Liver Session Summary

Time Devoted to Area

Long Term Findings

Tolerance

AMR

Consensus Document

Adult Long-Term PathologyM. Sebagh

20-year biopsied patients

20-year non-biopsied patients

Number of patients 91 56

Mean recipient age at LT 37.3 (+ 12.7) 39.9 (+ 13.0)

Mean donor age 26.5 (+ 12.8) 28.8 (+ 12.3)

Male gender 45 31

Initial IS: Cy/AZA/CT 91 56

LFTs and biopsy findings at 20 years

Normal LFTs33

Abnormal LFTs58

Normal histology 6 3

Abnormal histology 27 55

PPV= 6/33 (18%) NPV = 55/58 (95%)

Conclusion

• Rate of histological abnormalities (90%)

• Rate of combined insults (31%)

• Onset of hepatic structural abnormalities

• Both global and individual histological progression

Conclusion

• Most of the main histological abnormalities might warrant treatment

– Chronic rejection (23%): increase in immunosuppression

– Viral chronic hepatitis (46%): introduction of an antiviral treatment

– IPTH (8.8%): increase in immunosuppression

– LFD (11%): altered immunosuppression

– Recurrent diseases (8%): altered immunosuppression

– Hepatic structural abnormalities (20%): ?

Conclusion

• Partial unreliability of LFTs

• Partial unreliability of non-invasive markers (TE and FT-AT) despite of adequate samples

- for fibrosis but discriminative ability for significant fibrosis (> F2)

- for combined disorders

• Impact on immunosuppressive and antiviral therapies

Histological Findings in Paediatric Allograft Biopsies > 1 year Post-Transplant( Protocol Biopsies, > 50% have normal LFTs)

Centre Numberbiopsied

Time of biopsy

Abnormal histology

Main histological findings

Paris(Fouquet 2005)

67 >10 yrs 73% Chronic rejection (42%), centrilobular fibrosis (22%), biliary cirrhosis (4%), other (4%)

Birmingham(Evans 2006)

113,135,64 1,5,10 yrs 69%

(at 10 years)

Chronic hepatitis +/- fibrosis (64%), biliary fibrosis (2%), recurrent PSC (2%), other (2%) - at 10 years

London, KCH(Bachina 2008)

13 >10 yrs 91% Fibrosis (92%), lymphocytic infiltration (54%)

Chicago(Ekong 2008)

63 > 3yrs 97% Fibrosis (97%), inflammation (70%)

Groningen(Scheenstra 2009)

77,64, 66, 55 1,3,5,10 yrs 69%

(at 10 years)

Fibrosis (69%) - at 10 years

Birmingham, Groningen - prevalence & severity of abnormal histology increase with time

Histological Diagnosis 1 year (n=113)

5 years (n=135)

10 years (n=64)

Normal/ near normal 68.2% 45.2% 31.3%

Chronic hepatitis 22.1% 43.0% 64.0%

Rejection (acute or chronic) 2.7% 2.2% 0

Biliary obstruction 6.2% 7.4% 1.6%

Recurrent Disease 0.9% 0.7% 1.6%

Other 0 1.3% 1.6%

Progressive histological damage following paediatric liver transplantation Evans Hepatology 2006; 43: 1109-1117

Presence of chronic hepatitis associated with progressive fibrosis

• At 10 years - 37% bridging fibrosis, 15 % cirrhosis

Unexplained Portal Inflammation in Late Paediatric Allograft Biopsies

Birmingham (n=158)(Evans 2006)

Montreal (n=119)(Herzog 2008)

Chicago (n=63)(Ekong 2008)

Terminology Chronic Hepatitis Interface Hepatitis Graft Inflammation

Prevalence/time of presentation

22% at 1year43 % at 5 years,64 % at 10 years

24%( median 2 years)

28%(>3 years)

Association with fibrosis

37% -bridging fibrosis15% - cirrhosis

(at 10 years)

35% -bridging fibrosis35% - cirrhosis

(at 10 years)

22%-bridging fibrosis(> 3years)

Other findings 70-80% auto- antibodies(vs 10-13% in non-CH cases,

always in low titre)

Only 6% diagnosed as de novo AIH

(AST < 2x normal)

No association with auto-antibodies

55% - chronic rejection(risk factors for CR in 100%)

