The detrimental effects of Donor Specific HLA alloantibodies (DSA) on outcomes following liver organ transplantation have been known for many years. Liver transplantation is an exception but some evidence has been recently highlighted, showing that DSA could be associated with acute antibody-mediated rejection, chronic rejection, plasma cell hepatitis, anastomotic biliary stricture, NRH, fibrosis progression... The prevalence of preformed donor specific DSA is about 20% and the incidence of de novo DSA is about 10% in Liver transplantation (LT). DSA are associated with several graft diseases, mainly AMR but diagnosis was made on histological features+/-C4d staining. De novo DSA and preformed class II DSA, especially with high MFI, seem to pejoratively influence outcomes after LT. When associated with HCV, DSA worsen fibrosis progression. Thanks to antiviral IFN-free regimen, therapeutic strategies of DSA positivity and/or AMR will not differ from HCV- recipients, but need to be evaluated in prospective studies.

1. Breakthrough to achieve long-term survival
DSA & HCV in liver transplantation
Audrey Coilly, MD
Centre Hepato-Biliaire
Paul Brousse Hospital, Villejuif, France

2. DISCLOSURES
Audrey Coilly, MD
Centre Hepato-Biliaire
AP-HP Hopital Paul-Brousse, Villejuif, France
I have relevant financial relationships with respect to the content of this
session as follows:
1.Speaker Bureau: Gilead France
2.Grant: Novartis France
3.Consultancy and Teaching: Astellas, MSD, BMS, Janssen

3. How to improve long term survival in liver transplantation?How to improve long term survival in liver transplantation?
Watt, KD. Am J Transpl. 2010Watt, KD. Am J Transpl. 2010
% death according to time after liver
transplantation
% death according to time after liver
transplantation
50%
20%
• Register study, n=798

4. How to improve long term survival in liver transplantation?How to improve long term survival in liver transplantation?
Watt, KD. Am J Transpl. 2010Watt, KD. Am J Transpl. 2010
• Register study, n=798
• From 8-years post-LT, the
first cause of death is
liver-related

5. HCV recurrence is the first cause of death and graft loss when patients
are transplanted with a positive HCV RNA
HCV recurrence is the first cause of death and graft loss when patients
are transplanted with a positive HCV RNA
Forman, LM. Gastroenterology. 2002Forman, LM. Gastroenterology. 2002
Patient survival Graft survival
11,036 patients (UNOS registry)
11,791 LT from 1992 to 1998
1.00
0.75
0.50
0.25
0.00
1 2 3 4 5
Follow-up (years)
Proportionofpatientssurviving
Log-rank X2
=19.7
P<0.0001
HCV -
HCV +
1.00
0.75
0.50
0.25
0.00
1 2 3 4 5
Follow-up (years)
HCV -
HCV +
Log-rank X2
=52.85
P<0.0001Proportionofallograftssurviving
0 0

6. • The detrimental effects of DSA on outcomes following solid organ
transplantation have been known for many years1
• Liver transplantation is an exception but some evidence has been recently
highlighted, showing that DSA could be associated with2
:
Spectrum of diseases related to donor-specific HLA alloantibodies (DSA)
in liver transplantation
Spectrum of diseases related to donor-specific HLA alloantibodies (DSA)
in liver transplantation
1- Angaswamy, N. Human Immunology. 2013 2- O’Leary, JG. Am J Transpl. 20141- Angaswamy, N. Human Immunology. 2013 2- O’Leary, JG. Am J Transpl. 2014

7. 1. What are the prevalence and incidence of DSA after LT and
are they impacted by HCV replication?
2. How to diagnose acute antibody-mediated rejection (AMR)?
When associated with HCV recurrence?
3. What are the consequences of DSA on LT outcomes and on
HCV recurrence?
4. How to prevent? When and How to treat AMR?
AgendaAgenda

8. 1. What are the prevalence and incidence of DSA after LT and
are they impacted by HCV replication?
2. How to diagnose acute antibody-mediated rejection (AMR)?
When associated to HCV recurrence?
3. What are the consequences of DSA on LT outcomes and on
HCV recurrence?
4. How to prevent? When and How to treat AMR?
AgendaAgenda

