Clinical outcomes from the Assessing Donor-derived cell-free DNA Monitoring Insights of kidney Allografts with Longitudinal surveillance (ADMIRAL) study

Abstract

The use of routine monitoring of donor-derived cell-free DNA (dd-cfDNA) after kidney transplant may allow clinicians to identify subclinical allograft injury and intervene prior to development of clinically evident graft injury. To evaluate this, data from 1092 kidney transplant recipients monitored for dd-cfDNA over a three-year period was analyzed to assess the association of dd-cfDNA with histologic evidence of allograft rejection. Elevation of dd-cfDNA (0.5% or more) was significantly correlated with clinical and subclinical allograft rejection. dd-cfDNA values of 0.5% or more were associated with a nearly three-fold increase in risk development of de novo donor-specific antibodies (hazard ratio 2.71) and were determined to be elevated a median of 91 days (interquartile range of 30-125 days) ahead of donor specific antibody identification. Persistently elevated dd-cfDNA (more than one result above the 0.5% threshold) predicted over a 25% decline in the estimated glomerular filtration rate over three years (hazard ratio 1.97). Therefore, routine monitoring of dd-cfDNA allowed early identification of clinically important graft injury. Biomarker monitoring complemented histology and traditional laboratory surveillance strategies as a prognostic marker and risk-stratification tool post-transplant. Thus, persistently low dd-cfDNA levels may accurately identify allograft quiescence or absence of injury, paving the way for personalization of immunosuppression trials. The use of routine monitoring of donor-derived cell-free DNA (dd-cfDNA) after kidney transplant may allow clinicians to identify subclinical allograft injury and intervene prior to development of clinically evident graft injury. To evaluate this, data from 1092 kidney transplant recipients monitored for dd-cfDNA over a three-year period was analyzed to assess the association of dd-cfDNA with histologic evidence of allograft rejection. Elevation of dd-cfDNA (0.5% or more) was significantly correlated with clinical and subclinical allograft rejection. dd-cfDNA values of 0.5% or more were associated with a nearly three-fold increase in risk development of de novo donor-specific antibodies (hazard ratio 2.71) and were determined to be elevated a median of 91 days (interquartile range of 30-125 days) ahead of donor specific antibody identification. Persistently elevated dd-cfDNA (more than one result above the 0.5% threshold) predicted over a 25% decline in the estimated glomerular filtration rate over three years (hazard ratio 1.97). Therefore, routine monitoring of dd-cfDNA allowed early identification of clinically important graft injury. Biomarker monitoring complemented histology and traditional laboratory surveillance strategies as a prognostic marker and risk-stratification tool post-transplant. Thus, persistently low dd-cfDNA levels may accurately identify allograft quiescence or absence of injury, paving the way for personalization of immunosuppression trials. see commentary on page 676 The deployment of nucleic acid–based noninvasive biomarkers within routine clinical care reflects a paradigm shift in traditional monitoring after kidney transplant. Current clinical management of transplant relies on detection of functional injury (elevated creatinine), therapeutic drug monitoring, and selectively screening for harmful donor-specific antibodies (DSAs). In the absence of clinical signs, clinicians seeking to identify subclinical allograft injury and intervene prior to development of irreversible damage, were forced to rely on invasive allograft biopsies, which have inherent limitations from sampling error and variation in interpretation.1Martuszewski A. Paluszkiewicz P. Król M. et al.Donor-derived cell-free DNA in kidney transplantation as a potential rejection biomarker: a systematic literature review.J Clin Med. 2021; 10: 193Google Scholar Routine monitoring with donor-derived cell-free DNA (dd-cfDNA) after solid organ transplantation has been shown to accurately identify and characterize allograft injury,1Martuszewski A. Paluszkiewicz P. Król M. et al.Donor-derived cell-free DNA in kidney transplantation as a potential rejection biomarker: a systematic literature review.J Clin Med. 2021; 10: 193Google Scholar, 2Knight S.R. Thorne A. Lo Faro M.L. Donor-specific cell-free DNA as a biomarker in solid organ transplantation: a systematic review.Transplantation. 