11th Banff Conference on

Allograft Pathology
Carol F. Farver, MD
Cleveland Clinic
W. Dean Wallace, MD
UCLA
Co-Chairs, 11th Banff Conference-Lung
June 2011

Lung Session Topics

Antibody-mediated Rejection
Infections in Lung Transplant
Non-BOS Graft Dysfunction
Endothelial Cells in the Lung Allograft

Antibody-mediated Rejection
Lung
Clinical
Diagnostic criteria
Effect on survival
Role in etiology of BOS

CLINICAL FEATURES OF AMR

IN LUNG TRANSPLANTATION
Ramsey Hachem, MD
June 7, 2011

Humoral immunity
Rejection T-cell mediated
Role for antibodies in rejection
HLA antibodies in lung transplantation
AMR in kidney transplantation

AMR in lung transplantation is enigmatic

In 2006, no histologic features for
antibody-mediated rejection in the lung
were agreed upon
J Heart Lung Transplant 2007; 26: 1229

HLA antibodies

HLA AB & BOS

AB precede BOS
AB & lymphocytic
bronchiolitis
Underscore role of
humoral immunity

Transplantation 1998; 65: 648

Transplantation 1999; 67: 1155
J Heart Lung Transplant 2004; 23: 1135
Am J Transplant 2005; 5: 131

AMR study

Retrospective cohort study of acute AMR

Between 1/1/06 & 1/1/11
Follow-up through 4/1/11
AMR diagnosis fulfilling all 4 criteria
DSA, C4d, pathology, & graft dysfunction

19 patients

Donor-specific antibodies
19/19 had circulating DSA

Immunodominant DSA MFI

Neutrophilic pneumonitis: n=10

Capillaries injury with pneumonitis: n=9

+C4d staining: n=19

Early outcomes
14/19 (74%) improved & discharged
8/9 (89%) with capillary injury improved
6/10 (60%) without capillary injury improved

5/19 ( 26%) did not respond, died of

refractory AMR

Survival

DSA clearance
1.1
Cleared DSA
1.0
.9
.8
.7
.6
.5
Persistent DSA
.4
.3
.2
.1 log rank p = 0.005
0.0
0
365
730 1095

1460

Time after AMR diagnosis, d

1825

BOS
(Survived AMR: n=14)
1 pt: Pre-existing BOS
8/13 pts: Developed BOS within 1 year of
AMR diagnosis.

Overall Survival
(n=19 patients)
1.0

10/19 (53%) died

AMR, n = 5
BOS, n = 4
Pneumonia, n = 1

Survival

.8
.6
.4
.2
0.0
0

365

730

1095

1460

Time after AMR diagnosis, d

1825

Conclusions

AMR may cause acute graft failure

Index of suspicion
Less severe cases may be unrecognized
Subclinical humoral rejection & potential
impact on BOS
Unknown incidence

Subsequent BOS

Antibody-mediated Rejection
Lung
Clinical
Diagnostic criteria
Effect on survival
Role in etiology of BOS

Immunology
Role of DSA
HLA vs non-HLA antibodies

Immunology of AMR in
Lung Transplantation
Medhat Askar, MD, PhD
Director, Allogen Laboratories, Cleveland Clinic
Associate Professor, Department of Surgery
Cleveland Clinic Lerner College of Medicine at
Case Western Reserve University School of Medicine
11th Banff Conference | June 7, 2011

18

Outline
Pre-transplant Immunologic risk
assessment
Post-transplant immunological
monitoring
DSA
C4d

Non-HLA targets for humoral

immune responses

RESULTS
Pre-transplant DSA

RESULTS
Positive
Pre-Tx
DSA

Reference P
Group
Value

ACR ( ISHLT
grade II)

43%

29%

ACR/100 Pt./Yr

20.6

7.7

0.0009

(+)Post-Tx DSA with (+) C4d

>1000 consecutive lung transplant
biopsies at CCF were stained for C4d
and C3d (IF)
Surveillance and Clinical indication

