Transplant plenary II


7

De novo donor-specific anti-HLA antibodies are a risk factor for graft loss in DCD liver transplantation

Oya Andacoglu, Thomas Ellis, Anthony M. D'Alessandro, David Foley, Amy Powell, Glen E. Leverson, Michael R. Lucey, Alexandru I.Musat

University of Wisconsin School of Medicine and Public Health, Madison, WI

Donor shortage has led to an increasing use of liver grafts recovered from donors after circulatory death (DCD). In contrast to donation after brain death (DBD) grafts, DCD livers have inferior graft survival related mainly to ischemic cholangiopathy. Aim: In this study we explored the possible impact of humoral alloreactivity directed against donor HLA on the survival of DCD liver grafts. Methods: For this purpose we studied 20 consecutive ABO compatible, primary liver transplant recipients of DCD grafts transplanted between 5/1/2009 and 2/5/2012.Donor-specific antibodies (DSA) against HLA class I (A, B, C) and II (DR, DQ, DP) were determined pretransplant and at 3, 6, 12 months post-transplant using single antigen beads on a Luminex platform. Results: The patient follow-up was 6 months to 3.2 years. During the study period there were 4 graft losses, all due to ischemic cholangiopathy. None had hepatic artery thrombosis. There was no statistically significant difference between patients with or without preformed/pretransplant DSA in terms of patient and donor demographics, model for endstage liver disease (MELD) score, warm-, or cold ischemia times, or donor risk index. Cox proportional hazards model showed that patients who developed de novo anti-HLA DSA (i. e. as a direct consequence of sensitization to the graft) had a 10 times higher risk of ischemic cholangiopathy (p=0.003), the leading cause of DCD graft loss. Most de novo DSAs were directed against class II histocompatibility antigens. Conclusions: To our knowledge this is the first study to suggest that development of de novo humoral alloreactivity to the DCD liver contributes to graft loss, presumably due to ischemia caused by peribiliary plexus injury. Our observations raise the possibility that addressing ischemia-related generation of DSA, a potentially modifiable risk factor, may improve the DCD graft survival.

Disclosures:

Michael R. Lucey - Grant/Research Support: Vertex, Abbvie, Gilead, Salix; Speaking and Teaching: Roche

The following people have nothing to disclose: Oya Andacoglu, Thomas Ellis, Anthony M. D'Alessandro, David Foley, Amy Powell, Glen E. Leverson, Alexandru I. Musat

8

Ex-vivo hepatic perfusion with a new cell-free oxygen carrier solution upregulates hepatocyte associated gene response against ischemia-reperfusion injury and triggers protective and regenerative pathways

Paulo A. Fontes1, Shirish Paranjpe2, William R. Light3, George K. Michalopoulos2

1Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA; 2Pathology/ University of Pittsburgh Medical Center, Pittsburgh, PA; 3OPK Biotech, Cambridge, MA

Background: Machine perfusion (MP) with a newly developed cell-free oxygen carrier solution (CFOCS) has been shown to be superior to cold storage preservation (CSP) in a pre-clinical large animal model (porcine) for liver transplantation. This study aimed to evaluate the overall hepatocyte gene response to effective ex-vivo oxygenation at 21°C when compared to CSP at 4°C under anoxic conditions as the current standard of care for organ preservation. Methods: Two groups of 6 animals (60 kg) underwent orthotopic liver transplantation after a period of 8hours of cold ischemia time (CIT). The liver allografts from the control group were stored with CSP and the study group with MP (dual pressures and full oxygenation with Fi02=60%). Extensive clinical and laboratorial data was obtained, which included tissue oximetry and mitochondrial function assessment with oxygraph chamber. Affymetrix gene-arrays (swine oligoarray chip) were performed in both groups in 3 different time points: baseline, post reperfusion (9 hours) and at end-study necropsy (5days). Results: The MP group had 100% survival (5 days follow up) with excellent allograft function. The CSP group had 33% survival after moderate to severe ischemia/reperfusion injuries leading into irreversible liver allograft failure. A significant (p<0.004) benefit of MP was detected after 16 different variables were analyzed in comparison to CSP. Hepatic 0 2 consumption by the MP group was 0.096 mLO2/g/hour and 02 delivery was 0.78mL 0 2/g/hour. The top 100 most affected genes showed over-expression (fold change >1.5) of the genes associated with general metabolic functions, antiinflammatory, regenerative and protective mechanisms against free radicals. Activation of perfusion resulted in increased expressions of several genes associated with entry of hepatocytes into G1 phase of the cell cycle. Extensive retention of expression of many genes associated with hepatocyte differentiation was observed. Ingenuity® pathway analysis of functional groups affected by perfusion revealed that genes associated with liver damage were significantly down regulated in the MP group. The CSP group showed significant upregulation of genes associated with liver pathology when compared to the MP group. Conclusions: MP with a CFOCS promotes efficient hepatic oxygenation in a fully controlled exvivo environment minimizing I/R injuries. Enhanced oxygenation under subnormothermic conditions triggers seminal regenerative and cell protective gene responses that implicates directly on improved liver function after transplantation.

