Abstracts S83

but numbers are small. However, there was a significantly lower freedom from LVEF≤ 45% in the DSA+, C1q+ group compared to the DSA+, C1q- group (see table).Conclusion: Donor Specific Antibodies with the ability to bind complement is associated with complement fragment deposition on endomyocardial biopsy and cardiac dysfunction. This would suggest an activation of the complement system that may result in graft damage. Aggressive immuno-therapy is warranted in these pts.

Table 1. PRA screens with results from concomitant EMBs and Echos

No Ab (n= 53)Non-Specific Ab (n= 169)

DSA+, C1q- (n= 13)

DSA+, C1q+, (n= 8)

% C4d and/or C3d Positive by IF on Concomitant EMB

4% (20/489) 6% (8/140) 23% (3/13)1 29% (2/7)1

% Showing AMR (1,2,3) on Concomitant EMB

6% (30/484) 10% (15/149) 33% (4/12) 1,2 67% (4/6)1,2

% Showing Acute Cellular Rejection (1R,2R,3R) on Concomitant EMB

34% (175/523) 24% (40/167)1 46% (6/13) 75% (6/8)1,2

% Freedom from LVEF≤ 45

95% (498/523) 93% (139/150)100% (13/13) 50% (4/8)1,2,3

1p< 0.05 compared to No Ab group2p< 0.05 compared to Non-specific Ab group3p< 0.05 compared to DSA+, C1q- group

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Static Versus Dynamic Angiographic CAV Evaluation: Prognostic Stratification Beyond ISHLT GradingM. Masetti ,1 A. Aliabadi,2 M. Sabatino,1 G. Delle-Karth,3 P. Prestinenzi,1 S. Rödler,2 K. Uyanik Uenal,2 J. Gökler,2 G. Laufer,2 C. Rapezzi,1 F. Grigioni,1 A. Zuckermann,2 L. Potena.1 1DIMES, S.Orsola Hospital, University of Bologna, Bologna, Italy; 2Heart Transplantation, Cardiac Surgery Department, Vienna General Hospital, Vienna, Austria; 3Cardiology, Vienna General Hospital, Vienna, Austria.

Purpose: ISHLT grading of coronary allograft vasculopathy (CAV) provides a static definition of CAV severity that has recently been correlated with adverse outcome. Although this grading classifies even mild stenoses, as they may be a marker of adverse cardiovascular (CV) outcome, it may not detect the progression of subcritical lesions. Herein we aimed to analyze the prognostic implication of progressing vs. static angiographic lesions within ISHLT classification.Methods: All patients (pts) receiving heart transplant (HT) in 2 large centers between 2001-08, surviving > 1 year, having undergone 2 coronary angi-ographies (angio), entered this study. Angio progression (assessed by an operator blinded to the exam report) was defined as any stenosis increase or any new stenosis in any vessel. Study endpoints were freedom from CV death and from MACE combined occurrence (CV death, PCI, admissions for HF, re-HT) during 5 yrs after the second angio.Results: 161 pts (85% males, 16-70 yrs) were included. The 2 angios were performed respectively 13 and 61 months (median time) after HT. CAV occurred in 22.9% of the first angio (21.1% grade 1; 1.8% grade 2; 0% grade 3), and in 41.5% of the second (34.1% grade 1; 6.2% grade 2; 1.2% grade 3). ISHLT grading of both angios predicted MACE (p< 0.01). 24.8% of pts had an ISHLT-grading progression (75% from 0 to 1, 15% 1 to 2, 5% 0 to 2, 5% 1 to 3), 33.5% of pts had an angio progression. Grade progression predicted MACE (65.4±10.0% vs. 88.0±4.0%, p< 0.01) but not CV death (91.3±3.3% vs. 91.7±4.6% vs. p= 0.42). Any angio progression predicted both CV death (82.6±6.3% vs. 95.8±2.6%, p< 0.01) and MACE ( 59.4±9.2% vs 93.9±6.1% , p< 0.01). Of note, pts (8.9%) with angio progression within the same ISHLT grade, had a higher incidence of CV death and MACE as compared to the non-progression group (52.8±19.1 vs 95.8%±2.6% ,p< 0.01; 40.3±19.2% vs 93.9%±3.2%, p< 0.01).Conclusion: Both static and dynamic ISHLT CAV grading stratify CV prog-nosis post HT. However, analysis of progression of mild lesions not captured

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Protein Kinase C delta-Mediated Unfolded Protein Response and Necrotic Cell Death Contributes to Ischemia-Reperfusion Induced Injury in Lung TransplantationH. Kim , J. Zhao, D. Lee, X. Bai, M. Cypel, S. Keshavjee, M. Liu. Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto, ON, Canada.