Four (4) were + C4d

All in the setting of clinical disease
Acute and organizing DAD (x3); Capillary
injury (x1)

All 4 were + DSA (post-transplant)

None were + C3d

Pos C4d

Pos C4d

Pos C4d

Non-HLA Antibodies

MHC Class I-related chain A (MICA)

Antibodies
6/116 (5%)Reference were Pos for MICA
Ab
4/35 (11%) patients with Pos DSA were
also pos for MICA Ab
None were DSA MICA

Antibody-mediated Rejection
Lung
Clinical

Diagnostic criteria
Effect on survival
Role in etiology of BOS

Immunology

Role of DSA
HLA vs non-HLA antibodies

Pathology

Importance of C4d staining

Pathologic pattern of injury

Pathology of AntibodyMediated Rejection of the Lung

Allograft
W. Dean Wallace, M.D.
Associate Professor of Pathology
David Geffen School of Medicine
UCLA Medical Center

Putative Stages of Humoral Response to

an Organ Graft 2003 NIH
recommendations
I: Latent humoral response
Circulating antibody alone (but without biopsy findings or graft
dysfunction)
II: Silent humoral reaction (accommodation vs pre-rejection
state)
Circulating antibody, C4d deposition (but without histologic
changes or graft dysfunction)
III: Sub-clinical humoral rejection
Circulating antibody, C4d deposition, tissue pathology (but
without graft dysfunction)
IV: Humoral rejection
Circulating antibody, C4d deposition, tissue pathology, graft
dysfunction
Takemoto SK, et al. National conference to assess antibody-mediated rejection in solid organ
transplantation. Am J Transplant 2004;4:103341.

Complement deposition ISHLT

Lung Rejection Study Group
Historically associated with hyperacute rejection.
Serum anti-HLA antibodies and complement deposition
support the concept of AMR in the lung.
Some of the suggested clinical-pathologic features of
AMR, extrapolated from findings in other organ allografts
including deposition of C3d and C4d, may serve as a
marker AMR.
Low specificity limits use in protocol biopsies.
Stewart S, et al. Revision of the 1996 working formulation.lung
rejection. J Heart Lung
Transplant 2007; 26:1229-42.

C4d staining in lung transplant

biopsies
Saint Martin (1996)
C3, IgM, IgG

Magro (2003)

+C4d with septal injury

22 pts with (-) PRA

Magro (2003)

+C4d with septal injury

25 lung pts. With (-) PRA, (-) HLA-Abs

Magro (2003)

+C4d with bronchial epithelial injury

Girnita Al, (2006)

2 pts.
+DSA, +C4d

Wallace (2005)
+C4d

Hachem et al (unpublished)
C4d+
19 lung tx pts.

Nonspecific C4d staining

Nonspecific background staining
Post capillary venule

Nonspecific C4d staining

Nonspecific elastic staining

Hyaline membranes in DAD

Capillaritis in transplant
patient

with acute
pneumonia

the transbronchial biopsy

ISHLT study group recommends 5 pieces of alveolar
tissue
Unlike in the kidney, heart and liver, lung biopsies are
very easily crushed and distorted.
Biopsies are frequently inadequate.
Procedural hemorrhage can make the biopsy technically
difficult to perform and interpret.
Distinction between normal, infection, aspiration and
alloimmune rejection can be very difficult.

Putative Stages of Humoral Response to an

Organ Graft 2003 NIH recommendations
I: Latent humoral response
Circulating antibody alone (without biopsy findings or graft
dysfunction)
II: Silent humoral reaction (accommodation vs pre-rejection
state)
Circulating antibody,
(without histologic
changes or graft dysfunction)
III: Sub-clinical humoral rejection
C4d
Circulating antibody,
depositiontissue pathology (without
graft dysfunction)
IV: Humoral rejection
C4d
Circulating antibody,
tissue pathology, graft
deposition,
dysfunction

C4d
deposition,

Takemoto SK, et al. National conference to assess antibody-mediated rejection in solid organ
transplantation. Am J Transplant 2004;4:103341.