Abnormal LFTs

No auto-antibody data

10% - chronic rejectionNo correlation with

abnormal LFTs

Late Protocol Biopsies – Biopsy-directed Changes in Immunosuppression(Mells G, Mann C, Hubscher S, Neuberger J. Liver Transplantation 2009 ; 15 : 931-8)

235 protocol biopsies (> 1yr) from adult patients with normal LFTs

• 76 had change in immunosuppression after biopsy– 11 increased (active inflammation in protocol biopsy)

– 58 reduced (lack of inflammation in protocol biopsy)

– 7 switched to CNI-sparing regime (active inflammation and renal impairment)

Summary & Conclusions

• Histological abnormalities are commonly present in late-post transplant biopsies from paediatric liver allograft recipients.

• Many of changes seen (including graft fibrosis or cirrhosis) are present in children who appear to be clinically well with good graft function.

• In comparison with adults, children are more prone to develop late rejection biliary complications, de novo AIH, idiopathic chronic hepatitis and nodular changes.

• Late rejection, de novo AIH and idiopathic CH are probably part of a spectrum of late allo-immune graft injury

• Idiopathic chronic hepatitis is frequently sub-clinical, but is associated with the development of graft fibrosis and cirrhosis.

• Further studies are required to investigate the role of protocol liver biopsies in determining therapeutic strategies in children who appear to have good graft function using non-invasive investigations.

Liver transplantation & survival

Overall survival according to year of transplant in Europe (ELTR)

Liver transplantation & survival

Survival beyond year-1 in the UK 1994 - 2007 (n = 4483)

Mortality beyond year-1 in liver recipientssince 1994 (n = 206)

Graft failure 20.0%

Malignancy (lymphoid & non-lymphoid) 22.5%

Infection 12.5%

Multi-system failure (sepsis) 10.0%

Recurrent malignancy (HCC) 7.5%

Cardiac 5.0%

Renal failure 5.0%

‘Immune senescence’Factors that predispose to accelerated immune senescence

Chronic viral infections (cytomegalovirus, Epstein Barr Virus, HIV)Smoking and obesityStressDiabetes mellitusSarcoidosisJuvenile idiopathic arthritisTransplantation (via chronic antigenic stimulation)

Clinical consequences of immune senescence

Infections in the elderlyIncreased mortality from cardiovascular disease and infectionMalignancyDeath and dementia in stroke patientsAIDS

Immune senescence in ‘healthy’ liver recipients

Paris, June 2011

Normal 8

Unsuspected acute rejection 2

Unsuspected recurrent disease

15 7/11 PBC; 2/8 PSC; sarcoid

Expected recurrence 4 4 HCV

Mild non-specific hepatitis 25

Steatosis alone 6

siderosis 4 1 recipient HFE heterozygote

Α1AT globules 1 Donor SZ

‘Routine’ histology

Study group

Minimal inflammation & steatosis groups:

more likely to be:

olderaetiology related to alcohol, fatty liver, metabolic/genetic or acute

Less likely to have:

aetiology related to autoimmune liver disease

Paris, June 2011

Outcome of Kyoto Weaning Protocol (June, 1990- April, 2008)

540 survived patients

200 weaning attempted -152 protocol IS

weaning -48 non-elective IS off

84 Group-

tolerance

340Weaning never

been attempted

66under

weaning

50Group-intolerance

(24: rejection, 26: fibrosis)

675 pediatric LDLT

Ohe H, et al. 2011 Transplantation

Groups OT Maintenance IS (TAC b.i.d) P value

N 29 29

IS(immunosuppression)

OffTacrolimus or CsA

monotherapy b.i.d

Time(months) after LDLT at biopsy

121±43 52±22 mo P<0.01

ALT (IU/L)/AST (IU/L)γ-GTP (IU/L)T-Bil (mg/dl)

27.9/24.221.10.8

29.9/20.019.80.9

NSNSNS

Age(yr) at LDLTDonor/Recipient

32.7/1.0 34.5/4.2 NS/P<0.01

Operational Tolerance (OT) vs IS group

Fibrosis in the graft

(Evaluated by Masson-Trichrome)

