Evaluating threshold for donor fraction cell-free DNA using clinically available assay for rejection in pediatric and adult heart transplantation.

BACKGROUND: The aims of the study were to assess the performance of a clinically available cell-free DNA (cfDNA) assay in a large cohort of pediatric and adult heart transplant recipients and to evaluate performance at specific cut points in detection of rejection. METHODS: Observational, non-interventional, prospective study enrolled pediatric and adult heart transplant recipients from seven centers. Biopsy-associated plasma samples were used for cfDNA measurements. Pre-determined cut points were tested for analytic performance. RESULTS: A total of 487 samples from 160 subjects were used for the analysis. There were significant differences for df-cfDNA values between rejection [0.21% (IQR 0.12-0.69)] and healthy samples [0.05% (IQR 0.01-0.14), p < .0001]. The pediatric rejection group had a median df-cfDNA value of 0.93% (IQR 0.28-2.84) compared to 0.09% (IQR 0.04-0.23) for healthy samples, p = .005. Overall negative predictive value was 0.94 while it was 0.99 for pediatric patients. Cut points of 0.13% and 0.15% were tested for various types of rejection profiles and were appropriate to rule out rejection. CONCLUSION: The study suggests that pediatric patients with rejection show higher levels of circulating df-cfDNA compared to adults and supports the specific cut points for clinical use in pediatric and adult patients with overall acceptable performance.

3D-bioprinting of patient-derived cardiac tissue models for studying congenital heart disease.

Introduction: Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies. Methods: To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521. Results: Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture. Discussion: This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies.

Altered contractility, Ca2+ transients, and cell morphology seen in a patient-specific iPSC-CM model of Ebstein's anomaly with left ventricular noncompaction.

Patients with two congenital heart diseases (CHDs), Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. We investigated a familial EA/LVNC case associated with a variant (p.R237C) in the gene encoding Kelch-like protein 26 (KLHL26) by differentiating induced pluripotent stem cells (iPSCs) generated from affected and unaffected family members into cardiomyocytes (iPSC-CMs) and assessing iPSC-CM morphology, function, gene expression, and protein abundance. Compared with unaffected iPSC-CMs, CMs containing the KLHL26 (p.R237C) variant exhibited aberrant morphology including distended endo(sarco)plasmic reticulum (ER/SR) and dysmorphic mitochondria and aberrant function that included decreased contractions per minute, altered calcium transients, and increased proliferation. Pathway enrichment analyses based on RNASeq data indicated that the "structural constituent of muscle" pathway was suppressed, whereas the "ER lumen" pathway was activated. Taken together, these findings suggest that iPSC-CMs containing this KLHL26 (p.R237C) variant develop dysregulated ER/SR, calcium signaling, contractility, and proliferation.NEW & NOTEWORTHY We demonstrate here that iPSCs derived from patients with Ebstein's anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

Validation of donor fraction cell-free DNA with biopsy-proven cardiac allograft rejection in children and adults.

OBJECTIVES: Donor-specific cell-free DNA shows promise as a noninvasive marker for allograft rejection, but as yet has not been validated in both adult and pediatric recipients. The study objective was to validate donor fraction cell-free DNA as a noninvasive test to assess for risk of acute cellular rejection and antibody-mediated rejection after heart transplantation in pediatric and adult recipients. METHODS: Pediatric and adult heart transplant recipients were enrolled from 7 participating sites and followed for 12 months or more with plasma samples collected immediately before all endomyocardial biopsies. Donor fraction cell-free DNA was extracted, and quantitative genotyping was performed. Blinded donor fraction cell-free DNA and clinical data were analyzed and compared with a previously determined threshold of 0.14%. Sensitivity, specificity, negative predictive value, positive predictive value, and receiver operating characteristic curves were calculated. RESULTS: A total of 987 samples from 144 subjects were collected. After applying predefined clinical and technical exclusions, 745 samples from 130 subjects produced 54 rejection samples associated with the composite outcome of acute cellular rejection grade 2R or greater and pathologic antibody-mediated rejection 2 or greater and 323 healthy samples. For all participants, donor fraction cell-free DNA at a threshold of 0.14% had a sensitivity of 67%, a specificity of 79%, a positive predictive value of 34%, and a negative predictive value of 94% with an area under the curve of 0.78 for detecting rejection. When analyzed independently, these results held true for both pediatric and adult cohorts at the same threshold of 0.14% (negative predictive value 92% and 95%, respectively). CONCLUSIONS: Donor fraction cell-free DNA at a threshold of 0.14% can be used to assess for risk of rejection after heart transplantation in both pediatric and adult patients with excellent negative predictive value.

