Prof. Dr. med. Caner Süsal Department of Transplantation Immunology

University of Heidelberg Heidelberg, Germany

TTS Consensus Guidelines

27 Authors, 276 References

TTS Consensus Guidelines Support from…

� TTS

� DGI

� EFI

� BISHI

TTS Consensus Guidelines Groups

� The three groups were established in November 2011

� Convened for a ‘‘Consensus Conference“ in Rome, Italy, in May 2012

� The deliberations of the three groups meeting inde pendently and then together are the basis for this report

� Current knowledge, recommendations and future dire ctions are presented

TTS Consensus Guidelines Organization

Discussed Issues

1. Technical problems (prozone effect, false positive results)

2. Which test at what sensitivity level should be used for the determination of „Unacceptable“ HLA antigen mismatches

3. What is the impact of pre-Tx HLA DSA in different organ Tx ?

4. What is the impact of pre-Tx non-HLA antibodies in different organ Tx?

5. Risk assessment prior to Tx

6. Tx of highly sensitized patients (special programs, desensitization)

7. What is the impact of de novo or persistent DSA in the post-Tx phase?

8. Should we monitor all patients in the post-Tx phase for DSA?

a) SPI must be used for the detection of pre-Tx HLA Abs in solid organ transplant recipients and, in particular, the single- antigen bead assay which allows the detection of antibodies to HLA lo ci, such as Cw, DQA, DPA, and DPB, that are not readily det ected by other methods. [1]

b) The use of SPI for Ab detection should be supplemented with cell- based assays to examine the correlations between the two types of assays and to establish the likelihood of a pos itive crossmatch (XM). [1]

c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. [1]

Recommendations of the Technical Group Summary

Denatured Ag on SA Beads

� Morales-Buenrostro LE et al. “Natural” human leukocyte antigen antibodies found in non-alloimmunized healthy males. Transplantation 2008; 86: 1111.

� Cai J et al. Intact HLA not β-2-m free heavy chain specific HLA class 1 antibodies are predictors of graft failure. Transplantation 2009; 88: 226.

� Pereira et al. Donor-specific antibody against denatured HLA-A1: clinically non significant? Human Immunol 2011; 72: 492.

� Zoet et al. Challenging the golden standard of defining donor specific antibodies. Does the solid phase assay meet the expectations? Tissue Antigens 2011;79: 225.

� El-Awar et al. Epitopes of human leukocyte antigen class I antibodies found in sera of normal healthy males and cord blood. Human Immunol 2009; 70: 844.

� Poli et al. Heart transplantation with donor-specific antibodies directed toward denatured HLA-A*02:01 : a case report. Human Immunol 2011; 72: 1045.

� Zachary et al. Using real data for a virtual crossmatch. Human Immunol 2009; 70: 574.

“Phenotype beads (Luminex-PRA) was more predictive f or CDC and FC crossmatch results than were SAB.”

Luminex Single Antigen Bead Assay Non-Alloimmunized Patients

Heidelberg Kidney Waiting List

79%

0% 0% 0%

20%

40%

60%

80%

100%

Luminex SA ELISA Screening CDC

Per

cent

age

of P

atie

nts

MFI ≥1,000

Non-Alloi mmunized Waiting List Patients Luminex SA

HLA Specificities Median MFI (Range) Frequency in Patients

(%) Frequency in general population

(%)

A*24:02 4193 (1336-12197) 17.8 8.7 A*24:03 4043 (1010-11025) 15.6 0.1 A*23:01 6730 (3595-7108) 11.1 1.7 A*31:01 1967 (1036-3802) 11.1 2.4 A*25:01 2411 (1554-5004) 8.9 1.9 A*30:01 1725 (1253-3561) 8.9 1.3 A*30:02 1827 (1183-2783) 6.7 0.9 A*32:01 3887 (3312-7329) 6.7 3.1 A*34:01 1477 (1398-1509) 6.7 0.0

B*15:12 (B76) 1991 (1001-5505) 20.0 0.0 B*37:01 1599 (1026-5130) 17.8 1.3 B*08:01 2466 (1003-9862) 11.1 12.5 B*15:11 (B75) 5836 (1503-9723) 8.9 0.0 B*44:02 3100 (1654-8542) 6.7 9.0 B*45:01 1723 (1012-5452) 6.7 0.4 B*46:01 2225 (1102-12232) 6.7 0.0 B*49:01 7354 (1091-7569) 6.7 1.3 B*51:01 3606 (1643-4137) 6.7 4.5 B*59:01 4613 (2364-5882) 6.7 0.0 B*15:16 (B63) 2586 (1067-4852) 6.7 0.0

