Rejection Surveillance Research Articles

Use of Donor Derived Cell Free DNA for Long Term Rejection Surveillance in OHT Recipients

IntroductionThe advent of donor-derived cell free DNA (dd cf DNA) testing has transformed care for orthotopic heart transplant (OHT) recipients by reducing the number of invasive endomyocardial biopsies (EMB) for surveillance of acute rejection (AR). The dd cf DNA is elevated in any form of graft injury ranging from AR to ischemic injury from cardiac allograft vasculopathy (CAV). When used in combination with gene expression profiling, the AUC for the combination to rule out acute rejection is about 80.1% However, all the data about using dd cf DNA for AR surveillance is from studies done in the first few years after OHT. There is no data or recommendation on using dd cf DNA in long-term OHT survivors (>5 years). Here we present an interesting case of acute rejection detected by dd cf DNA in a patient who is 8 years post OHT.Case Report55 yo African American male with a history of OHT for non ischemic cardiomyopathy in 2013. He had no previous history of AR or CAV. As part of his routine post-transplant annual work-up, dd cf DNA was checked and it came back elevated at 0.95 % (Ref: >0.25% - significantly elevated, >1% - high probability of AR). Donor specific antibodies were negative. Echocardiogram revealed an ejection fraction of 55-60%. He had a right heart catheterization, which showed normal to low filling pressures and preserved cardiac index. EMB revealed Grade 1R cellular rejection as well as swollen endothelial cells, and >50% capillary positive staining with C4d consistent with acute antibody-mediated rejection (pAMR2). He was successfully treated with plasmapheresis/IVIG x 5 cycles and Rituximab. On further probing, the patient admitted to missing a few doses of his immunosuppression (IS) medications.SummaryThis case highlights the successful use of dd cf DNA for allograft rejection surveillance several years (>5 years) after OHT. However, there is sparse data in this population. Further studies are needed in large patient groups to assess the validity and reproducibility of dd cf DNA in long-term surveillance of rejection. The advent of donor-derived cell free DNA (dd cf DNA) testing has transformed care for orthotopic heart transplant (OHT) recipients by reducing the number of invasive endomyocardial biopsies (EMB) for surveillance of acute rejection (AR). The dd cf DNA is elevated in any form of graft injury ranging from AR to ischemic injury from cardiac allograft vasculopathy (CAV). When used in combination with gene expression profiling, the AUC for the combination to rule out acute rejection is about 80.1% However, all the data about using dd cf DNA for AR surveillance is from studies done in the first few years after OHT. There is no data or recommendation on using dd cf DNA in long-term OHT survivors (>5 years). Here we present an interesting case of acute rejection detected by dd cf DNA in a patient who is 8 years post OHT. 55 yo African American male with a history of OHT for non ischemic cardiomyopathy in 2013. He had no previous history of AR or CAV. As part of his routine post-transplant annual work-up, dd cf DNA was checked and it came back elevated at 0.95 % (Ref: >0.25% - significantly elevated, >1% - high probability of AR). Donor specific antibodies were negative. Echocardiogram revealed an ejection fraction of 55-60%. He had a right heart catheterization, which showed normal to low filling pressures and preserved cardiac index. EMB revealed Grade 1R cellular rejection as well as swollen endothelial cells, and >50% capillary positive staining with C4d consistent with acute antibody-mediated rejection (pAMR2). He was successfully treated with plasmapheresis/IVIG x 5 cycles and Rituximab. On further probing, the patient admitted to missing a few doses of his immunosuppression (IS) medications. This case highlights the successful use of dd cf DNA for allograft rejection surveillance several years (>5 years) after OHT. However, there is sparse data in this population. Further studies are needed in large patient groups to assess the validity and reproducibility of dd cf DNA in long-term surveillance of rejection.