9. Preformed DSA is quite common in LT contextPreformed DSA is quite common in LT context
1- Castillo-Rama, M. Liver Transpl. 2008 2- Kozlowski, T. Liver Transpl. 2011 3- Muro, M. Transpl Immuno.
2005 4- Fontana, M. Transpl Int. 2010 5- Taner, T. Am J Transpl. 2012
1- Castillo-Rama, M. Liver Transpl. 2008 2- Kozlowski, T. Liver Transpl. 2011 3- Muro, M. Transpl Immuno.
2005 4- Fontana, M. Transpl Int. 2010 5- Taner, T. Am J Transpl. 2012
• Several reports of monocentric, retrospective, cross-match
studies in liver transplant recipients showed prevalence of
DSA ranging from 5.2% to 24.2%1-4
• A prospective monocentric study5
was consistent with these
reports, as DSA was found in 22.2%

10. In most cases, preformed DSA disappear after liver transplantationIn most cases, preformed DSA disappear after liver transplantation
1- Taner, T. Am J Transpl. 2012 2- Dar, W. Am J Transpl. 20111- Taner, T. Am J Transpl. 2012 2- Dar, W. Am J Transpl. 2011
Among the 20 patients, only 3
had persistent DSA at 4 months
post-LT1
Some reports suggest that Class
I DSA were preferentially
cleared*2
*In recipients of SLKT

11. While preformed DSA disappear,
liver recipients may develop de novo DSA
While preformed DSA disappear,
liver recipients may develop de novo DSA
1- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 20141- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 2014
• In a large retrospective cohort study of 749 liver transplant recipients,
8.1% developed de novo DSA 1 year post-LT1
• Another large retrospective study enrolling 267 patients have shown an
incidence of de novo DSA of 9.1% (median follow-up: 110 months)2
• In both studies, de novo DSA were mainly Class II DSA (Anti-DQ)

12. Risk factors of de novo DSA appearanceRisk factors of de novo DSA appearance
1- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 20141- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 2014
At one year post-LT1
In the French study, the sole predictive factor for the emergence of de
novo DSAs was retransplantation (OR 3.75; 95% CI 1.28-11.05, p=0.025)2

13. Risk factors of de novo DSA appearanceRisk factors of de novo DSA appearance
1- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 20141- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 2014
At one year post-LT1
In the French study, the sole predictive factor for the emergence of de
novo DSAs was retransplantation (OR 3.75; 95% CI 1.28-11.05, p=0.025)2

14. Risk factors of de novo DSA appearanceRisk factors of de novo DSA appearance
1- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 20141- Kaneku, H. Am J Transpl. 2013 2- Del Bello, A. Am J Transpl. 2014
At one year post-LT1
In the French study, the sole predictive factor for the emergence of de
novo DSAs was retransplantation (OR 3.75; 95% CI 1.28-11.05, p=0.025)2
• None of these studies have shown differential prevalence or incidence of
DSA in HCV positive patients
• The use of pegylated interferon α was not a predictor of DSA appearence2

15. 1. What are the prevalence and incidence of DSA after LT and
are they impacted by HCV replication?
2. How to diagnose acute antibody-mediated rejection (AMR)?
When associated with HCV recurrence?
3. What are the consequences of DSA on LT outcomes and on
HCV recurrence?
4. How to prevent? When and How to treat AMR?
AgendaAgenda

16. The diagnosis of AMR is challengingThe diagnosis of AMR is challenging
• Abnormalities of liver test are inconstant and non specific
• In the majority of patients, the presence of DSA is not
associated with rejection
– Variability of DSA testing techniques
– Screening: Solid-phase immunoassays (Luminex®)
– The presence and specificity of antibodies is then detected
using a spectrophotometer
– The mean fluorescence intensity (MFI) allows a
normalization and a quantification of the results

17. There is no consensus concerning threshold of MFI to be considered
positive
There is no consensus concerning threshold of MFI to be considered
positive
Musat, AI. Liver Transpl. 2013Musat, AI. Liver Transpl. 2013
• Ranging from >300 to >10 000
• Prospective study enrolling 109 LT patients
– Aimed to determine cut-off of MFI with the best PPV and NPV for
acute rejection (<90d post-LT)
-- Class I or II MFI≥300
-- Class I and II MFI<300

