Rejection In Pediatric Heart Transplantation Research Articles

(297) - Micro-RNA Biomarkers of Allograft Rejection in Pediatric Heart Transplantation

(297) - Micro-RNA Biomarkers of Allograft Rejection in Pediatric Heart Transplantation

Histopathology, mRNA expression profile, and donor-derived cell-free DNA for assessment of rejection in pediatric heart transplantation.

The relationship between histopathologic and molecular ("MMDx"®) assessments of endomyocardial biopsy (EMB) and serum donor-derived cell-free DNA (ddcfDNA) in acute rejection (AR) assessment following pediatric heart transplantation (HT) is unknown. EMB sent for MMDx and histopathology from November 2021 to September 2022 were reviewed. MMDx and histopathology results were compared. DdcfDNA obtained ≤1 week prior to EMB were compared with histopathology and MMDx results. The discrimination of ddcfDNA for AR was assessed using receiver-operating curves. In this study, 177 EMBs were obtained for histopathology and MMDx, 101 had time-matched ddcfDNA values. MMDx and Histopathology displayed moderate agreement for T-cell-mediated rejection (TCMR, Kappa = 0.52, p < .001) and antibody-mediated rejection (ABMR, Kappa = 0.41, p < .001). Discordant results occurred in 24% of cases, most often with ABMR. Compared with no AR, ddcfDNA values were elevated in cases of AR diagnosed by both histopathology and MMDx (p < .01 for all). Additionally, ddcfDNA values were elevated in injury patterns on MMDx, even when AR was not present (p = .01). DdcfDNA displayed excellent discrimination (AUC 0.83) for AR by MMDx and/or histopathology. Using a threshold of ≥0.135%, ddcfDNA had a sensitivity of 90%, specificity of 63%, PPV of 52%, and NPV of 94%. Histopathology and MMDx displayed moderate agreement in diagnosing AR following pediatric HT, with most discrepancies noted in the presence of ABMR. DdcfDNA is elevated with AR, with excellent discrimination and high NPV particularly when utilizing MMDx. A combination of all three tests may be necessary in some cases.

Novel Diagnostics for Pediatric Heart Transplant Rejection

Novel Diagnostics for Pediatric Heart Transplant Rejection

Pediatric Heart Transplant Rejection After COVID-19 Infection

Infections have been associated with rejection episodes in solid organ transplant recipients. We report an association between COVID-19 infection and heart transplant (HT) rejection. The patient was 14 years old and 6.5 years post-HT. He developed symptoms of rejection within 2 weeks of COVID exposure and presumed infection. In this case, COVID-19 infection closely preceded significant rejection and graft dysfunction. Further study is needed to establish a correlation between COVID-19 infection and rejection in HT patients.

The teammate trial: Study design and rationale tacrolimus and everolimus against tacrolimus and MMF in pediatric heart transplantation using the major adverse transplant event (MATE) score

Currently there are no immunosuppression regimens FDA-approved to prevent rejection in pediatric heart transplantation (HT). In recent years, everolimus (EVL) has emerged as a potential alternative to standard tacrolimus (TAC) as the primary immunosuppressant to prevent rejection that may also reduce the risk of cardiac allograft vasculopathy (CAV), chronic kidney disease (CKD) and cytomegalovirus (CMV) infection. However, the two regimens have never been compared head-to-head in a randomized trial. The study design and rationale are reviewed in light of the challenges inherent in rare disease research. The TEAMMATE trial (NCT03386539, IND 127980) is the first multicenter randomized clinical trial (RCT) in pediatric HT. The primary purpose is to evaluate the safety and efficacy of EVL and low-dose TAC (LD-TAC) compared to standard-dose TAC and mycophenolate mofetil (MMF). Children aged <21 years at HT were randomized (1:1 ratio) at six months post-HT to either regimen, and followed for 30 months. Children with recurrent rejection, multi-organ transplant recipients, and those with an estimated glomerular filtration rate (eGFR) <30 ml/min/1.73m2 were excluded. The primary efficacy hypothesis is that, compared to TAC/MMF, EVL/LD-TAC is more effective in preventing 3 MATEs: acute cellular rejection (ACR), CKD and CAV. The primary safety hypothesis is that EVL/LD-TAC does not have a higher cumulative burden of 6 MATEs (antibody mediated rejection [AMR], infection, and post-transplant lymphoproliferative disorder [PTLD] in addition to the 3 above). The primary endpoint is the MATE score, a composite, ordinal surrogate endpoint reflecting the frequency and severity of MATEs that is validated against graft loss. The study had a target sample size of 210 patients across 25 sites and is powered to demonstrate superior efficacy of EVL/LD-TAC. Trial enrollment is complete and participant follow-up will be completed in 2023. The TEAMMATE trial is the first multicenter RCT in pediatric HT. It is anticipated that the study will provide important information about the safety and efficacy of everolimus- vs tacrolimus-based regimens and will provide valuable lessons into the design and conduct of future trials in pediatric HT.