Early Use of Donor Derived Cell-Free DNA (dd-cfDNA) in Heart Transplantation

Purpose Novel non-invasive cardiac allograft surveillance methods such as dd-cfDNA has emerged as a viable noninvasive tool for rejection surveillance, however, with limited data for early use. We sought to describe the incidence and outcomes of early dd-cfDNA use (< 14 days) for HT rejection surveillance. Methods Consecutive adult HT recipients were identified retrospectively from a single center (n=66). Patients were included if heart-only with at least one dd-cfDNA drawn within 14-days post HT between 8/2/2019 and 10/1/2021 (n=45). Baseline donor/recipient demographics, clinical characteristics, serial echocardiographic parameters, laboratory testing, medication regimens, and clinical course were collected over a 6-month period. The composite endpoint was defined as graft dysfunction (LVEF < 40% or >=25% decline in consecutive visit) triggering EMB, rejection >= 2R, development of de-novo donor specific antibodies (DSAs), or death. Results 45 HT recipients were identified. The cohort was predominantly male (71%), non-Caucasian (67%) with DM (40%) and HTN (38%), and had a mean follow up time of 273-days. Donors were male (33%) with mean ischemic time and donor age of 2.9h and 29.5y, respectively. Mean number of biopsies performed in the first 6 months was 1.6 and mean initial dd-cfDNA (obtained <14d) was 0.3 ± 0.2% (NL <0.12). Overall, there was no difference in baseline demographics by composite endpoint. Eleven patients (24%) met the composite endpoint (rejection >= 2R (n=4), de-novo DSAs (n=6), graft dysfunction (n=4)). Biopsy frequency was increased in those meeting the endpoint (2.4 vs 1.4, p<0.004). There were no deaths during the study period. No significant association was demonstrated between dd-cfDNA < 14 d and the composite endpoint p=0.547) at time of interim analysis. Conclusion Early use of dd-cfDNA (< 14 d) for rejection surveillance is effective in lieu of protocol endomyocardial biopsy. Interim analysis demonstrates significant reduction in protocol biopsy frequency with no deaths in this cohort to date. Completion of follow up is expected in March 2022. Novel non-invasive cardiac allograft surveillance methods such as dd-cfDNA has emerged as a viable noninvasive tool for rejection surveillance, however, with limited data for early use. We sought to describe the incidence and outcomes of early dd-cfDNA use (< 14 days) for HT rejection surveillance. Consecutive adult HT recipients were identified retrospectively from a single center (n=66). Patients were included if heart-only with at least one dd-cfDNA drawn within 14-days post HT between 8/2/2019 and 10/1/2021 (n=45). Baseline donor/recipient demographics, clinical characteristics, serial echocardiographic parameters, laboratory testing, medication regimens, and clinical course were collected over a 6-month period. The composite endpoint was defined as graft dysfunction (LVEF < 40% or >=25% decline in consecutive visit) triggering EMB, rejection >= 2R, development of de-novo donor specific antibodies (DSAs), or death. 45 HT recipients were identified. The cohort was predominantly male (71%), non-Caucasian (67%) with DM (40%) and HTN (38%), and had a mean follow up time of 273-days. Donors were male (33%) with mean ischemic time and donor age of 2.9h and 29.5y, respectively. Mean number of biopsies performed in the first 6 months was 1.6 and mean initial dd-cfDNA (obtained <14d) was 0.3 ± 0.2% (NL <0.12). Overall, there was no difference in baseline demographics by composite endpoint. Eleven patients (24%) met the composite endpoint (rejection >= 2R (n=4), de-novo DSAs (n=6), graft dysfunction (n=4)). Biopsy frequency was increased in those meeting the endpoint (2.4 vs 1.4, p<0.004). There were no deaths during the study period. No significant association was demonstrated between dd-cfDNA < 14 d and the composite endpoint p=0.547) at time of interim analysis. Early use of dd-cfDNA (< 14 d) for rejection surveillance is effective in lieu of protocol endomyocardial biopsy. Interim analysis demonstrates significant reduction in protocol biopsy frequency with no deaths in this cohort to date. Completion of follow up is expected in March 2022.

Don't Take Another Piece of My Heart: Substantial Reduction in Biopsies with Initiation of Non-Invasive Rejection Surveillance at One Month Post-Transplant

<h3>Purpose</h3> Historically, the standard of care for post heart transplant (HT) rejection surveillance has been endomyocardial biopsies (EMB), with the majority of biopsies in the first 6 months. <h3>Methods</h3> This was a retrospective analysis of adult HT recipients from 1/2018 to 12/2020 at a single institution. All patients without multiorgan transplants were considered for non-invasive monitoring starting at 28 days. Patients were excluded from analysis if they died within 55 days post HT or moved to another transplant center within first year post transplant. Patients were analyzed in six month cohorts based on transplant date. The primary outcomes were the number of EMB performed in first six months and one year survival. <h3>Results</h3> A total of 106 HT recipients were included and divided into six cohorts. Distribution of biopsies by cohort is shown in Figure 1. Patients in later cohorts had fewer EMB in the first six months post transplant compared to those at the initiation of protocol (p<0.01). The July 2018 and July 2019 cohorts each had two patients expire, making their one year survival 88% and 87% respectively. All other cohorts had 100% survival. There was no difference in one year survival between cohorts (p=0.07) (Figure 2). Baseline characteristics, immunosuppression and use of induction was similar between groups. <h3>Conclusion</h3> Initiation of non-invasive rejection at surveillance at 28 days post-transplant significantly reduces the number of biopsies and results in similar 1 year survival As providers increasingly use non-invasive monitoring, the number of early EMB continue to decline.

No Association Between Early Donor Specific Antibody and Subsequent Allograft Function at 3 Years Post-Pediatric Heart Transplantation. First Results of a Prospective Multi-Institutional Study