2019; 103: 273-283Google Scholar, 3Bloom R.D. Bromberg J.S. Poggio E.D. et al.Cell-free DNA and active rejection in kidney allografts.JASN. 2017; 28: 2221-2232Google Scholar correlate with pathologic findings,4Stites E. Kumar D. Olaitan O. et al.High levels of dd-cfDNA identify patients with TCMR 1A and borderline allograft rejection at elevated risk of graft injury.Am J Transplant. 2020; 20: 2491-2498Google Scholar, 5Huang E. Gillespie M. Ammerman N. et al.Donor-derived cell-free DNA combined with histology improves prediction of estimated glomerular filtration rate over time in kidney transplant recipients compared with histology alone.Transplant Direct. 2020; 6: e580Google Scholar, 6Kant S. Bromberg J. Haas M. Brennan D. Donor-derived cell-free DNA and the prediction of BK virus-associated nephropathy.Transplant Direct. 2020; 6: e622Google Scholar and assess response to therapy including treatment of rejection.7Wolf-Doty T.K. Mannon R.B. Poggio E.D. et al.Dynamic response of donor-derived cell-free DNA following treatment of acute rejection in kidney allografts.Kidney360. 2021; 2: 729-736Google Scholar,8Hinojosa R.J. Chaffin K. Gillespie M. et al.Donor-derived cell-free DNA may confirm real-time response to treatment of acute rejection in renal transplant recipients.Transplantation. 2019; 103: e61Google Scholar Importantly, evaluation in dd-cfDNA have been demonstrated to occur ahead of clinically apparent organ injury.9Agbor-Enoh S. Chan J.L. Singh A. et al.Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: evidence from cell-free DNA analysis.J Heart Lung Transplant. 2018; 37: 925-932Google Scholar,10Agbor-Enoh S. Shah P. Tunc I. et al.Cell-free DNA to detect heart allograft acute rejection.Circulation. 2021; 143: 1184-1197Google Scholar Consequently, allograft monitoring with plasma dd-cfDNA levels can support noninvasive identification of pathologies including cellular and humoral allograft rejection, viral injury, and drug toxicity.3Bloom R.D. Bromberg J.S. Poggio E.D. et al.Cell-free DNA and active rejection in kidney allografts.JASN. 2017; 28: 2221-2232Google Scholar,6Kant S. Bromberg J. Haas M. Brennan D. Donor-derived cell-free DNA and the prediction of BK virus-associated nephropathy.Transplant Direct. 2020; 6: e622Google Scholar dd-cfDNA can also be employed in the setting of acute allograft injury to guide further diagnostic testing and assess improvement following clinical intervention.7Wolf-Doty T.K. Mannon R.B. Poggio E.D. et al.Dynamic response of donor-derived cell-free DNA following treatment of acute rejection in kidney allografts.Kidney360. 2021; 2: 729-736Google Scholar The routine use of dd-cfDNA to detect, characterize, or exclude ongoing allograft injury is a valuable addition in current post-transplant surveillance. While the effectiveness of dd-cfDNA has been established in clinical trials, its utility in routine clinical practice has not been well described. The ADMIRAL study (Assessing AlloSure Dd-cfDNA, Monitoring Insights of Renal Allografts with Longitudinal Surveillance; NCT04566055), is a large, multicenter, observational cohort study of kidney transplant recipients monitored with dd-cfDNA for ≤3 years. The purpose of this study was to validate clinical trial data by documenting the effectiveness of dd-cfDNA in identifying allograft rejection and subclinical changes in a real-world setting and evaluate the relationship between dd-cfDNA measurements and nonimmune allograft injury. Additionally, ADMIRAL aimed to characterize the relationship between elevation in dd-cfDNA and important predictors of long-term graft survival, including estimated glomerular filtration rate (eGFR) and formation of de novo donor-specific antibodies (dnDSAs). A total of 1092 adult kidney transplant recipients across 7 transplant centers were monitored with AlloSure dd-cfDNA (CareDx Inc) as part of their standard of care. Data was collected between June 1, 2016, and January 31, 2020. An Institutional Review Board waiver of informed consent was obtained, and the study was performed in accordance with international standards and was not part of a larger study. Patients were managed prospectively with dd-cfDNA as part of post-transplant care where data captured was retrospectively examined. Clinical events (e.g., rejection, infection) and routine laboratory testing (creatinine, DSAs) were determined using the center’s electronic medical records. A full list of data collected is provided in Supplementary Table S1. Patients who had contraindications to dd-cfDNA monitoring were excluded. Exclusions include pregnancy, multiple organ recipients, monozygous twin-to-twin transplant, and patients with prior bone marrow transplantation. No exclusions from the analysis and no withdrawal of patients were made as the use of dd-cfDNA was medically necessary as part of the