Explainable synthetic image generation to improve risk assessment of rare pediatric heart transplant rejection

Expert microscopic analysis of cells obtained from frequent heart biopsies is vital for early detection of pediatric heart transplant rejection to prevent heart failure. Detection of this rare condition is prone to low levels of expert agreement due to the difficulty of identifying subtle rejection signs within biopsy samples. The rarity of pediatric heart transplant rejection also means that very few gold-standard images are available for developing machine learning models. To solve this urgent clinical challenge, we developed a deep learning model to automatically quantify rejection risk within digital images of biopsied tissue using an explainable synthetic data augmentation approach. We developed this explainable AI framework to illustrate how our progressive and inspirational generative adversarial network models distinguish between normal tissue images and those containing cellular rejection signs. To quantify biopsy-level rejection risk, we first detect local rejection features using a binary image classifier trained with expert-annotated and synthetic examples. We converted these local predictions into a biopsy-wide rejection score via an interpretable histogram-based approach. Our model significantly improves upon prior works with the same dataset with an area under the receiver operating curve (AUROC) of 98.84% for the local rejection detection task and 95.56% for the biopsy-rejection prediction task. A biopsy-level sensitivity of 83.33% makes our approach suitable for early screening of biopsies to prioritize expert analysis. Our framework provides a solution to rare medical imaging challenges currently limited by small datasets.

Short-Course Total Lymphoid Irradiation in Management of Pediatric Heart Transplant Rejection

<h3>Purpose/Objective(s)</h3> To evaluate short-course total lymphoid irradiation (TLI) as treatment for refractory heart transplant rejection in pediatric patients. <h3>Materials/Methods</h3> Pediatric patients who received short-course TLI for treatment of recurrent heart transplant rejection at a single institution were retrospectively reviewed. Radiotherapy consisted of 448 cGy in 4 fractions delivered once daily consecutively. This regimen was previously reported in a small series of adults but has not been reported in children. Potential advantages of short-course TLI compared to a more standard 800 cGy in 10 fractions delivered twice weekly (over 5 weeks) include fewer anesthesia sessions, lower cumulative dose, and patient convenience. <h3>Results</h3> Between 2019-2021, 3 pediatric patients (ages 4, 15, 17; 1 girl, 2 boys) received short-course TLI for chronic or recurrent rejection after cardiac allograft transplant. TLI was delivered between 5-51 months after heart transplantation, and time from initial rejection to TLI was 4-30 months. All patients were critically ill with limited medical options to manage rejection at time of TLI. They all received TLI as planned without need for modification due to acute toxicity. Two patients achieved resolution of rejection after TLI. One of these patients was rejection free on endomyocardial biopsy 3 months later and was alive 6 months after TLI with no complications related to TLI. The other patient's endomyocardial biopsy at 3 months also demonstrated no evidence of rejection and was able to undergo a second heart transplant 8 months after TLI for heart failure that was present prior to TLI. This patient was alive 3 years after TLI with no complications related to TLI. One patient died within 1 month of TLI due to multi-organ failure and thus the effects of TLI were not able to be fully assessed. <h3>Conclusion</h3> Short-course TLI was well-tolerated and significantly reduced the duration of the radiation treatment course in comparison to standard course; this is particularly advantageous in young patients who require anesthesia as well as acutely ill patients with poor performance status. Fortunately, alternative anti-rejection therapies are often effective and TLI is rarely requested, but if required a short-course TLI regimen remains an option.

Antibody Mediated Rejection in Pediatric Heart Transplantation: Should We Re-Biopsy Sooner or Later?