<h3>Purpose</h3> We have shown that sensitized and non-sensitized pediatric heart transplant (PHTx) recipients had similar 1 year patient and graft survival (CTOTC-04 study). In this follow-up study (CTOTC-09), we assess the impact of ‘preformed' (at Tx) and first year newly detected donor specific antibody (ndDSA) on allograft function at 3 years. <h3>Methods</h3> Consecutive children listed for Tx at 9 centers were prospectively enrolled. Management of sensitization, immunosuppression and rejection surveillance were standardized. The primary endpoint was PCWP at 3 years. Secondary endpoints included other hemodynamics, ejection fraction (EF), BNP, graft and patient survival and rejection. DSA was defined by single antigen Luminex testing with ≥1 antibody specific towards donor HLA antigens with MFI>1000. <h3>Results</h3> Of 407 enrolled, 370 achieved Tx (mean 8.7 years). Sensitization status at Tx: non-sensitized (n=169, 46%), sensitized/no DSA (n= 120, 32%), sensitized/DSA (n=76, 21%); 5 (1%) unknown. Subjects with and without early DSA (preformed and/or first year ndDSA) had similar PCWP at 3 years (table). There were no significant differences between the 2 groups for: other hemodynamics (PA, RA, RVEDP, LVEDP; all P > 0.08); cardiac index (P= 0.315); EF (P=0.682); and BNP (P=0.900). Subanalysis of the 3 sensitization groups at Tx stratified by presence/absence of first year ndDSA showed no significant impact of ndDSA on PCWP, EF or BNP (all P > 0.1). Graft and patient survival did not differ between groups, though freedom from all acute rejection types was inferior in subjects with DSA at Tx. <h3>Conclusion</h3> Sensitization status at Tx and development of first year ndDSA are not associated with worse allograft function or inferior survival at 3 years. Ongoing follow-up, including assessment of CAD, is in progress. This information is critical for defining Tx strategies and the long-term impact of DSA on graft outcomes.

Rationale and Design of a Randomized Controlled Trial of Donor-Derived Cell-Free DNA to Detect Rejection in Cardiac Transplantation (DETECT)

<h3>Purpose</h3> Endomyocardial biopsy (EMB) for rejection surveillance in cardiac transplantation is invasive, costly, associated with complications, variable in interpretation and thus an imperfect gold standard. Donor-derived cell-free DNA (dd-cfDNA) is an emerging biomarker for detection and surveillance of cardiac allograft rejection. Elevated circulating levels of dd-cfDNA are associated with acute cellular and antibody-mediated rejection in observational studies, but a clinical strategy trial to establish its usefulness in decreasing or eliminating EMB has not been conducted. We describe the rationale and design of the randomized clinical trial, Donor-Derived Cell-free <b>D</b>NA to D<b>ET</b>ect R<b>E</b>jection in <b>C</b>ardiac <b>T</b>ransplantation (DETECT) to test the hypothesis that dd-cfDNA monitoring is non-inferior to standard of care (SOC) surveillance EMB during year 1 post-transplant. <h3>Methods</h3> DETECT is a prospective, multicenter, open-label, randomized controlled trial in 600 adult heart transplant recipients at 40 transplant centers, randomized 1:1 30 days post-transplant to surveillance with dd-cfDNA (Prospera, Natera, San Carlos, CA) (Intervention) versus EMB surveillance (SOC) and followed for 1 year. Rejection surveillance in both arms will be performed per the institution's SOC EMB surveillance schedule. <h3>Results</h3> The primary endpoint is the composite of treated rejection +/- graft dysfunction, any graft dysfunction, listing for or undergoing re-transplantation or death. Secondary endpoints include number of heart biopsies performed, development of de novo donor specific antibodies and cost of care. The study has 80% power to demonstrate non-inferiority if the primary endpoint rate in the experimental arm is no greater than 0.27 (non-inferiority margin of 0.15, assuming a control rate of 0.22). Sample size re-estimation will occur once 80% of subjects are enrolled or 50% reach 1 year follow-up, whichever occurs first in order to maintain 80% power. If the non-inferiority hypothesis is met, analysis for superiority of surveillance with dd-cfDNA will be performed. <h3>Conclusion</h3> The DETECT trial will establish the efficacy and safety of dd-cfDNA as a primary clinical monitoring strategy for cardiac allograft rejection in an effort to decrease or eliminate invasive surveillance biopsies in adult heart transplant recipients.

Poke Not Prod: First Canadian Experience Using Donor-Derived Cell Free DNA to Replace Endomyocardial Biopsy During COVID-19

PurposeAfter a heart transplant (HT), non-invasive methods for rejection surveillance minimize the need for endomyocardial biopsies (EMBx). We describe the first experience with combined use of genetic expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) testing in Canada as part of a quality improvement project to minimize patient risk during the COVID pandemic.MethodsAdult outpatients at least 6 months after HT were screened from May 2021 to July 2021 to have their routine EMBx replaced by a combination of GEP and dd-cfDNA. Demographics, modification of immunosuppression (IS) and outcomes (hospital admission, rejection, and need for EMBx) were collected.ResultsAmong 90 patients, 31 (33%) were enrolled, and 37 non-invasive tests were performed. The median time after HT was 2 years and patients were predominantly Caucasian (52%) and male (68%). 53% had a history of acute cellular rejection during the first year and 32% had cardiac allograft vasculopathy. Of the tests performed, 23 (60%) were - GEP / - dd-cfDNA, 10 (27%) were + GEP / - dd-cfDNA, 4 (11%) were - GEP / + dd-cfDNA and none were + GEP / + dd-cfDNA. Being bridged with a VAD (OR = 5.5, p=0.034) and a history of a previously treated CMV (OR = 16.0, p=0.003) were associated with a positive GEP and a negative dd-cfDNA result. Having received a COVID vaccine in the last 3 months did not affect GEP results (GEP was positive in 23.8% after vaccination vs 33.3% in non-vaccinated patients, p=0.690; average GEP score 29.8 vs 30.7, p=0.673). The 4 patients with a + dd-cfDNA (range 0.19 - 0.81%) underwent an EMBx with no significant cellular or antibody mediated rejection, thus avoiding 89% of the EMBx. No unscheduled clinic visits, emergency department or hospital admissions were recorded. After non-invasive testing, the IS was reduced in 16 cases (43.2%). IS was reduced in in 59% of patients with negative concordant tests (- GEP / - dd-cfDNA), 30% in patients with + GEP / - dd-cfDNA and no reduction in IS occurred in those with + dd-cfDNA.ConclusionThe combination of GEP and dd-cfDNA for rejection surveillance allowed for a marked reduction in EMBx (89%) and for a personalized downtitration of IS without adverse events in the short term. The use of non-invasive rejection surveillance testing was an effective strategy to avoid hospital contact for HT recipients during the COVID-19 pandemic.

