Kiran K. Khush

Universal noninvasive detection of solid organ transplant rejection

It is challenging to monitor the health of transplanted organs, particularly with respect to rejection by the host immune system. Because transplanted organs have genomes that are distinct from the recipients genome, we used high throughput shotgun sequencing to develop a universal noninvasive approach to monitoring organ health. We analyzed cell-free DNA circulating in the blood of heart transplant recipients and observed significantly increased levels of cell-free DNA from the donor genome at times when an endomyocardial biopsy independently established the presence of acute cellular rejection in these heart transplant recipients. Our results demonstrate that cell-free DNA can be used to detect an organ-specific signature that correlates with rejection, and this measurement can be made on any combination of donor and recipient. This noninvasive test holds promise for replacing the endomyocardial biopsy in heart transplant recipients and may be applicable to other solid organ transplants.

Effect of High-Dose Atorvastatin on Hospitalizations for Heart Failure Subgroup Analysis of the Treating to New Targets (TNT) Study

Background— Statins reduce the rate of major cardiovascular events in high-risk patients, but their potential benefit as treatment for heart failure (HF) is less clear. Methods and Results— Patients (n=10 001) with stable coronary disease were randomized to treatment with atorvastatin 80 or 10 mg/d and followed up for a median of 4.9 years. A history of HF was present in 7.8% of patients. A known ejection fraction <30% and advanced HF were exclusion criteria for the study. A predefined secondary end point of the study was hospitalization for HF. The incidence of hospitalization for HF was 2.4% in the 80-mg arm and 3.3% in the 10-mg arm (hazard ratio, 0.74; 95% confidence interval, 0.59 to 0.94; P=0.0116). The treatment effect of the higher dose was more marked in patients with a history of HF: 17.3% versus 10.6% in the 10- and 80-mg arms, respectively (hazard ratio, 0.59; 95% confidence interval, 0.4 to 0.88; P=0.009). Among patients without a history of HF, the rates of hospitalization for HF were much lower: 1.8% in the 80-mg group and 2.0% in the 10-mg group (hazard ratio, 0.87; 95% confidence interval, 0.64 to 1.16; P=0.34). Only one third of patients hospitalized for HF had evidence of preceding angina or myocardial infarction during the study period. Blood pressure was almost identical during follow-up in the treatment groups. Conclusions— Compared with a lower dose, intensive treatment with atorvastatin in patients with stable coronary disease significantly reduces hospitalizations for HF. In a post hoc analysis, this benefit was observed only in patients with a history of HF. The mechanism accounting for this benefit is unlikely to be due primarily to a reduction in interim coronary events or differences in blood pressure.

Circulating Cell-Free DNA Enables Noninvasive Diagnosis of Heart Transplant Rejection

In a prospective cohort study of heart transplant recipients, sequencing-based quantification of donor-derived DNA in plasma of recipients noninvasively diagnosed transplant rejection. Donor DNA Indicates Transplant Rejection Not all heart transplants succeed, but early detection of organ rejection could spare the patient severe adverse events and graft dysfunction. De Vlaminck et al. devised a noninvasive, sequencing-based method to monitor and predict rejection, relying on the presence of donor DNA in recipient blood plasma. The fraction of donor DNA is naturally elevated 1 day after transplant (because organ transplants are essentially genome transplants), and these levels decline exponentially over the course of the week, if the organ is accepted. The authors noted that patients who rejected their new heart had high levels of donor DNA even months after transplant. In a prospective trial, elevated donor DNA was detected months before the rejection episode, suggesting that such noninvasive analysis tools could be used in lieu of an invasive biopsy, to let doctors know which patients are likely to reject their transplanted organ. Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga–base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy.