C*05:01 1340 (1005-1690) 11.1 9.1 C*17:01 2065 (1573-8586) 11.1 0.7 C*01:02 1482 (1217-1975) 6.7 2.9

C*03:03 (Cw9) 1679 (1268-2516) 6.7 5.5 DRB1*12:01 1532 (1183-2159) 6.7 1.5 DRB1*16:02 1169 (1114-1240) 6.7 0.1 DQA1*01:03/DQB1*06:03 1224 (1025-3175) 8.9 6.5 DQA1*03:02/DQB1*03:03 3548 (1117-10124) 8.9 4.5 DQA1*01:02/DQB1*06:04 1450 (1363-2742) 6.7 3.2 DQA1*02:01/DQB1*03:01 2201 (1295-3641) 6.7 18.5 DQA1*03:02/DQB1*03:02 2810 (2126-10081) 6.7 9.5 DQA1*02:01/DQB1*03:03 3957 (1486-8004) 6.7 4.5 DPA1*01:04/DPB1*11:01 1811 (1679-2607) 8.9 ns

Gombos AJT 2012 (in press)

Technical Group

Currently Used Algorithms for the Determination of Unacceptable HLA Antigen Mismatche s

in Kidney Transplant Recipients

C. Süsal, D.L. Roelen, G. Fischer, E.F. Campos, M. Gerbase-DeLima, G. Hönger, S. Schaub, N. Lachmann, J. Martorell, F. Claas

Tissue Antigens (in press)

1. Leiden

2. Vienna

3. São Paulo

4. Basel

5. Berlin

6. Barcelona

Methods for Specification of HLA Antibodies and Determination of Unacceptable HLA Antigen Mismatche s

� CDC-PRA (B-cell plates)

� ELISA-PRA

� Luminex-PRA (phenotype beads)

� (Immunization history of the patient)

� Luminex Single Antigen Bead

Luminex Single Antigen MFI

Acceptables

Pre-Tx Group

Süsal et al. Transplantation 2011

Pre-Tx Luminex SA Testing CDC- and ELISA -negative Kidney Tx Recipients

cut-off 1,000 MFI

5%11%

4%

14%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Class I Class II

Graft Loss Patients with DSA Non-Rejectors with DSA

Pre-Tx Luminex SA Testing CDC- or ELISA -positive Kidney Tx Recipients

cut-off 1,000 MFI

Pre-Tx Group

Impact of Luminex-Detected Pre-Tx DSA on Kidney Graft Survival

Otten et al AJT 2012

cut-off 1,000 MFI

Süsal, Döhler, Opelz Human Immunol 2009

Pre-Tx HLA Antibody Status (ELISA)

Pre-Tx HLA Antibody Status (ELISA) Deceased Donor Kidney Transplants 2000-2008

Süsal, Döhler, Opelz Human Immunol 2009

Pre-Tx Group

Pre-Tx Group

Table 6 continued

a) Risk categories should be established based on the antibody and the XM results obtained. [3]

b) DSA detected by CDC and a positive XM should be avo ided due to their strong association with antibody-medi ated rejection and graft loss. [1]

c) A renal transplantation can be performed in the absence of a prospective XM if single antigen bead screening for antibodies to all class I and II HLA loci is negat ive. [3]

d) The presence of HLA DSA should be avoided in heart and lung Tx and considered a risk factor for liver, intestinal, and islet cell Tx. [1]