In This Issue of the Journal

In This Issue of the Journal

The Gut Microbiome as a Marker of Early Cardiac Allograft Injury

<h3>Purpose</h3> Gut microbial dysbiosis has been correlated to solid organ and stem cell transplant rejection and death. The HeartCare lab test (CareDx) is increasingly used as routine surveillance for heart transplant (HT) injury and rejection. We investigated whether the microbiome can serve as a noninvasive marker of immune activation and allograft injury among HT recipients. <h3>Methods</h3> We conducted a single-center, prospective cohort study of single organ HT recipients from 2020-2021. Shotgun metagenomic sequencing was performed on peri-HT stool samples. Gut microbial diversity and composition were correlated with the highest abnormal AlloSure or AlloMap obtained within 6 months post-HT. <h3>Results</h3> Thirty-eight HT recipients were included. Stool samples were collected 5.3 ± 7.4 days from HT. The highest HeartCare lab values were measured 72 days (IQR [41, 103]) post-HT. Seven HT recipients had high AlloSure (median 0.2%, IQR [0.14, 0.26]) and 7 had high AlloMap (median 32, IQR [30, 33]) scores. The high and low AlloSure and AlloMap groups had similar within sample microbial i.e. α-diversity. Low AlloSure scores significantly correlated with an increased abundance of phylum <i>Bacteroidetes</i> (34.5% vs 14.4%, p=0.04), and class <i>Bacteroidia</i> (34.3% vs 14.3%, p=0.04); and tended to have lower abundance of the phylum <i>Proteobacteria</i> (5.0% vs 23.3%, p=0.06) (Fig. 1A). Higher AlloMap scores correlated with higher abundance of class <i>Deltaproteobacteria</i> (1.3% vs 0.10%, p=0.04) and order <i>Desulfovibrionales</i> (1.25% vs 0.07%, p=0.02), and a trend towards lower abundance of <i>Firmicutes</i> (36.8% vs 57.3%, p=0.08) (Fig. 1B). Intergroup β-diversity analysis showed clustering based on AlloSure but not AlloMap scores (Fig. 1C). <h3>Conclusion</h3> In our cohort, increased abundance of certain gut microbial taxa in the peri-HT period correlated with increased HeartCare values. The gut microbiome may play a role in immune activation and allograft injury or serve as a biomarker. Further investigation into the mechanistic ties between the gut microbiome and alloimmunity is needed.

Low AlloSure® in HeartCare® Associated with Low Risk of Significant Allograft Rejection: An Analysis of SHORE

<h3>Purpose</h3> HeartCare is a non-invasive method of surveillance for cardiac allograft rejection comprised of gene expression profiling (AlloMap, AM) and donor derived cell-free DNA (AlloSure-Heart, AS). AM scores inform immune quiescence whereas AS reflects presence of tissue injury. <h3>Methods</h3> Surveillance HeartCare Outcomes Registry (SHORE) is a multicenter, observational registry assessing the clinical utility of AS/AM. AM score >30 (<6 months) or > 34 (≥6 months) was deemed positive (AM+). AS result >0.15% was defined as positive (AS+). The presence of acute cellular rejection ≥2R or antibody mediated rejection (AMR 1, 2, or 3) was assessed at 14 and 30 days following AS/AM. <h3>Results</h3> Of the 4927 samples analyzed, 2507 (50.9%) were concordant low AS and AM (AS-/AM-), 558 (11.3%) were AS+/AM+. Discordance was noted in 1245 (25.3%) with AS+/AM- and 617 (12.5%) with AS-/AM+. The AS+/AM+ cohort had significantly more rejection events when compared with the AS-/AM- group at 14 days (1.25% vs 0.4%, p = 0.032) with a trend toward more rejection at 30 days (1.61% vs 0.68%, p = 0.055). The AS-/AM+ group had fewer rejection episodes when compared with those with AS+/AM+ at 30 days (0.16% vs 1.61%, p = 0.017) with a trend toward fewer rejection events at 14 days (0.16% vs 1.25%, p = 0.055). The AS-/AM- group had the fewest number of endomyocardial biopsies performed when compared to all other cohorts at 14 days (p < 0.05). <h3>Conclusion</h3> AS/AM provides complementary information regarding probability of clinically significant allograft rejection. In those with discordant AM and AS results, presence of a low AS was associated with lower risk of rejection in the subsequent 14 and 30 days. A concordantly low AS and AM may safely obviate a surveillance endomyocardial biopsy in the appropriately selected.