Summary1) The frequency of both conventional and naïve

Tregs was high in OT

2) Both Tregs exerted donor-specific suppressive activity in OT only

3) OT in this population was non-deletional

4) Strikingly, the number of naïve Tregs increased with time after cessation of IS, but not conventional Tregs

OT : operational tolerance

Summary

Not intragraft FOXP3mRNA level, but the increased number of FOXP3 protein positive CD4+ cells characterized OT

These cells are accumulated in the portal area

Summary

1) In the presence of reintroduction of minimal maintenance IS, most patients with fibrosis exhibited improvement or no progression of fibrosis

2) In single case, fibrosis rapidly progressed in the absence of IS, but such a case was not observed in patients given minimal maintenance IS

3) Thus, fibrosis in IS free patients or patients during weaning process may be at least in part antigen-dependent

ITN030: Tapering Status of HCV Recipients

ITN030: Tapering Status of Non-HCV Recipients

12 of 20 Participants Met the Primary Endpoint:Off Immunosuppression for 30.0 – 50.7 Months

Clinical Factors Associated with Tolerance (1)

CharacteristicTolerant n = 12

Non-Tolerant n = 7

P value

Recipient Age (yrs)

At Transplant 0.75 (0.61) 1.62 (2.59) 0.41

At Study Entry 9.03 (2.27) 7.43 (3.53) 0.24

Recipient Male Gender 8 (66.7%) 3 (42.9%) 0.38

Donor Age 34.95 (5.43) 33.28 (6.90) 0.57

Donor Male Gender 3 (25.0) 3 (42.9) 0.62

Interval Between Transplant and Study Entry (yrs)

99.46 (29.97) 69.70 (13.67) 0.025

History of Rejection 7 (58.3%) 4 (57.1%) > 0.99

Baseline ALT (U/mL) 31 (18 - 48) 30 (22 - 38) 0.85

Baseline GGT (U/mL) 27 (12 – 88) 16.0 (10 – 69) 0.82

BaselineCNI

Tacrolimus 7 (58.3) 5 (71.4) 0.66

Cyclosporine 5 (41.7) 2 (28.6)

CharacteristicTolerant n = 12

Non-Tolerant n = 7

P value

HLA Antibody(Flow PRA; Class I / Class II)

Positive 5 5

0.15Negative 7 1

Unknown 0 1

Donor Specific Antibody

Yes 4 5

0.13No 8 1

Unknown 0 1

HLA A+B+DRMismatch

3 8 30.44

<3 4 4

HLA Class I Mismatch

3 7 30.81

<3 5 4

HLA Class II Mismatch

3 9 50.82

<3 3 2

Clinical Factors Associated with Tolerance (2)

Histologic Factors Associated with Tolerance (1)H and E Findings

CharacteristicNon-Tolerant

(n=7)Tolerant

(n=12)P

value

Overall intensity of portal inflammation

None 3 0.43 11 0.920.04

Mild 4 0.57 1 0.08

Steatosis

None 6 0.86 5 0.42

0.15Micro, Macro, or Mixed

1 0.14 7 0.58

Histologic Factors Associated with Tolerance (2)Total C4d Score

P = 0.019

C4d / CD31

Correlation of Baseline Total C4d Score and DSA Strength

Tota

l C4d

Sco

re

DSA Strength (MFI)

0

4

8

12

16

20

5K 10K 15K 20K 25K 30K 35K 40K0

Pearson Coefficient = 0.752P = <0.001

Non - Tolerant

Tolerant

ALT and GGT Profiles of Tolerant Participants: Group A

Fibrosis Over Time

Pt IDPortal Fibrosis Disse Fibrosis Central Fibrosis

Yr 0 Yr 1 Yr 3 Yr 0 Yr 1 Yr 3 Yr 0 Yr 1 Yr 3

001001

001002

001004

001008

001010

001012 N/A N/A N/A

001014

002001

002002

002004

002006

002007

Alloantibody Evolution During Withdrawal:Five Sensitized and Successful Participants

Pt ID

PRIOR TO W/DRAWAL

DURINGW/DRAWAL

AFTER WITHDRAWAL

1st VISIT 1 YEAR 2 YEARS 3 YEARS

Class I / II PRA

DSAClass

I / II PRA

DSAClass I / II PRA

DSAClass

I / II PRA

DSAClass

I / II PRA

DSAClass I / II PRA

DSA

001002 0/92 DQ5 5/88

001010 0/96DR51DQ6

16/93 B7 13/98 X

001014 12/0 16/45 DQ7 46/69 DQ7 33/88 DQ7 X

002002 0/58DR15DR51

6/50

002007 14/99DR51 DQ6

Cw10 X

5 of 10 sensitized participants achieved the 1º endpoint.