<h3>Purpose</h3> The intent of this study was to examine if there are any factors associated with clearance of antibody mediated rejection (AMR) and to identify the optimal time for follow up biopsy after a diagnosis of AMR. <h3>Methods</h3> A retrospective single-center cohort analysis of patients < 18 years of age who underwent heart transplant (HTx) between 08/2013 - 9/2021 was performed. AMR was defined as positive C4d on endomyocardial right ventricular biopsy (EMB). Concurrent ACR was recorded. Time until clearance was measured in days until follow up EMB was C4d negative. The number of biopsies performed until clearance was also recorded to account for clinical variation in timing for follow up biopsies. <h3>Results</h3> Thirty-five unique patients developed AMR with a total of 41 unique episodes of AMR. All patients received pulse steroids. Additional treatments included IVIG, rituximab, daratumumab, eculizumab, and plasmapheresis, depending on the degree of illness and persistence of AMR. The cohort was 62% male, 27% non-Hispanic White, 51% Hispanic, 20% non-Hispanic Black, and 2% Other. Median age at HTx was 4.6 years (IQR 0 - 12.3), and median days from HTx until AMR was 80 days (IQR 16 - 765 days). The median duration to clear AMR was 28 days (IQR: 17 - 56), 56% of AMR episodes cleared on the first biopsy; and the mean number of biopsies to clear AMR was 1.8. AMR did not resolve in 10% (4/41). Patients with unresolved AMR tended to have a history of prior AMR (50% vs 11%, p = 0.095) and be older at HTx (median age 15yrs, IQR 12.5 - 17.1 vs median 4.3yr, IQR 0.7 to 11.4 yrs, p = 0.01). Analyzing those who had resolution of AMR, there was no association of sex, age at HTx or time since HTx with time to resolution. Those with recurrent AMR took longer to resolve (median 162 days vs 28 days, p = 0.01). Patients with concurrent ACR had a shorter time to AMR resolution (median 16 days vs 33 days, p = 0.01). <h3>Conclusion</h3> There does not appear to be an association between time to clear AMR with patient demographics or time after transplant of the index case of AMR. Patients with prior resolved AMR take longer to clear recurrent cases of AMR. Concurrent ACR was associated with quicker time to clear AMR for reasons that remain unclear. The optimal time period to reassess for AMR clearance, based on this single-center data, is 1 month.

Impact of induction strategy change on first-year rejection in pediatric heart transplantation at a single center-From postoperative basiliximab to either postoperative anti-thymocyte globulin or preoperative basiliximab.

Our pediatric heart transplant center transitioned from post-bypass basiliximab (BAS) induction to either anti-thymocyte globulin (ATG) or pre-bypass BAS. The purpose of this study was to compare first-year rejection rates before and after this change. A single-center retrospective analysis was conducted of pediatric heart transplant recipients from 2010 to 2019. Primary outcome was first-year rejection. Bivariate analysis, Kaplan-Meier curves, and multivariable regression were performed across eras. Forty-three early era patients (55%) received post-bypass BAS, and 35 late era patients (45%) received pre-bypass BAS (n=17) or ATG (n=18). First-year rejection decreased in the late era (31% vs 53%, p=.05). This finding was more pronounced after excluding infants (38% vs 73%, p=.006). Late era was associated with a decreased likelihood of rejection (all cohort OR 0.19, 95% CI 0.05-0.66; infants excluded OR 0.17, 95% CI 0.04-0.61). No differences in post-transplant lymphoproliferative disease, donor-specific antibody, or infection were observed. Fewer late era patients receiving ATG or pre-bypass BAS induction had first-year rejection compared to the early era patients receiving standard post-bypass BAS induction. This programmatic shift in induction strategy was readily achievable and potentially effective in reducing first-year rejection.

