C4d in pancreas transplant biopsies:the Leiden experience

Ingeborg BajemaIngeborg BajemaIngeborg Bajema

Introduction

• Taking biopsies from pancreas transplants was uncommon until the early 1990s when a technique of percutaneous needle biopsy was introduced

• Each year, circa 3000 pancreas transplants are performed worldwide, 75% in the setting of a SPKT, mostly for patients with diabetes type 1

• Some hospitals follow serum creatinine as a surrogate markers for pancreas rejection

Pancreas rejection: Drachenberg et al, Am J Transpl 2008

Working proposal for grading pancreas allograft rejection

1. Normal

2. Indeterminate

3. Cell-mediated rejection (grade I, II, III)

4. Antibody-mediated rejection

5. Chronic allograft rejection/graft sclerosis

6. Other histological diagnosis (e.g. CMV pancreatitis, PTLD)

Pancreas rejection: Drachenberg et al, Am J Transpl 2008

Working proposal for grading pancreas allograft rejection

1. Normal

2. Indeterminate

3. Cell-mediated rejection (grade I, II, III)

4. Antibody-mediated rejection

5. Chronic allograft rejection/graft sclerosis

6. Other histological diagnosis (e.g. CMV pancreatitis, PTLD)

• The role of a C4d staining in relation to AMR is only beginning to be described

Pancreas rejection: Drachenberg et al, Am J Transpl 2008

Working proposal for grading pancreas allograft rejection

Antibody-mediated rejection:• Complement deposition in vessels (i.e. capillary C4d deposition)

which can be accompanied by monocyte/macrophage and

neutrophil margination within interstitial capillaries

• Graft dysfunction

• Donor-specific antibodies (DSA) in serum

Melcher in 2006 were the first to report a patient with a SPKT

who developed AMR with donor-specific HLA-DR allo-

antibodies and had positive C4d staining in the pancreas one

month after a SPKT. The renal transplant biopsy of this

patient taken at day 10 was also positive for C4d.

Overview of literature: Melcher, Am J Transplant, 2006

pancreas kidney

Carbajal et al a year later reported a patient with pancreas after

kidney transplantation (PAK) who developed AMR of the

pancreas with C4d positivity in the biopsy. The kidney, which

was from a different donor, remained unaffected, but the

pancreas graft was lost

Overview of literature: Carbajal et al, NDT 2007

Gaber reported one case of a C4d positive surveillance biopsy

taken 2 weeks after transplantation from a patient who was

sensitized by two previous islet transplants and had

detectable anti-HLA antibodies

Overview of literature: Gaber, Arch Pathol Lab Med 2007

Pascual reported 13 patients with an acute rejection of the

pancreas of which 2 were biopsy-proven, and these were

positive for C4d staining.

In one patient, two biopsies of the pancreas were taken. DSA

were negative at the time of the first biopsy, which was C4d

positive. Three months later, a second biopsy was taken

which was C4d positive as well, and by that time DSA were

also positive

Overview of literature: Pascual, JASN 2008

Torrealba et al reported on the potential role of C4d in pancreas

transplant biopsies with AMR in a group of 18 patients

27 biopsies from 18 patients

Correlation between C4d+, development of DSA and graft dysfunction

There is potential value of C4d staining in the diagnosis of AMR of

pancreas allografts

Overview of literature: Torrealba et al, Transplantation 2008

C4d+ can be encountered in various clinical settings:

2/4 patients with C4d+ had typical AMR

In one patient, C4d+ was associated with recurrent diabetes

Another patient (with circulating DSA), remained euglycemic

after 12 months of follow-up and without treatment of AMR

Overview of literature: comment by Munivenkatappa et al. to Torrealba et al, Transplantation 2009

…. there is perhaps a spectrum of clinical scenarios, tissue

evidence of antibody activation, and serologic patterns that

still need to be defned to better understand AMR as an entity

in pancreas allograft.

Overview of literature: reply from Torrealba et al. To comment by Munivenkatappa et al., Transplantation 2009

Whole pancreas transplantation at LUMC

• 109 pancreas transplants between 1991 – 2001

• All SPKT

• Pancreas transplant biopsies were taken on clinical indication until

2001

• 19 biopsies from pancreas transplants in this study

• 26 pre-transplant biopsies

Pancreas transplant biopsies from LUMC

• 19 biopsies from 18 patients

• 17/19 biopsies taken because of clinically suspected rejection

• 10 renal biopsies taken at the same time

• H&E slides scored according to BANFF working proposal

• C4d immunostaining was performed on 4 μm paraffin sections

• C4d positivity: • Diffuse: > 50% of interacinar capillaries positive staining

• Focal: 5 to 50% of interacinar capillaries positive staining

• Minimal: < 5% of interacinar capillaries positive staining

• Of 13/18 patients, DSA assessment was available

C4d staining patterns

negative diffusely +

focally +

Recipient characteristics

• Mean age: 38.9 years (SD: 8.3)

• Gender, % male: 67%

• Years of DM1 at Tx: 6.9 years

• % of women pregnant before Tx: 67%

• Biopsy taken at day (mean, range): 97 (12-481)

• DSA: negative in 6, positive in 7

Results

2 / 24 pre-transplant biopsies showed diffuse

C4d staining pattern

R=rejectiono=no rejection

n =negativeF=focal positiveD=diffuse positive

Rest (83%)

> 5yr graft survival

ResultsBanff C4d DSA Outcomepancreas kidney pancreas kidney

1 o o n n -

1 o F +

2 o o n n -

3 R n -

4 o o n n -

5 o o n n -

6 o n +

7 R o n n

8 o o F D +

9 R R F n

10 o o D F † 45 days

11 R R D F † 31 days

12 R D +

13 R R D n

14 o D -

15 R D +

16 R D + 13,5 months

17 o D +

18 R D -

R=rejectiono=no rejection

n =negativeF=focal positiveD=diffuse positive

Rest (83%)

