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BackgroundThe Molecular Microscope (MMDx) system classifies heart transplant endomyocardial biopsies as No-rejection (NR), Early-injury, T cell-mediated (TCMR), antibody-mediated (ABMR), mixed, and possible rejection (possible TCMR, possible ABMR). Rejection-like gene expression patterns in NR biopsies have not been described. We extended the MMDx methodology, using a larger data set, to define a new "Minor" category characterized by low-level inflammation in non-rejecting biopsies.MethodsUsing MMDx criteria from a previous study, molecular rejection was assessed in 1,320 biopsies (645 patients) using microarray expression of rejection-associated transcripts (RATs). Of these biopsies, 819 were NR. A new archetypal analysis model in the 1,320 data set split the NRs into NR-Normal (N = 462) and NR-Minor (N = 359).ResultsCompared to NR-Normal, NR-Minor were more often histologic TCMR1R, with a higher prevalence of donor-specific antibody (DSA). DSA positivity increased in a gradient: NR-Normal 24%; NR-Minor 34%; possible ABMR 42%; ABMR 66%. The top 20 transcripts distinguishing NR-Minor from NR-Normal were all ABMR-related and/or IFNG-inducible, and also exhibited a gradient of increasing expression from NR-Normal through ABMR. In random forest analysis, TCMR and Early-injury were associated with reduced LVEF and increased graft loss, but NR-Minor and ABMR scores were not. Surprisingly, hearts with MMDx ABMR showed comparatively little graft loss.ConclusionsMany heart transplants currently diagnosed as NR by histologic or molecular assessment have minor increases in ABMR-related and IFNG-inducible transcripts, associated with DSA positivity and mild histologic inflammation. These results suggest that low-level ABMR-related molecular stress may be operating in many more hearts than previously estimated. (ClinicalTrials.gov #NCT02670408).

Immune checkpoint inhibitor (ICI)‐associated myocarditis is a rare, potentially life‐threatening complication of immunotherapy. We report a case of a 60‐year‐old female with a history of colorectal cancer treated with nivolumab immunotherapy who presented with new cardiomyopathy complicated by cardiogenic shock and ventricular arrhythmias. Treatment of ICI‐associated myocarditis requires aggressive immunosuppression and supportive therapy. In this case, the patient required advanced mechanical circulatory support as a bridge to recovery. This case highlights the complexity of diagnosis, haemodynamic management, and treatment of fulminant ICI myocarditis.

STEM disciplines are the dominant culture in K-12 education. With its study of organs and diseases that afflict patients’ bodies, Western evidence-based medicine is seen and understood in the modern cultural paradigm as a science and as the practice in which a subject, the doctor, acts on an object; the patient’s body—a dominant culture in the patient’s journey. However, with the continually evolving high-technological and medical knowledge, life-saving therapeutic options are life-changing. They can range from changes in the diet, requiring structural and cultural changes in family life, to changes related to the experiences of learning to live tethered to a machine that is partly inside and partly outside one’s body or with somebody else’s heart. In this article, we show how competing needs to personalize care for the patient as a person forcefully emerge in response to evidence-based medicine’s global cultural dominance. We highlight two fundamental issues emerging in decision-making processes: (1) Framing evidence-based knowledge, uncertainties of the course of the disease and options, and (2) working with different, equally important, and often at odds conceptions of time in the care for the Other. Through the longitudinal analysis of moment-to-moment interactions in high-tech medicine encounters of a patient, his family, and the team caring for them, we show how framing and different conceptions of time emerge as issues, are profoundly interconnected, and are addressed by participants to care for a patient confronting existential decisions.

The only potentially curative treatment for lung adenocarcinoma patients remains complete resection of early-stage tumors. However, many patients develop recurrence and die of their disease despite curative surgery. Underlying mechanisms leading to establishment of systemic disease after complete resection are mostly unknown. We therefore aimed at identifying molecular signatures of resected lung adenocarcinomas associated with the risk of an early relapse. The study comprised 89 patients with totally resected stage IA–IIIA lung adenocarcinomas. Patients suffering from an early relapse within two years after surgery were compared to patients without a relapse in two years. Patients were clinically and molecular pathologically characterized. Tumor tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34, by Nanostring nCounter PanCancer Immune Profiling Panel as well as a comprehensive methylome profiling using the Infinium MethylationEPIC BeadChip. We detected differential DNA methylation patterns as well as significantly differentially expressed genes associated with an early relapse after complete resection. Especially, CD1A was identified as a potential biomarker, whose reduced expression is associated with an early relapse. These findings might help to develop biomarkers improving risk assessment and patient selection for adjuvant therapy as well as establish novel targeted therapeutic strategies.

Biological determinants of survival in advanced heart failure (AdHF) are linked to systems biological properties of disease severity including age, comorbidities, and frailty. We hypothesize that a...

INTRODUCTION: In parallel to the clinical maturation of heart transplantation over the last 50 years, rejection testing has been revolutionized within the systems biology paradigm triggered by the Human Genome Project. AREAS COVERED: We have co-developed the first FDA-cleared diagnostic and prognostic leukocyte gene expression profiling biomarker test in transplantation medicine that gained international evidence-based medicine guideline acceptance to rule out moderate/severe acute cellular cardiac allograft rejection without invasive endomyocardial biopsies. This work prompted molecular re-classification of intragraft biology, culminating in the identification of a pattern of intragraft myocyte injury, in addition to acute cellular rejection and antibody-mediated rejection. This insight stimulated research into non-invasive detection of myocardial allograft injury. The addition of a donor-organ specific myocardial injury marker based on donor-derived cell-free DNA further strengthens the non-invasive monitoring concept, combining the clinical use of two complementary non-invasive blood-based measures, host immune activity-related risk of acute rejection as well as cardiac allograft injury. EXPERT OPINION: This novel complementary non-invasive heart transplant monitoring strategy based on leukocyte gene expression profiling and donor-derived cell-free DNA that incorporates longitudinal variability measures provides an exciting novel algorithm of heart transplant allograft monitoring. This algorithm’s clinical utility will need to be tested in an appropriately designed randomized clinical trial which is in preparation.