Putative Stages of Humoral Response to

an Organ Graft 2003 NIH
recommendations
I: Latent humoral response/Silent humoral reaction
Circulating antibody alone (without biopsy findings or graft
dysfunction)

III: Sub-clinical humoral rejection

Circulating antibody,
(without graft
dysfunction)
tissue pathology
IV: Humoral rejection
Circulating antibody,
graft dysfunction

tissue pathology,

I: Latent humoral response/Silent humoral reaction/Subclinical humoral rejection

Circulating antibody alone (without biopsy findings or
graft dysfunction)

Where is the patholo

II: Humoral rejection
Circulating antibody, graft dysfunction

Clinical-Pathologic Diagnosis of
AMR in the Lung Allograft
Matt DeNicola, Sam Weigt, W. Dean Wallace.
UCLA David Geffen School of Medicine

Objective
Determine if there is a specific histopathologic or
immunohistochemical finding that is characteristic of
patients with de-novo DSA and AMR.

Criteria for AMR

Detection of circulating DSA
Compatible tissue pathology
Intravascular neutrophils
C3d/C4d deposition?

Graft dysfunction

Study design
Case control study:
Since 1/1/2006, lung txp recipients screened
for DSA at surveillance time-points.
Inclusion required Tbbx within 30 days of denovo DSA or negative screening test for DSA:
16 cases with de-novo anti-HLA DSA
30 controls never developing DSA bx selected to
achieve a similar range of duration post-transplant
9 with de-novo anti-HLA, not donor specific (Non-DSA)
21 without any anti-HLA ab

Study Design
Biopsies assessed by pathologist blind to any clinical
data including the presence or absence of DSA.
Capillary neutrophilia graded on 0-4 scale

0 Normal
1+ Mild increase in PMN
2+ Increase of PMNs with groups of 2
3+ Increase of PMNs with groups of 3 or more
4+ Capillaritis

DAD
Pathology suspicious for AMR was defined as > 2+ neutrophilia,
or unexplained DAD.

Biopsies also stained for C4d and C3d and scored by

pathologist
Any positive staining was used for correlation analyses

2+ capillary neutrophilia

C4d

C 4D coded

C4d does not correlate with pathology

Fisher's
Exact Test
Left
Right
2-Tail

Sus pect AMR (path)

Count no
yes
Total %
Col %
Row %
neg
19
8
47.50
20.00
63.33
80.00
70.37
29.63
pos
11
2
27.50
5.00
36.67
20.00
84.62
15.38
30
10
75.00
25.00

Prob
0.2861
0.9182
0.4507

27
67.50

13
32.50

40

Alternative Hypothesis
Prob(Suspect AMR (path)=yes) is greater for C4D coded=neg than pos
Prob(Suspect AMR (path)=yes) is greater for C4D coded=pos than neg
Prob(Suspect AMR (path)=yes) is different across C4D coded

Pathology does correlate with DSA

Suspect AMR (path)

DSA
negative

Fisher's
Exact Test
Left
Right
2-Tail

Prob
0.0010*
0.9999
0.0016*

Count DSA
Total %
Col %
Row %
no
6
13.04
37.50
18.75
yes
10
21.74
62.50
71.43
16
34.78

26
56.52
86.67
81.25
4
8.70
13.33
28.57
30
65.22

32
69.57

14
30.43

46

Alternative Hypothesis
Prob(DSA=negative) is greater for Suspect AMR (path)=no than yes
Prob(DSA=negative) is greater for Suspect AMR (path)=yes than no
Prob(DSA=negative) is different across Suspect AMR (path)