Relationship of Noninvasive Detection of Allograft Rejection and Injury (Donor-Derived Cell Free DNA and Gene Expression Profiling) and Tissue-Based Molecular Microscopic Diagnosis After Heart Transplantation

<h3>Purpose</h3> Non-invasive quantitative detection of allograft injury through donor-derived cell-free DNA (dd-cfDNA; AlloSure) and gene expression profile (GEP; AlloMap) are used for surveillance of antibody-mediated rejection (AMR) and cell-mediated rejection (CMR) in heart transplant (HTx) patients. While dd-cfDNA and GEP correlate well with the histologic assessment of endomyocardial biopsy (EMB), the relationship with molecular microscopic transcript (MMDx) remains unknown in HTx. <h3>Methods</h3> We retrospectively reviewed HTx patients from 2020-2021 who underwent dd-cfDNA, GEP and EMB with histology and MMDx results. The results were compared using non-parametric analyses with Kruskal-Wallis test. CMR ISHLT ≥ Grade 2R and AMR ≥1 from histology and MMDx were considered as rejection. <h3>Results</h3> A total of 108 patients were included in the analysis. Through MMDx, there were 9 patients with CMR, 39 with AMR, 2 with mixed rejection (AMR/CMR), and 58 with no rejection. Elevations in dd-cfDNA occurred in the CMR, AMR, mixed rejection groups, when compared to no rejection (Median: 0.24%, 1.20%, 2.90%, 0.12%, respectively, P<0.001). GEP was not different between different groups of MMDx. The AUC for dd-cfDNA and rejection by MMDx was 0.89 [95% C.I.: 0.83-0.96]. <h3>Conclusion</h3> The non-invasive quantitative detection of dd-cfDNA correlated well with the tissue molecular microscopic transcript in both AMR and CMR in heart transplant patients. Further study of how the addition of dd-cfDNA/GEP and MMDx would complement the diagnosis and prognosis of allograft injury in heart transplantation should be performed.

Non-Invasive Surveillance for Cardiac Allograft Rejection Using an Echocardiographic Rejection Score in Pediatric Heart Transplant Recipients

<h3>Purpose</h3> Cardiac allograft rejection post heart transplant (HT) remains a significant cause of morbidity and mortality in pediatric HT recipients. Endomyocardial biopsy is the gold standard for diagnosis, but is invasive, expensive, and not without risks. We examine the correlation between a non-invasive echocardiographic ejection score (ERS) and clinical and echocardiographic (echo) evidence of rejection. <h3>Methods</h3> All pediatric HT patients followed at our institution who had surveillance echo using the ERS were retrospectively enrolled. Rejection is defined as any changes in clinical or echo parameters resulting in augmented immunosuppression or administration of steroids, biologics, or plasmapheresis. ERS, left ventricular relaxation index (LVRI), RR interval, diastolic time, and atrioventricular valve regurgitation were obtained from the echo reports. The ERS is scored from 0-10 and is stratified as follows: 0-4: no rejection; 5-6: possible rejection necessitating closer follow up or consideration of other clinical factors; ≥7: automatic rejection. <h3>Results</h3> Sixty-six patients were followed over an 8 year period. Mean age at HT was 6.81 years (range 22 days - 21 years). Thirty-five (53%) patients were male. Primary indication for HT was cardiomyopathy in 54.5% (n=36), congenital heart disease in 44% (n=29), and other in 1.5% (n=1). Fifty-eight episodes of acute or ongoing rejection were treated based on clinical evidence. The ERS correlated weakly with LVRI (r = -.191, p < .01), RR interval (r = .117, p <.01), diastolic time (r = .159, p <.01), and new onset mitral or tricuspid regurgitation (MR/TR) (r = .168, p <.01). The ERS did not correlate with rejection identified by changes in clinical status alone (r = .015, p = .428). <h3>Conclusion</h3> ERS appears to be a useful non-invasive diagnostic tool for early detection of rejection in heart transplant pediatric patients. A larger study is needed to determine the diagnostic utility of this non-invasive ERS.

Unilateral vs Bilateral Lower Lobe Surveillance Transbronchial Biopsies in Patients with Bilateral Lung Transplant Patients

<h3>Purpose</h3> Transbronchial biopsies are routinely performed in surveillance of subclinical rejection after lung transplantation. Sensitivity of transbronchial biopsies can be increased by sampling lower lobes and multiple lobes. Due to the inherent risks of the procedure, biopsies are usually limited to one lung. It is not known if sampling bilateral lower lobes improves diagnostic sensitivity for ACR or affects the outcomes. At our institution, one of the transplant pulmonologist routinely biopsies both the lower lobes hence, this provided us with an opportunity to study and compare the diagnostic yield of biopsies of unilateral vs bilateral lower lobes. <h3>Methods</h3> Retrospective analysis of charts was performed on 83 patients transplanted with lungs bilaterally over 3.5 years. Details of surveillance transbronchial biopsies at 1, 3, 6, 9 (Optional) and 12 months after transplantation were collected for analysis. <h3>Results</h3> 83 lung transplant recipients studied over 3.5 years with a total of 427 transbronchial biopsies. Out of these, 116 procedures were performed bilaterally and 195 procedures were performed unilaterally. Among the 116 patients biopsied bilaterally, 2 samples were inadequate and 25 patients (21.73%) had evidence of ACR including minimal (A1) in 31 samples and A2 rejection in 1 sample. Only 6 out of 25 patients had evidence of ACR in both the lower lobes and 4 out of those procedures were performed in the same patient at different times. Only one of these patient with A2 rejection and another with persistent A1 rejection were treated with pulsed dose steroids for ACR. Among the 195 samples obtained unilaterally, 1 sample was inadequate and 30 biopsy specimens had evidence of ACR (15.38%) including minimal (A1) ACR in 24 samples and mild (A2) rejection in 6 samples. All patients with A2 rejection were treated with pulsed dose steroids for ACR. There were no reported episodes of life threatening hemoptysis or pneumothorax associated any of the above procedures. <h3>Conclusion</h3> In bilateral lung transplant recipients, bilateral lower lobe surveillance transbronchial biopsies may increase the yield for detection of ACR. Detection of ACR simultaneously in both the lower lobes is rare. Unilateral ACR was minimal in most of the biopsies and did not alter the management. Bilateral lower lobe biopsies was not associated with increased risk of pneumothorax