Nuclear Cell-Free DNA Predicts Adverse Events After Pediatric Cardiothoracic Surgery.

BACKGROUND: Preoperative risk stratification in cardiac surgery includes patient and procedure factors that are used in clinical decision-making. Despite these tools, unidentified factors contribute to variation in outcomes. Identification of latent physiologic risk factors may strengthen predictive models. Nuclear cell-free DNA (ncfDNA) increases with tissue injury and drops to baseline levels rapidly. The goal of this investigation is to measure and to observe ncfDNA kinetics in children undergoing heart operations with cardiopulmonary bypass (CPB), linking biomarkers, organ dysfunction, and outcomes. METHODS: This is a prospective observational study of 116 children <18 years and >3 kg undergoing operations with CPB. Plasma ncfDNA samples were collected and processed in a stepwise manner at predefined perioperative time points. The primary outcome measure was occurrence of postoperative cardiac arrest or extracorporeal membrane oxygenation. RESULTS: Data were available in 116 patients (median age, 0.9 years [range, 0-17.4 years]; median weight, 7.8 kg [range, 3.2-98 kg]). The primary outcome was met in 6 of 116 (5.2%). Risk of primary outcome was 2% with ncfDNA <20 ng/mL and 33% with ncfDNA >20 ng/mL (odds ratio, 25; CI, 3.96-158; P = .001). Elevated ncfDNA was associated with fewer hospital-free days (P < .01). CONCLUSIONS: This study analyzes ncfDNA kinetics in children undergoing operations with CPB for congenital heart disease. Elevated preoperative ncfDNA is strongly associated with postoperative arrest and extracorporeal membrane oxygenation. Further studies are needed to validate this technology as a tool to predict morbidity in children after cardiac surgical procedures.

Significance of α-Myosin Heavy Chain (MYH6) Variants in Hypoplastic Left Heart Syndrome and Related Cardiovascular Diseases.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with complex genetic inheritance. HLHS segregates with other left ventricular outflow tract (LVOT) malformations in families, and can present as either an isolated phenotype or as a feature of a larger genetic disorder. The multifactorial etiology of HLHS makes it difficult to interpret the clinical significance of genetic variants. Specific genes have been implicated in HLHS, including rare, predicted damaging MYH6 variants that are present in >10% of HLHS patients, and which have been shown to be associated with decreased transplant-free survival in our previous studies. MYH6 (α-myosin heavy chain, α-MHC) variants have been reported in HLHS and numerous other CHDs, including LVOT malformations, and may provide a genetic link to these disorders. In this paper, we outline the MYH6 variants that have been identified, discuss how bioinformatic and functional studies can inform clinical decision making, and highlight the importance of genetic testing in HLHS.

A Systematic Review of Ebstein's Anomaly with Left Ventricular Noncompaction.

Traditional definitions of Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), two rare congenital heart defects (CHDs), confine disease to either the right or left heart, respectively. Around 15-29% of patients with EA, which has a prevalence of 1 in 20,000 live births, commonly manifest with LVNC. While individual EA or LVNC literature is extensive, relatively little discussion is devoted to the joint appearance of EA and LVNC (EA/LVNC), which poses a higher risk of poor clinical outcomes. We queried PubMed, Medline, and Web of Science for all peer-reviewed publications from inception to February 2022 that discuss EA/LVNC and found 58 unique articles written in English. Here, we summarize and extrapolate commonalities in clinical and genetic understanding of EA/LVNC to date. We additionally postulate involvement of shared developmental pathways that may lead to this combined disease. Anatomical variation in EA/LVNC encompasses characteristics of both CHDs, including tricuspid valve displacement, right heart dilatation, and left ventricular trabeculation, and dictates clinical presentation in both age and severity. Disease treatment is non-specific, ranging from symptomatic management to invasive surgery. Apart from a few variant associations, mainly in sarcomeric genes MYH7 and TPM1, the genetic etiology and pathogenesis of EA/LVNC remain largely unknown.

Relationship between donor fraction cell-free DNA and clinical rejection in heart transplantation.