Pathology is associated with lung

allograft survival

Percent survival

100
80
60
2+ Neutrophilia

40
20
0

Log-Rank p = 0.02
0

365

730

1095

1460

Days Post-Transplant

1825

Pathology + DSA is associated with lung

allograft survival

Percent survival

100
80
60
Histopathology + DSA

40
20
0

Log-Rank p = 0.02
0

365

730

1095

1460

Days Post-Transplant

1825

Suspected AMR (Pathology + DSA +

dysfunction) is associated with lung
allograft survival

Percent survival

100
80
60
Suspected AMR

40
20
0

Log-Rank p = 0.04
0

365

730

1095

1460

Days Post-Transplant

1825

Wallace Pathology Summmary

Pathology may be graded capillary injury
with capillaritis at one end of spectrum
Capillary neutrophils (>Grade +2) correlate
with post-tx DSA, but not with +C4d
Almost all biopsies can demonstrate some form
of C4d staining; therefore, C4d deposition is
nonspecific and is probably not sensitive
All features are nonspecific in isolation

11th Banff Conference---Lung

Session Discussion Points
DSA antibodies
Pre-transplant (memory) Abs vs post-transplant (de
novo) antibodies
represent different processes/risks in the development of AMR
this needs to be explicit in literature/future studies

Timing of Abs appearance with the pathology of the

biopsy is important
protocols that time blood sampling with lung biopsy are essential

Non-HLA antibodies need further study in lung

transplantation patients

11th Banff Conference---Lung

Session Discussion Points
Pathology
Methodologies for staining C4d are NOT
standardized
Interpretation of C4d staining in the lung is
probably subject to inter-observer variability
Defining (naming) the pathologic pattern of
injury in biopsies of possible AMR in the lung is
not consistent

Capillaritis
Capillary injury
Acute pneumonitis
Acute lung injury with neutrophils

11th Banff Conference---Lung

Session Discussion Points
Pathology
Given the overlap of pathologic features of
AMR with infection, the role of infection in the
pathology has not been adequately assessed
in many published studies
Incidence of biopsies with possible AMR in the
lung (graft dysfunction, +DSA, +C4d) may be
quite low

Conclusions
Published data to date are difficult to
interpret given
Lack of standardization of methodology
Antibody (DSA) measurement
C4d staining/interpretation

Inconsistent pathologic descriptions

Other etiologies (infection, drug toxicity) not
excluded

THE LUNG IS A MESS!

Background Studies Needed

Pathology:
Consensus for basic definitions of pathologic
patterns of injury
Education around terminology

Standardization of methodology for staining and

for pathologic interpretation of C4d

Immunology
Standardization of antibody measurement
methodology
Routine measurement of both pre- and postDSAs

Background Studies Needed

Clinical
Protocols that coordinate clinical evaluation
(graft function), Ab measurement and biopsy
results for accurate comparison of data from
same time point.
Rigorous screening for other causes must be
routinely done
Infections (cultures and tissue stains)
Drug toxicity

The journey of a thousand miles

begins with a single step
Lao-tzu,
Chinese philosopher 604 BC - 531 BC

First Steps
Organize lung transplant pathologists (Farver
and Wallace) with clinicians (Levine and
Glanville) into AMR working group
Survey for interest
ISHLT
Banff
Pulmonary Pathology Society

12 Banff Conference
th

Working group of AMR in Lung Transplant

Collection of biopsy slides from possible
AMR patients from pathologists
Digital pathology platforms

Standard methods for

C4d staining and interpretation
Standardization of pathology pattern of injury
in AMR
? grading system..

Additional Topics
Infections in Lung Transplant (Stewart)
New infections with an update of molecular diagnostic
techniques
Always in the differential diagnosis (esp. AMR)

Progressive, fibrosing parenchymal lung

diseases post-transplantation (Iversen)
Is there parenchymal form of chronic rejection

Mechanism of endothelial injury in transplanted

lungs (Belperio)
Chronic vascular rejection: small and large vessels

Conference Presenters
Lung Session
Medhat Askar, Cleveland Clinic (USA)
John Belperio, UCLA (USA)
Ramsey Hachem, Washington University (USA)
Martin Iversen, Copenhagen University Hospital,
(Denmark)
Susan Stewart, Papworth Hospital (UK)
W. Dean Wallace, UCLA (USA)
Plenary Speakers
Andrew Fisher, Newcastle University (UK)
Adriana Zeevi, University of Pittsburgh (USA)