Evaluation of Safety and Effectiveness of the Paragonix™ SherpaPak Cardiac Transport System in Donation After Circulatory Death Heart Transplantation (clinicaltrials.gov Identifier: Nct05038943)

Purpose To evaluate the safety and effectiveness of The Paragonix SherpaPak™ Cardiac Transport System (CTS) in the transportation of cardiac allografts recovered from donors after circulatory death (DCD) with thoracoabdominal normothermic regional perfusion (TA-NRP). Methods Institutional Review Board (IRB#0478-21-FB) approved this single center pilot study for a total number of 5 heart transplants. The study is designed as a single arm feasibility study. The study opened for enrollment on September 15th 2021, the anticipated duration is one year. All patients included in DCD NRP study (NCT04615182) will be consented to participate in DCD NRP Paragonix study (NCT05038943). Inclusion and exclusion criteria are summarized in Table 1. Donor organs will be procured after no more than 30 minutes of warm ischemia (defined as a time period commencing from systolic blood pressure < 50 mmHg to the start of reperfusion) and resuscitated in-situ with TA-NRP in accordance with the approved institutional protocol. Criteria for donor acceptance for transplantation are depicted in Table 2. Allografts will be transported from a donor site to recipient center with the SherpaPak™ CTS. All implantation procedures will be performed at our institution. Recipients will receive standardized immunosuppression and infection prophylaxis and will undergo rejection surveillance with serial endomyocardial biopsies. Endpoints Primary outcome is primary graft dysfunction (PGD) at 24, 48, and 72 hours (using ISHLT definition). Secondary outcomes are: need for cardioversion or pacing to restart transplanted heart; vasoactive-inotropic score at 24, 48, and 72 hours (dopamine dose (µg/kg/min) + dobutamine dose (µg/kg/min) + 100 x epinephrine dose (µg/kg/min) + 100 x norepinephrine dose (µg/kg/min); intensive care unit and hospital length of stay; and 30-day, 90-day, 1-year survival. To evaluate the safety and effectiveness of The Paragonix SherpaPak™ Cardiac Transport System (CTS) in the transportation of cardiac allografts recovered from donors after circulatory death (DCD) with thoracoabdominal normothermic regional perfusion (TA-NRP). Institutional Review Board (IRB#0478-21-FB) approved this single center pilot study for a total number of 5 heart transplants. The study is designed as a single arm feasibility study. The study opened for enrollment on September 15th 2021, the anticipated duration is one year. All patients included in DCD NRP study (NCT04615182) will be consented to participate in DCD NRP Paragonix study (NCT05038943). Inclusion and exclusion criteria are summarized in Table 1. Donor organs will be procured after no more than 30 minutes of warm ischemia (defined as a time period commencing from systolic blood pressure < 50 mmHg to the start of reperfusion) and resuscitated in-situ with TA-NRP in accordance with the approved institutional protocol. Criteria for donor acceptance for transplantation are depicted in Table 2. Allografts will be transported from a donor site to recipient center with the SherpaPak™ CTS. All implantation procedures will be performed at our institution. Recipients will receive standardized immunosuppression and infection prophylaxis and will undergo rejection surveillance with serial endomyocardial biopsies. Primary outcome is primary graft dysfunction (PGD) at 24, 48, and 72 hours (using ISHLT definition). Secondary outcomes are: need for cardioversion or pacing to restart transplanted heart; vasoactive-inotropic score at 24, 48, and 72 hours (dopamine dose (µg/kg/min) + dobutamine dose (µg/kg/min) + 100 x epinephrine dose (µg/kg/min) + 100 x norepinephrine dose (µg/kg/min); intensive care unit and hospital length of stay; and 30-day, 90-day, 1-year survival.