BACKGROUND: Clinical rejection (CR) defined as decision to treat clinically suspected rejection with change in immunotherapy based on clinical presentation with or without diagnostic biopsy findings is an important part of care in heart transplantation. We sought to assess the utility of donor fraction cell-free DNA (DF cfDNA) in CR and the utility of serial DF cfDNA in CR patients in predicting outcomes of clinical interest. METHODS: Patients with heart transplantation were enrolled in two sequential, multi-center, prospective observational studies. Blood samples were collected for surveillance or clinical events. Clinicians were blinded to the results of DF cfDNA. RESULTS: A total of 835 samples from 269 subjects (57% pediatric) were included for this analysis, including 28 samples associated with CR were analyzed. Median DF cfDNA was 0.43 (IQR 0.15, 1.36)% for CR and 0.10 (IQR 0.07, 0.16)% for healthy controls (p < .0001). At cutoff value of 0.13%, the area under curve (AUC) was 0.82, sensitivity of 0.86, specificity of 0.67, and negative predictive value of 0.99. There was serial decline in DF cfDNA post-therapy, however, those with cardiovascular events (cardiac arrest, need for mechanical support or death) showed significantly higher levels of DF cfDNA on Day 0 (2.11 vs 0.31%) and Day 14 (0.51 vs 0.22%) compared to those who did not have such an event (p < .0001). CONCLUSION: DF cfDNA has excellent agreement with clinical rejection and, importantly, serial measurement of DF cfDNA predict clinically significant outcomes post treatment for rejection in these patients.

Elevated nuclear and mitochondrial cell-free deoxyribonucleic acid measurements are associated with death after infant cardiac surgery.

OBJECTIVES: Mortality rates following pediatric cardiac surgery with cardiopulmonary bypass have declined over decades, but have plateaued in recent years. This is in part attributable to persistent issues with postoperative global inflammation and myocardial dysfunction, commonly manifested by systemic inflammatory response syndrome and low cardiac output syndrome, respectively. Quantified cell-free DNA (cfDNA), of nuclear or mitochondrial origin, has emerged as a biomarker for both inflammation and myocardial injury. Recent data suggest that nuclear cfDNA (ncfDNA) may quantify inflammation, whereas mitochondrial cfDNA (mcfDNA) may correlate with the degree of myocardial injury. We hypothesize that threshold levels of ncfDNA and mcfDNA can be established that are sensitive and specific for postoperative mortality mediated through independent pathways, and that association will be enhanced with combined analysis. METHODS: Prospective observational study of infants younger than age 1 year undergoing planned surgery with cardiopulmonary bypass. The study received institutional review board approval. Samples were drawn before skin incision, immediately after completion of cardiopulmonary bypass, and subsequently at predetermined intervals postoperatively. Association of early postoperative ncfDNA and mcfDNA levels with mortality were assessed by logistic regression with cut-points chosen by receiving operating characteristic curve exploration. RESULTS: Data were available in 59 patients. Median age and weight were 122 days (interquartile range, 63-154 days) and 4.9 kg (interquartile range, 3.9-6.2 kg). Median STAT category was 3 (interquartile range, 1-4). The primary outcome of death was met in 3 out of 59 (5%). Combined analysis of ncfDNA and mcfDNA levels at 12 hours after the initiation of cardiopulmonary bypass with death at a threshold of 50 ng/mL ncfDNA and 17 copies/µL mcfDNA yielded 100% sensitivity and negative predictive value. The specificity (91%) and positive predictive value (38%) increased through combined analysis compared with univariate analysis. Combined analysis exhibited high specificity (93%) and negative predictive value (78%) for prolonged (>30 postoperative days) hospitalization. CONCLUSIONS: Combined analysis of early postoperative ncfDNA and mcfDNA can stratify risk of mortality and prolonged hospitalization following infant cardiac surgery. Evaluation of both ncfDNA and mcfDNA to identify states of generalized inflammation and myocardial injury may allow for targeted interventions and improved outcomes.

Increase in nuclear cell-free DNA is associated with major adverse events in adult and pediatric heart transplant recipients.

BACKGROUND: Cell-free DNA is an emerging biomarker. While donor fraction may detect graft events in heart transplant recipients, the prognostic value of total nuclear cell-free DNA (ncfDNA) itself is largely unexplored. OBJECTIVE: Explore the relationship between ncfDNA and clinical events in heart transplant recipients. METHODS: We conducted a multi-center prospective study to investigate the value of cell-free DNA in non-invasive monitoring following heart transplantation. Over 4000 blood samples were collected from 388 heart transplant patients. Total ncfDNA and donor fraction were quantified. Generalized linear models with maximum likelihood estimation for repeated measures with subjects as clusters were used to explore the relationship of ncfDNA and major adverse events. Receiver operating characteristic curves were used to help choose cutpoints. RESULTS: A ncfDNA threshold (50 ng/ml) was identified that was associated with increased risk of major adverse events. NcfDNA was elevated in patients who suffered cardiac arrest, required mechanical circulatory support or died post heart transplantation as well as in patients undergoing treatment for infection. CONCLUSIONS: Elevated ncfDNA correlates with risk for major adverse events in adult and pediatric heart transplant recipients and may indicate a need for enhanced surveillance after transplant.