standard of care. dd-cfDNA was measured at regular intervals based on each center’s standard of care practice and was used both as part of surveillance testing and acutely as a diagnostic aid in patients with clinically evident graft dysfunction. A list of center management protocols is provided in Supplementary Table S2. Venous blood was collected in Streck Cell-Free DNA BCT tubes and shipped to the central Clinical Laboratories Improvements Act–certified laboratory at CareDx, Inc. Details of the standardized specimen processing and analytical methods to determine the percentage of dd-cfDNA (AlloSure) have been published.11Grskovic M. Hiller D.J. Eubank L.A. et al.Validation of a clinical-grade assay to measure donor-derived cell-Free DNA in solid organ transplant recipients.J Mol Diagn. 2016; 18: 890-902Google Scholar The targeted next-generation sequencing assay employs highly polymorphic single nucleotide polymorphisms to quantify dd-cfDNA without need for separate genotyping of the recipient or the donor.11Grskovic M. Hiller D.J. Eubank L.A. et al.Validation of a clinical-grade assay to measure donor-derived cell-Free DNA in solid organ transplant recipients.J Mol Diagn. 2016; 18: 890-902Google Scholar Results of protocol surveillance and for-cause kidney transplant biopsies were captured. Indications for for-cause biopsy included change in development of or a of clinical management was performed based on biopsy at the of the transplant were by a single to the dd-cfDNA for study biopsy were using the were and between and central were no or clinical was provided on the biopsy or pathologies were were for the of the rejection In of central was included in the rejection was captured and as antibody-mediated rejection and the rejection not include borderline A of the biopsy is provided in Supplementary Table pathologic as or acute injury or acute were also captured and used for the injury were not included in the rejection patients with allograft rejection, the to was made to each center’s clinical and rejection events were also with dd-cfDNA levels were each center’s standard and after acute A biopsy was as a biopsy days after dd-cfDNA period reflects the of patients for and allograft were included in the analysis was no performed between the time of the dd-cfDNA and A of days between dd-cfDNA sampling and biopsy is shown in Supplementary S1. were used for and of dd-cfDNA measurements from blood at the time of clinical In the the was at of 0.5% and R.D. Bromberg J.S. Poggio E.D. et al.Cell-free DNA and active rejection in kidney allografts.JASN. 2017; 28: 2221-2232Google E. Kumar D. Olaitan O. et al.High levels of dd-cfDNA identify patients with TCMR 1A and borderline allograft rejection at elevated risk of graft injury.Am J Transplant. 2020; 20: 2491-2498Google Scholar to the of the assay patients were as dd-cfDNA low dd-cfDNA for further between the and low dd-cfDNA were for and for of dd-cfDNA between were The the was used to determine the of dd-cfDNA and of were used to the relationship between dd-cfDNA and the clinical for the allograft biopsy was used to determine which were of dd-cfDNA measurements Supplementary Table for were to was analysis was performed in Patients and were included for the purpose of analysis based on the data as shown in the in The of the ADMIRAL cohort included in the for each of the has been in this including the total biopsies and the of used for was determined by using the of in Renal dd-cfDNA and for each was where and was as part of to the relationship between and dd-cfDNA using as to was each the was et M. S. et and of donor-derived cell-free DNA testing in 2020; Scholar provided to the analytical variation and variation of AlloSure was used to the change between dd-cfDNA associated with using the methods by et et of for changes in or more of a biomarker based on a Clin Scholar variation was as variation and the of M. S. et and of donor-derived cell-free DNA testing in 2020; Scholar A. A. M. et to study the of kidney following kidney J Scholar was time allowed the formation of time from to years post-transplant. The data were by the of using as at transplant, evidence of BK dd-cfDNA of allograft rejection, and strategies were to exclude of detection was used to measure the of association between and with the to determine the of the The provided time for to to and to is provided in Supplementary The relationship between dd-cfDNA and development of was in patients with dd-cfDNA and testing at the patients with a Patients were as was evidence of at a as by the transplant as part of the post-transplant surveillance. were of was to be for both and and was used for this D. et of donor-specific antibodies and in renal transplant patients with a standardized Transplant. 