4 of 9 participants with DSA achieved the 1º endpoint.

102 Patients were enrolled

395 Patients were excluded 160 had been transplanted for < 3 years 58 had medical disorders incompatible with the safe conduct of the study and/or interpretation of the results. 35 declined to participate 30 were included in another clinical trial 29 had history of autoimmune liver disease 19 had abnormal liver function tests exceeding pre-specified criteria 19 exhibited abnormalities in basal liver biopsy exceeding pre-specified criteria 18 could not be closely followed-up 14 had history of rejection in the previous 12 months 9 had no side effects of IS drugs 4 were already receiving no immunosuppressive drugs

497 Patients were screened

41 Patients were TOLERANT 57 Patients REJECTED

4 Patients withdrawn from study during dose reduction classified as Non-TOLERANT in the ITT analysis

Patient demographics

Characteristic Tolerant (N=41)

Non-tolerant (N=57)

p-value

Age at weaning start (years) 61±10 52±10.8 0.0001

Gender (% male) 83 58 0.009

Indication for liver transplantation (no. of patients)     0.154

  HCV cirrhosis 9 15  

  HBV cirrhosis 11 12  

  Alcoholic cirrhosis 15 11  

  Cryptogenic cirrhosis 5 2  

  Amyloidotic polyneuropathy 0 5  

  Fulminant hepatitis 1 4  

  Other causes 0 8  

Time from transplant to weaning start (months) 131±43 83±40 <0.0001

Donor age (years) 40±18.3 44±19.7 0.859

Previous episodes of rejection (%) 29 26 0.824

Active HCV infection (no. of patients) 5 5 0.738

Total HLA-A,B,DR mismatches 4.6±1.2 4.8±1.1 0.343

Immunosuppressive therapy at weaning start     0.003

  Tacrolimus 8 30  

  Cyclosporin A 12 14  

  Mycophenolate 12 5  

  Azathioprine 1 0  

  Tacrolimus + MMF 4 1  

  Cyclosporin A + MMF 3 7  

Adjusted odds ratio

P-value

Time from transplantation to enrolment (per year) 1.35 0.000

Age at transplantation (per year) 1.37 0.009

Recipient gender (male) 4.36 0.016

Search for the immunological signature of operational tolerance in liver transplantation(clinicaltrials.gov NCT00647283)

