Can Blood Gene Expression Profile and Donor-Derived Cellfree DNA Guide Postrejection Management among Kidney Transplant Recipients?

Since the origins of kidney transplantation, rejection has been a significant challenge. Initially, the rejection mechanisms were poorly understood, leading to various experimental strategies, such as total body irradiation, 6-mercaptopurine, and azathioprine as solo agents, followed by the introduction of steroids in the 1960s.1 The development of cytotoxic crossmatching and the discovery of cyclosporine in the 1970s marked a new era in maintaining patients on multidrug regimens for immunosuppression. Despite these advancements, allograft survival remained poor because of acute rejection. With modern immunosuppressive therapies and a deeper understanding of the pathophysiology of rejection, rejection rates have significantly decreased—from 50% to 60% in the 1980s to 9% or less in 2021, depending on age and the use of lymphocyte-depleting agents.1,2 In addition, the median long-term allograft survival has improved for both deceased and living donors since the 1990s (from 8.2 to 11.7 years for deceased donors and 12.1 to 19.2 years for living donors).3 This progress is due to multiple interventions, including enhanced pretransplant and post-transplant management, improved HLA testing, and increased living donor rates through paired kidney exchange programs, even with the increasing complexity of recipients and less HLA matching.3 Despite these advances, acute rejection within the first year is still associated with worse graft outcomes, including lower eGFR and increased mortality due to cardiovascular disease and cancer.4 Significant graft failure after an acute rejection episode is often due to death with a functioning graft, or death-censored graft failure, mainly due to transplant glomerulopathy, particularly in cases with persistent rejection. Kidney biopsy is the gold standard test for diagnosing acute rejection. However, advances have now been made in noninvasive diagnostics biomarkers, such as donor-derived cellfree DNA (dd-cfDNA), which is more sensitive to acute antibody-mediated rejection (AMR), although its efficacy in detecting various degrees of T-cell–mediated rejection (TCMR) remains variable.5 Another study highlighted the use of gene expression profiles (GEP) in monitoring for acute rejection, with a reported negative predictive value (NPV) of 88%.6 Combining dd-cfDNA with GEP may further improve the diagnostic accuracy of rejection.7 Persistent rejection after an initial episode is a significant concern, occurring in up to 37% of cases.8 Diagnosing persistent rejection can be challenging because many transplant centers rely on clinical parameters, like serum creatinine levels, rather than repeat biopsies, and data on the use of noninvasive biomarkers to guide treatment response are limited.9 This is where this study published in this issue of Kidney360 by Heilman et al. gains importance in advancing our understanding of persistent rejection and the role of noninvasive biomarkers.10 In this study, patients from the CTOT-08 trial were analyzed by Heilman and colleagues to assess the role of noninvasive biomarkers in detecting persistent rejection. The analysis included patients who had paired biopsy samples within 9 months of their initial rejection with a subanalysis of repeat biopsies within 90 days of rejection, which is more likely to be clinically actionable. Both initial and persistent rejection were confirmed through biopsy, and the diagnostic performance of GEP and/or dd-cfDNA was evaluated. The study included 58 patients with 64 index rejections, with almost equal representation between AMR/mixed rejection and TCMR. Follow-up biopsies were available in 44 patients. Most of the follow-up biopsies were part of surveillance biopsies and were clinically stable. Persistent rejection was found in 61% of follow-up biopsies. GEP had good positive predictive value (PPV) of 79% with a NPV of 54% for persistent rejection within 9 months. Similarly, dd-cfDNA had a PPV and NPV of 88% and 56%, respectively. When combined together, they would correctly identify whether persistent rejection was present or not in 76% of the cases. Interestingly, GEP was more likely to be positive in TCMR cases, while dd-cfDNA was more sensitive to AMR/mixed rejection, highlighting the complementary roles of these tests in identifying different types of rejection. In the subanalysis cohort with repeat biopsies within 90 days, they retained their PPV and NPV, but were limited by sample size. Remarkably, in clinically stable patients, both GEP and dd-cfDNA were positive in persistent