Pathologist Interrater Reliability and Clinical Implications of Elevated Donor-Derived Cell-Free DNA beyond Heart Transplant Rejection.

BACKGROUND: There is significant variability amongst pathologists in the histopathological interpretation of the endomyocardial biopsy (EMB) for acute cellular rejection (ACR) and assessment of variability in the interpretation of antibody-mediated rejection (AMR) has not been reported. In contemporary practice, the strategy of allograft surveillance with donor-derived cell-free DNA (dd-cfDNA) as compared to EMB has not been compared with a focus on long-term clinical outcomes beyond acute rejection (AR). METHODS: The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter, prospective cohort study that enrolled patients from 2015 to 2020. The center pathologist read was compared to two blinded core cardiac pathologists. ACR and AMR were graded based on the International Society for Heart and Lung Transplantation (ISHLT) criteria. Weighted Cohen's kappa (κ) was used to evaluate interrater reliability between the center and core reads. To assess long-term outcomes, we evaluated a composite of AR, allograft dysfunction, and mortality within 1 year. RESULTS: The study included 94 patients (median age 55 years [IQR 45, 62]), 30% female, 41% Black race) with a total of 429 EMBs and paired dd-cfDNA measures. The concordance rate between center and core pathologists was 77% for ACR (95%CI: 66% - 89%) and 63% for AMR (95%CI: 53% - 74%). 46 patients had an elevation in dd-cfDNA without AR by EMB. The median dd-cfDNA was 0.49% (IQR: 0.35, 1.01) and subsequent AR, allograft dysfunction, or mortality occurred in 59% of these patients at 1 year. In patients with AR by EMB and negative dd-cfDNA (n=5) the composite outcome occurred in 20% of patients at 1 year. At baseline, the positive likelihood ratio (LR+) of dd-cfDNA to detect AR by the center pathologist was 3.74 (95% CI 3.01 - 4.64) and core pathologist was 2.59 (95%CI: 1.95 - 3.45). If the composite outcome was included as a true positive, the LR+ of dd-cfDNA improved to 9.82 (95%CI: 7.04, 13.69) and7.63 (95% CI: 5.61, 10.38) at 1-year, respectively. CONCLUSIONS: Pathologists interrater reliability is limited in both ACR and AMR. The positive LR of dd-cfDNA when compared to traditional histopathology is limited, but when longitudinal clinical outcomes are included to assess diagnostic performance, the LR+ improves significantly. The value of dd-cfDNA extends beyond the diagnosis of AR to include other clinically meaningful outcomes for patients after heart transplant.

Sex-specific patterns of donor-derived cell-free DNA in heart transplant rejection: An analysis from the Genomic Research Alliance for Transplantation (GRAfT).

BACKGROUND: Noninvasive methods for surveillance of acute rejection are increasingly used in heart transplantation (HT), including donor-derived cell-free DNA (dd-cfDNA). As other cardiac biomarkers differ by sex, we hypothesized that there may be sex-specific differences in the performance of dd-cfDNA for the detection of acute rejection. The purpose of the current study was to examine patterns of dd-cfDNA seen in quiescence and acute rejection in male and female transplant recipients. METHODS: Patients enrolled in the Genomic Research Alliance for Transplantation who were ≥18 years at the time of HT were included. Rejection was defined by endomyocardial biopsy with acute cellular rejection (ACR) grade ≥2R and/or antibody-mediated rejection ≥ pAMR 1. dd-cfDNA was quantitated using shotgun sequencing. Median dd-cfDNA levels were compared between sexes during quiescence and rejection. The performance of dd-cfDNA by sex was assessed using area under the receiver operator characteristic (AUROC) curve. Allograft injury was defined as dd-cfDNA ≥0.25%. RESULTS: One hundred fifty-one unique patients (49 female, 32%) were included in the analysis with 1,119 available dd-cfDNA measurements. Baseline characteristics including demographics and comorbidities were not significantly different between sexes. During quiescence, there were no significant sex differences in median dd-cfDNA level (0.04% [IQR 0.00, 0.16] in females vs 0.03% [IQR 0.00, 0.12] in males, p = 0.22). There were no significant sex differences in median dd-cfDNA for ACR (0.33% [0.21, 0.36] in females vs 0.32% [0.21, 1.10] in males, p = 0.57). Overall, median dd-cfDNA levels were higher in antibody-mediated rejection (AMR) than ACR but did not significantly differ by sex (0.50% [IQR 0.18, 0.82] in females vs 0.63% [IQR 0.32, 1.95] in males, p = 0.51). Elevated dd-cfDNA detected ACR/AMR with an AUROC of 0.83 in females and 0.89 in males, p-value for comparison = 0.16. CONCLUSIONS: There were no significant sex differences in dd-cfDNA levels during quiescence and rejection. Performance characteristics were similar, suggesting similar diagnostic thresholds can be used in men and women for rejection surveillance.