Purpose: Ischemia-reperfusion (IR) induced lung injury is one of the major causes of morbidity and mortality after lung transplantation. We have reported that inhibition of protein kinase C delta (PKCδ ) reduces cell death and cytokine production in rat lung transplantation. However, its underlying cellular mechanism is not yet determined. Necrosis, not apoptosis, correlates with prolonged cold ischemic time (CIT) and contributes to detrimental clini-cal outcome. We hypothesized that PKCδ activation mediates IR-induced necrosis via specific intracellular pathways.Methods: To determine the IR-induced cellular mechanisms, we used a cell culture model that simulates lung transplantation procedure. Using various molecular and biochemical techniques, we determined the IR-induced cel-lular changes, which include unfolded protein response (UPR), necrosis and apoptosis. A rat model was used to test the efficacy of PKCδ inhibition in attenuating IR-induced lung injury.Results: In the cell culture, prolonged CIT followed by simulated reperfusion induced UPR, as shown by the expression level of multiple UPR markers in human lung epithelial cells. Both necrotic and apoptotic cell death were also observed. The physical interaction of p53 and cyclophilin D, the hallmark of the intracellular signals leading to necrosis, was detected by co-immunopre-cipitation. Treatment of δ V1-1, a PKCδ peptide inhibitor, reduced UPR and converted the mode of cell death from necrosis to apoptosis. δ V1-1 treatment reduced HMGB1 release and p53/cyclophilin D interaction. Lastly, admin-istration of δ V1-1 during pulmonary IR effectively protected the lung func-tions (PaO2/FiO2: control= 325 ± 70 mmHg; treatment= 522 ± 19 mmHg, p< 0.05), and reduced cell death and lung injury. The treatment inhibited the intracellular pathways related to necrosis in the lung tissues.Conclusion: During hypothermic IR, PKCδ translocates to endoplasmic reticulum and mitochondria, and subsequently promotes apoptotic and necrotic cell death. Administration of PKCδ inhibitor switches the mode of cell death from necrosis to apoptosis. This mode of cell death causes less lung injury and leads to a better clinical outcome.

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Does the Ability for Donor Specific Antibodies to Bind Complement Always Result in C4d Staining in the Endomyocardial Biopsy After Heart Transplant?N. Lipson-Altman ,1 N. Reinsmoen,1 M. Kittleson,1 D. Luthringer,2 J. Patel,1 F. Liou,1 Z. Yu,1 B. Kearny,1 L. Czer,1 D.H. Chang,1 D. Ramzy,1 J. Kobashigawa.1 1Cedars- Sinai Heart Institute, Los Angeles, CA; 2Cedars-Sinai Medical Center, Los Angeles, CA.

Purpose: Donor Specific Antibodies (DSA) after heart transplantation (HTx) is seen in approximately 25% of patients (pts) according to a recent study. The ability to bind complement suggests that these antibodies are cytotoxic and therefore have the ability to injure the donor graft. We sought to prove this point by evaluating our HTx pts who had DSA with the ability to bind complement (positive C1q assay). Concomitant endomyocardial biopsy performed at the time of antibody assay was assessed for the deposition of C4d staining.Methods: Between 2007 and 2013, we evaluated 713 PRA screens with con-comitant endomyocardial biopsies from 260 HTx pts. Blood draws with con-comitant biopsies were divided into those that showed DSA+ C1q-, DSA+ C1q+, non-specific antibodies only, and no antibodies. Each concomitant heart biopsy was specifically assessed for cellular rejection, complement staining through C4d and C3d, as well as for ISHLT AMR 1, 2, and 3. Left ventricular ejection fraction (LVEF) on concomitant echocardiograms was also assessed.Results: The PRA screens that were DSA+ and C1q+ had a significantly greater incidence of C4d/C3d positivity, AMR, cellular rejection on concomi-tant biopsies compared to the no antibody group. When the DSA+ C1q+ group was compared to the DSA+ C1q- group, there was no difference in C4d/C3d positivity, AMR, and cellular rejection on concomitant biopsies,

