Heart Sessions Summary 11th Banff Conference on Allograft

Pathology

June 6 -10, 2011

Enghien les Bains, France

10 years of Heart Sessions

• 2001 Banff

• 2003 Aberdeen

• 2005 Edmonton

• 2007 A Coruña

• 2009 Banff

• 2011 Enghien les Bains

2001 Banff – 6th Banff Conference √-First time a Heart Symposium was formally convened√ -ISHLT WF had withstood the test of time ISHLT WF had withstood the test of time (11 years or worldwide use)(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

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

ISHLT 2004

• Convenes panel for revision of the working formulation

• Cellular rejection – Revised• Humoral Rejection – Recommendations made• AMR 0 and AMR1• AMR requires:

1. Allograft dysfunction. 1. Allograft dysfunction. 2. Immunostain positive - several possible 2. Immunostain positive - several possible

markers markers3. DSA3. DSA

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*

2007 La Coruña – 9th Banff conference- Review of the updated ISHLT WF 2004√ - What about kids?- Patient with severe allograft dysfunction, no Patient with severe allograft dysfunction, no evidence of CR or AMR on biopsyevidence of CR or AMR on biopsywhat can pathology (anatomic, clinical, what can pathology (anatomic, clinical, molecular) offermolecular) 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?

2007 – 9th 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 (reproduciblereproducible) 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?

Using ISHLT 2004/2006 Criteria we Using ISHLT 2004/2006 Criteria we looked prospectively at CC patientslooked prospectively at CC patients

• 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.

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

Mayo < 24 h C4das stained by all centers

Tissue ArrayCleveland C3d

Center Cases Rcd C4d Rcd C3d

1 Cleveland 2 1 12 Rigshospitalet 1 1 13 Pompidou 1 1 14 Harefield 2 1 15 Columbia 2 1 16 Mayo 1 1 07 Padova 1 1 18 U Maryland 2 0 09 Birmingham UK 1 1 1

10 Brigham & Women's 0 1 011 U Alberta 0 1 012 Stanford 0 1 013 U la Coruña 0 1 014 Montefiore 0 1 115 U Minnesota 0 1 116 Herzzentrum 0 1 117 Sentara 0 1 018 UPMC 0 1 019 U Colorado 0 1 020 U Arizona 0 1 121 CC Immunology 0 1 022 U Chicago 0 1 23 UCLA 0 1 24 Cedars Sinai 0 1 13 23 11

Cent

ers

in th

e C4

d C3

d IH

C Re

prod

ucib

ility

Stu

dy

Is IF equivalent to IHC?

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

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

Miller DV et al Arch Pathol Lab Med 2010: 134; 1679-1684Miller 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+)

2.IF is sensitive, specific and quick•IHC is reproducible amongst centers•Optimization of staining protocols and appropriate controls available• IF and IHC are highly comparableIF and IHC are highly comparable

Is IF equivalent to IHC?

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 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

2009 – Banff – Consensus Serology and Reproducibility (II)

Recommendations for the technical aspects:Obviate the need of numerous immunostains that do not have sensitivity or specificityUse 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

Proposed ISHLT 2011 GRADING SCHEME FOR AMR

• 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

Berry GJ, et al J Heart Lung Transplant 2011: 30; 601-611Berry GJ, et al J Heart Lung Transplant 2011: 30; 601-611

Proposed ISHLT 2011 GRADING SCHEME FOR AMR

• 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

Berry GJ, et al J Heart Lung Transplant 2011: 30; 601-611Berry GJ, et al J Heart Lung Transplant 2011: 30; 601-611

Proposed ISHLT 2011 GRADING SCHEME FOR AMR

• It suggests the use of C4d and for centers using CD68 continue to use it

• IF C4d, C3d and if needed HLA for capillaries

• Evaluate only capillaries

Wait until you have Q wave MI approach

• Chest pain?• Sweat?• Radiation to arm?• ST changes in ECG?• Mild elevation of Troponin?

• ….. Wait until you see Q waves to make the diagnosis

11th Banff Conference on Allograft Pathology – Heart Session

• 1:00 - 1:30 Towards a new ISHLT antibody mediated rejection criteria. Rene Rodriguez

• 1:30 - 2:00 Are histological lesions needed for diagnosis of cardiac AMR before further injury occurs? Marny Fedrigo/Annalisa Angelini

• 2:00 - 2:30 Symptomatic and asymptomatic cardiac antibody mediated rejection. Maryl Johnson

• 2:30 - 3:00 C4d positive biopsy without dysfunction: accommodation or subclinical rejection?. Carmela D. Tan

• 3:00 - 3:30 Coffee Break • 3:30 - 4:00 Redefining patterns and significance of late rejection in

the era of AMR. Jean Paul Duong van Huyen • 4:00 - 4:30 New serologic markers in cardiac AMR: Incidence and

diagnostic utility. Marlene Rose • 4:30 - 5:00 Improving diagnostic specificity and sensitivity of EMB

by biopsy-based gene expression profiling. Michael Mengel• 5:00 – 6:00 Discussion

Conclusions 1• Histological lesions are not always present in Asymptomatic

AMR

– Endothelial swelling is an aspecific histological criteria for AMR

– Intravascular mononuclear cells are present in only 1/3 of C4d and DSA positive pts

• Combinig these parameters did not significantly improve their sensitivity and specificity.

• Histology is not a screening tool.

• Sensitivy and specificity of histological lesions are not modified by C4d + C3d together.

• Preliminary results do not show correlation between antibody title and histological lesions.

Drs. Fedrigo/Angelini

Where do We go From Here?Allograft Dysfunction

Without Cellular Rejection

• Need registry of treatments/outcomes• Could consider study of most promising therapies when they

are defined

Likely treat as AMRTreat as AMR

(-) Biopsy AMR(-) DSA(-) CAV

(+) DSA(Other antibodies?)

(+) Biopsy AMR

Dr. Maryl Johnson

C4d positive biopsy without dysfunction:C4d positive biopsy without dysfunction:Accommodation vs subclinical rejection?Accommodation vs subclinical rejection?

Carmela D. Tan, M.D.Carmela D. Tan, M.D.Cleveland ClinicCleveland Clinic

08 June 201008 June 2010

11th Banff Conference on Allograft PathologyEnghien-les-Bains, France

ConclusionsConclusions• Diffuse C4d capillary staining alone should not be

equated with AMR.

• Majority of C4d+ only episodes are single occurrences and asymptomatic.

• Presence of C4d staining and DSA without allograft dysfunction may indicate accommodation.

• Only a minority of patients with C4d staining alone may develop AMR on follow-upon follow-up.

• C4d alone is neither diagnostic or prognostic.

• C4d and C3d are highly correlative with DSA and allograft dysfunction (i.e. AMR as defined by ISHLT 2004) and appears late after transplant.

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

Outstanding issues

Late protocol biopsies?

Criteria for mixed rejection?

Relevance of new entities: MI+ C4d- DSA+?

Risk stratification/therapeutic intervention?

86

67

43

23 40

70

56

23

21 3572

56

3823

35

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

% A

ntib

ody

posi

tive

Vimentin IgM non-HLA HLA

Dr. John Smith

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.

ConclusionsDr. John Smith

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 workPediatric focused issues with AMRCause effect of anything and CAV What triggers AMR as a late (de novo) phenomenon?

Working Groups on Cardiac AMR

• ISHLT

• AHA

• Banff

• AECVP

• Dx COP - AST

• The molecular microscope vs. the light microscope

• Either one needs a thinking human brain to interpret