Circulating microRNAs in cellular and antibody-mediated heart transplant rejection.

BACKGROUND: Noninvasive monitoring of heart allograft health is important to improve clinical outcomes. MicroRNAs (miRs) are promising biomarkers of cardiovascular disease and limited studies suggest they can be used to noninvasively diagnose acute heart transplant rejection. METHODS: The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter prospective cohort study that phenotyped heart transplant patients from 5 mid-Atlantic centers. Patients who had no history of rejection after transplant were compared to patients with acute cellular rejection (ACR) or antibody-mediated rejection (AMR). Small RNA sequencing was performed on plasma samples collected at the time of an endomyocardial biopsy. Differential miR expression was performed with adjustment for clinical covariates. Regression was used to develop miR panels with high diagnostic accuracy for ACR and AMR. These panels were then validated in independent samples from GRAfT and Stanford University. Receiver operating characteristic curves were generated and area under the curve (AUC) statistics calculated. Distinct ACR and AMR clinical scores were developed to translate miR expression data for clinical use. RESULTS: The GRAfT cohort had a median age of 52 years, with 35% females and 45% Black patients. Between GRAfT and Stanford, we included 157 heart transplant patients: 108 controls and 49 with rejection (50 ACR and 38 AMR episodes). After differential miR expression and regression analysis, we identified 12 miRs that accurately discriminate ACR and 17 miRs in AMR. Independent validation of the miR panels within GRAfT led to an ACR AUC 0.92 (95% confidence interval [CI]: 0.86-0.98) and AMR AUC 0.82 (95% CI: 0.74-0.90). The externally validated ACR AUC was 0.72 (95% CI: 0.59-0.82). We developed distinct ACR and AMR miR clinical scores (range 0-100), a score ≥ 65, identified ACR with 86% sensitivity, 76% specificity, and 98% negative predictive value, for AMR score performance was 82%, 84% and 97%, respectively. CONCLUSIONS: We identified novel miRs that had excellent performance to noninvasively diagnose acute rejection after heart transplantation. Once rigorously validated, the unique clinical ACR and AMR scores usher in an era whereby genomic biomarkers can be used to screen and diagnose the subtype of rejection. These novel biomarkers may potentially alleviate the need for an endomyocardial biopsy while facilitating the initiation of targeted therapy based on the noninvasive diagnosis of ACR or AMR.

Cell-Free DNA to Detect Heart Allograft Acute Rejection.

BACKGROUND: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients. METHODS: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. RESULTS: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P<0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. CONCLUSIONS: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.

Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.

Monitoring pharmacologically induced immunosuppression by immune repertoire sequencing to detect acute allograft rejection in heart transplant patients: a proof-of-concept diagnostic accuracy study.

BACKGROUND: It remains difficult to predict and to measure the efficacy of pharmacological immunosuppression. We hypothesized that measuring the B-cell repertoire would enable assessment of the overall level of immunosuppression after heart transplantation. METHODS AND FINDINGS: In this proof-of-concept study, we implemented a molecular-barcode-based immune repertoire sequencing assay that sensitively and accurately measures the isotype and clonal composition of the circulating B cell repertoire. We used this assay to measure the temporal response of the B cell repertoire to immunosuppression after heart transplantation. We selected a subset of 12 participants from a larger prospective cohort study (ClinicalTrials.gov NCT01985412) that is ongoing at Stanford Medical Center and for which enrollment started in March 2010. This subset of 12 participants was selected to represent post-heart-transplant events, with and without acute rejection (six participants with moderate-to-severe rejection and six without). We analyzed 130 samples from these patients, with an average follow-up period of 15 mo. Immune repertoire sequencing enables the measurement of a patient's net state of immunosuppression (correlation with tacrolimus level, r = -0.867, 95% CI -0.968 to -0.523, p = 0.0014), as well as the diagnosis of acute allograft rejection, which is preceded by increased immune activity with a sensitivity of 71.4% (95% CI 30.3% to 94.9%) and a specificity of 82.0% (95% CI 72.1% to 89.1%) (cell-free donor-derived DNA as noninvasive gold standard). To illustrate the potential of immune repertoire sequencing to monitor atypical post-transplant trajectories, we analyzed two more patients, one with chronic infections and one with amyloidosis. A larger, prospective study will be needed to validate the power of immune repertoire sequencing to predict rejection events, as this proof-of-concept study is limited to a small number of patients who were selected based on several criteria including the availability of a large number of samples and the absence or presence of rejection events. CONCLUSIONS: If confirmed in larger, prospective studies, the method described here has potential applications in the tailored management of post-transplant immunosuppression and, more broadly, as a method for assessing the overall activity of the immune system.

Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection.

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.

Utility of gene expression profiling score variability to predict clinical events in heart transplant recipients.

BACKGROUND: Gene expression profiling test scores have primarily been used to identify heart transplant recipients who have a low probability of rejection at the time of surveillance testing. We hypothesized that the variability of gene expression profiling test scores within a patient may predict risk of future events of allograft dysfunction or death. METHOD: Patients from the IMAGE study with rejection surveillance gene expression profiling tests performed at 1- to 6-month intervals were selected for this cohort study. Gene expression profiling score variability was defined as the standard deviation of an individual's cumulative test scores. Gene expression profiling ordinal score (range, 0-39), threshold score (binary value=1 if ordinal score ≥ 34), and score variability were studied in multivariate Cox regression models to predict future clinical events. RESULTS: Race, age at time of transplantation, and time posttransplantation were significantly associated with future events in the univariate analysis. In the multivariate analyses, gene expression profiling score variability, but not ordinal scores or scores over threshold, was independently associated with future clinical events. The regression coefficient P values were <0.001, 0.46, and 0.773, for gene expression profiling variability, ordinal, and threshold scores, respectively. The hazard ratio for a 1 unit increase in variability was 1.76 (95% CI, 1.4-2.3). DISCUSSION: The variability of a heart recipient's gene expression profiling test scores over time may provide prognostic utility. This information is independent of the probability of acute cellular rejection at the time of testing that is rendered from a single ordinal gene-expression profiling test score.

Identification and classification of acute cardiac rejection by intragraft transcriptional profiling.

BACKGROUND: Treatment of acute rejection (AR) in heart transplantation relies on histopathological grading of endomyocardial biopsies according to International Society for Heart and Lung Transplantation guidelines. Intragraft gene expression profiling may be a way to complement histological evaluation. METHODS AND RESULTS: Transcriptional profiling was performed on 26 endomyocardial biopsies, and expression patterns were compared with the 1990 International Society for Heart and Lung Transplantation AR grades. Importantly, transcriptional profiles from settings with an equivalent AR grade appeared the same. In addition, grade 0 profiles could not be distinguished from 1A profiles, and grade 3A profiles could not be distinguished from 3B profiles. Comparing the AR groupings (0+1A, 1B, and 3A+3B), 0+1A showed more striking differences from 1B than from 3A+3B. When these findings were extrapolated to the 2005 revised guidelines, the combination of 1A and 1B into a single category (1R) appears to have brought together endomyocardial biopsies with different underlying processes that are not evident from histological evaluation. Grade 1B was associated with upregulated immune response genes, as 1 categorical distinction from grade 1A. Although grade 1B was distinct from the clinically relevant AR grades 3A and 3B, all of these grades shared a small number of overlapping pathways consistent with common physiological underpinnings. CONCLUSION: The gene expression similarities and differences identified here in different AR settings have the potential to revise the clinical perspective on acute graft rejection, pending the results of larger studies.

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 recipient's 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.

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.

Gene-expression profiling for rejection surveillance after cardiac transplantation.