Recommendations of the Pre-Tx Group Summary

No recommendation was made for the detection of nonHLA antibodies

a) Unacceptable HLA antigens should be a part of kidney allocation algorithms. [2]

b) Accurate XM prediction depends on complete HLA typing . To minimize the incidence of unexpected positive XM in paired exchange registries, the donor should be typed at HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLADRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DPA, and HLA-DPB loci. [2]

c) A renal Tx can be performed without a prospective pre-Tx CDC or flow XM if SAB testing indicates the consistent absence of DSA

d) Risk assessment should include HLA antibody specificities identified in historic sera. [3]

e) If DSA is present but the CDC XM against donor T and B cells is negative , this should be regarded as an increased immunologic risk but not necessarily a contraindication to Tx, especially after elimination of DSA by desensitization. [2]

f) To optimize access to transplantation of highly sensitized reci pients , inclusion of patients in special programs, such as kidney paired donation, AM, or desensitization , should be considered. [1]

g) HLA matching should be part of the allocation pro cedures to reduce the probability of developing HLA antibody, rejection, and graft loss. [2]

h) ABO incompatibility is no longer an absolute cont raindication in kidney Tx and ABO- incompatible transplants can be successfully performed in recipients in whom isoagglutinin titers have been lowered to acceptable levels. [1]

i) Based on current evidence, no recommendation can be made for routine pre-Tx testing for non-HLA antibodies other than ABO. [2]

Recommendations of the Pre-Tx Group Kidney Tx

1. Transplantation risk stratification categories sh ould be developed based on antibody identification and XM results. [3]

2. Information regarding prior sensitizing events sh ould be considered in interpreting antibody testing results. [2]

3. DSA detected by CDC antibody screening and crossm atching in the most recent serum collected must be avoided because they are associa ted with a high risk for AMR and graft loss. [1]

4. To minimize risk of sensitization and antibody-me diated allograft damage, administration of blood products pre-Tx should be avoided if possible. [1]

5. When a patient is sensitized, precise characterization of HLA antibodies and com plete HLA typing of the donor pre-Tx must be performed. [1]

6. HLA antibody screening should be performed at a f requency that accommodates the likelihood of an imminent transplantation based on local waiting times and the immunologic risk of adverse outcome such as in hig hly sensitized patients. [3]

7. A minimum of two sera obtained at different time points should be tested to confirm presence or absence of HLA antibodies. [3]

8. Sera should be tested after known sensitizing eve nts, proinflammatory events, and at regular intervals once listed for transplantation. [1]

Recommendations of the Pre-Tx Group All Organs

Post-Tx Antibody Monitoring

Humoral Theory of Transplantation

Paul I. Terasaki

“antibodies are responsible for all graft losses”

Post-Tx Monitoring, Detection and Elimination of

Persistent or de novo DSA

Post-Tx Ab Monitoring - Critical Issues -

1. It is expensive; is it clinically relevant?

2. If yes, magnitude?

How many grafts do we lose?

How many of these graft losses are due to AMR?

3. There is no validated therapy once DSA are detected in a stable patient.

Deceased Donor Kidney Transplants

3.6% per year

Collaborative Transplant Study (www.ctstransplant.o rg)

Causes of Late Graft Failure - Halloran et al. -

1. AMR (C4d+ or C4d -) (>60%)

2. Recurrent disease

3. CNI toxicity (<5%)

Under-Immunosuppression as Cause of Chronic Rejection

� Non-compliance (22%, Hansen 2007)

� Minimization of IS

→→→→ T cell ↑↑↑↑ →→→→ de novo DSA ↑↑↑↑ →→→→ AMR

Effect of Withdrawing or Reducing Maintenance IS After the Second Post-Tx Year

Opelz G, Döhler B, Transplantation 2008;86:371-376.

after mo 6 at 4.6 yr

15% 32% class I 94% class II

>300 MFI

Risk factors for de novo DSA:

1. HLA DRß1 mismatches

2. Non-adherence to IS

3. Previous clinical rejections (before mo 6)

14 graft losses

8 graft losses

Post-Tx de novo DSA

Luminex SA Testing 500-2,000 MFI

22%

12%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Graft Loss with dnDSA Non-Rejectors with dnDSA

p=0.005

n=51 n=51

Süsal C, et al. TTS Berlin MON.CO16.01

Risk factors for de novo DSA (MFI ≥≥≥≥1000):

1. HLA DQ mismatches

2. Deceased donor

3. 18-35 years of age

4. Pre-Tx non-DSA (immunoreactive patient or DSA by epitope spreading)

5. Physician directed IS reduction/change

1. 11% of 189 patients developed dnDSA in the first post-Tx year.