Disclosures:

The following people have nothing to disclose: Paulo A. Fontes, Shirish Paranjpe, William R. Light, George K. Michalopoulos

9

Prognostic cell-based assay predicts acute cellular rejection in pre- and post-transplant testing of children with liver and intestine transplantation (LTx, ITx)

Chethan Ashokkumar, Rafia Khan, Kyle A. Soltys, Geoffrey Bond, George V. Mazariegos, Brandon W. Higgs, Rakesh Sindhi

Children's Hospital of Pgh of UPMC, Pittsburgh, PA

Background and Purpose: Children with LTx demonstrate lower rejection-risk and immunosuppression requirements compared with ITx recipients. Accurate rejection-risk assessment could enable early minimization and reduced drug toxicity in either recipient type. Allospecific CD154+T-cytotoxic memory cells (CD154+TcM) predict rejection in small cohorts of pediatric LTx and ITx and require validation. Methods: Between 2006-2012, screening-validation testing of CD154+TcM was undertaken at our center under protocol NCT#01163578 in children <21 years who receive LTx or ITx. In this cell-culture-based test, donor-specific immune response is expressed as the ratio of donor- and third-party-induced CD154+TcM. The ratio is termed immunoreactivity index or IR. In general, an IR > 1 implies increased donor-specific immune response and increased risk of rejection. Results: 218 children provided 283 samples for testing. Donor- or third-party-induced CD154+TcM did not exceed background in 37 samples from 23 children and were discarded (Poisson statistic). A screening cohort of 147 samples from 120 subjects was tested with research-grade reagents and flow cytometers. In this cohort, an IR ≥ 1.1 in 98 post-transplant samples predicted rejection within 60 days after a test with performance (performance metrics are sensitivity, specificity, positive predictive value, and negative predictive values) of 92%, 84%, 65%, and 97%, respectively (AUC 0.878). In 49 pre-transplant samples, an IR >1.23 predicted rejection within the first 60 days after LTx or ITx with performance of 80%, 71%, 74%, and 77%, respectively (AUC 0.820). A validation cohort of 99 samples from 71 children was tested with a standardized test format using cGMP-manufactured reagents and FDA-approved instruments. Performance of the abovementioned pre- and post-transplant rejection-risk thresholds for predicting rejection was replicated in the validation cohort. Specifically, test performance in 67 post-transplant samples was 84%, 81 %, 64%, and 93%, respectively (AUC 0.792). Test performance in 32 pre-transplant samples was 54%, 89%, 78%, and 74%, respectively (AUC 0.848). Conclusions: In screening-replication testing of pre-transplant and post-transplant blood samples, allospecific CD154+T-cytotoxic memory cells predict acute cellular rejection after liver or intestine transplantation with high sensitivity and specificity. This performance can permit safe minimization of immunosuppression if confirmed in multicenter testing.

Disclosures:

The following people have nothing to disclose: Chethan Ashokkumar, Rafia Khan, Kyle A. Soltys, Geoffrey Bond, George V. Mazariegos, Brandon W. Higgs, Rakesh Sindhi

10

Putative miRNA signatures indicative of Fatal acute liver failure and Survival in HEV infection in pregnancy

Nirupma Trehanpati1, Rashi Sehgal1, Paul David1, Ashish Vyas1, Ritu Khosla1, Shiv K. Sarin1 ,2

1Research, Institute of Liver and Biliary Sciences, New Delhi, India; 2Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India