Cytomegalovirus (CMV) causes mostly asymptomatic but lifelong infection. Primary infection or reactivation in immunocompromised individuals can be life-threatening. CMV viremia often occurs in solid organ transplant (SOT) recipients and associates with decreased graft survival and higher mortality. Furthering understanding of impaired immunity allowing CMV reactivation is critical to guiding anti-viral therapy and examining CMV's impact on outcomes of SOT. This study characterized longitudinal immune responses to CMV in 31 kidney transplant recipients with CMV viremia and matched, non-viremic recipients. Subjects were sampled three- and twelve-months post-transplant, with additional samples one-week and one-month post-viremia. Peripheral blood mononuclear cells (PBMC) were stained for NK and T cell markers. PBMC transcriptomes were characterized by RNA-Seq. Plasma proteins were quantified by Luminex. CD8 T cell transcriptomes were characterized by single-cell RNA-Seq. Pre-viremia, patients had high levels of IL-15 with concurrent expansion of immature CD56bright NK cells. Post-viremia, mature CD56dim NK cells and CD28- CD8 T cells upregulating inhibitory and NK-associated receptors were expanded. Phenotype of NK cells and CD28- CD8 T cells were associated with control of viremia. These findings suggest signatures of innate activation may be prognostic for CMV reactivation post-transplant, while CD8 T cell functionality is critical for effective control of CMV.

Noninvasive immunologic analysis of peripheral blood holds promise for explaining the mechanism of development of adverse clinical outcomes, and may also become a method for patient risk stratification before or after mechanical circulatory support device (MCSD) implantation. Dysregulation of the innate immune system is associated with increased patient age but has yet to be evaluated in the older patient with advanced heart failure undergoing MCSD surgery. Patients pre- and post-MCSD implantation had peripheral blood mononuclear cells (PBMC) and serum isolated. Multiparameter flow cytometry was used to analyze markers of innate cell function, including monocyte subtypes. Multiplex cytokine analysis was performed. MELD-XI and SOFA scores were utilized as surrogate markers of outcomes. Increased levels of pro-inflammatory cytokines including IL-15, TNF-α, and IL-10 were associated with increased MELD-XI and SOFA scores. IL-8, TNF- α, and IL-10 were associated with risk of death after MCSD implantation, even with correction for patient age. Increased frequency of ‘classical’ monocytes (CD14 + CD16−) were associated with increased MELD-XI and SOFA scores. This suggests that inflammation and innate immune system activation contribute to progression to multiorgan system failure and death after MCSD surgery. Development of noninvasive monitoring of peripheral blood holds promise for biomarker development for candidate selection and patient risk stratification.

Purpose The INTERHEART study previously used microarray assessment of 889 heart transplant biopsies to develop the Molecular Microscope Diagnostic System (MMDx) based on expression of rejection-associated transcripts (RATs). The present study reclassified the rejection-related states in an expanded set of 1320 prospectively collected biopsies from 645 patients from 13 centers. Methods Biopsies were classified by ensembles of classifiers and analyzed for left ventricular ejection fraction (LVEF) and survival. Results New algorithms identified 853 No rejection (NR), 179 ABMR, 76 TCMR, 13 Mixed, 161 possible ABMR (pABMR), and 38 possible TCMR (pTCMR). No rejection was subclassified as NR-Normal 462, NR-Minor 359, and NR-Early-injury 32 (Figure 1A). Compared to NR-Normal, NR-Minor biopsies had mild elevation of many inflammation transcripts (e.g. IFNG-inducible genes) and to a lesser extent parenchymal injury transcripts. NR-minor biopsies were often designated as TCMR1R by histology. In all NR biopsies, NR-Minor scores and histologic TCMR1R increased through the first year, peaking about one year, suggesting that Minor inflammation is a late response to injury, unrelated to rejection. LVEF was similar in NR-Normal and NR-Minor but depressed in TCMR and Early injury. In a 3-year post-biopsy survival analysis, NR-Minor and NR-Normal had similar survival. TCMR and Early-injury were associated with increased graft loss, but many losses were not related to rejection. Surprisingly, 76 hearts with ABMR (149 biopsies) and follow-up data had only 3 losses within 3 years post-biopsy (Figure 1B). Conclusion Many biopsies with molecular no rejection develop minor increases in rejection-related transcripts in the first year, often called TCMR1R by histology, with no apparent effects on function or survival. An unexpected finding was that molecular ABMR was associated with very few graft losses over three years. (ClinicalTrials.gov #NCT02670408).

Purpose of the Review This is a comprehensive update on failing Fontan physiology and the role of heart and combined heart and liver transplantation in the current era. Recent Findings Single ventricle physiology encompasses a series of rare congenital cardiac abnormalities that are characterized by absence of or hypoplasia of one ventricle. This effectively results in a single ventricular pumping chamber. These abnormalities are rarely compatible with long-term survival if left without surgical palliation in the first few years of life. Surgical treatment of single ventricle physiology has evolved over the past 60 years and is characterized by numerous creative innovations. These include the development of arteriopulmonary shunts, the evolution of partial cavopulmonary connections, and the eventual development of the “Fontan” operation. Regardless of the type of Fontan modification, the long-term consequences of the Fontan operation are predominantly related to chronic central venous hypertension and the multi-organ consequences thereof. Atrial arrhythmias can further compromise this circulation.Patients with single ventricle physiology represent a special sub-segment of congenital cardiac transplants and are arguably the most challenging patients considered for transplantation. Summary This review describes in detail the challenges and opportunities of heart and liver transplantation in Fontan patients, as viewed and managed by the experienced team at the Ahmanson/UCLA Adult Congenital Heart Center.