18. Pathological examination is important to assess diagnosis of AMRPathological examination is important to assess diagnosis of AMR
O’Leary, JG. Liver Transpl. 2014O’Leary, JG. Liver Transpl. 2014
• Until recently, there was no consensus concerning histological fearures of
AMR
• Most of the time, AMR is followed by ACR
AMR - ACRAMR - ACR
Persistent ARPersistent AR
Chronic rejectionChronic rejection
Graft LossGraft Loss

19. « acute-AMR score » « acute-AMR score »
O’Leary, JG. Liver Transpl. 2014O’Leary, JG. Liver Transpl. 2014
3 LT centers, 4 blinded pathologists

20. « acute-AMR score » « acute-AMR score »
O’Leary, JG. Liver Transpl. 2014O’Leary, JG. Liver Transpl. 2014
3 LT centers, 4 blinded pathologists

21. Does C4d staining assist the histopathological diagnosis in liver
transplantation?
Does C4d staining assist the histopathological diagnosis in liver
transplantation?
1- Colvin, RB. J Am Soc Nephrol 2007 2- Bellamy CO. Histopathology. 2007 3- Jain, A. Clin transp. 2006
4- Bouron-Dal Soglio, D. Hum Pathol 2008 5- Lunz, J. Am J Transplant. 2012
1- Colvin, RB. J Am Soc Nephrol 2007 2- Bellamy CO. Histopathology. 2007 3- Jain, A. Clin transp. 2006
4- Bouron-Dal Soglio, D. Hum Pathol 2008 5- Lunz, J. Am J Transplant. 2012
• C4d staining is well validated to
diagnose AMR in kidney
transplantation1
• In LT, interpretation of C4d staining
and practical utility is less clear2
– More sensitive in fresh tissue
– C4d deposits are often seen in other
liver insults as HCV recurrence3-4
• Only obvious and diffuse staining
(strong portal vein, capillary,
periportal sinusoids)5 O’Leary, JG. Liver Transplant. 2014

22. Finally, the diagnosis of AMR should be based on 4 criteriaFinally, the diagnosis of AMR should be based on 4 criteria
O’Leary, JG. Am J Transpl. 2014 – Meeting reportO’Leary, JG. Am J Transpl. 2014 – Meeting report
Expert opinion
1. Presence of DSA in serum
2. Histopathologic evidence of diffuse microvascular
endothelial cell injury and microvasculitis
3. Strong and diffuse positivity of C4d staining in
tissue
4. Exclusion of other causes of injury, including HCV
related-injury

23. HCV recurrence could complicate the diagnosis of acute rejection and
AMR
HCV recurrence could complicate the diagnosis of acute rejection and
AMR
HCV Acute rejection
Portal injuries
Infiltrates ++ ++
Bile duct injury +/- ++
Endotheliitis - ++
Fibrosis +/++ -
Parenchymal injuries
Inflammation ++ +
Necrosis + -
Cholestasis - +
Central vein endotheliitis - +
Steatosis + -
Acknowledgements to Dr Sebagh MAcknowledgements to Dr Sebagh M

24. 1. What are the prevalence and incidence of DSA after LT and
are they impacted by HCV replication?
2. How to diagnose acute antibody-mediated rejection (AMR)?
When associated with HCV recurrence?
3. What are the consequences of DSA on LT outcomes and on
HCV recurrence?
4. How to prevent? When and How to treat AMR?
AgendaAgenda

25. De novo DSAs decrease both patient and graft survivalDe novo DSAs decrease both patient and graft survival
• Patients with de novo DSA develop AMR in 23.8%1
1- Del Bello, A. Am J Transpl. 2014 2- Kaneku, H. Am J Transpl. 20131- Del Bello, A. Am J Transpl. 2014 2- Kaneku, H. Am J Transpl. 2013
Patient survival2
Graft survival2

26. Available data concerning the impact of preformed DSA on graft and
overall survival are contradictory
Available data concerning the impact of preformed DSA on graft and
overall survival are contradictory
1- Gordon, RD. Surgery. 1986 2- Moore, SB. Transplant Proc. 1987 3- Ogura, K. Transplant Proc. 1992
4- Takaya, S. . Transplant Proc. 1991 5- Goh, A. Liver Transplant. 2010
6- Castillo-Rama, M. Liver Transpl. 2008
1- Gordon, RD. Surgery. 1986 2- Moore, SB. Transplant Proc. 1987 3- Ogura, K. Transplant Proc. 1992
4- Takaya, S. . Transplant Proc. 1991 5- Goh, A. Liver Transplant. 2010
6- Castillo-Rama, M. Liver Transpl. 2008
• Several ancient reports found that a positive cross-match was
unrelated to LT outcomes1,2
• … But many others demonstrated an increased graft loss rates3-6