> 5yr graft survival

ResultsBanff C4d DSA Outcomepancreas kidney pancreas kidney

1 o o n n -

1 o F +

2 o o n n -

3 R n -

4 o o n n -

5 o o n n -

6 o n +

7 R o n n

8 o o F D +

9 R R F n

10 o o D F † 45 days

11 R R D F † 31 days

12 R D +

13 R R D n

14 o D -

15 R D +

16 R D + 13,5 months

17 o D +

18 R D -

R=rejectiono=no rejection

n =negativeF=focal positiveD=diffuse positive

Rest (83%)

> 5yr graft survival

ResultsBanff C4d DSA Outcomepancreas kidney pancreas kidney

1 o o n n -

1 o F +

2 o o n n -

3 R n -

4 o o n n -

5 o o n n -

6 o n +

7 R o n n

8 o o F D +

9 R R F n

10 o o D F † 45 days

11 R R D F † 31 days

12 R D +

13 R R D n

14 o D -

15 R D +

16 R D + 13,5 months

17 o D +

18 R D -

R=rejectiono=no rejection

n =negativeF=focal positiveD=diffuse positive

Rest (83%)

> 5yr graft survival

ResultsBanff C4d DSA Outcomepancreas kidney pancreas kidney

1 o o n n -

1 o F +

2 o o n n -

3 R n -

4 o o n n -

5 o o n n -

6 o n +

7 R o n n

8 o o F D +

9 R R F n

10 o o D F † 45 days

11 R R D F † 31 days

12 R D +

13 R R D n

14 o D -

15 R D +

16 R D + 13,5 months

17 o D +

18 R D -

R=rejectiono=no rejection

n =negativeF=focal positiveD=diffuse positive

Rest (83%)

> 5yr graft survival

ResultsBanff C4d DSA Outcomepancreas kidney pancreas kidney

1 o o n n -

1 o F +

2 o o n n -

3 R n -

4 o o n n -

5 o o n n -

6 o n +

7 R o n n

8 o o F D +

9 R R F n

10 o o D F † 45 days

11 R R D F † 31 days

12 R D +

13 R R D n

14 o D -

15 R D +

16 R D + 13,5 months

17 o D +

18 R D -

C4d staining in simultaneous pancreas and kidney biopsies (case 8)

focally +

A cooperation between LUMC and UMMC

UMMC:

• 148 pancreas transplantations between 2000 and 2007

• 37 pancreas transplant biopsies, taken on clinical indication

Results

G BANFF C4d DSA Acute Rejection 1 1 Indeterminate Negative Negative 1 2 Normal Focal Positive HLA class I 2 1 Indeterminate Negative Negative 3 1 ACR grade III (severe) Negative Negative 4 1 Normal Negative Negative 5 1 Normal Negative Negative 6 1 Normal Negative Negative 7 1 Normal Negative Negative 8 1 Normal Negative Negative 9 1 Normal Negative Negative

10 1 Indeterminate Negative Negative 11 1 Indeterminate Negative Negative 12 1 Indeterminate Negative Negative 13 1 ACR grade III (severe) Negative Negative 14 2 Normal Negative Positive HLA class 1 15 2 ACR grade II (mild) Negative Positive HLA class I 16 2 Normal Focal Positive HLA class II 17 2 ACR grade II (moderate) Diffuse Negative 18 2 Indeterminate Diffuse Negative 19 3 ACR grade II (moderate) Diffuse Positive HLA class II 20 3 ACR grade I (mild) Diffuse Positive HLA class II 21 3 ACR grade I (mild) Diffuse Positive HLA class I 22 3 Normal Diffuse Positive HLA class I 23 3 Normal Diffuse Positive HLA class I & II 24 3 Normal Diffuse Positive HLA class I & II 25 3 Normal Diffuse Positive HLA class I 26 3 Normal Diffuse Positive HLA class I & II 27 3 ACR grade III (severe) Diffuse Positive HLA class I

AMR and graft survival

27 patients with C4d and DSA results available were categorized into 3 groups:

I: minimal or no C4d staining, no DSA (N=13)

II: diffuse C4d staining, no DSA (N=2);

focal C4d staining and DSA (N=2);

minimal/no C4d staining and DSA (2)

III: Diffuse C4d staining and DSA (N=9)

Results

Conclusion

• C4d+ in the pancreas is independent of Banff classification

• C4d staining patterns can differ per organ (kidney/pancreas)

• C4d positivity alone, has no effect on pancreas survival

• 1 patient DSA, Banff and C4d positive pancreas lost

Conclusions

- Diffuse C4d positivity with concurrent DSA had poor graft

survival, most likely related to AMR

- Patients did well who had:

- either DSA or C4d alone

- or no DSA or C4d

- The sole occurrence of either C4d or DSA poses no additional

risk for poor outcome of the pancreas graft

Conclusions

The results from our study emphasize the importance of taking

into account a combination of C4d staining pattern,

histological diagnosis and presence of DSA before a

diagnosis of AMR of the pancreas is made

Acknowledgements

LUMC, the Netherlands

Department of Pathology

Hanneke de Kort

Jan A. Bruijn

Emile de Heer

Department of Nephrology

Eelco de Koning

Cees van Kooten

Stefan Berger

Department of ImmunoHematology

and Blood Transfusion (IHB)

Michael Eikmans

Univ. of Maryland, School of Medicine, Baltimore, USA

Department of Pathology

Raghava Munivenkatappa

Cinthia Drachenberg

Benjamin Philosophe

All doctors who contributed patients