COVID-19 is an evolutionarily unprecedented natural experiment testing human immunological fitness and the potential to recover from a virus-activated ‘cytokine storm syndrome’ [1], a manifestation...

The infectious disease Coronavirus Disease 2019 (COVID‐19) was declared a pandemic by the World Health Organization in March 2020. The impact of COVID‐19 on solid organ transplantations, including heart transplantation, is currently unclear. Many transplant programs have been forced to swiftly re‐evaluate and adapt their practices, leading to a marked decrease in transplants performed. This trend has been due to various factors, including increased donor COVID‐19 screening scrutiny and recipient waiting list management in anticipation of COVID‐19 critical care surge capacity planning. In the face of these unknown variables, determining when and how to proceed with transplantation in our population of patients with end‐stage cardiomyopathies is challenging. Here, we describe our center’s experience with orthotopic heart transplantation (OHT) in one of the country’s pandemic epicenters, where we performed eight OHTs in the first two months after community spread began in late February 2020.

For most patients with end-stage heart failure, heart transplantation is the treatment of choice. Allograft rejection is one of the major post-transplantation complications affecting graft outcome and survival. Recent advancements in science and technology offer an opportunity to integrate genomic and other omics-based biomarkers into clinical practice, facilitating noninvasive evaluation of allograft for diagnostic and prognostic purposes. Omics, including gene expression profiling (GEP) of blood immune cell components and donor-derived cell-free DNA (dd-cfDNA) are of special interest to researchers. Several studies have investigated levels of dd-cfDNA and miroRNAs in blood as potential markers for early detection of allograft rejection. One of the achievements in the field of transcriptomics is AlloMap, GEP of peripheral blood mononuclear cells (PBMC), which can identify 11 differentially expressed genes and help with detection of moderate and severe acute cellular rejection in stable heart transplant recipients. In recent years, the utilization of GEP of PBMC for identifying differentially expressed genes to diagnose acute antibody-mediated rejection and cardiac allograft vasculopathy has yielded promising results. Advancements in the field of metabolomics and proteomics as well as their potential implications have been further discussed in this paper.

Background Mitochondrial dysfunction is associated with poor allograft prognosis. Mitochondrial-related gene expression (GE) in endomyocardial biopsies (EMBs) could be useful as a nonimmune functional marker of rejection. We hypothesize that acute cardiac allograft rejection is associated with decreased mitochondrial-related GE in EMBs. Methods We collected 64 routines or clinically indicated EMB from 47 patients after heart transplant. The EMBs were subjected to mRNA sequencing. We conducted weighted gene coexpression network analysis to construct module-derived eigengenes. The modules were assessed by gene ontology enrichment and hub gene analysis. Modules were correlated with the EMBs following the International Society of Heart and Lung Transplantation histology-based criteria and a classification based on GE alone; we also correlated with clinical parameters. Results The modules enriched with mitochondria-related and immune-response genes showed the strongest correlation to the clinical traits. Compared with the no-rejection samples, rejection samples had a decreased activity of mitochondrial-related genes and an increased activity of immune-response genes. Biologic processes and hub genes in the mitochondria-related modules were primarily involved with energy generation, substrate metabolism, and regulation of oxidative stress. Compared with International Society of Heart and Lung Transplantation criteria, GE-based classification had stronger correlation to the weighted gene coexpression network analysis-derived functional modules. The brain natriuretic peptide level, ImmuKnow, and Allomap scores had negative relationships with the expression of mitochondria-related modules and positive relationships with immune-response modules. Conclusions During acute cardiac allograft rejection, there was a decreased activity of mitochondrial-related genes, related to an increased activity of immune-response genes, and depressed allograft function manifested by brain natriuretic peptide elevation. This suggests a rejection-associated mitochondrial impairment.

Within a participatory research project, we investigate how forms of touch we call caring touch are enacted in AdHF medical encounters. Through the theoretical lens of Relational Ontology (Raia, 2018), grounding multimodality in phenomenology, we identify various forms of caring touch. When occurring in conjunction with medical/diagnostic touch, especially in situations of a perceived patient’s vulnerability, caring touch facilitates passages from the person level to the organ, tissue, and gene levels and then back to the whole-person level in an uninterrupted movement, maintaining the person-person relation between doctor and patient. Gentle shepherding (Cekaite, 2010) is used to guide the patient body, and comforting touch (Goodwin & Cekaite, 2018) accompanies invitations to enter a space where death is part of living. We show the existential grounding power of caring touch, which constitutes forms of reciprocal sharing of existential experiences in caring-for-the-Other. All these forms of caring touch are employed by an AdHF doctor whose work centers on the practice of Relational Medicine (Raia and Deng, 2015b), in which the starting and returning point is the patient in his/her life. While providing a single case analysis, the research builds from a corpus of 500 hours of recorded medical encounters with 125 patients in high-tech medicine.