2017; Scholar from was using and patients were Patients were as dd-cfDNA or a low dd-cfDNA dd-cfDNA in A and was used to evaluate the association of with the development of Supplementary Table for The of dd-cfDNA as a marker of quiescence was retrospectively using both biopsy and quiescence was as the absence of injury. included BK allograft rejection, or as by biopsy days after dd-cfDNA The of the 1092 ADMIRAL study patients the adult transplant to the of The ADMIRAL cohort was of a percentage of recipients and was also a of donor recipients in this study compared with in the of the ADMIRAL cohort compared to at of AlloSure AlloSure Dd-cfDNA, Monitoring Insights of Renal Allografts with Longitudinal Surveillance; estimated glomerular filtration not of in a AlloSure Dd-cfDNA, Monitoring Insights of Renal Allografts with Longitudinal Surveillance; estimated glomerular filtration not of The included dd-cfDNA measurements from 1092 the study The association between dd-cfDNA levels and the of allograft rejection was using biopsies from patients with dd-cfDNA the biopsies, were for-cause biopsies and the were surveillance biopsies performed clinical The biopsies for-cause and from patients were as acute rejection The of the acute rejection study patients in Table data on rejection is in Supplementary Table the biopsies, were by the central of the ADMIRAL rejection cohort compared to the no rejection rejection at of AlloSure AlloSure Dd-cfDNA, Monitoring Insights of Renal Allografts with Longitudinal Surveillance; after after estimated glomerular filtration not in a AlloSure Dd-cfDNA, Monitoring Insights of Renal Allografts with Longitudinal Surveillance; after after estimated glomerular filtration not was no in the median in patients with a no rejection biopsy range and patients with rejection The for was In the median dd-cfDNA patients with a no rejection biopsy was which was significantly than the median dd-cfDNA in patients with biopsies cellular or humoral rejection The for rejection dd-cfDNA was which was significantly than the of The for dd-cfDNA was dd-cfDNA levels significantly between patients with and was in biopsies from compared to the median dd-cfDNA was TCMR was in biopsies from Patients with compared to had a median dd-cfDNA of The median dd-cfDNA in patients with borderline TCMR was on and of rejection is provided in Supplementary and Clinical for biopsy was determined to have a on measured dd-cfDNA of pathologic In patients without rejection, dd-cfDNA levels were significantly in patients a for-cause biopsy than in patients a surveillance biopsy median dd-cfDNA in patients with in the for-cause biopsies was than in patients in the surveillance biopsy The median dd-cfDNA in patients with TCMR biopsy was in the for-cause biopsy and in the surveillance biopsy surveillance biopsies rejection had significantly dd-cfDNA than biopsies Supplementary for dd-cfDNA biopsies no rejection, rejection, and TCMR biopsies by diagnostic (0.5% and rejection, A increase of dd-cfDNA was associated with a increase in the risk of rejection with rejection ratio of of AlloSure dd-cfDNA to allograft rejection rejection antibody-mediated donor-derived cell-free pathologies were excluded. data rejection biopsies with in a antibody-mediated donor-derived cell-free rejection. pathologies were excluded. data rejection biopsies with The median of and dd-cfDNA was and over the were with no of kidney between and demonstrated a between the elevation of dd-cfDNA and decline in kidney in dd-cfDNA were associated with decline at years post-transplant. Persistently elevated dd-cfDNA result nearly the risk of a 25% decline in The patients with dd-cfDNA and had no The median antibody was The median of and dd-cfDNA was were in patients with and with of also had histologic evidence of allograft rejection. dd-cfDNA was associated with a nearly elevation in the risk of formation In a increase in the dd-cfDNA was associated with a increase in the risk of of dd-cfDNA values in patients who demonstrated a median increase in dd-cfDNA from prior dd-cfDNA which a median of 91 days days) detection of dd-cfDNA elevated in with A of graft injury as the following BK allograft rejection, or was in dd-cfDNA was measured to days ahead of injury of patients without of events or evidence of kidney allograft injury were the in the median dd-cfDNA in the patients was the median dd-cfDNA for patients with active injury was dd-cfDNA had of The for dd-cfDNA was The median in patients with quiescence was which was not from patients graft injury The for was in Table a dd-cfDNA of 0.5% has a of and of for graft