Abstract
Background
While endomyocardial biopsy (EMB) remains the gold standard for detection of acute allograft rejection (AR), its limitations have led many pediatric programs to utilize adjunctive non-invasive methods of rejection surveillance.
Objectives
This study compares the procedural burden, detection of AR, and severity of AR episodes between an EMB-based protocol and a surveillance protocol utilizing donor-derived cell free DNA (dd-cfDNA).
Methods
Patients who underwent orthotopic heart transplantation (OHT) before age 18 followed at our center from 2019 to 2023 were retrospectively reviewed. Two protocols and time periods were compared: Era 1 (01/2019–03/2020, EMB-based) and Era 2 (07/2022–06/2023, dd-cfDNA based). Patients with multi-organ transplants, post-transplant lymphoproliferative disorder, and bone marrow transplants were excluded.
Results
Biopsies, anesthesia, and intubations had a statistically significant decrease in Era 2, with a 46 % reduction in biopsies per patient-year in Era 2. When screening with dd-cfDNA in Era 2, there was a significant increase in the number of positive EMB (32/312, 10.1 %), compared to 19/719 (2.6 %) positive EMB found in Era 1 ( p < 0.001). Cases of rejection were significantly more likely to be associated with a positive EMB (72.9 % in Era 2 vs. 45.9 % in Era 1) and less likely to present as clinical rejection ( p = 0.017).
Conclusion
dd-cfDNA surveillance is associated with a significant decrease in surveillance EMB and related procedures without a change in clinical allograft rejection. Our limited experience suggests that integrating dd-cfDNA may also lead to earlier detection and lesser severity of rejection in pediatric populations.
Highlights
- •
Acute rejection is common in pediatric heart transplant.
- •
Endomyocardial biopsy is invasive and resource-intensive.
- •
Incorporation of new technologies can lower the number of biopsies.
- •
Surveillance for rejection can be safely done despite major decrease in biopsies.
1
Introduction
Orthotopic heart transplantation (OHT) has become standard of care in the pediatric population for indications such as inoperable congenital heart disease (CHD) and end-stage heart failure, which can occur secondary to cardiomyopathy, myocarditis, or CHD, among others [ ]. Advances in the field have yielded median post-transplant survival beyond 15 years [ ]. However, one of the leading morbidities associated with transplantation is acute rejection (AR), occurring in up to 30 % of patients in the first year following OHT [ , ]. While endomyocardial biopsy (EMB) is considered the gold standard for diagnosis of rejection and a staple in most surveillance protocols, it is an invasive procedure that requires anesthesia in smaller children [ ]. Additionally, EMB interpretation by pathologists varies significantly, and it can be falsely negative in cases of patchy distribution of rejection [ , ].
There is also significant variation between pediatric transplant centers with regard to surveillance protocols [ ]. Recent studies have shown that acute rejection with hemodynamic compromise (AR HC) was less common in pediatric patients diagnosed using EMB as the primary surveillance tool; however, in multivariate analysis, the number of biopsies performed was not independently associated with freedom from AR HC [ ]. A transition to a non-invasive protocol is desirable, proven in adults, and adopted by an increasing number of pediatric heart transplant centers across the country. Recently, we have integrated donor-derived cell-free DNA (dd-cfDNA) into our surveillance protocols through a commercially available assay, a test proven to have a high negative predictive value for ruling out AR. This study aims to compare the procedural burden and the detection and severity of AR episodes between our former biopsy-based protocol and the current predominantly non-invasive surveillance protocol.
2
Materials and methods
2.1
Study population and follow-up
This is a single-center retrospective chart review conducted at the New York-Presbyterian Morgan Stanley Children’s Hospital (Columbia University Irving Medical Center). This study was approved by the Morgan Stanley Children’s Hospital of New York’s Institutional Review Board with waiver of consent.
Pediatric and young adult patients who underwent OHT before the age of 18 and were followed between January 2019 and June 2023 by our Program for Pediatric Cardiomyopathy, Heart Failure, and Transplantation were included. Patients with multiple organ transplants, post-transplant lymphoproliferative disorder, and bone marrow transplants were excluded, given that dd-cfDNA is not interpretable in patients with multiple sources of non-recipient DNA. Exclusion criteria were applied to both eras for uniformity. Data from the Congenital Cardiac Catheterization Project on Outcomes (C3PO) were obtained to assess the procedural burden and complications of EMB. Two periods of surveillance were examined: January 2019 – March 2020 (Era 1) and July 2022 – June 2023 (Era 2). Data were not examined from April 2020 – June 2022 during the COVID-19 pandemic due to local restrictions on procedures, which could introduce bias. In July 2022, we transitioned to a non-invasive rejection surveillance protocol that integrated dd-cfDNA. As a cohort, patients from Era 1 who were still followed by our service also appear in Era 2. These patients in Era 2 were included to evaluate the validity of the dd-cfDNA screening in patients >2 years post-OHT. Because of the time elapsed between eras, a patient’s first two years post-OHT can only potentially be observed in a single era. There were no institutional changes to the immunosuppression regimen, treatment thresholds for AR, or treatment protocols for AR between the two eras.