Background Survival after heart transplantation (HTx) is limited by complications related to alloreactivity, immune suppression, and adverse effects of pharmacologic therapies. We hypothesize that time-dependent phenomapping of clinical and molecular data sets is a valuable approach to clinical assessments and guiding medical management to improve outcomes. Methods We analyzed clinical, therapeutic, biomarker, and outcome data from 94 adult HTx patients and 1,557 clinical encounters performed between January 2010 and April 2013. Multivariate analyses were used to evaluate the association between immunosuppression therapy, biomarkers, and the combined clinical end point of death, allograft loss, retransplantation, and rejection. Data were analyzed by K-means clustering (K = 2) to identify patterns of similar combined immunosuppression management, and percentile slopes were computed to examine the changes in dosages over time. Findings were correlated with clinical parameters, human leucocyte antigen antibody titers, and peripheral blood mononuclear cell gene expression of the AlloMap (CareDx, Inc., Brisbane, CA) test genes. An intragraft, heart tissue gene coexpression network analysis was performed. Results Unsupervised cluster analysis of immunosuppressive therapies identified 2 groups, 1 characterized by a steeper immunosuppression minimization, associated with a higher likelihood for the combined end point, and the other by a less pronounced change. A time-dependent phenomap suggested that patients in the group with higher event rates had increased human leukocyte antigen class I and II antibody titers, higher expression of the FLT3 AlloMap gene, and lower expression of the MARCH8 and WDR40A AlloMap genes. Intramyocardial biomarker-related coexpression network analysis of the FLT3 gene showed an immune system–related network underlying this biomarker. Conclusions Time-dependent precision phenotyping is a mechanistically insightful, data-driven approach to characterize patterns of clinical care and identify ways to improve clinical management and outcomes.

Immunologic impairment may contribute to poor outcomes after implantation of mechanical circulatory support device (MCSD), with infection often as a terminal event. The study of immune dysfunction is of special relevance given the growing numbers of older patients with heart disease. The aim of the study was to define which immunologic characteristics are associated with development of adverse clinical outcomes after MCSD implantation. We isolated peripheral blood mononuclear cells (PBMC) from patients pre- and up to 20 days post-MCSD implantation and analyzed them by multiparameter flow cytometry for T cell dysfunction, including terminal differentiation, exhaustion, and senescence. We used MELD-XI and SOFA scores measured at each time point as surrogate markers of clinical outcome. Older patients demonstrated increased frequencies of terminally differentiated T cells as well as NKT cells. Increased frequency of terminally differentiated and immune senescent T cells were associated with worse clinical outcome as measured by MELD-XI and SOFA scores, and with progression to infection and death. In conclusion, our data suggest that T cell dysfunction, independently from age, is associated with poor outcomes after MCSD implantation, providing a potential immunologic mechanism behind patient vulnerability to multiorgan dysfunction and death. This noninvasive approach to PBMC evaluation holds promise for candidate evaluation and patient monitoring.

Heart failure (HF) is a complex clinical syndrome that results from structural or functional cardiovascular disorders causing a mismatch between demand and supply of oxygenated blood and consecutive failure of the body’s organs. For those patients with stage D HF, advanced therapies, such as mechanical circulatory support (MCS) or heart transplantation (HTx), are potentially life-saving options. The role of risk stratification of patients with stage D HF in a value-based healthcare framework is to predict which subset might benefit from advanced HF (AdHF) therapies, to improve outcomes related to the individual patient including mortality, morbidity and patient experience as well as to optimize health care delivery system outcomes such as cost-effectiveness. Risk stratification and subsequent outcome prediction as well as therapeutic recommendation-making need to be based on the comparative survival benefit rationale. A robust model needs to (I) have the power to discriminate (i.e., to correctly risk stratify patients); (II) calibrate (i.e., to show agreement between the predicted and observed risk); (III) to be applicable to the general population; and (IV) provide good external validation. The Seattle Heart Failure Model (SHFM) and the Heart Failure Survival Score (HFSS) are two of the most widely utilized scores. However, outcomes for patients with HF are highly variable which make clinical predictions challenging. Despite our clinical expertise and current prediction tools, the best short- and long-term survival for the individual patient, particularly the sickest patient, is not easy to identify because among the most severely ill, elderly and frail patients, most preoperative prediction tools have the tendency to be imprecise in estimating risk. They should be used as a guide in a clinical encounter grounded in a culture of shared decision-making, with the expert healthcare professional team as consultants and the patient as an empowered decision-maker in a trustful safe therapeutic relationship.

Purpose In order to create a clinical assay that preoperatively predicts 1-year survival status of AdHF patients following surgical/interventional therapies and to select the appropriate commercial platform, we compared the properties of Next Generation Sequencing (NGS) to NanoString (NS). Methods 8ml of blood was collected in CPT tubes from 25 AdHF patients (11 heart failure control, 4 mechanical circulatory support surgery, and 10 heart transplant surgery) at UCLA Medical Center 2015-2016. Purified RNA samples were aliquoted in two sets: one was processed using NGS transcriptome analysis (Illumina HiSeq 2500) and compared to 770 genes represented on NS pre-existing PanCancer IO 360 Gene Expression research panel. Prior to statistical analysis, expression values were log transformed. Pearson correlation coefficients were computed for each sample. Gene expression values were compared between NS and NGS across the set of matched genes and for each of the matched genes across the set of samples. Genes were grouped by average NGS expression, and the NS-NGS correlation for each group was computed. Results Out of 770 genes, 734 overlapped between both platforms and showed high intrasample correlation (R^2 = 0.82). Genes with higher expression demonstrated higher correlation across both platforms (Figure). NS demonstrated high robustness: Gene signature scores were similar across different RNA input concentrations, cartridges, and outcomes; sensitivity: Probe counts between 100 and 500 detected and quantified. The RNA input of 100 ng yielded the most precise result and were used for downstream analysis; and reproducibility: Technical replicates with equal concentration (100ng input) showed extremely high correlation (R^2 > 0.99), thus demonstrating high reproducibility. Conclusion Both platforms have meaningful, complementary roles, NGS for discovery and NanoString for commercialization and can be used for AdHF-biomarker test development.