Correction: Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

[This corrects the article DOI: 10.1371/journal.pgen.1009679.].

Estimate of the contemporary live-birth prevalence of recurrent 22q11.2 deletions: a cross-sectional analysis from population-based newborn screening.

BACKGROUND: Although pathogenic 22q11.2 deletions are an important cause of developmental delays and lifelong disease burden, their variable and complex clinical expression contributes to under-recognition, delayed molecular diagnosis and uncertainty about prevalence. We sought to estimate the contemporary live-birth prevalence of typical 22q11.2 deletions using a population-based newborn screening sample and to examine data available for associated clinical features. METHODS: Using DNA available from an unbiased sample of about 12% of all dried blood spots collected for newborn screening in Ontario between January 2017 and September 2018, we prospectively screened for 22q11.2 deletions using multiplex quantitative polymerase chain reaction assays and conducted independent confirmatory studies. We used cross-sectional analyses to compare available clinical and T-cell receptor excision circle (TREC, used in newborn screening for severe combined immunodeficiency) data between samples with and without 22q11.2 deletions. RESULTS: The estimated minimum prevalence of 22q11.2 deletions was 1 in 2148 (4.7 per 10 000) live births (95% confidence interval [CI] 2.5 to 7.8 per 10 000), based on a total of 30 074 samples screened, with 14 having confirmed 22q11.2 deletions. Of term singletons, samples with 22q11.2 deletions had significantly younger median maternal age (25.5 v. 32.0 yr, difference -6.5 yr, 95% CI -7 to -2 yr), a greater proportion with small birth weight for gestational age (odds ratio 7.00, 95% CI 2.36 to 23.18) and lower median TREC levels (108.9 v. 602.5 copies/3 µL, p < 0.001). INTERPRETATION: These results indicate that the 22q11.2 deletion syndrome is one of the most common of rare genetic conditions and may be associated with relatively younger maternal ages and with prenatal growth abnormalities. The findings support the public health importance of early - prenatal and neonatal - diagnosis that would enable prompt screening for and management of well-known actionable features associated with 22q11.2 deletions.

Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.

Total Cell-Free DNA Predicts Death and Infection Following Pediatric and Adult Heart Transplantation.

BACKGROUND: Elevated total cell-free DNA (TCF) concentration has been associated with critical illness in adults and elevated donor fraction (DF), the ratio of donor specific cell-free DNA to total cell-free DNA present in the recipient's plasma, is associated with rejection following cardiac transplantation. This study investigates relationships between TCF and clinical outcomes after heart transplantation. METHODS: A prospective, blinded, observational study of 87 heart transplantation recipients was performed. Samples were collected at transplantation, prior to endomyocardial biopsy, during treatment for rejection, and at hospital readmissions. Longitudinal clinical data were collected and entered into a RedCAP (Vanderbilt University) database. TCF and DF levels were correlated with endomyocardial biopsy and angiography results, as well as clinical outcomes. Logistic regression for modeling and repeated measures analysis using generalized linear modeling was used. The standard receiver operating characteristic curve, hazard ratios, and odds ratios were calculated. RESULTS: There were 257 samples from 87 recipients analyzed. TCF greater than 50 ng/mL were associated with increased mortality (P = .01, area under the curve 0.93, sensitivity 0.44, specificity 0.97) and treatment for infection (P < .005, area under the curve 0.68, sensitivity 0.45, specificity 0.96). Increased DF was not correlated with treatment for infection. DF was associated with rejection and cardiac allograft vasculopathy (P < .001), but TCF was not. CONCLUSIONS: TCF elevation predicted death and treatment for infection. DF elevation predicted histopathologic acute rejection and cardiac allograft vasculopathy. Surveillance of TCF and DF levels may inform treatment after heart transplantation.

Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 µm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 µm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent's iPSCs recapitulated the phenotype of the proband's iPSC-CMs, and conversely, correction of the R443P variant in the proband's iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.

Novel KLHL26 variant associated with a familial case of Ebstein's anomaly and left ventricular noncompaction.

