technical aspects: 1. Who orders the test - is it based on clinical suspicion of the clinicians or histologic screening by the pathologists, any other scenario 2. Which antibodies to use - Immunofluorescence - Immunoperoxidase 3. Testing schedule - When is testing done? - Routinely? - Only during the first 6 weeks 4. Are there false negatives? The goals for the meeting were categorized as follows:

19. HOW DO WE MAKE THE PATHOLOGIC DIAGNOSIS OF AMR? Interpretation of tests for complement deposition 1. Which vascular territories to interpret? 2. Is intensity graded? how? do we need it? does it correlate? 3. What is the extent of staining? - focal vs diffuse? 4. How often are endothelial swelling and intravascular macrophages seen? Do they correlate with severity? 5. Artifacts - interstitial staining, endocardial staining, arterial staining, myocytes?

20. HOW DO WE MAKE THE PATHOLOGIC DIAGNOSIS OF AMR? Reporting 1. How is AMR reported? what is or should be reported as considered positive? 2. ISHLT definition (i.e. histologic markers + complement + DSA + clinical dysfunction)? 2.1. C4d alone? (there are papers reporting asymptomatic AMR). 3. What are the changes seen after therapy? 4. How often do you see clear cellular rejection greater than grade 1R along with markers of AMR?

21. HOW DO WE MAKE THE PATHOLOGIC DIAGNOSIS OF AMR? Serologic aspects in the diagnosis of AMR 1. When DSA is present, how often do we determine the titers? One time? More than one? Does you center follow the titers to monitor therapy? 2. How often does a drop in DSA titer correlate with improvement in function of the graft? Or changes in the histopathologic markers? 3. Does your center test for Non-HLA antigens? Routinely? Reflexive testing after HLA is negative?

22. OK… What happened during the discussion session?

23. Complement Immunohistochemical Staining Reproducibility Exercise: Invitation to submit autopsy tissue from patients who had AMR 1.Formalin fixed paraffin embedded autopsy tissue requested 1.2. Provide larger blocks, 1.3. No IRB or HIPPA issues in the USA 2.Tissue blocks to be divided and distributed back to participating centers 3. Centers that contributed tissue did not object to sharing of extra block with centers that did not have autopsy tissue available

24. Center# Blocks# CasesArraySlide MethodPermissionLateClinical Information Recd Sent Rcd C4d Rcd C3dRcdto ShareTissue Cleveland - Control 111 Y Non-ischemic explanted heart 1 Fixation < 24 hr Cleveland - Control 211 Y Non-ischemic explanted heart 2 Fixation < 24 hr 1Cleveland22111 Y Cleveland1 = ACR & AMR Fixation < 24 hr Y Cleveland2 = AMR Fixation < 24 hr 2Rigshospitalet111111Y No History 3Pompidou111111Y No History 4Harefield221111Y Harefield1 = ACR & AMR in it as well as GVD Harefield2 = Hyperacute rejection 5Columbia221111Y Columbia1 = + Control for C4d Columbia2 = + Control for C4d 6Mayo211101Y Mayo1 = Florid AMR 2 weeks after Transplant Fixation < 24 hr Mayo2 = Florid AMR 2 weeks after Transplant Fixation > 24 hr 7Padova21111 Y No History 8U Maryland22100 2 9Birmingham UK11111 1 10 Brigham & Women's0 0 110 11U Alberta0 0 110 12Stanford0 0 110 13U la Coruña0 110 171513127 2

26. C4d IHC

27. Tissue Array Rigshospitalet C4d

28. Cleveland 01 C4d as stained by all centers

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

30. Mayo < 24 h C3d as stained by all centers

31. Communication / collaboration of the teams (pathology immunology cardiology) is paramount History of the Pt is crucial - sensitization, surgeries, pregnancies, other instances of AMR Discussion - I

32. DSA Pre tx - Solid phase (whatever is best for your center) Bank serum Intervals? Consider doing virtual cross matches Pre Tx Then: 1 st month, 3 rd Month, 6 months, 1 year? When biopsying for cause Very important in pediatric transplants since biopsies are a difficult consideration Discussion - II

33. Consensus: Histology Issues Good assessable myocardium ISHLT-Working Formulation should define: - How often should AMR be evaluated? -Modified as a function of risk? -For how long? vs. Continuously? Discussion - III

34. -Adequacy of biopsy -What structure to interpret: CAPILLARIES ONLY – Diffuse > 50% - Focal < 50% -What to report?: Negative, Focal, Diffuse (Intensity of stain? Scale? Will need further discussion due to differences in technical aspects) -Presence of macrophages? -Edema of EC? -Mixed inflammatory infiltrates? Discussion - IV

35. Discussion - V -1 st Tier of stains for evaluation -Markers C4d (diffuse capillary pattern) and perhaps both C4d and C3d -Not needed to evaluate IgG, IgM …. -2 nd Tier of stains for evaluation CD68 Optional: CD20 and CD3 if Mixed Rejection -Optional: CD55, CD59 -IF vs IHC (at least two centers in the US are evaluating these two techniques for equivalency) **If C4d is positive, recommend DSA and consider doing C3d for confirmation

36. Conclusion Consensus was achieved on most of the goals set 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 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 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 With standardization of criteria for diagnosing clinical AMR it will be then possible to start evaluating “in a standartized” manner subclinical AMR and the impact of this on allograft longevity or epicardial coronary disease (allograft vasculopathy).