Multivariable analysis

Intra-graft expression differences between tolerant and non-tolerant grafts

Non-TOL TOL

4,90-1,29hypothetical protein FLJ40722FLJ40722

4,90-1,34premature ovarian failure, 1BPOF1B

4,90-1,27DTW domain containing 2DTWD2

2,95-1,27component of oligomeric golgi complex 5COG5

2,95-1,29tubulin alpha 4ATUBA4A

2,95-1,33similar to T25G3.1LOC651316

0-1,27dipeptidyl-peptidase 4 (CD26)DPP4

0-1,26sarcoma antigen 1SAGE1

0-1,34acyl-CoA synthetase long-chain family member 1ACSL1

0-1,36similar to heterogeneous nuclear ribonucleoprotein A1LOC648210

0-1,40ferritin, heavy polypeptide-like 12FTHL12

0-1,45ferritin, heavy polypeptide-like 8FTHL8

0-1,49uracil-DNA glycosylaseUNG

0-1,99transferrin receptorTFRC

01,26mucolipin 1MCOLN1

01,27similar to golgi autoantigen, golgin subfamily a LOC729266

01,28myosin XIXMYO19

01,43tumor protein p53 inducible protein 3TP53I3

03,92hepcidin antimicrobial proteinHAMP

FDRfold changenamegene

4,90-1,29hypothetical protein FLJ40722FLJ40722

4,90-1,34premature ovarian failure, 1BPOF1B

4,90-1,27DTW domain containing 2DTWD2

2,95-1,27component of oligomeric golgi complex 5COG5

2,95-1,29tubulin alpha 4ATUBA4A

2,95-1,33similar to T25G3.1LOC651316

0-1,27dipeptidyl-peptidase 4 (CD26)DPP4

0-1,26sarcoma antigen 1SAGE1

0-1,34acyl-CoA synthetase long-chain family member 1ACSL1

0-1,36similar to heterogeneous nuclear ribonucleoprotein A1LOC648210

0-1,40ferritin, heavy polypeptide-like 12FTHL12

0-1,45ferritin, heavy polypeptide-like 8FTHL8

0-1,49uracil-DNA glycosylaseUNG

0-1,99transferrin receptorTFRC

01,26mucolipin 1MCOLN1

01,27similar to golgi autoantigen, golgin subfamily a LOC729266

01,28myosin XIXMYO19

01,43tumor protein p53 inducible protein 3TP53I3

03,92hepcidin antimicrobial proteinHAMP

FDRfold changenamegene

FDR <25%

In stable liver recipients operational tolerance is much more prevalent than previously estimated at late time points after transplantation.

The most significant molecular differences between tolerant and non-tolerant recipients are related to genes involved in the regulation of iron metabolism.

Hepcidin and hepatocyte iron deposition are likely to be involved in the attenuation of liver-directed alloimmune responses.

Other mechanisms must also be implicated in the maintenance of the tolerant state (CD26, ADORA3, ADDSL1)

Liver biopsy-derived transcriptional markers are currently the most promising strategy to identify tolerant recipients before immnosuppressive drugs are discontinued.

CONCLUSIONS

Histology indices IFNG induced

transcripts

T cell transcripts

Macrophage transcripts

NK cell transcripts

B cell transcripts

Ig transcripts

Endothelial transcripts

Liver transcripts

Injury up transcripts

Hepatitis Activity GRIT1 QCAT QCMAT NKST BAT IGT ENDAT LT IRITD5

Metavir Activity score 0.478 0.415 NS NS NS NS NS NS NS

Ishak Activity score 0.687 0.781 0.578 NS NS NS NS -0.573 0.515

Fibrosis Stage

Metavir Fibrosis Stage NS 0.530 NS NS NS 0.596 NS -0.619 NS

Ishak Fibrosis Stage NS 0.638 NS NS NS 0.602 NS -0.655 0.410

Lesions

Metavir lobular necrosis/inflammation 0.586 NS 0.422 NS NS NS NS NS NS

Ishak focalnecrosis/inflammation 0.503 0.552 NS NS NS NS NS NS NS

Ishak confluent necrosis NS NS NS NS NS NS NS NS NS

Metavir interface hepatitis 0.447 0.506 NS NS NS NS NS NS NS

Ishak interface hepatitis 0.444 0.557 NS NS NS NS NS NS 0.421

Ishak portal inflammation 0.538 0.448 0.443 NS NS NS NS -0.538 NS

Values represent Spearman's correlation coefficient, p<0.05Correlation coefficients >+/- 0.50 are in bold. NS, non significant.

Histological necro-inflammatory activity correlated with IFN-inducible transcripts and T cell transcripts

Histology indices IFNG induced

transcripts

T cell transcripts

Macrophage transcripts

NK cell transcripts

B cell transcripts

Ig transcripts

Endothelial transcripts

Liver transcripts

Injury up transcripts

Hepatitis Activity GRIT1 QCAT QCMAT NKST BAT IGT ENDAT LT IRITD5

Metavir Activity score 0.478 0.415 NS NS NS NS NS NS NS

Ishak Activity score 0.687 0.781 0.578 NS NS NS NS -0.573 0.515

Fibrosis Stage

Metavir Fibrosis Stage NS 0.530 NS NS NS 0.596 NS -0.619 NS

Ishak Fibrosis Stage NS 0.638 NS NS NS 0.602 NS -0.655 0.410

Lesions

Metavir lobular necrosis/inflammation 0.586 NS 0.422 NS NS NS NS NS NS

Ishak focalnecrosis/inflammation 0.503 0.552 NS NS NS NS NS NS NS

Ishak confluent necrosis NS NS NS NS NS NS NS NS NS

Metavir interface hepatitis 0.447 0.506 NS NS NS NS NS NS NS

Ishak interface hepatitis 0.444 0.557 NS NS NS NS NS NS 0.421

Ishak portal inflammation 0.538 0.448 0.443 NS NS NS NS -0.538 NS

Values represent Spearman's correlation coefficient, p<0.05Correlation coefficients >+/- 0.50 are in bold. NS, non significant.