rejection and negative with resolution of rejection. These findings suggest that noninvasive testing could play a significant role in monitoring clinically stable patients with a history of rejection. The study also checked GEP and dd-cfDNA at different time points between the initial biopsy for rejection and repeat biopsy. Again, the sample size was limited, but they observed a decreasing trend in biomarker levels corresponding with effective therapy, whereas persistent rejection was indicated by biomarker levels exceeding threshold test characteristics. This suggests a potential role for monitoring GEP and dd-cfDNA postrejection to identify patients who may benefit from noninvasive testing to rule out persistent rejection. However, questions remain about the optimal frequency of testing and the timing for repeat biopsies based on noninvasive biomarkers. Notably, there was no difference in graft survival between patients with resolved versus persistent rejection. Where Do We Go from Here? In the current era of kidney transplantation, striking a balance between optimal immunosuppression to prevent rejection and avoiding the risks of overimmunosuppression, such as opportunistic infections, is crucial. Unfortunately, we lack a precise tool—a so-called Immuno-Meter—that allows us to dial immunosuppression needed for each patient on the basis of their clinical status. Traditionally, allograft biopsy has been the standard for diagnosing rejection. However, with the advent of noninvasive biomarkers, the landscape of kidney transplantation is evolving. These biomarkers offer the potential for surveillance monitoring to detect rejection or to assess the efficacy of treatment after a rejection episode, particularly in identifying patients at risk for persistent rejection. While promising, caution is needed when interpreting these markers beyond the first-year post-transplant because new data are still emerging, and they may be elevated in patients without rejection. In addition, providers may need to use a combination of tests, guided by the pretest probability of the type of rejection. However, access to these tests can be limited by insurance coverage, especially in light of recent Centers for Medicare & Medicaid Services guidelines on reimbursement. In addition, several critical questions remain unanswered, and we have summarized some of the potential solutions on the basis of our clinical practice and experiences in Table 1. Table 1 - Unanswered questions with some potential solutions Question Potential Solutiona What should be the frequency and duration of testing in clinically stable patients? Monthly starting at month 2 for the first 6 mo, then at 9, 12 mo, and then annually or clinically indicated Once elevated then how often should these biomarkers be repeated 1. Clinically stable • Low PreTest probability: Repeat in 1 mo, if stable, then plan as above in clinically stable patients • High PreTest probability: Repeat in 2 wk, if stable, then plan as above in clinically stable patients2. For causeSuggest biopsy, if elevated When should a biopsy be triggered? Suggest biopsy once the test threshold is met irrespective of clinical status How should we manage immunosuppression in participants on immunotherapy for cancer? Establish baseline (if not done before) before starting immunotherapy, then monthly for as long as on immunotherapy Can these biomarkers guide immunosuppressive drug dosing in clinically stable patients at risk for complications or during recovery from infections like BK or CMV, with or without DSA monitoring? Possibly may adjust immunosuppression dosing based on biomarkers trends Change in immunosuppressive regimen? Establish a baseline (if not done before) before conversion, then follow trends monthly for first 3 mo What about programs with surveillance biopsies? Establish baseline and follow trends with surveillance biopsies, and repeat as clinically indicated CMV, cytomegalovirus; DSA, donor specific antibody.aPotential solutions are solely based on the author's clinical experience and practices. The next chapter in kidney transplantation is certainly exciting as we develop newer therapies in the field of rejection, especially AMR, understand the role of Torque teno virus in immunosuppression, and understand the utility of noninvasive biomarkers in different settings. This study helps in furthering the role of noninvasive biomarkers after initial rejection within the first year and will need to be replicated in larger studies. It raises the potential of using combining biomarkers to diagnose different types of rejection, after rejection therapy, and rule out persistent rejection.