27. Class II DSA (preformed or de novo) are independent predictor of
patient death and allograft loss
Class II DSA (preformed or de novo) are independent predictor of
patient death and allograft loss
• Preformed class II DSA have been shown to be associated with early AR1
• In a monocentric retrospective study analyzing 86 SLKT patients2
, class II DSA
(preformed or de novo) increased AMR and ACR of the liver and kidney
grafts
1- O’Leary, JG. Liver Transpl. 2013 2- O’Leary, JG. Am J Transpl. 20131- O’Leary, JG. Liver Transpl. 2013 2- O’Leary, JG. Am J Transpl. 2013

28. DSA are associated with fibrosis progression in HCV positive recipientsDSA are associated with fibrosis progression in HCV positive recipients
• In this monocentric retrospective study analyzing 507 LT patients
• Preformed Class I and II DSAs were associated with lower survival
• de novo DSA are limit but unsignificant regarding fibrosis progression
O’Leary, JG. Liver Transplant. 2014O’Leary, JG. Liver Transplant. 2014

29. 1. What are the prevalence and incidence of DSA after LT and
are they impacted by HCV replication?
2. How to diagnose acute antibody-mediated rejection (AMR)?
When associated with HCV recurrence?
3. What are the consequences of DSA on LT outcomes and on
HCV recurrence?
4. How to prevent? When and How to treat AMR?
AgendaAgenda

30. « Prevention is better than cure »: Before liver transplantation« Prevention is better than cure »: Before liver transplantation
1- Curry, MP. ILTS 2014. Oral #137 2- O’Leary, JG. Am J Transpl. 20131- Curry, MP. ILTS 2014. Oral #137 2- O’Leary, JG. Am J Transpl. 2013
HCV: Achieve SVR before LT
• Not always possible with
IFN-based regimen
• Most of patients will be
able to achieve SVR before
LT
• Post-LT SVR12: 70%
Preformed DSA
Liver transplant
(up to 48 weeks)
SOF 400 mg/day +
RBV 1000–1200 mg/day
SOF 400 mg/day +
RBV 1000–1200 mg/day
Undergoing LT
for HCC
2° to HCV1
N=61
Undergoing LT
for HCC
2° to HCV1
N=61
• Evidence does not advocate
to contraindicate LT when
there is a positive
crossmatch, nor to delay LT
awaiting the results
• Avoid preformed Class II
DSA in combined liver-
kidney transplantation
(MFI>10,000)2

31. « Prevention is better than cure »: After liver transplantation« Prevention is better than cure »: After liver transplantation
• In my opinion, treating HCV recurrence based on IFN-free regimen is
mandatory without waiting a significant fibrosis on the liver graft
Preformed DSA
Strict monitoring:
-Liver test when tapering IS
-Retest DSA: timing?
-Protocol liver biopsies: 1, 2 and 5 years

32. « Prevention is better than cure »: After liver transplantation« Prevention is better than cure »: After liver transplantation
• In my opinion, treating HCV recurrence based on IFN-free regimen is
mandatory without waiting a significant firbrosis on the liver graft
De novo or persistent DSA
*Anti-humoral agents and techniques as plasmapheresis, IV immune globuline, rituximab, bortezomib*Anti-humoral agents and techniques as plasmapheresis, IV immune globuline, rituximab, bortezomib

33. • The prevalence of preformed DSA is about 20% and the
incidence of de novo DSA is about 10% in LT
• DSA are associated with several graft diseases, mainly AMR
but diagnosis was made on histological features+/-C4d
staining
• De novo DSA and preformed class II DSA, especially with high
MFI, seem to pejoratively influence outcomes after LT
• When associated with HCV, DSA worsen fibrosis progression
• Thanks to antiviral IFN-free regimen, therapeutic strategies of
DSA positivity and/or AMR will not differ from HCV-
recipients, but need to be evaluated in prospective studies
ConclusionConclusion