Higher fibrosis stages related to increased Ig, mast cell, T cell transcripts and decreased liver transcripts

High fibrosis stage (p=0.008) and high plasma cell transcripts (p=0.070) were both related to late time post transplant.

Conclusions -1

• Increased expression of IFNG induced transcripts correlates with Hepatitis C activity and abnormal LFTs.

• High fibrosis stages and cirrhosis correlate with increased plasma cell and mast cell transcripts.

• It is yet to be determined whether transcripts can predict progression to fibrosis. (only three progressors present in this dataset)

Conclusions -2

• As expected, increased expression of IFNG-induced transcripts and T cell transcripts was shared across recurrent HCV, ACR, cirrhosis, and hepatocellular carcinoma.

• However, NK cell transcripts were selectively increased in recurrent HCV or HCV-related cirrhosis, but not in ACR, dysplasia or hepatocellular carcinoma.

• In addition, liver transcripts were strikingly lower in ACR and HCC than in hepatitis C and cirrhosis.

• These preliminary data suggest that transcript measurements (i.e. high NK transcripts without significant loss of liver transcripts) may help to distinguish recurrent hepatitis C from ACR.

K TinckamTx Review2009: 80

C4d staining decreased with the distance away from the afferent blood

supply

Portal Tract

BDHA

PVCV

Portal Capillaries

Sinusoids

Percentage of early (<3 weeks post-Tx) biopsies irregardless of crossmatch status with either Focal or Diffuse C4d in each anatomical compartment. A similar C4d staining pattern was also seen in late (>3 weeks) biopsies.

Hepatic Artery - 16%Portal Vein - 24%Portal Capillary - 23%

Sinusoids – 4% Central Vein – 2%

Lunz 2011

Correlation of C4d staining early (<3 weeks) after transplant with histopathological

findings

Total C4d score exhibited a positive correlation with components of the Rejection Activity Index (RAI) score. C4d+ biopsies also showed an insignificant trend (p=0.26) toward having a higher incidence of acute cellular rejection (63% vs. 55%).

Total C4d Score Increases with Crossmatch Score

Crossmatch Score 1 2 4 6, 8

Mean Total C4d Score (sd) 2[ 2.1] 2.8[2.6] 3.7[3.8] 5.0[4.4]

Median Total C4d

Score 2 2 4 4

Range 0-12 0-6 0-9 0-14

P=0.02

Lunz et al in preparation 2011

Negative Stromal

Endothelial Endothelial

C4d Stain Pattern in Liver

Nonspecific injury

AMR

AMR

AMR

Summary: Use of C4d

Grading of AMRSevere/potentially irreversible AMR is suspected1) Very high postoperative isoagglutinin titer2) Use of both CDC and AHG-CDC positive donor3) Stromal ± endothelial C4d staining

Mild/potentially reversible AMR is suspected4) High postoperative isoagglutinin titer5) Use of CDC or AHG-CDC positive donor6) Endothelial only C4d staining

Detection of de novo DSA rejectionEnd of the Slides

Ductopenic rejection

C4d

71 y/o man OLT for A1AT deficiency cirrhosis. POD 22: ACR, resistant to steroids and ATG.POD 43: ductopenia. High DSA class I and II. Diffuse portal C4d.

Summary

Based on detection of DSA in conjunction with C4d deposition:

Humoral alloreactivity

-accompanies at least 50% of ACR/ does not require treatment beyond the usual -appears significantly associated with

-steroid and even thymoglobulin resistant rejection-ductopenic rejection

-identifies a group of patients that are at risk of losing the allograft due to unrelenting, rapidly progressing cholestasis and ductopenia especially early post-transplantation (1.5 % of liver transplants)

DSA may serve as a donor-specific marker of immune reactivity to the graft,In assessing the risk of rejection and need for augmented IS and to decide who may tolerate weaning and potentially elimination of immunosuppression