2. 20% within 5 years and

3. 25% within 10 years

From DSA appearance,

1. 9% of patients lost their graft at 1-year

2. 24% at 3-years

Recommendations of the Post-Tx Group

Four risk categories

First year and thereafter

Low-risk patients (non-sensitized, first transplant ation) 1. Screen for DSA under the following circumstances:

a) at least once 3 to 12 months after Tx. [2]

b) whenever significant change in maintenance immunosuppression is considered (e.g., minimization/withdrawal/conversion ). [2]

c) suspected non-adherence . [2]

d) graft dysfunction . [2]

e) before transfer of care to a remote center outside the Tx center . [3]

2. If DSA present, then perform a biopsy.

a) If the biopsy is positive for rejection the objective is to treat . Efficacy of treatment is reflected as normal graft function and is associated with a reduction of DSA levels. [2]

b) If the biopsy is negative, monitoring should be considered within the first year. [3]

3. Patients with a DSA in the absence of biopsy-proven rejection should not be considered for reduction in immunosuppression. [3]

4. If no DSA present, then no additional testing in the first year is recommended in the absence of circumstances listed under point 1. [2]

Recommendations of the Post-Tx Group Post-Tx months 0-12

Intermediate-risk patients (history of sensitization to donor antigen(s) but currently negative)

1. Monitor for DSA within the first month. [2]

2. If a DSA present, then perform a biopsy. A biopsy is recommended because of data that document an association between DSA and clinical or subclinical rejection. [2]

a) If biopsy is positive for rejection, the objective is to treat. [2]

b) If biopsy is negative, additional DSA monitoring should be considered within the first year. [3]

3. Patients with a DSA in the absence of biopsy-proven rejection should not be considered for reduction in immunosuppressi on [3]

4. In the absence of a DSA follow-up as if low risk . [2]

Recommendations of the Post-Tx Group Post-Tx months 0-12

High-risk patients (DSA positive/XM negative)

1. Monitor DSA and conduct a protocol biopsy in the first 3 months after Tx. [1]

2. If biopsy is positive for AMR , the objective is to treat . [2]

3. If there is a rapidly increasing level of DSA accompanied by a biopsy showing no rejection, initiation of therapy to reduce the DSA levels should be considered. [3]

4. If DSA persists in the absence of biopsy proven reject ion , immunosuppression should not be reduced and additional monitoring should be considered. [3]

5. If the DSA and biopsy are negative , follow as if low risk unless there is an inflammatory event, in which case additional monitoring for DSA is recommended. [2]

Recommendations of the Post-Tx Group Post-Tx months 0-12

Very high-risk patients (desensitized DSA+/XM+) 1. Monitor DSA and conduct protocol biopsies in the fi rst 3 months

after Tx.[1]

2. If there is evidence of clinical or subclinical A MR, the patient should be treated . [2]

3. If there is a rapidly increasing level of DSA accompanied by a biopsy showing no rejection , initiation of therapy to reduce the DSA levels should be considered. [3]

Recommendations of the Post-Tx Group Post-Tx months 0-12

Recommendations of the Post-Tx Group After month 12

Applies to all risk categories

1. Store at least one serum sample per year . [3]

2. Evaluate DSA in a current serum if any of the following conditions occur:

a) Significant change in maintenance immunosuppre ssion is considered (e.g., minimization/withdrawal/conversion). [2]

b) Suspected nonadherence. [2]

c) Graft dysfunction. [2]

d) Before transfer of care to a remote center out side the Tx center. [3]

3. If de novo DSA present or if there is an increase in previous DSA levels, perform a biopsy . [2]

a) If biopsy is positive for AMR , the objective is to treat . Efficacy of treatment is reflected as normal graft function a nd is associated with a reduction of DSA levels (253, 266). [2]

b) If biopsy is negative (no sign of rejection) monitor the DSA and monitor for a change in graft function. [3]

4. Patients with a DSA even without biopsy proven rejection should not be considered for reduction in immunosuppression.[ 3]

Thank you very much for your attention!