injury. In addition to the the change in dd-cfDNA was associated with allograft injury. A median increase of between is of graft injury of dd-cfDNA as a marker of injury where the absence of injury is as dd-cfDNA donor-derived cell-free is a of patients with injury patients who were in a donor-derived cell-free is a of patients with injury patients who were The large, multicenter, ADMIRAL cohort study the dd-cfDNA both clinically evident and subclinical and TCMR in a real-world of dd-cfDNA dd-cfDNA was significantly more of ongoing graft injury than the current of In elevated dd-cfDNA was associated with and the development of low dd-cfDNA predicted allograft which can the need for protocol The of injury used was not with pathologies allograft patients monitored with dd-cfDNA (AlloSure) had levels of which correlated with both and of injury. using dd-cfDNA is as early identification of injury in post-transplant surveillance is for of and the utility of dd-cfDNA as injury surveillance tool has potential to clinical Patients with had levels of dd-cfDNA were in patients with both clinical rejection and subclinical rejection patients with clinically evident and subclinical TCMR had in dd-cfDNA compared to patients without evidence of rejection. utility of dd-cfDNA in subclinical rejection to be a of injury, where the elevation of allograft injury in absence of clinical changes is compared to dd-cfDNA correlated with TCMR than borderline with the median dd-cfDNA with the of rejection 1A The median dd-cfDNA in patients with borderline TCMR was with injury within this borderline by transplant without evidence of clinical have been with histology significantly more borderline TCMR than and median dd-cfDNA of I. et of donor-derived cell-free DNA with histology and of kidney transplant Scholar The between dd-cfDNA was also associated with clinically events including formation and allograft injury. the need to a from in with elevation of dd-cfDNA above a of to identify graft injury. has been shown by et E. Kumar D. Olaitan O. et al.High levels of dd-cfDNA identify patients with TCMR 1A and borderline allograft rejection at elevated risk of graft injury.Am J Transplant. 2020; 20: 2491-2498Google Scholar While a measured or the increase of from not injury, changes patients have further diagnostic potential or a of the for allograft rejection was determined at the change of dd-cfDNA is important to in with the The ADMIRAL study the between dd-cfDNA and rejection established by the Cell-Free DNA in for in study R.D. Bromberg J.S. Poggio E.D. et al.Cell-free DNA and active rejection in kidney allografts.JASN. 2017; 28: 2221-2232Google Scholar In the a was used to between rejection and no rejection. ADMIRAL of change in dd-cfDNA is also important in the of injury. data a median dd-cfDNA elevation of from a change from quiescence to potential injury. patients this to be elevation from of In et S. S. M. et al.Donor-derived cell-Free DNA in kidney transplant patients to J Transplant. 2020; Scholar demonstrated increase in dd-cfDNA of was associated with the data routine post-transplant surveillance with which both changes and (e.g., increase the to detect injury in a and in the absence of clinical S. J. et of donor-derived cell-free DNA (dd-cfDNA) in kidney transplant patients by Transplant. 2021; Scholar injury is with than in dd-cfDNA can result from or each of which Therefore, the to from clinical and subclinical injury is important and is as dd-cfDNA was correlated with allograft the need for invasive in dd-cfDNA were specific and of the study of allograft injury. Thus, routine monitoring with dd-cfDNA may allow clinicians to risk post-transplant identify with graft injury in need of potential further and without injury who may from in immunosuppression to long-term N. et in renal the 2020; Scholar of has been correlated with allograft survival, in the absence of clinically evident S. K. A. et and clinical of de novo donor specific antibodies after kidney Scholar patients not result in allograft injury. In a study of patients who were after the development of in the absence of clinical rejection, had pathologic evidence of humoral D. P. M. et biopsies in patients with subclinical de novo donor-specific antibodies after kidney transplantation: a 2020; D. et of de novo by assay and antibodies not of subclinical after kidney 2021; Scholar In a study from the patients were monitored with screening and protocol et of protocol biopsies to identify patients with de novo donor-specific antibody at risk for allograft J Transplant. 