Hepatitis E is known to be more serious with fatal acute liver failure (FALF) and death in 15-20% of infected pregnant females. There is paucity of knowledge in the underlying mechanisms distinct to HEV infection during pregnancy. AimWe investigated the identification of putative miRNA signatures indicative of fatal acute liver Failure, survival and HEV infection during pregnancy. Methods: Next generation sequencing(NGS) and microarray analysis was performed using total RNA and small RNA isolated from peripheral blood mononuclear cells in fatal ALF pregnant infected with HEV (Gr. A, n=5), pregnant with acute viral hepatitis (Gr. B, AVH-E, n=5) and healthy pregnants (Gr. C, n=5). Deep sequencing of miRNA and mRNA Profiling was performed using lllumina NGS platform and lllumina Human HT12-V4 whole genome microarray. miRNA-Target coregulation analysis, PCA, gene ontology and biological Analysis Network was performed to identify miRNA signatures indicative of Death, Survival and Infection. Results804 miRNA were expressed in all groups, 85 miRNA were differentially expressed by ≥ 2 fold in either one of the Gr. miRNA death signature with target genes analysis revealed that miR 450b; 450a1; 450a2; 4772; 188; 504 were >2 fold expressed (P = 0.005) only in Gr. A and not in Gr. B and Gr. C(Fig. A). There were putative miRNA signature i. e miR 1468, 4435, 431-5p indicative of natural survival signal which were again more than two fold expressed in Gr. C pregnant uninfected subjects but not in FALF(Fig. B). There were miRNA signature for triggered (infection induced) survival signal upon infection in Gr. B which were not present in Gr. A and C. (Fig. C). Conclusions: miRNAs identified by NGS in HEV infected pregnant patients can be used as markers for development of fatal acute liver failure. There is potential of enhancing putative miRNAs indulging in survival to improve patient outcomes

Disclosures:

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The following people have nothing to disclose: Nirupma Trehanpati, Rashi Sehgal, Paul David, Ashish Vyas, Ritu Khosla, Shiv K. Sarin

11

Intention-to-treat Outcome of T1 Hepatocellular Carcinoma Using the Approach of “Wait and not Ablate” until Meeting T2 Criteria for Liver Transplant Listing

Neil Mehta1, Jennifer L. Dodge2, Nicholas Fidelman3, John P. Roberts2, Francis Y. Yao1

1Medicine, UCSF, San Francisco, CA; 2Surgery, UCSF, San Francisco, CA; 3Radiology, UCSF, San Francisco, CA

Background: Patients with T1 hepatocellular carcinoma (HCC) (1 lesion <2 cm) are not eligible for priority listing for liver transplantation (LT) under current MELD allocation. A common practice is to wait without loco-regional therapy (LRT) until tumor growth from T1 to T2 (1 lesion 2-5 cm or 2-3 lesions < 3 cm) to be eligible for listing with MELD exception. The risk of tumor progression to beyond LT criteria using this approach is unknown. We aimed to evaluate the intention-to-treat outcome of this strategy of “wait and not ablate” for non-resection candidates with T1 HCC 1.0 to 1.9 cm. Methods: We identified 138 patients with a new diagnosis of T1 HCC by abdominal CT or MRI between 2004 and 2012.Among them, 24 patients underwent definitive LRT. The other 114 undertook the “wait and not ablate” approach of close observation with serial CT or MRI every 3 months until meeting T2 criteria and formed the study cohort. Two of the investigators performed independent review of the imaging studies to confirm the diagnosis of HCC. Competing risks (CR) regression was used to determine predictors of exclusion from LT. Results: The median age was 60 and 75% were male. There were 47.4% Caucasian, 31.6% Asian, 14.0% Hispanic, 4.4% Black, and 2.6% other race. The most common etiologies were hepatitis C (57.0%), hepatitis B (22.8%), fatty liver (10.5%) and alcohol (8.8%). The median initial tumor diameter was 1.4 cm; 91% were hypervascular and 74% displayed washout. The median time to progression beyond T1 was 6.9 months (IQR 4.6-12.0). Probabilities of progression from T1 to directly beyond T2 without LT listing were 4.4% at 6 months and 9.0% at both 12 and 24 months. Median increase in total tumor diameter was 0.14 cm per month (IQR 0.08-0.29). The 1- and 3-year survival was 94.5% and 75.5%. In multivariate analysis, predictors of rapid tumor progression, defined as a >1 cm increase in total tumor diameter over 3 months, included alcoholic liver disease (HR 6.32, 95% CI 1.3-30.84; p = 0.02) and Hispanic race (HR 3.81, 95% CI 1.01-14.4; p = 0.049), whereas hepatitis B appeared to be protective (HR 0.08, 95% CI 0.01-0.85; p=0.04). Predictors of exclusion from LT (with or without listing for LT under T2) by CR were AFP > 500 ng/mL (HR 12.7, 95% CI 2.8-57.0; p = 0.001) and rapid tumor progression (HR 5.7, 95% CI 2.2-14.6; p < 0.001). Conclusions: The “wait and not ablate” approach for T1 HCC until tumor grows to T2 stage is associated with a <10% risk of exclusion from LT due to progression to directly beyond T2 at 2 years. Those with initial AFP > 500 ng/mL and rapid tumor progression are at high risk for wait-list dropout and should receive early intervention with LRT.