Recommendations of the Pre-Tx Group Heart, Lung, Liver Tx

Heart a. In both pediatric and adult heart transplantation, determination of pre-Tx DSA must be performed because it is critical to improve short-term outcomes and preventing early acute rejection. [1]

b. Desensitization therapy should be considered in sensitized heart transplant recipients. [2]

Lung a. Pre-Tx DSA in recent serum should be avoided in lung transplantation whenever possible. [1]

Liver a. The liver allograft may be partially resistant to antibody mediated damage; however, high-level DSA antibody may be associated with inferior outcomes and should be considered as a risk factor for graft dysfunction. [2]

b. Pre-Tx screening for HLA antibodies is recommended in liver transplant recipients for risk stratification. [3]

c. Donor tissue should be collected and stored in live r transplantation. [3]

d. A XM should be performed in sensitized liver transp lant recipients. [2]

e. In sensitized recipients of combined liver-kidney Tx, the liver may not confer full protection for preventing AMR in the kidney and should be included in risk assessment. [2]

Pancreas a. Recommendations for kidney Tx should apply to the pancreas for simultaneous pancreas- kidney Tx. [1]

b. Pancreas is at risk for AMR and pre-Tx DSA should b e avoided whenever possible . [1]

c. In pancreas Tx, AMR should be considered in the differential diagnosis of early graft thrombosis and graft dysfunction. [2]

Intestine a. In intestinal Tx, pre-Tx HLA antibodies should be determined. The risk assessment should be based on the level of DSA. [2]

Islets a. Based on the available literature, pre-Tx DSA are associated with impaired islet cell function post-Tx and should be avoided . [2]

Recommendations of the Pre-Tx Group Pancreas, Intestine, Islets Tx

a) High-risk patients (i.e., desensitized or DSA positive/XM negative)

should be monitored by measurement of DSA and protocol biopsies in the first 3 months after Tx .

b) Intermediate-risk patients (history of DSA but currently negative)

should be monitored for DSA within the first month.

If DSA is present, a biopsy should be performed.

c) Low-risk patients (nonsensitized first Tx)

should be screened for DSA at least once 3 to 12 months after Tx. If DSA is detected, a biopsy should be performe d.

In all three categories, the recommendations for su bsequent treatment are based on the biopsy results.

Recommendations of the Post-Tx Group Summary

Future Directions Technical Group

1. A coordinated search for a selected bank of HLA r eference antibodies to be used for assessing the interlaboratory variability in SPI antibody testing should be undertaken. Antibodies to MICA and relev ant non-HLA antigens should be included.

2. The C1q modified single-antigen antibody identification requires more research and validation to understand ist application to ri sk assessment and monitoring efficacy of treatment.

3. If production of the SPI donor-specific XM kit is continued, further research will be required to establish the clinical role of such a test. Specifically, the sensitivity of the XM in relation to SAB testing r equires further investigation.

4. The clinical endothelial cell XM for the detection of antiendothelial antibodies needs further investigation.

5. SPI for the detection of non-HLA anti-endothelial antibodies require development.

6. Multicenter studies are required to establish the clinical utility of testing for antibodies to tissue-specific non-HLA antigens such as the angiotensin II type 1 receptor . Further development of SPI for the detection of t hese antibodies is warranted based on initial findings.

7. The role of antibodies to epitopes found on HLA-E merits further investigation.

8. The short-term and long-term clinical effect of low levels of HLA antibodies detected by SPI requires further investigation.

Future Directions Pre-Tx Group

1. More systematic studies for immunologic risk asse ssment that include long- term outcome data are required.

2. Randomized controlled studies to analyze the effica cy and safety of different desensitization protocols are required.

3. Additional studies are needed to determine the ro le of preexisting complement fixing versus noncomplement fixing anti bodies to HLA and their role in organ Tx.

4. Efficacy of desensitization in achieving good long -term graft survival needs to be established in heart and lung transplan tation.

5. More systematic studies are required on the impac t of DSA on pancreas transplant alone or in combination with a kidney, on intestine and multivisceral Tx, islet cell Tx.

6. Standardization of the methodology for determining ABO isoagglutinin titers is required.

7. Future studies are required to define the role of preexisting antibodies to non-HLA and self- antigen on the outcome of solid organ and cellular Tx.

Future Directions Post-Tx Group

1. Serial screening of serum to determine timing of onset of de novo DSA before onset of graft dysfunction.

2. Protocol biopsies at first appearance of de novo DSA to document pathologic correlation.

3. Assessment of DSA for complement fixing activity and correlation with clinical events (e.g., DSA C1q binding and Ig G subclass specificity of DSA).

4. Clinical trials that include serial DSA monitorin g posttreatment and posttreatment biopsies to correlate DSA levels wit h histologic response to therapy and long-term outcome.

5. Clinical trials to prevent production of DSA .

Technical Group