2.2
Protocol
The Era 1 protocol (included in Table 1 A ) utilizes EMB as the primary method of screening for AR. Frequency of EMB is highest in the immediate post-transplant period and decreases in frequency after 1-year post-OHT. The current rejection surveillance protocol (Era 2, Table 1 , B, and C) for all patients integrates commercial dd-cfDNA tests into a protocol with EMB, coronary angiography, donor-specific antibodies (DSAs), echocardiogram, and ECG. In both eras, outpatient visits are scheduled twice a week for the first month post-OHT, weekly in the second month, biweekly in the third month, and then monthly until 12 months post-OHT. All protocols in Era 1 and Era 2 incorporated non-invasive imaging with an echocardiogram every month in the first year of transplant before decreasing in frequency in year 2. Annual coronary angiography is included indefinitely in both eras. In Era 2, patients are divided into two categories: standard risk for rejection and high risk for rejection. By default, patients were sorted into the standard risk protocol. Patients transplanted with a positive retrospective crossmatch, positive DSAs, a history of protein losing enteropathy (PLE), and suspected or proven non-adherence were classified as high-risk patients. Both standard and high-risk protocols incorporated routine EMBs, while the high-risk protocol increased dd-cfDNA testing frequency. We utilize Allosure® (CareDx, Inc., Brisbane, CA) as our dd-cfDNA assay, a test that quantifies the fraction of circulating dd-cfDNA, and samples were analyzed by CareDx, Inc. A threshold of >0.2 % was considered a positive test for the prediction of AR [ ], and a positive test triggered a reflex EMB if obtained at a timepoint without a scheduled EMB.
| A. EMB rejection screening protocol (2019–2020) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1–2 weeks | 1 mo | 2 mo | 3 mo | 5–6 mo | 7–8 mo | 9–10 mo | 12 mo | 15 mo | 18 mo | 21 mo | 2 yr | |
| EMB | x | x | x | x | x | x | x | x | x | x | x | x |
| Angio | x | x | ||||||||||
| DSA | x | x | x | x | x | x | x | |||||
| Echo | Monthly | Q2–3 mo | ||||||||||
| ECG | Every Visit | |||||||||||
| C. dd-cfDNA High Risk | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1–2 weeks | 6–8 weeks | 3 mo | 4 mo | 5 mo | 6 mo | 12 mo | 14 mo | 16 mo | 18 mo | 21 mo | 2 yr | 2.5 yr | 3 yr | |
| dd-cfDNA | (x) † | x | x | x | x | Q1–2 mo | x | x | x | x | x | x | x | x |
| EMB | x | x | x | x | x | x | x | x | ||||||
| Angio | x | x | x | |||||||||||
| DSA | x | x | x | x | x | x | x | x | x | |||||
| Echo | Monthly | Q2 mo | Q3 mo | |||||||||||
| ECG | Every Visit | |||||||||||||
† Initial dd-cfDNA taken as a baseline, clinical interpretation before 2 months post-transplant is limited.
‡ Biopsy at 18 months is optional but considered in the presence of other risk factors.
2.3
Definitions
AR was defined as an episode of clinical rejection or biopsy-proven rejection. Biopsy-proven rejection was defined as acute cellular rejection (ACR) grade 1B or higher according to the 1990 International Society for Heart and Lung Transplantation grading (1R/1B) and antibody-mediated rejection (AMR) grade pAMR1i and higher [ ]. The pathology department at our institution independently graded and reviewed all biopsy samples without prior knowledge of the patients or timing of EMB. ACR grade 1R/1B was considered and treated as AR based on published prognostic data [ ]. Clinical rejection was defined as an episode of rejection based on clinical findings without corresponding histopathologic findings that prompted an augmentation in immunosuppression. A positive dd-cfDNA test was not sufficient for or considered in the diagnosis of clinical rejection. AR HC was defined as any episode of rejection that required intravenous inotropic agents or mechanical support.
2.4
Statistical analysis
Clinical and demographic variables were described with standard descriptive statistics. Continuous measures were reported as median and interquartile range and compared with the Mann-Whitney U test. Categorical variables were compared with chi-squared and Fisher’s exact test as applicable. Odds ratios (OR) and 95 % confidence intervals (CI) were calculated. Incidence rates and 95 % CIs of all newly diagnosed episodes of AR per person-year were calculated for each cohort and were compared using Poisson regression.
3
Results
3.1
Study population
Clinical and demographic data are summarized in Table 2 . A total of 215 patients were identified in Era 1, and 205 patients were identified in Era 2. Thirteen patients in Era 1 and 7 patients in Era 2 met the exclusion criteria and were excluded from analysis. The median age at transplant was 6.7 years in Era 1 and 4.6 years in Era 2. There were no statistically significant differences between the two populations in the two study periods. The most common reason for transplant was cardiomyopathy (61 % in Era 1 and 54 % in Era 2). The percentage of patients on mechanical support at the time of transplant was 29.2 % in Era 1 and 33.3 % in Era 2. A total of 21.8 % of patients in Era 1 and 16.7 % in Era 2 were in their first year post-OHT.