Circulating microRNA Biomarkers in Cellular and Antibody-Mediated Heart Transplant Rejection

<h3>Purpose</h3> Non-invasive monitoring of heart allograft health is an important clinical goal and circulating microRNAs (miRs) have been associated with acute rejection in limited studies. <h3>Methods</h3> The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter prospective study. Patients with no history of rejection after transplant were selected as controls. Cases were 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 analysis and LASSO regression was used to develop ACR and AMR miR panels while adjusting for clinical covariates. The panels were validated in an independent sample from GRAfT and Stanford University. Receiver operating characteristic curves were generated and area under the curve (AUC) statistics are reported. Distinct ACR and AMR clinical scores were developed to translate gene expression data for clinical use. <h3>Results</h3> The GRAfT cohort had a median age of 52 years, with 35% females and 45% Blacks. We included 85 controls and 34 patients with rejection (20 ACR, 14 AMR). In ACR and AMR, using differential gene expression and regression analysis, we identified 11 and 15 miRs that accurately discriminated ACR and AMR (p<0.05). Independent validation of the miR panels within GRAfT led to excellent diagnostic accuracy for ACR AUC 0.92 (95% CI: 0.86-0.98) and AMR AUC 0.82 (95% CI: 0.74-0.90). The externally validated ACR AUC was 0.91 (95% CI: 0.82-0.99). Distinct ACR and AMR miR clinical scores were developed ranging from 0-100; using a threshold of 65 identified ACR with a sensitivity of 86%, specificity of 78% and negative predictive value of 98%, for AMR the respective performance was 82%, 84% and 97% (Figure). <h3>Conclusion</h3> The unique clinical ACR and AMR scores usher in an era whereby genomic biomarkers are used not only for rejection surveillance but permit accurate, non-invasive diagnosis of ACR and AMR.

Poke Not Prod: Improving Quality of Life Through Non-Invasive Rejection Surveillance for Heart Transplant Recipients

<h3>Purpose</h3> Gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) provide effective non-invasive rejection surveillance for heart transplant (HT) recipients with a trend toward improved quality of life. During the COVID-19 pandemic, rejection monitoring and titration of mediations in HT patients was difficult due to limited health-care resources for endomyocardial biopsy (EMBx). This is the first Canadian study to assess non-invasive rejection surveillance in improving patient satisfaction and reducing anxiety during HT rejection screening. <h3>Methods</h3> Adult HT recipients, at least 6 months post-transplant, were enrolled to have surveillance EMBx replaced by non-invasive rejection testing with GEP and dd-cfDNA. Patients with multiorgan transplant, dialysis, or high rejection risk (recent acute cellular rejection ≥ grade 2R, new graft dysfunction, or heart failure) were excluded. All patients completed the Medical Outcomes Study 12-item Short Form Health Survey (SF-12) and a patient satisfaction survey. Thematic analysis was performed for open-ended responses. <h3>Results</h3> Out of 90 patients screened, 31 had their routine EMBx replaced by non-invasive rejection testing. Based on test results, 89% of EMBx were safely eliminated. On the SF-12, participants had a median physical health score of 43 (40-53) and mental health score of 53 (46-58) out of 100 . Patients' self-reported satisfaction was 90%. Median self-reported anxiety score prior to EMBx was 50 (10-71) versus 2.5 (0-7.5) out of 100 prior to GEP/dd-cfDNA. Four codes ("emotions" (pain, anxiety, fear), "time", "biopsy", "accuracy") were used to uncover two themes of "Superiority to Biopsy" and "Mental or Physical Stress". Patients described feeling much more satisfied and less emotionally distressed with the non-invasive screening compared to EMBx. HT patients reported less fear and anxiety, reduced pain, and enjoyed the simplicity of non-invasive testing. <h3>Conclusion</h3> Non-invasive rejection surveillance screening can positively impact patients' mental health. In this study, non-invasive rejection surveillance eliminated the recovery time and risk of an invasive procedure for HT recipients while reducing anxiety, improving patient satisfaction, and providing an alternative way to screen patients during a period of limited resources due to a global pandemic.

Early Outcomes of Donation After Circulatory Death Heart Transplantation with Thoracoabdominal Normothermic Regional Perfusion

<h3>Purpose</h3> To evaluate the early outcomes of donation after circulatory death (DCD) heart transplantation using thoracoabdominal normothermic regional perfusion (TA-NRP). <h3>Methods</h3> The study is designed as a single center prospective study with single arm. Institutional Review Board approved this pilot for a total number of 60 potential DCD heart recipients using grafts recovered with TA-NRP technique (IRB#460-20-FB). The first patient was enrolled on January 3rd 2021. The anticipated study duration is one year for a total of 10 transplant procedures. Recipient inclusion criteria are age ≥ 19 years, signed informed consent, and eligibility for heart transplantation based on standardized institutional criteria. Patients listed for dual organ transplantation and patients with previous cardiac operation including durable LVAD implantation will also be included. All hearts will be recovered with TA-NRP after no more than 30 minutes of warm ischemia (defined as the time period commencing from SBP < 50 mmHg to in-situ reperfusion). TA-NRP protocol is summarized in Figure 1. Acceptance for transplantation will be based upon the functional assessment with TEE and invasive hemodynamic measurements combined with the visual inspection after separation from mechanical support. Grafts will be recovered using 2,000mL of Celsior® for cardio protection, and transported with colds storage. All implantation procedures will be performed at our institution. Recipients will receive standardized immunosuppression and infection prophylaxis and will undergo rejection surveillance with serial endomyocardial biopsies. <h3>Endpoints</h3> Primary outcome is 30-day patient survival. Secondary outcomes are primary graft dysfunction and inotropic score at 72 hours, ICU and hospital length of stay, ventilation duration, and the need for renal replacement therapy.

Acute rejection and post lung transplant surveillance.