S84 The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2014

P. Macdonald,1 J. Moore.1 1Heart and Lung Transplant Unit, St. Vincent’s Hospital, Darlinghurst, Australia; 2Dept of Haematology, St. Vincent’s Hospital, Darlinghurst, Australia; 3Dept of Anatomical Pathology, St. Vincent’s Hospital, Darlinghurst, Australia.

Purpose: Heart and lung transplant recipients have one of the highest incidences of post-transplant lymphoproliferative disease (PTLD). There is still a paucity of data specific to this group of patients, in particular, on factors associated with improved survival and on the efficacy of newer therapeutic strategies. We conducted the largest analysis to date on heart (H), lung (L) and heart-lung (HL) transplant recipients with PTLD, in order to identify unique features of PTLD in thoracic organ transplant recipients, to identify specific prognostic factors and thereby improve treatment.Methods: 70 cases (H [43], L [21] and HL [6]) of PTLD were identified from a single institution between 1984-2011. Data were retrospectively analysed in detail for demographics, immunosuppression, rejection, treatment, response, complications and survival. Univariate and multivariate Cox regression anal-yses were performed to identify factors associated with survival outcomes.Results: Univariate analysis identified poorer survival in patients > 50 years at diagnosis of PTLD (hazard ratio 2.22, p= 0.004), bone marrow (BM) involve-ment (HR 5.34, p< 0.001), serum albumin < 30g/l (HR 2.90, p= 0.02) and Ann Arbor stage ≥ 3 (HR 2.78, p= 0.001). Improved survival was associated with age between 18-50 years at PTLD diagnosis (HR 0.44, p= 0.004) and when complete response (CR) was achieved after the initial course of treatment (HR 0.06, p< 0.001). This analysis did not show prognostic significance for factors such as early (430U/l), anaemia (Hb < 100g/l), allograft involvement (lung only) or treatment with rituximab (n= 18). Multivariate analysis identified a significantly poorer prognosis in patients with BM involvement (HR 11.23, p< 0.001) and serum albumin < 30g/l (HR 3.70, p= 0.02) and significantly improved survival in those with CR after the initial course of treatment (HR 0.07, p< 0.001). Three-year overall survival (OS) was 33% and 10-year OS 20% post-PTLD, consistent with figures quoted in the literature.Conclusion: This study is the first to identify prognostic markers unique to H, L and HL transplant recipients with PTLD. Hypoalbuminaemia and BM involvement are identified as significant markers of poorer survival post-PTLD, while CR after initial treatment is a significant marker of improved survival. Larger studies are required to determine the effects of rituximab on survival in this group of patients.

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Circulating Cell-Free DNA Is a Non-Invasive Marker of Heart Transplant RejectionI. De Vlaminck ,1 H.A. Valantine,2 H. Luikart,2 D. Weisshaar,3 D. Bernstein,4 S.R. Quake,1 K.K. Khush.2 1Bioengineering, Stanford University, Palo Alto, CA; 2Cardiovascular Medicine, Stanford University, Palo Alto, CA; 3Heart Transplant Services, Kaiser Permanente Northern California, Santa Clara, CA; 4Pediatric Cardiology, Stanford University, Palo Alto, CA.