BACKGROUND: Endomyocardial biopsy is the standard method of monitoring for rejection in recipients of a cardiac transplant. However, this procedure is uncomfortable, and there are risks associated with it. Gene-expression profiling of peripheral-blood specimens has been shown to correlate with the results of an endomyocardial biopsy. METHODS: We randomly assigned 602 patients who had undergone cardiac transplantation 6 months to 5 years previously to be monitored for rejection with the use of gene-expression profiling or with the use of routine endomyocardial biopsies, in addition to clinical and echocardiographic assessment of graft function. We performed a noninferiority comparison of the two approaches with respect to the composite primary outcome of rejection with hemodynamic compromise, graft dysfunction due to other causes, death, or retransplantation. RESULTS: During a median follow-up period of 19 months, patients who were monitored with gene-expression profiling and those who underwent routine biopsies had similar 2-year cumulative rates of the composite primary outcome (14.5% and 15.3%, respectively; hazard ratio with gene-expression profiling, 1.04; 95% confidence interval, 0.67 to 1.68). The 2-year rates of death from any cause were also similar in the two groups (6.3% and 5.5%, respectively; P=0.82). Patients who were monitored with the use of gene-expression profiling underwent fewer biopsies per person-year of follow-up than did patients who were monitored with the use of endomyocardial biopsies (0.5 vs. 3.0, P<0.001). CONCLUSIONS: Among selected patients who had received a cardiac transplant more than 6 months previously and who were at a low risk for rejection, a strategy of monitoring for rejection that involved gene-expression profiling, as compared with routine biopsies, was not associated with an increased risk of serious adverse outcomes and resulted in the performance of significantly fewer biopsies. (ClinicalTrials.gov number, NCT00351559.)

Frequent occult infection with Cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis.

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

Validation of a screening protocol for identifying low-risk candidates with type 1 diabetes mellitus for kidney with or without pancreas transplantation.

BACKGROUND: Certain clinical risk factors are associated with significant coronary artery disease in kidney transplant candidates with diabetes mellitus. We sought to validate the use of a clinical algorithm in predicting post-transplantation mortality in patients with type 1 diabetes. We also examined the prevalence of significant coronary lesions in high-risk transplant candidates. METHODS: All patients with type 1 diabetes evaluated between 1991 and 2001 for kidney with/without pancreas transplantation were classified as high-risk based on the presence of any of the following risk factors: age >or=45 yr, smoking history >or=5 pack years, diabetes duration >or=25 yr or any ST-T segment abnormalities on electrocardiogram. Remaining patients were considered low risk. All high-risk candidates were advised to undergo coronary angiography. The primary outcome of interest was all-cause mortality post-transplantation. RESULTS: Eighty-four high-risk and 42 low-risk patients were identified. Significant coronary artery stenosis was detected in 31 high-risk candidates. Mean arterial pressure was a significant predictor of coronary stenosis (odds ratio 1.68; 95% confidence interval 1.14-2.46), adjusted for age, sex and duration of diabetes. In 75 candidates who underwent transplantation with median follow-up of 47 months, the use of clinical risk factors predicted all eight deaths. No deaths occurred in low-risk patients. A significant mortality difference was noted between the two risk groups (p = 0.03). CONCLUSIONS: This clinical algorithm can identify patients with type 1 diabetes at risk for mortality after kidney with/without pancreas transplant. Patients without clinical risk factors can safely undergo transplantation without further cardiac evaluation.

Recurrence of iron deposition in the cardiac allograft in a patient with non-HFE hemochromatosis.

We report the case of a 36-year-old woman with a diagnosis of idiopathic dilated cardiomyopathy who underwent cardiac transplantation. The results of her initial iron studies were normal, but hemochromatosis was suspected after microscopy of the explanted heart revealed iron deposition. By 6 months post-transplantation, iron deposition was detected in her surveillance endomyocardial biopsy specimens and studies then confirmed the existence of non-HFE hemochromatosis. The patient has been stable on treatment with regular phlebotomies and a low vitamin C diet.

The role of viruses in cardiac allograft vasculopathy.