Conclusions

Modern techniques of detection of DSA and C4d deposition provide further support to earlier observations that humoral alloreactivity

-is frequently intertwined with cellular mechanisms of acute and chronic rejection and -may on its own destroy the liver allograft

Prospective studies on consecutive patients with protocol biopsies / C4d stainingand determination of DSA complement fixing and DSA subclasses are needed to better gauge the contribution of humoral alloreactivity to liver graft damage

17 individual endothelial genes are increased in ABMR

Red arrows indicate genes that are known to be involved in endothelial cell activation

N ABMR TCMRGene Symbol Normalized Signal Normalized Signal Normalized Signal

VWF 1.05 115.36 6.39 738.30 3.39 419.08CAV1 1.17 206.07 5.35 857.90 2.99 494.11RHOJ 0.99 27.60 2.94 85.27 1.65 53.58MCAM 1.12 192.50 2.93 476.93 2.02 326.45CDH5 1.00 75.34 2.58 201.54 1.61 123.20SELE 1.06 23.25 2.23 55.26 1.20 28.66

PALMD 0.93 78.15 2.12 182.35 1.36 122.61PECAM1 0.93 294.26 2.00 638.35 1.60 514.29

KLF4 1.11 236.31 1.52 318.70 1.00 216.59CYYR1 1.04 162.34 1.52 238.68 1.15 182.55CD34 1.05 138.33 1.50 198.87 1.12 148.89TEK 1.04 190.45 1.47 275.89 1.04 198.66

SOX18 1.02 13.48 1.46 19.92 1.08 14.41ZNF521 0.92 37.82 1.38 55.56 0.80 33.75RASIP1 0.98 80.80 1.37 114.53 0.97 80.99HOXD4 1.02 372.75 1.33 495.27 1.02 378.73RAI14 0.95 273.72 1.21 347.63 0.87 256.99

PODXL 1.11 1489.49 1.06 1404.16 0.74 1067.37DLC1 0.95 273.83 0.98 287.39 0.74 221.40FGD5 0.88 212.06 0.97 233.58 0.71 170.90FOXF2 1.02 12.59 0.86 10.61 0.99 12.25EMCN 1.01 700.89 0.85 600.32 0.64 459.15KDR 0.94 307.06 0.77 244.78 0.60 197.55

CETP 1.04 26.61 0.72 18.27 1.06 29.67MAOB 0.97 1450.68 0.69 1031.30 0.52 795.14

Welch t testFDR 0.05

Sis et al. AJT 2009;9:2312-23

Also not in our strictdefinition of ENDAT list, but increased in ABMR:

CDH13Duffy blood groupSOX7THBDMALL

probe set gene symbol gene name expression by cell type normal nephrectomy TG non TG208335_s_at FY DARC endothelium, RBC 0.90 5.25 2.57204006_s_at FCGR3A ; FCGR3B FCGR3B NK macrophages neutrophils 1.05 5.04 2.19

1554899_s_at FCER1G FCER1G mast cell, eosinophils, DC 0.96 4.97 2.64202112_at VWF VWF endothelium 1.05 4.94 2.86204122_at TYROBP TYROBP NK cells 1.02 3.49 2.20232617_at CTSS CTSS macrophages 1.07 3.35 2.11

214511_x_at FCGR1A ; LOC440607 FCGR1B macrophages, neutrophils 1.14 3.14 1.87223343_at MS4A7 MS4A7 macrophages 0.89 3.00 1.44204232_at FCER1G FCER1G mast cell, eosinophils, DC 0.95 2.81 1.76226517_at BCAT1 BCAT1 0.90 2.71 1.64204438_at MRC1 ; MRC1L1 MRC1 macrophages 0.95 2.61 1.56

201858_s_at PRG1 SRGN monocytes, CTL 1.25 2.48 1.62225285_at BCAT1 BCAT1 1.04 2.39 1.49209949_at NCF2 NCF2 macrophages, neutrophils 0.92 2.34 1.49

214022_s_at IFITM1 IFITM2 0.94 2.19 1.60210869_s_at MCAM MCAM endothelium 1.15 2.18 1.53223344_s_at MS4A7 MS4A7 macrophages 1.07 2.17 1.31201859_at PRG1 SRGN monocytes, CTL 1.07 2.13 1.49