Disclosures:

Nicholas Fidelman - Grant/Research Support: Bayer, Inc., Nordion

The following people have nothing to disclose: Neil Mehta, Jennifer L. Dodge, John P. Roberts, Francis Y. Yao

12

Increased Rates of Liver Transplant Wait-Listing for Hepatocellular Carcinoma in Individuals with Hepatitis C from 2003 – 2010 in the United States

Jennifer A. Flemming1, W. Ray Kim2, Eric Vittinghoff3, Carol L. Brosgart4, Kris V. Kowdley5, Norah Terrault1

1Medicine, University of California San Francisco, San Francisco, CA; 2Medicine, Mayo Clinic, Rochester, MN; 3Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA; 4Gllead Sciences, Foster City, CA; 5Virginia Mason Medical Center, Seattle, WA

Prior projection studies predicted a rapid rise in end-stage liver disease (ESLD) and hepatocellular carcioma (HCC) as Americans with hepatitis C (HCV) infection grow older with a longer duration of infection. We examined the nationwide liver transplant (LT) wait-list (WL) to determine longitudinal trends in ESLD and HCC secondary to HCV in the US. Methods: The Scientific Registry for Transplant Recipients database between 2003 and 2010 was used to develop a cohort of adult patients listed for LT secondary to HCV. The indication for listing was defined as ESLD if the MELD score was ≥15 at the initial WL date, or HCC if they were listed with HCC or had an HCC MELD exception application within 180 days of WL. Incidence rates (IR) of WL in HCV individuals were standardized using the direct method to the 2002 US census population. Comparison of IR between years of listing and indication for listing was implemented using the Poission regression. Results: 20, 325 individuals with HCV (75% male, median age 54 years) were WL for LT during the study period. Of those, 12, 724 (62.6%) were listed for ESLD and 7, 601 (37.4%) for HCC. Those listed with HCC were more likely to be older (56 years vs 52 years, p < 0.001) and be male (79% vs. 73%, p < 0.001) than those with ESLD. After adjusting for age and sex, the overall rate of listing for HCV increased over time (6.9 per 100, 000 in 2003 vs. 10.2 per 100, 000 in 2010, p < 0.001). Importantly, this increase was entirely attributable to HCC, which, on average, rose by 12% per year (IR ratio 1.12, 95% CI 1.11-1.13, p < 0.001), whereas listing for ESLD remained constant over the study period (IR ratio 1.01, 95% CI 0.99 −1.01, p = 0.09, figure). Conclusion: While there may be a number of possible explanations for the lack of increase in WL registration for ESLD, these data highlight urgent need for primary and secondary prevention for HCC.

Disclosures:

W. Ray Kim - Advisory Committees or Review Panels: Salix; Consulting: Bristol Myers Squibb, Gilead

Carol L. Brosgart - Board Membership: Tobira, Juvaris, Tobira, Juvaris, Tobira, Juvaris, Tobira, Juvaris; Management Position: Alios BioPharma, Inc., Alios BioPharma, Inc., Alios BioPharma, Inc., Alios BioPharma, Inc.; Stock Shareholder: Alios BioPharma, Inc., Gilead Sciences, Inc., Tobira, Juvaris, Alios BioPharma, Inc., Gilead Sciences, Inc., Tobira, Juvaris, Alios BioPharma, Inc., Gilead Sciences, Inc., Tobira, Juvaris, Alios BioPharma, Inc., Gilead Sciences, Inc., Tobira, Juvaris

Kris V. Kowdley - Advisory Committees or Review Panels: Abbott, Gilead, Merck, Novartis, Vertex; Grant/Research Support: Abbott, Beckman, Boeringer Inqelheim, BMS, Gilead Sciences, Ikaria, Janssen, Merck, Mochida, Vertex

Norah Terrault - Advisory Committees or Review Panels: Eisai, Biotest; Consulting: BMS; Grant/Research Support: Eisai, Biotest, Vertex, Gilead, AbbVie, Novartis

The following people have nothing to disclose: Jennifer A. Flemming, Eric Vittinghoff

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