PurposeThe purpose of this review is to summarize the current evidence on the evaluation and treatment of acute rejection after lung transplantation.ResultsDespite significant progress in the field of transplant immunology, acute rejection remains a frequent complication after transplantation. Almost 30% of lung transplant recipients experience at least one episode of acute cellular rejection (ACR) during the first year after transplant. Acute cellular rejection, lymphocytic bronchiolitis, and antibody-mediated rejection (AMR) are all risk factors for the subsequent development of chronic lung allograft dysfunction (CLAD). Acute cellular rejection and lymphocytic bronchiolitis have well-defined histopathologic diagnostic criteria and grading. The diagnosis of antibody-mediated rejection after lung transplantation requires a multidisciplinary approach. Antibody-mediated rejection may cause acute allograft failure.ConclusionsAcute rejection is a risk factor for development of chronic rejection. Further investigations are required to better define risk factors, surveillance strategies, and optimal management strategies for acute allograft rejection.

Rejection surveillance in pediatric heart transplant recipients: Critical reflection on the role of frequent and long-term routine surveillance endomyocardial biopsies and comprehensive review of non-invasive rejection screening tools.

Despite significant medical advances in the field of pediatric heart transplantation (HT), acute rejection remains an important cause of morbidity and mortality. Endomyocardial biopsy (EMB) remains the gold-standard method for diagnosing rejection but is an invasive, expensive, and stressful process. Given the potential adverse consequences of rejection, routine post-transplant rejection surveillance protocols incorporating EMB are widely employed to detect asymptomatic rejection. Each center employs their own specific routine rejection surveillance protocol, with no consensus on the optimal approach and with high inter-center variability. The utility of high-frequency and long-term routine surveillance biopsies (RSB) in pediatric HT has been called into question. Sources for this comprehensive review were primarily identified through searches in biomedical databases including MEDLINE and Embase. The available literature suggests that the diagnostic yield of RSB is low beyond the first year post-HT and that a reduction in RSB intensity from high-frequency to low-frequency can be done safely with no impact on early and mid-term survival. Though there are emerging non-invasive methods of detecting asymptomatic rejection, the evidence is not yet strong enough for any test to replace EMB. Overall, pediatric HT centers in North America should likely be doing fewer RSB than are currently performed. Risk factors for rejection should be considered when designing the optimal rejection surveillance strategy. Noninvasive testing including emerging biomarkers may have a complementary role to aid in safely reducing the need for RSB.

Sparing the Prod: Providing an Alternative to Endomyocardial Biopsies With Noninvasive Surveillance After Heart Transplantation During COVID-19

BackgroundThe COVID-19 pandemic has reduced access to endomyocardial biopsy (EMB) rejection surveillance in heart transplant (HT) recipients. This study is the first in Canada to assess the role for noninvasive rejection surveillance in personalizing titration of immunosuppression and patient satisfaction post-HT. MethodsIn this mixed-methods prospective cohort study, adult HT recipients more than 6 months from HT had their routine EMBs replaced by noninvasive rejection surveillance with gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) testing. Demographics, outcomes of noninvasive surveillance score, hospital admissions, patient satisfaction, and health status on the medical outcomes study 12-item short-form health survey (SF-12) were collected and analyzed, using t tests and χ2 tests. Thematic qualitative analysis was performed for open-ended responses. ResultsAmong 90 patients, 31 (33%) were enrolled. A total of 36 combined GEP/dd-cfDNA tests were performed; 22 (61%) had negative results for both, 10 (27%) had positive GEP/negative dd-cfDNA results, 4 (11%) had negative GEP/positive dd-cfDNA results, and 0 were positive on both. All patients with a positive dd-cfDNA result (range: 0.19%-0.81%) underwent EMB with no significant cellular or antibody-mediated rejection. A total of 15 cases (42%) had immunosuppression reduction, and this increased to 55% in patients with negative concordant testing. Overall, patients’ reported satisfaction was 90%, and on thematic analysis they were more satisfied, with less anxiety, during the noninvasive testing experience. ConclusionsNoninvasive rejection surveillance was associated with the ability to lower immunosuppression, increase satisfaction, and reduce anxiety in HT recipients, minimizing exposure for patients and providers during a global pandemic.

The role of the electrocardiogram in the recognition of cardiac transplant rejection: A systematic review and meta-analysis.

BackgroundIn cardiac transplant recipients, the electrocardiogram (ECG) is a noninvasive measure of early allograft rejection. The ECG can predict an acute cellular rejection, thus shortening the time to recognition of rejection. Earlier diagnosis has the potential to reduce the number and severity of rejection episodes.MethodologyA systematic literature review was conducted to identify and select the original research reports on using electrocardiography in diagnosing cardiac transplant rejection in accordance with the PRISMA guidelines. Studies included reported sensitivity and specificity of ECG readings in heart transplant recipients during the first post‐transplant year. Data were analyzed with Review manager version 5.4. p‐value was used in testing the significant difference.ResultsAfter the removal of duplicates, 98 articles were eligible for screening. After the full‐text screening, a total of 17 papers were included in the review based on the above criteria. A meta‐analysis of five studies was done.ConclusionIn heart transplant recipients, a noninvasive measure of early allograft rejection has the potential to reduce the number and severity of rejection episodes by reducing the time and cost of surveillance of rejection and shortening the time to recognition of rejection.