Plasma Induced Transcriptional Analysis Assessment of Rejection in Pediatric Heart Transplantation

Graft rejection after heart transplantation (HT) is a key clinical issue. The diagnostic gold standard of cardiac catheterization with myocardial biopsies is an invasive procedure that may underdiagnose rejection. A possible novel noninvasive alternative is using plasma to perform transcriptional analysis (TA) to assess gene expression levels associated with rejection. Studies on TA in pediatric HT are lacking. This study aimed to investigate plasma induced TA as an assessment of acute cellular rejection (ACR) in pediatric HT. Plasma collected from 25 pediatric HT recipients at the time of catheterization was used to induce a transcriptional response, measured by Affymetrix U133+2.0 array, in commercially available peripheral blood mononuclear cells. Additional data collected included age, gender, time since transplant (TST), cardiac index (CI), right ventricular end diastolic pressure (RVEDP), wedge pressure (WP), echo ejection fraction (EF), and NT Pro BNP. Subjects with ACR (defined as grade ≥ 1R on biopsy) were compared to those with no rejection (NR). 10 patients had ACR and 15 had NR. There were no significant differences between ACR and NR in age (8.5 vs 9.3 years, p=0.76), gender (60% vs 53% male, p=0.74), TST (3.4 vs 3.7 years, p=0.75), CI (3.8 vs 3.2 L/min/m2, p=0.08), RVEDP (7.8 vs 7.3 mmHg, p=0.77), WP (11.6 vs 10.5 mmHg, p=0.39), EF (65% vs 66%, p=0.49), and NT Pro BNP (831 vs 1083 pg/mL, p=0.67), respectively. Between ACR and NR, TA revealed 494 unique probesets with a false discovery rate of ≤10% (Figure 1). Genes associated with T-cell activation and migration were upregulated in the ACR group. These included CX3CR1, TNFRSF13C, TNFAIP8, ITGA4, TRDC, IL6ST, IL23A, and CD84. TA produced signatures specific to ACR in pediatric HT recipients. The identified genes were consistent with current understanding of ACR. TA can shed insight into the mechanisms of rejection in pediatric HT recipients and should be investigated further as a noninvasive diagnostic tool.

Two-dimensional systolic speckle tracking echocardiography provides a noninvasive aid in the identification of acute pediatric heart transplant rejection.

Acute rejection is an important cause of morbidity and mortality in the pediatric heart transplant (HT) population. A reliable noninvasive method for diagnosis of clinical rejection could substantially reduce these negative outcomes. Evaluate left ventricular (LV) global longitudinal strain (GLS), and global circumferential strain (GCS) as early noninvasive indicators of acute pediatric HT rejection. An 18-month prospective cohort study involving 61 patients evaluated absolute change in peak global systolic strain (GLS and GCS) from enrollment (baseline) to next planned clinical encounter (follow-up) or rejection. Acute rejection defined as a biopsy of grade≥2R or treatment with enhanced immunosuppression by the transplant team, blinded to strain analysis. Two patient cohorts three months post HT without evidence of rejection at enrollment were identified. The study cohort experienced rejection. The control cohort remained free from rejection on follow-up. Two-way analysis of variance (ANOVA) models evaluated change in GLS and GCS by cohort group and time. Applying exclusion criteria, 51 patients enrolled in the control cohort and 10 in the study cohort. The study cohort's mean GLS declined 33% from baseline to rejection (P<.001) and mean GCS declined 16.6% (P=.021). No significant change from baseline to follow-up was seen in the control cohort. A threshold absolute GLS value of 16.1% identified acute rejection with 100% sensitivity and 98% specificity (Likelihood Ratio, [LR] 51). Noninvasive global longitudinal strain was sensitive and specific in the identification of acute clinical rejection in pediatric HT recipients.

Using Label-free Proteomics to Elucidate Affected Pathways in Pediatric Heart Transplant Rejection

Using Label-free Proteomics to Elucidate Affected Pathways in Pediatric Heart Transplant Rejection

P061 Donor-specific anti-human leukocyte antibodies of immunoglobulin G3 subtype are associated with high-grade rejection in pediatric heart transplantation