Effect of pulmonary hypertension on clinical outcomes in advanced heart failure: Analysis of the Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness (ESCAPE) database

BACKGROUND Pulmonary hypertension has been shown to predict hospitalizations and mortality in patients with heart failure. We aimed to define the prevalence of mixed pulmonary hypertension (MPH; mean pulmonary artery pressure > or = 25 mm Hg, pulmonary capillary wedge pressure >15 mm Hg, and pulmonary vascular resistance > or = 3 Wood units), identify clinical predictors of MPH, and determine whether MPH predicts adverse outcomes in patients hospitalized with severe heart failure. METHODS This is a subgroup analysis of patients assigned to pulmonary artery catheter placement in the Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness (ESCAPE) trial. Patients with and without MPH were compared with respect to baseline characteristics and clinical outcomes, including NYHA class, 6-minute walk distance, quality of life, days hospitalized, and 6-month mortality. RESULTS Of the 171 patients studied, 80 (47%) had MPH. Older age was the only significant predictor of MPH. MPH patients had lower cardiac index (1.8 +/- 0.5 L/min vs 2.1 +/- 0.5 L/min, P = .001) and higher systemic vascular resistance index (3,179 +/- 1,454 vs 2,550 +/- 927 dynes x s/cm5 x m2, P < .001) compared to those without MPH. Importantly, right ventricular function was relatively preserved (median RVSWI 8.7 gm-m/m2/beat) in MPH patients. There were no significant differences in clinical outcomes between the two groups. CONCLUSIONS Mixed pulmonary hypertension is common in patients hospitalized with advanced heart failure and is not associated with adverse short-term clinical outcomes over and above the poor prognosis of ADHF patients without MPH.

Differentially Expressed RNA from Public Microarray Data Identifies Serum Protein Biomarkers for Cross-Organ Transplant Rejection and Other Conditions

Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers.

National decline in donor heart utilization with regional variability: 1995-2010.

The severe shortage of donor hearts limits the availability of transplantation for the growing population of patients with end‐stage heart disease. We examined national trends in donor heart acceptance for transplant. OPTN data were analyzed for all potential adult cardiac organ donors between 1995 and 2010. Donor heart disposition was categorized as transplanted, declined for transplant or other. We studied changes in the probability of donor heart acceptance according to demographic and clinical characteristics, nationwide and by UNOS region. Of 82 053 potential donor hearts, 34% were accepted and 48% were declined (18% used for other purposes). There was a significant decrease in donor heart acceptance from 44% in 1995 to 29% in 2006, and subsequent increase to 32% in 2010. Older donor age, female sex and medical co‐morbidities predicted non‐acceptance. Donor age and co‐morbidities increased during the study period, with a concomitant decrease in acceptance of hearts from donors with undesirable characteristics. Overall, predictors of heart non‐use were similar across UNOS regions, although utilization varied between regions. Regional variation suggests a potential to improve heart acceptance rates in under‐performing regions, and supports research and policy efforts aimed at establishing evidence‐based criteria for donor heart evaluation and acceptance for transplantation.

Noninvasive monitoring of infection and rejection after lung transplantation.

Significance Over 3,500 patients receive life-saving lung transplants every year. Nonetheless, complications due to infection and rejection occur frequently and undermine the long-term benefits of lung transplantation. Although clinicians strive to carefully monitor patients, diagnostic options are often limited. Rejection monitoring currently relies on invasive tissue biopsies, and tests of infection are predominately limited to testing one pathogen at a time. This manuscript describes a noninvasive assay based on sequencing of circulating cell-free DNA that simultaneously enables diagnosis of rejection and broad screening of infections. The survival rate following lung transplantation is among the lowest of all solid-organ transplants, and current diagnostic tests often fail to distinguish between infection and rejection, the two primary posttransplant clinical complications. We describe a diagnostic assay that simultaneously monitors for rejection and infection in lung transplant recipients by sequencing of cell-free DNA (cfDNA) in plasma. We determined that the levels of donor-derived cfDNA directly correlate with the results of invasive tests of rejection (area under the curve 0.9). We also analyzed the nonhuman cfDNA as a hypothesis-free approach to test for infections. Cytomegalovirus is most frequently assayed clinically, and the levels of CMV-derived sequences in cfDNA are consistent with clinical results. We furthermore show that hypothesis-free monitoring for pathogens using cfDNA reveals undiagnosed cases of infection, and that certain infectious pathogens such as human herpesvirus (HHV) 6, HHV-7, and adenovirus, which are not often tested clinically, occur with high frequency in this cohort.