BACKGROUND: Ebstein's anomaly (EA) is a rare congenital heart disease of the tricuspid valve and right ventricle. Patients with EA often manifest with left ventricular noncompaction (LVNC), a cardiomyopathy. Despite implication of cardiac sarcomere genes in some cases, very little is understood regarding the genetic etiology of EA/LVNC. Our study describes a multigenerational family with at least 10 of 17 members affected by EA/LVNC. METHODS: We performed echocardiography on all family members and conducted exome sequencing of six individuals. After identifying candidate variants using two different bioinformatic strategies, we confirmed segregation with phenotype using Sanger sequencing. We investigated structural implications of candidate variants using protein prediction models. RESULTS: Exome sequencing analysis of four affected and two unaffected members identified a novel, rare, and damaging coding variant in the Kelch-like family member 26 (KLHL26) gene located on chromosome 19 at position 237 of the protein (GRCh37). This variant region was confirmed by Sanger sequencing in the remaining family members. KLHL26 (c.709C > T p.R237C) segregates only with EA/LVNC-affected individuals (FBAT p < .05). Investigating structural implications of the candidate variant using protein prediction models suggested that the KLHL26 variant disrupts electrostatic interactions when binding to part of the ubiquitin proteasome, specifically Cullin3 (CUL3), a component of E3 ubiquitin ligase. CONCLUSION: In this familial case of EA/LVNC, we have identified a candidate gene variant, KLHL26 (p.R237C), which may have an important role in ubiquitin-mediated protein degradation during cardiac development.

Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability.

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay's conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

Donor fraction cell-free DNA and rejection in adult and pediatric heart transplantation.

BACKGROUND: Endomyocardial biopsy (EMB) is the current standard for rejection surveillance in heart transplant recipients. The quantification of donor-specific cell-free DNA (cfDNA) may be an appropriate biomarker for non-invasive rejection surveillance. A multicenter prospective blinded study (DNA-Based Transplant Rejection Test, DTRT) investigated the value of donor fraction (DF), defined as the ratio of cfDNA specific to the transplanted organ to the total amount of cfDNA present in a blood sample. METHODS: A total of 241 heart transplant patients were recruited from 7 centers. Age at transplant ranged from 8 days to 73 years, with 146 subjects <18 years and 95 ≥18 years. All the patients were followed for at least 1 year, with blood samples drawn at routine and for-cause biopsies. A total of 624 biopsy-paired samples were included for analysis through a commercially available cfDNA assay (myTAIHEART, TAI Diagnostics Inc.). A blinded analysis of repeated measures compared the outcomes using receiver operating characteristic (ROC) curves. All primary clinical end-points were monitored at 100%. All analysis and conclusions were reviewed by both an independent external oversight committee and the National Institutes of Health-mandated DTRT steering committee. RESULTS: DF in acute cellular rejection (ACR) 1R/2R (n = 15) was higher than ACR 0R (n = 42) (p = 0.02); DF in antibody-mediated rejection pAMR1 (n = 8) and pAMR2 (n = 12) (p = 0.05) were higher than pAMR0 (n = 466) (p = 0.04 and p = 0.05 respectively). An optimal DF threshold was determined by the use of an ROC analysis, which ruled out the presence of either ACR or antibody-mediated rejection. CONCLUSIONS: The cell-free DNA DF holds promise as a non-invasive diagnostic test to rule out acute rejection in both adult and pediatric heart transplant populations.

Early changes in cell-free DNA levels in newly transplanted heart transplant patients.

Heart transplantation is a well-established therapy for end-stage heart failure in children and young adults. The highest risk of graft loss occurs in the first 60 days post-transplant. Donor fraction of cell-free DNA is a highly sensitive marker of graft injury. Changes in cell-free DNA levels have not previously been studied in depth in patients early after heart transplant. A prospective study was conducted among heart transplant recipients at a single pediatric heart center. Blood samples were collected from children and young adult transplant patients at three time points within 10 days of transplantation. DF and total cell-free DNA levels were measured using a targeted method (myTAIHEART ). In 17 patients with serial post-transplant samples, DF peaks in the first 2 days after transplant (3.5%, [1.9-10]%) and then declines toward baseline (0.27%, [0.19-0.52]%) by 6-9 days. There were 4 deaths in the first year among the 10 patients with complete sample sets, and 3 out of 4 who died had a late rise or blunted decline in donor fraction. Patients who died trended toward an elevated total cell-free DNA at 1 week (41.5, [34-65] vs 13.6, [6.2-22] P = .07). Donor fraction peaks early after heart transplant and then declines toward baseline. Patients without sustained decline in donor fraction and/or elevated total cell-free DNA at 1 week may have worse outcomes.