Considerable evidence suggests a role for viruses in transplant arteriosclerosis (TA), including observational data, experimental models and therapeutic trials implicating human cytomegalovirus (HCMV) in the progression to TA. In pediatric heart transplant patients, adenoviral genome in endomyocardial biopsies (EMB) is an important predictor of TA and graft loss. During CMV viremia, EMBs from adult patients demonstrate endothelialitis and vascular smooth muscle cell proliferation. These changes are predictors of subsequent diffuse TA. HCMV immediate early proteins (IE-1 and IE-2) increase the constitutive expression of intercellular adhesion molecule-1 (ICAM-1) independent of other intracellular cytokines. Likewise, viral chemokines such as US28 have been implicated in vascular disease because of their ability to induce smooth muscle cell migration. Recent data suggests that CMV might accelerate TA through its ability to abrogate the vascular protective effects of the endothelium-derived nitric oxide system (eNOS). Confirmation of causality requires clinical trials demonstrating that antiviral agents such as ganciclovir inhibit TA. Such studies in patients though limited to retrospective analyses, suggest that ganciclovir prophylaxis early after heart transplantation reduces the risk of TA. These observations emphasize the need for randomized controlled clinical trials to confirm a causal role for CMV (and other viruses) in TA.

Analysis of survivors more than 10 years after heart transplantation in the cyclosporine era: Stanford experience.

BACKGROUND: Truly long term survival post heart transplantation has become increasingly frequent over the past two decades. METHODS: We analyzed multiple clinical outcomes in the cohort of 140 patients in the Stanford database who underwent heart transplantation after the introduction of cyclosporine-based immunosuppression in 1980 and survived >10 years after transplantation. RESULTS: We found generally excellent functional status in these patients, but a high incidence of hypertension, renal dysfunction, and graft CAD as well as malignancy. CONCLUSION: With continued improvement in post-transplant survival rates, providing complex care for such long-term recipients as these will assume increasing clinical importance in the everyday practice of transplant medicine and these data highlight the problems to be anticipated.

Longer-term risks associated with 10-year survival after heart transplantation in the cyclosporine era.

BACKGROUND: Long-term survival after heart transplantation is common in the cyclosporine era. However, there are few data documenting pre-transplant/peri-operative factors predictive of truly long-term survival (>10 years). The purpose of this study is to identify factors associated with 10-year survival after heart transplantation. METHODS: Our study population included 197 adults who survived >6 months and died <10 years after heart transplant (medium-term group) and 140 adults who survived >10 years after heart transplant (long-term group) between December 1980 and May 2001. A comparison was done between the two groups and we used multivariate analysis to identify which factors predicted 10-year survival. RESULTS: The long-term group had younger recipient and donor age, lower recipient body mass index at transplant, shorter waiting time and lower percentages of ischemic etiology/male recipient/non-white recipient. Kaplan-Meier plots of freedom from graft coronary artery disease and malignancy showed later onset patterns in the long-term group compared with the medium-term group. Multivariate analysis showed that white recipient, younger recipient and lower recipient body mass index at heart transplant were factors significantly associated with 10-year survival. CONCLUSIONS: Several pre-transplant/peri-operative factors were associated with survival beyond 10 years after heart transplantation. Stratified/tailored strategies based on these factors may be helpful to attain longer-term survival of recipients with higher risks.

Mycophenolic acid concentrations in long-term heart transplant patients: relationship with calcineurin antagonists and acute rejection.

BACKGROUND: When used in conjunction with steroids and cyclosporin, mycophenolate mofetil (MMF) has been shown to significantly reduce mortality and incidence of rejection in the first year after heart transplantation. It also appears that in this early post-transplantation period, the monitoring of immunosuppressive therapies may be warranted. The current study was undertaken to determine if such monitoring is still useful more than 1 yr after heart transplantation. METHODS: Twenty-six patients who had survived the first year after orthotopic heart transplantation and had been on MMF therapy for more than 3 months were prospectively followed. At the time of their routine endomyocardial biopsy blood samples were taken to monitor immunosuppressive therapy. Most patients had two samples taken, on average 109 d apart. RESULTS: There were 22 episodes of asymptomatic rejection documented on a total of 48 biopsies. Of these, only two were of ISHLT (International Society for Heart and Lung Transplantation) grade 3A the remainder being of ISHLT grades 1 or 2. There was no relation between immunosuppressive regimen (tacrolimus and MMF or cyclosporin and MMF) and rejection. There was no relation between monitored immunosuppressive levels and rejection. Patients with the combination of MMF and tacrolimus had significantly higher plasma mycophenolic acid levels despite significantly lower daily MMF dose. CONCLUSION: There does not appear to be a benefit in continued monitoring of plasma mycophenolic acid levels beyond the first year of heart transplantation. There were significant differences in plasma mycophenolic acid levels depending on the type of calcineurin inhibitor concomitantly used.