Influence of donor and recipient sex mismatch on heart transplant outcomes: Analysis of the International Society for Heart and Lung Transplantation Registry

BACKGROUND Prior studies have presented contradictory results after analyzing associations between donor and recipient sex on survival after heart transplantation and causes of death such as acute rejection (AR) and cardiac allograft vasculopathy (CAV). We used the International Society for Heart and Lung Transplantation (ISHLT) Registry, the largest repository of heart transplant outcomes worldwide, to comprehensively address these questions. METHOD We studied 60,584 adult recipients of heart transplants performed between 1990 and 2008. Outcomes of interest were overall survival, death-censored allograft survival, AR, and CAV, which were studied using regression models. To assess whether donor/recipient sex mismatch affected outcomes, the experience of male recipients with female vs male donors was compared with that of female recipients with female vs male donors through inclusion of an interaction term between donor and recipient sex. RESULTS Significant differences were observed between male and female recipients in overall survival and death-censored allograft survival for female vs male donors. Male recipients of female allografts had a 10% increase in adjusted mortality relative to male recipients of male allografts, whereas female recipients of female allografts had a 10% decrease in adjusted mortality relative to female recipients of male allografts (p < 0.0001). Findings were similar for death-censored allograft survival. Differences in the effect of donor sex on AR or CAV between male and female recipients were not significant. CONCLUSIONS Analysis of the ISHLT data set has demonstrated a strong association between donor/recipient sex mismatch and reduced survival after heart transplantation.

Donor Predictors of Allograft Use and Recipient Outcomes After Heart Transplantation

Background—Despite a national organ-donor shortage and a growing population of patients with end-stage heart disease, the acceptance rate of donor hearts for transplantation is low. We sought to identify donor predictors of allograft nonuse, and to determine whether these predictors are in fact associated with adverse recipient post-transplant outcomes. Methods and Results—We studied a cohort of 1872 potential organ donors managed by the California Transplant Donor Network from 2001 to 2008. Forty-five percent of available allografts were accepted for heart transplantation. Donor predictors of allograft nonuse included age>50 years, female sex, death attributable to cerebrovascular accident, hypertension, diabetes mellitus, a positive troponin assay, left-ventricular dysfunction and regional wall motion abnormalities, and left-ventricular hypertrophy. For hearts that were transplanted, only donor cause of death was associated with prolonged recipient hospitalization post-transplant, and only donor diabetes mellitus was predictive of increased recipient mortality. Conclusions—Whereas there are many donor predictors of allograft discard in the current era, these characteristics seem to have little effect on recipient outcomes when the hearts are transplanted. Our results suggest that more liberal use of cardiac allografts with relative contraindications may be warranted.

Donor Predictors of Allograft Use and Recipient Outcomes After Heart TransplantationClinical Perspective

Background—Despite a national organ-donor shortage and a growing population of patients with end-stage heart disease, the acceptance rate of donor hearts for transplantation is low. We sought to identify donor predictors of allograft nonuse, and to determine whether these predictors are in fact associated with adverse recipient post-transplant outcomes. Methods and Results—We studied a cohort of 1872 potential organ donors managed by the California Transplant Donor Network from 2001 to 2008. Forty-five percent of available allografts were accepted for heart transplantation. Donor predictors of allograft nonuse included age>50 years, female sex, death attributable to cerebrovascular accident, hypertension, diabetes mellitus, a positive troponin assay, left-ventricular dysfunction and regional wall motion abnormalities, and left-ventricular hypertrophy. For hearts that were transplanted, only donor cause of death was associated with prolonged recipient hospitalization post-transplant, and only donor diabetes mellitus was predictive of increased recipient mortality. Conclusions—Whereas there are many donor predictors of allograft discard in the current era, these characteristics seem to have little effect on recipient outcomes when the hearts are transplanted. Our results suggest that more liberal use of cardiac allografts with relative contraindications may be warranted.