Purpose: The ability to monitor allograft health is a critically important compo-nent of post-transplant therapy. The endomyocardial biopsy is the current gold standard for cardiac allograft monitoring, but requires an expensive and invasive procedure. Proof of principle of a universal non-invasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort.Methods: We performed a prospective cohort study (63 adult and pediatric heart transplant recipients, 562 samples) that tested the utility of cfdDNA for detection of acute rejection after heart transplantation. Circulating cell-free DNA was puri-fied from serial heart transplant recipient plasma samples and sequenced (mean depth 1.2 Gbp) to quantify the fraction of donor-derived DNA.Results: Through a comparison with endomyocardial biopsy results we dem-onstrate that cfdDNA enables the diagnosis of acute rejection [area under the receiver operating characteristic curve (AUC) 0.825, sensitivity 0.72, specificity 0.75, threshold cfdDNA 0.075%] after heart transplantation. The concordance of the cfdDNA levels and biopsy data depends on patient age and time post- transplant; it is highest for patients younger than 60 years old and for samples collected ≥ 4 months post-transplant.Conclusion: We conclude that this non-invasive “genome transplant dynam-ics” approach is a powerful and accurate method for routine surveillance of allograft health. Our data support the extension of these studies to larger, multi-center clinical trials to determine whether cell-free DNA can be used in lieu of invasive biopsies.

by changes in ISHLT grading appears to improve angio accurancy in stratify prognosis long term after HT, underlining CAV dynamic features and its negative prognostic impact. These data support the inclusion of the concept of “progression” in CAV grading system, identifying mild progressing lesions as potential therapeutic targets to improve prognosis after HT.

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Clinical Importance of Flow Cytometry Crossmatch in the Context of Complement-Dependent Cytotoxicity Crossmatch Results Following Heart TransplantationB.C. Keeshan ,1 M.J. O’Connor,1 K.Y. Lin,1 D.S. Monos,2 C.T. Lind,2 C.E. Mascio,3 T.L. Spray,3 R.E. Shaddy,1 J.W. Rossano.1 1Division of Cardiology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Immunogenetics Laboratory, Children’s Hospital of Philadelphia, Philadelphia, PA; 3Division of Cardiothoracic Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA.

Purpose: The complement-dependent cytotoxicity crossmatch (CDC-XM) has historically been used for evaluation of preformed antibodies in patients undergo-ing heart transplantation. Flow cytometry crossmatch (FC-XM) is a more sensi-tive assay and is used with increasing frequency. As it remains largely unknown, we sought to determine the clinical relevance of a positive FC-XM for T-cells and/or B-cells in the context of negative CDC-XM in heart transplantation.Methods: We analyzed the United Network for Organ Sharing (UNOS) data for all heart transplants between 1987 and 2013. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess graft survival for four different patient groups defined by crossmatch.Results: T-cell and B-cell CDC-XM+ (Group 1), CDC-XM- but T-cell and B-cell FC-XM+ (Group 2), CDC-XM- with B-cell FC-XM+/ T-cell FC-XM- (Group 3), and T-cell/B-cell CDC-XM- and FC-XM- (Group 4).Results: Of the 48,392 heart transplants during the study period, 2,690 met inclusion criteria [10.7% (Group 1), 7.4% (Group 2), 11.4% (Group 3), 65.5% (Group 4)]. Patients with a positive CDC-XM (Group 1) had significantly decreased graft survival compared to all other groups (p< 0.05 for all) (Figure 1). Patients with a negative CDC-XM but a positive T-cell and/or B-cell FC-XM (Groups 2 & 3) had similar graft survival to patients with a negative XM (Group 4) (p> 0.1 for both). In multivariate analysis only being CDC-XM+ was inde-pendently associated with decreased graft survival [HR 1.55, 95% CI 1.30-1.85].Conclusion: Heart transplant recipients with a positive T-cell and B-cell CDC-XM have decreased graft survival compared patients with a positive T-cell and/or B-cell FC-XM, or those with a negative XM. However, a positive FC-XM in the context of a negative CDC-XM does not confer an increased risk of graft failure. Further study is needed to understand the role and impli-cations of CDC-XM and FC-XM testing in heart transplant recipients.

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Defining Prognostic Markers in Heart and Lung Transplant Recipients with PTLDG. Kumarasinghe ,1 O. Lavee,2 I. Nivison-Smith,2 A. Parker,3 M. Malouf,1 A. Keogh,1 S. Milliken,2 A. Dodds,2 M. Plit,1 C. Hayward,1 D. Ma,2 K. Fay,2 J. Joseph,2 E. Kotlyar,1 A. Havryk,1 A. Jabbour,1 A. Glanville,1