Background Cytomegalovirus (CMV) is one of the most common infections after transplantation and continues to cause significant morbidity and mortality. Current guidelines recommend 3-6 months of post-transplant prophylaxis with 900mg daily of valganciclovir in heart transplant recipients. At our institution, however, the protocol is to use 450mg daily of valganciclovir for 6-12 months for intermediate risk (R+) patients and 900 mg daily for high risk (D+/R-) patients. In this study we aimed to identify underlying patient characteristics associated with detectable viral load above the quantifiable threshold. Table 1. Comparison of patients with a CMV viral above and below 137. Methods We retrospectively reviewed medical records of adult (≥ 18 years) heart transplant recipients with detectable CMV viremia from 2016-2018 resulted during routine clinical screening. Results Ninety-seven heart transplant recipients with a detectable CMV viral load were identified. Of those, 38 (37.2%) had a quantifiable viral load above the 137 IU/mL threshold. When compared to the individuals with a detectable viral load below the threshold (< 137 IU/mL), they had similar age at time of transplant, increased likelihood of donor/recipient CMV IgG mismatch, and were more frequently on 900mg daily of prophylaxis at time of viremia. Of the individuals with CMV DNAemia above the threshold, the median time to viremia was 271.4 days and the median peak viral load was 701 IU/mL. When limiting analysis to only recipients who were CMV IgG positive, patients with viremia had similar age and more likely to be on 900mg daily of valganciclovir as prophylaxis when compared to individuals with CMV viremia < 137 IU/mL. When comparing CMV D+/R- patients, age and rates of 900mg valganciclovir as prophylaxis were similar (Table 1). Conclusion We found that despite receipt of CMV prophylaxis, an appreciable number of both R+ and D-/R+ heart transplant recipients developed breakthrough DNAemia despite being on prophylaxis of valganciclovir as recommended by guidelines. Despite receipt of the higher 900 mg daily dose, high risk patients had higher rates of breakthrough DNAemia at our institution compared with R+ intermediate risk patients. More research is needed to evaluate the optimal dose and duration for prophylaxis in heart transplant patients against CMV. Disclosures All Authors: No reported disclosures

Purpose AlloSure test is a non-invasive blood test designed to detect the presence of donor cell-free DNA in the recipients blood as a measure of the probability of rejection in heart transplant recipients. The result is displayed as a percent of donor-derived cell-free DNA (dd-cfDNA) in the totalcell-free DNA (cfDNA). Recommended threshold of 0.2% dd-cfDNA has been predictive of low likelihood of rejection. There is no data looking at its value and validation in multi-organ transplant. We aimed to study this area by reviewing our transplant database. Methods All patients who received more than one solid organ transplant at our center and had AlloSure testing were included. Study outcomes included one year survival, rejection grade 2R or more, and hemodynamically significant graft dysfunction. Results There were (7) patients included in the study, average AlloSure score was 0.93% ±0.61% with an average AlloMap score of 33.8±2.14. Four patients (57.1%) had heart and kidney transplant, 3 patients (42.9%) had heart and liver transplantation. Average AlloSure score for heart/kidney transplant recipients was 0.52±0.17% with an average AlloMap score of 33.6±2.28. The while average AlloSure score for heart/liver transplant recipients was 1.21±0.55% mean AlloMap score of 33.93±2.32. None of our patients had graft dysfunction or biopsy-proven rejection >=2R. Survival rate was 100% for the first year in our patients Conclusion AlloSure levels were higher in heart-liver vs. heart-kidney recipients, and in both cohorts higher than the 0.2% threshold recommended for heart transplant prediction of rejection, however did not predict rejection. In our cohort, while AlloMap scores were consistent in both groups, (33.93±2.32 vs. 33.6±2.28 respectively). Elevated AlloSure was not predictive of rejection in multiorgan heart transplant recipients.

We analyzed humoral immune responses to nonhuman leukocyte antigen (HLA) after cardiac transplantation to identify antibodies associated with allograft rejection. Protein microarray identified 366 non-HLA antibodies (>1.5 fold, P1R) with an area under the curve of 0.87 (P&nbsp

Background Allosensitization during mechanical circulatory support (MCS) is a well-described phenomenon, although its mechanism remains unknown. Although immune-mediated interactions from devices or blood transfusions have been proposed, the role of inflammation in this development is less clear. This study was undertaken to further investigate the temporal association of cytokines and B-cell phenotypes in the MCS population. Methods Adult patients who received the Heartmate II (Thoratec, Pleasanton, Calif) at our center between September 2012 and March 2015 were prospectively followed after device implantation. Blood draws for anti-human leukocyte antigen (HLA) antibody, cytokine expression, and B-cell immunophenotyping were performed before implantation and for 3 weeks postoperatively. Time courses for cytokines and B-cell subsets were expressed using visual representations of median levels as heat maps, and mixed modeling analysis was used to model changes with time and patient factors. Results Twenty patients who received the Heartmate II (Thoratec) were analyzed during the study period. Four patients showed measureable levels of anti-HLA antibody during the follow-up period, although 3 of these had evidence of antibodies preoperatively. Analysis of cytokine trends revealed early (interleukin [IL]-6, IL-8, and IL-10) and late peaking (IL-3, IL-4, fibroblast growth factor 2, and CD40L) patterns. Upregulation of switched memory, transitional, and plasma blast B cells occurred over time. Right ventricular assist device use and low Interagency Registry for Mechanically Assisted Circulatory Support score were associated with decreased mature naive and increased antibody-secreting cells. Conclusions MCS device implantation was associated with increased inflammatory cytokines and maturation of B-cell phenotypes. No patients developed de novo HLA antibodies, whereas several showed increases in anti-HLA antibody levels detected before implantation. This suggests that inflammation and maturation of existing sensitized B cells might play an important role in the pathogenesis of allosensitization in MCS.