210427_x_at ANXA2 ANXA2 endothelium 1.10 2.03 1.52204924_at TLR2 TLR2 macrophages 1.01 1.96 1.43

224358_s_at MS4A7 MS4A7 macrophages 0.97 1.89 1.18203561_at FCGR2A FCGR2A macrophages neutrophils 1.10 1.79 1.20

202638_s_at ICAM1 ICAM1 endothelium 0.92 1.68 1.09229510_at NYD-SP21 MS4A7 macrophages 1.01 1.64 1.20217757_at A2M 1.00 1.63 1.28

201315_x_at IFITM2 IFITM2 0.93 1.62 1.23228461_at SH3MD4 SH3MD4 1.00 1.59 1.31209906_at C3AR1 C3AR1 T cells 0.97 1.58 1.11204007_at FCGR3B FCGR3B NK macrophages neutrophils 0.96 1.47 1.08228754_at SLC6A6 SLC6A6 macrophages 0.99 1.42 1.10212501_at CEBPB CEBPB 0.98 1.40 0.99220088_at C5R1 C5AR1 macrophages 1.09 1.37 1.06

203887_s_at THBD THBD endothelium 1.08 1.24 0.82204466_s_at SNCA SNCA 1.00 0.94 0.73205910_s_at CEL CEL 1.11 0.61 0.86

1553970_s_at CEL CEL 1.05 0.59 0.86

Differentially expressed transcripts between TG vs. non TG(p<0.05 FDR=0.05)

CD56 A

BMR

CD56 T

CMR

CD68 A

BMR

CD68 T

CMR

CD3 ABM

R

CD3 TCM

R

0

1

2

3A. B.

C. D. E.

C4d+ ABMR

C4d- ABMR

TCMR

CD56+ CD68+ CD3+ M

ean

nu

mb

er o

f p

osi

tive

cel

ls

in f

ive

per

itu

bu

lar

cap

illar

ies

p=0.006

p=0.03 p=0.09

CD3CD68CD56

NK cells and macrophages in antibody mediated peritubular capillaritis

Hidalgo et al. AJT 2010; 10: 1812–1822

Consensus Document

   

Portal inflammation and interface activity

Preferably absent, but minimal to focal mild portal mononuclear inflammation may be present. Interface necro-inflammatory activity is absent or equivocal/minimal and, if present, involves a minority of portal tracts.

Centrizonal/Perivenular inflammation

Preferably absent, but minimal/mild perivenular mononuclear inflammation around a minority of central veins without hepatocyte necrosis without endothelitis.

Bile duct changesAbsence of lymphocytic bile duct damage, ductopenia and biliary epithelial senescence changes, unless there is an alternative, non-immunologic explanation (e.g. biliary strictures).

FibrosisFibrosis, if present, should be mild overall and not more than rare portal-to-portal bridging. Perivenular fibrosis should not be more than mild according to Banff Criteria.

Arteries Negative for obliterative or foam cell arteriopathy.

Table 3. BASELINE OR PRE-WEANING BIOPSY FINDINGS CONDUCIVE TO MINIMIZATON OF IS*Does not include patients with underlying AIH, HCV, PBC or PSC (see text).

Consensus Document

   

Portal inflammation and interface activity

Increased portal inflammation compared to pre-weaning biopsy especially when associated with histopathologic evidence of focally worsening or more prevalent lymphocytic bile duct damage, interface hepatitis, or appearance of venous endothelitis.

Centrizonal/Perivenular inflammation

Increased perivenular inflammation compared to pre-weaning biopsy associated with necro-inflammatory activity.

Bile duct changesNew onset biliary epithelial cell senescence changes or ductopenia where sampling problems and/or an alternative, non-immunologic explanation (e.g. biliary stricture) are reasonably excluded.

FibrosisIncrease of fibrosis over consecutive biopsies (see text) without an alternative explanation (e.g. biliary strictures). New onset or increase of perivenular fibrosis.

Arteries Any evidence of foam cell or obliterative arteriopathy.

Table 4. FOLLOW-UP BIOPSY FINDINGS THAT MERIT CONCERN AND CONSIDERATION OF CLOSE FOLLOW-UP DURING OR AFTER WEANING*Does not include patients with underlying AIH, HCV, PBC or PSC (see text).