2017; Scholar a median of of the patients of patients had biopsy evidence of borderline or more acute cellular rejection, and had evidence of active or active at time of the et S. Bromberg J.S. et al.Donor-derived cell-free DNA antibody-mediated rejection in donor specific antibody kidney transplant Direct. 2018; Scholar patients with kidney with clinically biopsies with dd-cfDNA and testing In patients with with the dd-cfDNA was compared with in patients with without and in patients without In this observational of patients not have elevated dd-cfDNA and not to have evidence of antibody-mediated allograft injury. where long-term allograft was not in the setting of A. D. et antibodies and J Med. Scholar data dd-cfDNA may monitoring, by identifying clinical and subclinical in patients with kidney as the injury antibody formation as the of antibodies by the allograft by S. I. on for donor-derived cell-free 2020; Scholar the between dd-cfDNA and further to assess both the of S. I. on for donor-derived cell-free 2020; Scholar and the potential for therapeutic et E. S. A. et clinical using donor-derived cell-free DNA to detect rejection in kidney transplant J Transplant. 2019; Scholar demonstrated histologic of in patients with in dd-cfDNA not have In transplantation by antibodies has been well with et M. D. et antibodies and in kidney transplant recipients with antibody-mediated 2021; Scholar the of in patients with who transplantation by Scholar Thus, the utility of dd-cfDNA in the of to be not performed in this is from patients with the of the use of dd-cfDNA in its may be a tool for S. Kumar D. I. et of donor-derived cell-free DNA in in patients with 2021; 6: Scholar et et between decline and after kidney 2016; Scholar and et et of biomarkers within the years and kidney transplant from clinical in organ 2018; Scholar have a decline in is to of long-term kidney transplant A decline in between years and after kidney transplant is associated with of and allograft et between decline and after kidney 2016; et of biomarkers within the years and kidney transplant from clinical in organ 2018; Scholar ADMIRAL of the between changes in dd-cfDNA and long-term graft levels of dd-cfDNA were correlated with early identification of injury traditional functional changes occur graft The of injury elevated dd-cfDNA may identify patients who from further using from routine clinical the cohort of kidney transplant recipients surveillance with dd-cfDNA to The limitations of this study its real-world with data clinical not of patients across 7 centers and this cohort the transplant clinicians were with to dd-cfDNA measurements and clinical clinical treatment may have the of and the In to dd-cfDNA levels and biopsies not To for allowed biopsies days after dd-cfDNA levels to be as While is subclinical rejection may have prior to this have the study the and not the is a as biopsies were performed and not or on with data this the identify for values in the absence of a in the prediction analysis is also a the this, where allow patients to be their potential is testing is more performed in the of transplant. Therefore, is a and as injury and more this this the routine reflects is to the of monitoring as is dd-cfDNA as part of the clinical of the of dd-cfDNA levels between of the of association between dd-cfDNA and clinical evidence need to be In the evaluation of the clinical and current to be in to the of this of dd-cfDNA in clinical its use in post-transplant histology and traditional surveillance strategies as important prognostic marker and risk-stratification allograft quiescence is to long-term as both and injury to graft further the of on of dd-cfDNA levels in clinical utility as a ahead of clinical of allograft injury, formation of and subclinical rejection. N. et in renal the 2020; Scholar to the provided by dd-cfDNA can be used to guide clinical practice and management of treatment of of rejection, and or the formation of on the of and has support from CareDx, of of and and and the has from has from has and from has and from and in is a of the Board and as a for and has and from and of has from CareDx, and the of has and from the no data from the study on to for of and the and for support in of the and or the and in the and or the and in the and data and to and in the the with Supplementary Donor-derived cell-free DNA in kidney transplantation: and potential et elevated donor-derived cell-free DNA on measurements performed for both surveillance and of kidney allograft was associated with rejection, de novo donor-specific and decline in estimated glomerular filtration data donor-derived cell-free DNA may be a of kidney allograft In this the of donor-derived cell-free DNA for allograft surveillance and potential

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