Purpose Disease associated long non-coding RNAs (lncRNAs) may yield biomarkers to assess the risk of disease progression, facilitating treatment decisions. We hypothesize that identification of lncRNA genes related to recovery/survival outcome in Advanced Heart Failure (AdHF) patients after various surgical/interventional therapies (S/IT), and integration of these lncRNAs into our existing independently validated outcome prediction algorithm further improves the survival prediction precision vs clinical parameters alone. Methods Study was conducted on 77 AdHF patients undergoing various S/IT. Clinical data was collected within 30 days before and 1 year post S/IT. All patients were assigned survivor/non survivor status based on 1 year outcome. Blood samples were collected within 30 days pre-intervention. Total RNA was isolated from peripheral blood mononuclear cells (PBMC) for RNAseq genomic/transcriptomic analysis. Raw RNAseq data was aligned to Hg19 build and subjected to DESeq normalization in StrandNGS software (3.2). Genes with fold change of 2.0 were included in the analysis. Biological significance, functional characterization, pathway analysis were assessed using GO annotations/KEGG/DAVID/BioCarta/GeneCards/LNCipedia databases. Results Out of 161 differentially expressed and patented AdHF outcome prediction genes we identified 13 lncRNAs related to 1 year survival/functional recovery. SEPT5-GP1BB was upregulated in AdHF patients surviving after S/IT and its neighbor protein coding genes (SEPT5, GP1BB, synphilin1, PARK2) involved in proteolysis pathway responsible for degrading regulatory proteins, have a role in immune/inflammatory responses. HIF1A-AS2 was upregulated in surviving patients and can be predictive based on its effect on the organ failure/hypoxia induced angiogenesis. XIST was upregulated in surviving patients, it is known to co-occur with at least 14 coding genes. The predictive information of XIST on survival might be linked to its immunomodulatory effect. Conclusion Differentially expressed lncRNA genes from PBMCs are correlated with recovery/1 year survival outcomes in AdHF patients and can be systematically identified by a phenotype guided NGS strategy. Incorporation of lncRNA data will allow for optimization of our existing multidimensional outcome prediction algorithm.

Purpose We previously reported that preoperative differentially expressed genes can predict short-term recovery and long-term survival in patients after mechanical circulatory support (MCS)-surgery. Here, we aimed to train and independently validate an algorithm in a larger mixed-intervention cohort. This algorithm is based on preoperative clinical and transcriptomic data that can predict 1-year survival after various interventions. Methods A retrospective study was conducted on 96 AdHF-patients undergoing MCS-surgery to identify clinical predictors of 1-year survival. Subsequently, a study was conducted on 77 patients evaluated for various AdHF-interventions to train (n=29) and validate (n=48) the algorithm (Table, Figure). In this prospective cohort, blood samples were collected within 30-days pre-intervention. In all studies, clinical data was collected within 30-days before and 1-year post-S/IT. All patients were assigned survivor/non-survivor status based on 1-year post-intervention outcome. Results Age, white blood cell count, respiratory rate, and minimum diastolic blood pressure yielded the highest correlation with 1-year survival status. An algorithm was trained by combining clinical and transcriptomic predictors on 29 samples from AdHF-patients undergoing MCS-surgery. The algorithm was validated on an independent set of 48 samples from patients undergoing various AdHF-interventions. Prediction of 1-year survival using 4 clinical parameters alone achieved an AUC=0.69. Adding 12 differentially expressed genes to the clinical model improved the AUC=0.90 and demonstrated 71% sensitivity, 90% specificity, 56% positive predictive value, and 95% negative predictive value. Conclusion The algorithm can increase accurate survival prediction by AUC>0.1 compared to clinical data alone and warrants further study in larger longitudinal cohorts.

With ongoing advancements in cancer-related treatments, the number of cancer survivors continues to grow globally, with numbers in the United States predicted to reach 18 million by 2020. As a result, it is expected that a greater number of patients will present with chemotherapy-related side effects. One entity in particular, chemotherapy-related cardiomyopathy (CCMP), is a known cardiotoxic manifestation associated with agents such as anthracyclines, trastuzumab, and tyrosine kinase inhibitors. Although such effects have been described in the medical literature for decades, concrete strategies for screening, prevention, and management of CCMP continue to be elusive owing to limited studies. Late recognition of CCMP is associated with a poorer prognosis, including a lack of clinical response to pharmacologic therapy, and end-stage heart failure. A number of advanced cardiac therapies, including cardiac resynchronization therapy, ventricular assist devices, and orthotopic cardiac transplantation, are available to for end-stage heart failure; however, the role of these therapies in CCMP is unclear. In this review, management of end-stage CCMP with the use of advanced therapies and their respective effectiveness are discussed, as well as clinical characteristics of patients undergoing these treatments. The relative paucity of data in this field highlights the importance and need for larger-scale longitudinal studies and long-term registries tracking the outcomes of cancer survivors who have received cardiotoxic cancer therapy to determine the overall incidence of end-stage CCMP, as well as prognostic factors that will ultimately guide such patients toward receiving appropriate end-stage care.