Aim Factors that determine histopathologic phenotype of antibody mediated heart allograft rejection (AMR) are not known. The purpose of this study was to evaluate associations between histopathology and IgG subtype of anti-HLA donor-specific antibody (DSA) in a single-center pediatric heart transplantation program. Methods We analyzed data from 66 consecutive patients with biopsy proven rejection. Serum samples were timely matched with heart allograft biopsies, which were graded for cellular rejection. DSAs were identified by single-antigen multi-bead array, both by generic IgG, and IgG1-2-3-4 subtype. Rejection was classified as ISHLT higher grade (HR, grade 2) or no/low grade (NLR, grade 1B). We compared the prevalence of IgG subclasses between those with HR and those with NLR. Results Out of 66 cases, we identified DSAs in 28 patients, while 38 were negative. In the DSA-positive group (table), 15 patients had HR and 13 patients had NLR. IgG3 subtype was more prevalent in the HR group, compared with NLR group (14/15 HR vs 1/13 NLR, p p p . Conclusions The prevalence of DSAs of IgG3 subtype was significantly elevated in patients with HR. High-grade rejectors were characterized by multiple IgG subtypes, while low-grade rejectors exhibited DSAs of a single subtype (non-IgG3). The determination of the IgG subclass in DSA-positive patients may be a less invasive method for identifying patients at risk for higher grade cellular rejection. Download high-res image (71KB) Download full-size image

(181) - Circulating Cell-Free DNA as a Non-Invasive Marker of Pediatric Heart Transplant Rejection and Immunosuppressive Treatment

(181) - Circulating Cell-Free DNA as a Non-Invasive Marker of Pediatric Heart Transplant Rejection and Immunosuppressive Treatment

Abstract 14567: Detection of Acute Cellular Rejection in Pediatric Heart Transplantation: A Pilot Cardiac Magnetic Resonance Imaging Study

Introduction: The diagnosis of significant acute cellular rejection (ACR) after heart transplantation (HT) prompts an immediate change in management and is associated with adverse outcome. Endomyocardial biopsy (EMB) is the gold standard for the detection of ACR but has important limitations. The purpose of this study was to evaluate cardiac magnetic resonance imaging (CMR) as a non-invasive tool to detect ACR in pediatric patients after HT. Methods: In this single centre prospective cohort study, 30 pediatric HT recipients underwent CMR at the time of surveillance EMB. Their CMR results were compared to 14 non-HT pediatric controls. Ventricular volumes and ejection fraction, native T1 times and T2-weighted signal intensities were compared between patients and controls and between patients with (grade 2 R) and without (grade 0 R/1 R) significant ACR according to standard criteria. Extracellular volumes (ECV) and the presence of late gadolinium enhancement (LGE) were compared between the two HT groups. Results: There were no significant demographic differences between the patient groups. Transplant patients were on average 11 ± 6.1 years of age and were 31.5 ± 41.3 months post-transplant. Significant (grade 2 R) ACR was an infrequent event in our population (5/30, 17%). Compared to controls, heart rate (100.3 ± 16.4 bpm) and brain natriuretic peptide (60.8 ± 100.7 ng/L) were significantly elevated post-HT but did not differentiate between grades of ACR. Ventricular volumes, ejection fractions, LGE prevalence, ECVs, native T1 times and T2 signal intensities were not significantly altered by ACR. Conclusions: We describe the myocardial characteristics of pediatric HT recipients by CMR. Although limited by the small number of patients with significant ACR the results of this pilot study suggest that current CMR imaging protocols are insufficient to reliably identify ACR-related changes in pediatric HT.

Global left ventricular relaxation: A novel tissue Doppler index of acute rejection in pediatric heart transplantation

Serial invasive cardiac catheterization with endomyocardial biopsies (EMBs) is the current standard of reference for evaluation after orthotopic heart transplant (OHTx). We developed a novel, non-invasive echocardiographic index of global left ventricular relaxation (LVRI) and assessed its sensitivity, specificity and predictive value for detecting rejection compared with EMB results in a prospective study conducted from September 2012 through May 2014. LVRI was calculated as the sum of diastolic tissue Doppler imaging (TDI) velocities (E') of the left ventricular lateral, septal and posterior walls divided by the percentage of left ventricular posterior wall (LVPW) thinning by M-mode. LVRI was measured in 47 OHTx patients and 50 patients with normal hearts. Of the 33 patients who underwent clinically indicated EMB, 22 patients had Grade 0R EMB, 6 had Grade 1R and 5 had Grade 2R to 3R biopsy results. Sensitivity, specificity and predictive value of LVRI for discriminating Grade 1R to 3R EMB were calculated. The LVRI was compared before and after OHTx rejection treatment and during the early and late post-transplant period. To characterize LVRI, 1-way analysis of variance was used to compare all groups, including non-OHTx patients. LVRI was lower in patients with Grade 0R EMBs compared with non-OHTx patients. Patients with Grade 1R to 3R EMBs had lower LVRI than those with Grade 0R EMBs. LVRI recovered after treatment for rejection. LVRI appeared to normalize between 40 and 90 days post-transplantation. After 90 days, sensitivity was 100% and specificity was 90.9% for detecting patients with Grade 1R to 3R EMBs using an LVRI of 0.8. LVRI, a novel, non-invasive TDI index of global left ventricular diastolic dysfunction, appears to be useful for detecting rejection in children beyond 3 months post-OHTx.

