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The routine surveillance of kidney transplant allografts has relied on imperfect non-invasive biomarkers such as creatinine and urinary indices, while the gold standard allograft biopsy is associated with risk of bleeding, organ injury and sampling errors. Donor derived cell free DNA (dd-cfDNA) is being employed as a biomarker that addresses limitations of these surveillance methods, albeit has inherent drawbacks. This review provides an update on the enhanced understanding of dd-cfDNA and its expanded use beyond the conventional indication of detecting allograft rejection.
In the past 5 decades of the successful practice of kidney transplantation, a biomarker for monitoring of allograft rejection continued to elude the field. Donor derived cell free DNA (dd-cfDNA) has gained widespread utility as that biomarker in the transplant community since its introduction. After the initial demonstration of its use in detecting T-cell mediated and antibody mediated rejection in kidney transplantation (
A multitude of clinical studies have documented the efficacy of dd-cfDNA in detecting rejection, however, is role in surveillance of kidney allografts in routine clinical practice has not been well elucidated. The ADMIRAL study (Assessing AlloSure Dd-cfDNA, Monitoring Insights of Renal Allografts with Longitudinal Surveillance; NCT04566055) looked to address this aspect through a large, multicenter, observational cohort study of kidney transplant recipients monitored with dd-cfDNA for ≤3 years (
In a cohort of nearly 1,100 patients from over seven major transplant centers in the United States, dd-cfDNA measurements were done at regular intervals done as part of surveillance and for-cause in the setting of graft dysfunction to examine its “real world” application. Transplant kidney biopsies were performed as a part of the study in the setting of worsening creatinine, proteinuria and/or development of
Circulating Donor-Derived Cell-Free DNA in Blood for Diagnosing Acute Rejection in Kidney Transplant Recipients (DART) study demonstrated that a dd-cfDNA threshold of >1% aided in discerning presence of rejection (
A decline in eGFR 1–3 years post kidney transplantation portends an increased risk of graft failure and death (
While the ADMIRAL study expanded the repertoire of dd-cfDNA interpretation, it is important to address its limitations. Many dd-cfDNA and biopsy samples were not truly paired, with the investigators allowing for biopsies to be done within 30 days of dd-cfDNA measurements. It is possible that many early disease processes may have been missed or a new pathology may have arisen in the interim. This confounding cannot be accounted for, and future studies should endeavor to limit the duration elapsed between the dd-cfDNA measurement and subsequent kidney biopsy. There could also be observer bias since all biopsies were read locally and lacked centralized reporting. Given that this study was designed to assess the “real world” utility of dd-cfDNA, the investigators could have also assessed the correlation of dd-cfDNA with proteinuria. The presence of proteinuria is strongly associated with reduced graft survival (
The Molecular Microscope Diagnostic System (MMDx) is a method of elucidating various pathologies on allograft biopsy sample by utilizing automatic genome-wide microarray measurements and interprets disease states by machine learning–derived classifiers and archetype scores (
The correlation of multigene measurement scores (transcript sets) with dd-cfDNA were strongest with ABMR and all-rejection scores, while being moderate with TCMR scores, and weak with recent parenchymal injury, dedifferentiation, and atrophy-fibrosis scores. The investigators performed a principal component analysis (PCA), in which the dd-cfDNA vector highly approximated the peritubular capillaritis molecular classifier vector in all three dimensions (all rejection, ABMR and early stage ABMR). Dd-cfDNA, therefore, correlated with an important component of the Banff classification used for diagnosis of ABMR-peritubular capillaritis.
Active rejection based on molecular measurements had the highest dd-cfDNA levels, while biopsies with no molecular or histologic evidence of rejection has the lowest values. Importantly, the molecular scores predicted dd-cfDNA ≥1.0% better than histologic scores. This finding adds further to accumulating evidence that histology, while regarded as the gold standard for diagnosing a vast array of allograft pathologies, may not correlate with extent of damage.
The Trifecta study findings of lower dd-cfDNA levels in TCMR in comparison to ABMR is in line with that of the DART study (
Histologic lesions of borderline and TCMR 1A can exhibit considerable overlap, with clinical relevance of either lesions and optimal treatment continues to debated (
Previous studies elucidating the use of dd-cfDNA in kidney allograft rejection demonstrated that a proportion of patients with TCMR 1A did not have elevated levels (
The generation of dnDSA is associated with adverse consequences, including development of antibody mediated rejection and eventual graft loss. Patients with dnDSA have a significant reduction in 10-year graft survival in comparison to those who do not (57% vs. 96%) (
The ADMIRAL study provided further granularity to relationship between dd-cfDNA and dnDSA (
Another study investigated the diagnostic value of dd-cfDNA when added to DSA in detecting ABMR in two independent cohorts of kidney transplant patients (one cohort with subclinical cases identified with DSA testing >180 days post transplantation and the other with indication biopsies >1 month post transplantation) (
Given dd-cfDNA is calculated as percentage of the total circulating DNA (donor derived and recipient derived cell free DNA), any change in background cell free DNA may result in a false result. High body mass index (BMI) and increasing age may result in higher background cell free DNA given association with increased inflammation and escalated cell senescence respectively (
The COVID-19 pandemic necessitated dramatic changes in delivery of healthcare. From a transplant perspective, measures to reduce exposure to the virus and augmenting vaccine response have been the most essential initiatives to mitigate the viral infection in the vulnerable transplant population. Telemedicine and remote home phlebotomy were employed as methods to minimize healthcare associated exposure the virus.
Two studies demonstrated that dd-cfDNA drawn via remote home phlebotomy could be utilized for surveillance of allografts (
The utilization of dd-cfDNA has been extensively validated in TCMR and HLA antibody induced antibody mediated rejection (ABMR). It is now being employed beyond these conventional indications ( (1) Angiotensin-1 receptor (AT1R) antibody mediated rejection: the presence of AT1R antibodies has been demonstrated to be independently associated with high risk for development of ABMR and decreased long term graft survival ( (2) Anti-programmed cell death-1 (PD-1) inhibitor induced rejection: Immune check point inhibitors are being increasingly used to treated numerous cancers. In addition to being associated with multi-systemic adverse effects, allograft rejection can be a devastating consequence of these agents ( (3) Distinguishing BK virus associated nephropathy (BKVAN) from BK viremia (BKV): it can be challenging to discern progression of BKV to BKVAN- especially with reliance on the often debated cut off viral load of >10,000 copies/mL and eventual allograft biopsy, which itself is associated with discordant reads (
The expanding paradigm of donor derived cell free DNA (dd-cfDNA). ABMR-antibody mediated rejection; AT1R, angiotensin 1 receptor; BKVAN, BK virus associated nephropathy; dnDSA,
As dd-cfDNA continues to integrate into surveillance regimes of kidney allografts, some aspects continue to remain unanswered. There is yet to be a defined frequency of dd-cfDNA testing substantiated by a robust clinical study (
Writing–original draft preparation, SK; Writing—review and editing, DB.
SK has received a fellowship funded by CareDx. DB has received honoraria and consulting fees from CareDx, and Natera, and Johns Hopkins; has received research support from CareDx and Natera; and funding to support a transplant nephrology fellowship from CareDx.
This work was supported by a generous gift from Raymond and Melody Ranelli.