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking. We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

Purpose Kidney disease is a common comorbidity seen in heart disease, and so the number of patients undergoing combined heart-kidney transplantation (HKT) continues to increase, as well as the number of waitlisted patients for HKT. Recent evidence suggests a favorable immunomodulatory effect for combined HKT, making these patients ideal candidates for non-invasive monitoring for rejection and injury. The use of gene-expression profile testing (AlloMap®) in HKT recipients vs heat transplant (HT) alone has remained uncertain with earlier reports suggesting higher values in HKT, while more recent reports suggesting equivocal results between the two. Additionally, the profile of donor-derived cell-free DNA (dd-cfDNA) (AlloSure®) in HKT has not yet been evaluated. Methods The Utility of dd-cfDNA in Association with Gene Expression Profiling (D-OAR) registry is a prospective, observation study of HT recipients monitored with AlloMap and AlloSure. The D-OAR registry and prospectively collected data from UCLA Medical Center were assessed for patients who underwent HKT with associated AlloSure and AlloMap scores and compared to patients who underwent HT alone. The cumulative distribution functions of AlloMap and AlloSure scores were stratified by HT vs. HKT. Patients with CMV and acute cellular rejection (≥2R) were excluded from the analysis. Results There were 300 HT recipients and 14 HKT recipients. Baseline characteristics were similar between groups. The median AlloMap and AlloSure scores for HT recipients (N=300) were 29 and 0.09% respectively, while for HKT recipients (N=14) AlloMap and AlloSure scores were 33.5 and 0.41% respectively. See table 1 Conclusion There were slightly higher AlloMap scores in HKT vs HT patients, while AlloSure results in HKT were significantly higher. Further work to establish baseline parameters and understand changes to these parameters during rejection episodes would benefit HKT patients by allowing non-invasive monitoring for rejection and graft injury.

As immunotherapy use in various malignancies expands, immune checkpoint inhibitor (ICI) myocarditis is a rare but potentially fatal complication requiring a high degree of clinical suspicion for early diagnosis and treatment. A 60 year-old female with colon cancer status post 3 months of nivolumab

In preparation to create a clinical assay that predicts 1-year survival status of advanced heart failure (AdHF) patients before surgical/interventional therapies and to select the appropriate clinical assay platform for the future assay, we compared the properties of next generation sequencing (NGS) used in the gene discovery phase to the NanoString platform used in the clinical assay development phase. In 25 AdHF patients in a tertiary academic medical center from 2015 to 2016, PBMC samples were collected and aliquoted for NGS RNA whole transcriptome sequencing and compared to 770 genes represented on NanoString’s PanCancer IO 360 Gene Expression research panel. Prior to statistical analysis, NanoString and NGS expression values were log transformed. We computed Pearson correlation coefficients for each sample, comparing gene expression values between NanoString and NGS across the set of matched genes and for each of the matched genes across the set of samples. Genes were grouped by average NGS expression, and the NanoString-NGS correlation for each group was computed. Out of 770 genes from the NanoString panel, 734 overlapped between both platforms and showed high intrasample correlation. Within an individual sample, there was an expression-level dependent correlation between both platforms. The low- vs. intermediate/high-expression groups showed NGS average correlation 0.21 vs. 0.58–0.68, respectively, and NanoString average correlation 0.07–0.34 vs. 0.59–0.70, respectively. NanoString demonstrated high reproducibility (R2 > 0.99 for 100 ng input), sensitivity (probe counts between 100 and 500 detected and quantified), and robustness (similar gene signature scores across different RNA input concentrations, cartridges, and outcomes). Data from NGS and NanoString were highly correlated. These platforms play a meaningful, complementary role in the biomarker development process.

We describe a simple bioinformatics method for biomarker discovery that is based on the analysis of global transcript levels in a population of inbred mouse strains showing variation for disease-related traits. This method has advantages such as controlled environment and accessibility to heart and plasma tissue in the preclinical selection stage. We illustrate the approach by identifying candidate heart failure (HF) biomarkers by overlaying mouse transcriptome and clinical traits from 91 Hybrid Mouse Diversity Panel (HMDP) inbred strains and human HF transcriptome from the Myocardial Applied Genomics Network (MAGNet) consortium. We found that some of the top differentially expressed genes correlated with known human HF biomarkers, such as galectin-3 and tissue inhibitor of metalloproteinase 1. Using ELISA assays, we investigated one novel candidate, Glycoprotein NMB, in a mouse model of chronic β-adrenergic stimulation by isoproterenol (ISO) induced HF. We observed significantly lower GPNMB plasma levels in the ISO model compared to the control group (p-value = 0.007). In addition, we assessed GPNMB plasma levels among 389 HF cases and controls from the METabolic Syndrome In Men (METSIM) study. Lower levels of GPNMB were also observed in patients with HF from the METSIM study compared to non-HF controls (p-value < 0.0001). In summary, we have identified several candidate biomarkers for HF using the cardiac transcriptome data in a population of mice that may be directly relevant and applicable to human populations.

BACKGROUND. Because injury is universal in organ transplantation, heart transplant endomyocardial biopsies present an opportunity to explore response to injury in heart parenchyma. Histology has limited ability to assess injury, potentially confusing it with rejection, whereas molecular changes have potential to distinguish injury from rejection. Building on previous studies of transcripts associated with T cell–mediated rejection (TCMR) and antibody-mediated rejection (ABMR), we explored transcripts reflecting injury. METHODS. Microarray data from 889 prospectively collected endomyocardial biopsies from 454 transplant recipients at 14 centers were subjected to unsupervised principal component analysis and archetypal analysis to detect variation not explained by rejection. The resulting principal component and archetype scores were then examined for their transcript, transcript set, and pathway associations and compared to the histology diagnoses and left ventricular function. RESULTS. Rejection was reflected by principal components PC1 and PC2, and by archetype scores S2TCMR, and S3ABMR, with S1normal indicating normalness. PC3 and a new archetype score, S4injury, identified unexplained variation correlating with expression of transcripts inducible in injury models, many expressed in macrophages and associated with inflammation in pathway analysis. S4injury scores were high in recent transplants, reflecting donation-implantation injury, and both S4injury and S2TCMR were associated with reduced left ventricular ejection fraction. CONCLUSION. Assessment of injury is necessary for accurate estimates of rejection and for understanding heart transplant phenotypes. Biopsies with molecular injury but no molecular rejection were often misdiagnosed rejection by histology. TRAIL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02670408","term_id":"NCT02670408"}}NCT02670408 FUNDING. Roche Organ Transplant Research Foundation, the University of Alberta Hospital Foundation, and Alberta Health Services.