(424) - Identification of Candidate MicroRNA as Pathological Markers of Pediatric Heart Transplant Rejection

Rejection of a transplanted heart by the recipient is the most common cause of death within 5 years in the pediatric orthotopic heart transplant (OHT) population. The gold standard for diagnosing rejection is direct pathologic analysis of endomyocardial biopsy samples (EMB). Current methods of hematoxylin and eosin staining and immunohistochemistry-based diagnosis are non-quantitative, subjective, have limited early prognostic value, and, in up to 20% of cases, do not correlate with clinical status.

(100) - Global Left Ventricular Relaxation: A Novel Tissue Doppler Index of Acute Rejection in Pediatric Heart Transplantation

(100) - Global Left Ventricular Relaxation: A Novel Tissue Doppler Index of Acute Rejection in Pediatric Heart Transplantation

Left ventricular myocardial performance index change for detection of acute cellular rejection in pediatric heart transplantation

EMB, the gold standard for diagnosis of ACR, poses unique risks in children. Limited cross-sectional data have associated LV MPI with ACR. We hypothesize that a relative change in MPI from baseline without ACR to the time of ACR will better detect ACR than an absolute threshold LV MPI value. We identified 40 children with ACR ≥60days post-transplant matching them by age and time from transplantation to 40 children without ACR. There was a significant increase in LV MPI at time of ACR vs. baseline (0.59±0.17 vs. 0.41±0.11; p<0.001). There was no difference in LV MPI between baseline and follow-up (0.41±0.11 vs. 0.42±0.11; p=0.65). An absolute increase in LV MPI of ≥0.47 had 82.5% sensitivity and 85% specificity for ACR, whereas an increase in LV MPI from baseline of ≥20.4% was 90% sensitive and 100% specific. Serial measurement of LV MPI appears to be a sensitive and specific marker of ACR. LV MPI shows good interobserver agreement and increases at the time of EMB-proven ACR with subsequent resolution to baseline measurements upon EMB-proven resolution of ACR. Future studies in larger, prospective cohorts should be undertaken to validate these findings.

Gene Expression Profiling in Pediatric Heart Transplant Rejection

Purpose Gene expression profiling (GEP) has demonstrated utility in identifying heart transplant (HTx) patients at increased risk for acute rejection (AR) in adult populations. GEP has not been well studied in pediatric HTx patients whose immune system function may differ from adults. Improved understanding of alterations in gene expression between AR and quiescence may be beneficial in pediatric HTx recipients. Methods and Materials Blood samples were prospectively collected from pediatric HTx recipients (aged 1-18 years) who had undergone transplant at least six months prior to enrollment. Samples were collected over one year, at six month intervals and at time of AR. Acute rejection was defined as clinically indicated augmentation of immunosuppression which in some patients was accompanied by abnormal echocardiogram or biopsy. GEP of 84 genes in the inflammatory cascade (chemokines, cytokines and their receptors) was performed on peripheral mononuclear lymphocytes by commercially available PCR (Illumina Biosciences) array. Expression profiles were analyzed with the commonly used Significant Analysis of Microarray (SAM). Comparison was within individual at time of AR and quiescence. The genes that differentially expressed with and without AR were identified by controlling the false discovery rates at 5%. Results Gene expression profiling was conducted on 17 blood samples (AR and quiescent) from five subjects. A total of 47331 transcripts from the 84 genes were analyzed. Expression levels of defensin family of genes were significantly higher at the time of at AR than at the time of quiescence. This pattern was consistent in 3 of 5 subjects. Conclusions Gene expression profiling holds promise in identifying specific regulatory pathways or gene families with differential activity at times of AR in pediatric HTx recipients. The gene family identified in this study is different from those identified in adult HTx recipients. Further study could lead to improved ability to modulate immunosuppression and/or predict impending AR.