BackgroundDonor-specific HLA antibodies (DSA) are associated with increased rates of rejection and of graft failure in cardiac transplantation. The goal of this study was to determine the association of preformed and posttransplant development of newly detected DSA (ndDSA) with antibody-mediated rejection (AMR) and characterize the clinical relevance of complement-activating DSA in heart allograft recipients.MethodsThe study included 128 adult and 48 pediatric heart transplant patients transplanted between 2010 and 2013. Routine posttransplant HLA antibody testing was performed by IgG single-antigen bead test. The C3d single-antigen bead assay was used to identify complement-activating antibodies. Rejection was diagnosed using International Society for Heart and Lung Transplantation criteria.ResultsIn this study, 22 patients were transplanted with preexisting DSA, and 43 patients developed ndDSA posttransplant. Pretransplant (P < 0.05) and posttransplant (P < 0.001) ndDSA were associated with higher incidence of AMR. Patients with C3d + DSA had significantly higher incidence of AMR compared with patients with no DSA (P < 0.001) or patients with C3d-DSA (P = 0.02). Nine (36%) of 25 patients with AMR developed transplant coronary artery disease compared with 17 (15.9%) of 107 patients without AMR (P < 0.05). Among the 47 patients who received ventricular assistant device (VAD), 7 of 9 VAD+ patients with preformed DSA experienced AMR compared with 7 of 38 VAD+ patients without preformed DSA, indicating presensitization to donor HLA significantly increased the risk of AMR (P < 0.01).ConclusionsPreformed and posttransplant ndDSA were associated with AMR. C3d + DSA correlates with complement deposition on the graft and higher risk of AMR which may permit the application of personalized immunotherapy targeting the complement pathway.

Heart failure (HF) is a complex clinical syndrome that causes systemic hypoperfusion and failure to meet the body’s metabolic demands. In an attempt to compensate, chronic upregulation of the sympathetic nervous system and renin-angiotensin-aldosterone leads to further myocardial injury, HF progression and reduced O2delivery. This triggers progressive organ dysfunction, immune system activation and profound metabolic derangements, creating a milieu similar to other chronic systemic diseases and presenting as advanced HF with severely limited prognosis. We hypothesize that 1-year survival in advanced HF is linked to functional recovery potential (FRP), a novel clinical composite parameter that includes HF severity, secondary organ dysfunction, co-morbidities, frailty, disabilities as well as chronological age and that can be diagnosed by a molecular biomarker.

Background As the population of patients with a Fontan palliation grows so does, the number of patients with cardiac failure necessitating orthotopic heart transplant (OHT) and combined heart-liver transplant (CHLT). There is recent evidence that current era cardiac transplant in Fontan patients has improved outcomes, but most studies have a preponderance of pediatrics patients in their cohorts. We examine our institutional experience with adult OHT and CHLT transplantation for failed Fontan physiology. Methods and results Retrospective analysis of patients at the Ahmanson/UCLA Adult Congenital Heart Disease Center who underwent OHT or CHLT for failing Fontan physiology from January 1, 2002 to May 31, 2017. We identified 20 patients with single-ventricle physiology and Fontan palliation who underwent OHT or CHLT. The median age was 29.5 years (range 19-44). Five patients underwent CHLT because of biopsy proven hepatic cirrhosis. The median length of hospital stay was 23 days (range 8-76) post-OHT and 51 days (range 26-77) post-CHLT. During a median follow-up of 56 months (range 2-178), there was one mortality occurring at 34 months post-OHT due to coronary vasculopathy. Most frequent early postoperative complications included bleeding and infection (55% and 20%, respectively) and surgical reintervention for bleeding complications (n = 8, 40%). One CHLT patient experienced clinically significant hepatic rejection requiring admission and steroid treatment. Conclusions Despite inherent risks and complexities of OHT or CHLT in patients with a failed Fontan, transplant is a reasonable therapy. Peri- and postoperative complications are common and may require surgical reintervention. Continued observation of practices and unifying themes may help improve patient selection, pre- and postoperative treatment and ultimately outcomes.

Purpose In heart and kidney transplants, rejection is a major cause of graft loss. In kidneys, antibody-mediated rejection (ABMR) is more important than T cell-mediated rejection (TCMR), and molecules predict graft loss better than histology (JASN 26 (7):1711-1720, 2015). We examined the relative importance of ABMR vs TCMR in heart transplant endomyocardial biopsies (EMBs), and the molecules predicting graft survival. Methods The INTERHEART population includes 1219 transplant biopsies from 8 centers in Canada, USA, Australia and Europe. Gene expression was studied using microarrays, selecting the most recent biopsy per patient. Random forest classifiers were used to assess predictive accuracy and determine the importance of molecular predictors, including gene sets and scores from analyses in a reference set of 889 EMBs. Results We studied 3-year survival in 484 patients with follow-up times. Graft failure occurred in 60 patients. Median follow-up was 435 days; biopsies were mainly for indications. Surprisingly, TCMR was a greater short-term hazard than ABMR. The molecular archetype clusters for TCMR and injury (Fig. 1) had the highest risk of graft failure. Fig. 2 combines these clusters since they have similar characteristics. Conclusion Unlike kidneys, graft loss (particularly within one year) after EMB is highly associated with TCMR but not ABMR. TCMR may reflect failure of immunosuppression or non-adherence. This difference between the heart and renal transplant populations raises the possibility that TCMR is relatively more destructive, and ABMR less destructive, in heart than in kidney transplants. ClinicalTrials.gov # NCT02670408