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Despite advances in transplant immunosuppression, long-term renal allograft outcomes remain suboptimal because of the occurrence of rejection, recurrent disease, and interstitial fibrosis with tubular atrophy. This is largely due to limitations in our understanding of allogeneic processes coupled with inadequate surveillance strategies. The concept of donor-derived cell-free DNA as a signal of allograft stress has therefore rapidly been adopted as a noninvasive monitoring tool. Refining it for effective clinical use, however, remains an ongoing effort. Furthermore, its potential to unravel new insights in alloimmunity through novel molecular techniques is yet to be realized. This review herein summarizes current knowledge and active endeavors to optimize cell-free DNA-based diagnostic techniques for clinical use in kidney transplantation. In addition, the integration of DNA methylation and microRNA may unveil new epigenetic signatures of allograft health and is also explored in this report. Directing research initiatives toward these aspirations will not only improve diagnostic precision but may foster new paradigms in transplant immunobiology.

Author(s): Amara, Dominic; Wisel, Steven A; Braun, Hillary J; Collisson, Eric A; Friedlander, Terence; Worner, Giulia; Roll, Garret R; Hirose, Ryutaro; Stock, Peter G | Abstract: Stopping immunosuppression in a transplant patient with donor-derived malignancy offers the theoretical benefit that reconstitution of the patient's immune system will allow "rejection" of the malignancy, as the malignancy also originates from allogeneic tissue. However, this option exists with the caveat that the patient's allograft(s) will likely be rejected too. In simultaneous pancreas-kidney (SPK) recipients, the normal continued functioning and possible absence of malignancy in either the unaffected kidney or pancreas further complicate this decision.MethodsThe charts of 3 patients with donor-derived metastatic malignancies after SPK were retrospectively reviewed in detail. We provide treatment and management recommendations based on successful outcomes and a review of the existing literature.ResultsConsistent with a broad review of the literature, in all 3 cases, complete immunosuppression cessation, removal of both grafts, and in 1 case treatment with an immune checkpoint inhibitor to augment the immune response was successful. One patient is doing well 1 year after successfully undergoing kidney retransplantation, while a second patient is active on the waitlist for SPK retransplantation after no evidence of metastatic disease for 2 years.ConclusionThe successful management of metastatic donor-derived malignancies requires allograft removal, immunosuppression cessation, and adjuvant therapy that includes occasional use of checkpoint inhibitors to augment the immune response.

Background. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive plasma biomarker to evaluate for transplant allograft rejection. The relationship between infectious complications in kidney allografts and dd-cfDNA has received cursory attention in prior publications. Methods. Retrospective review of all renal transplant recipients who underwent dd-cfDNA testing between November 2017 and August 2019. Results. We report on 7 cases in whom infections affecting the transplanted kidney were associated with elevation in dd-cfDNA without concomitant rejection or elevation in serum creatinine. Five patients had BK viremia, and 2 patients had urinary tract infection associated with elevated dd-cfDNA levels. Conclusions. These observations suggest that elevations in dd-cfDNA are not specific to kidney allograft rejection and can be associated with infections affecting the transplanted kidney. This biomarker may be valuable in evaluating infectious complications of kidney allografts.

Supplemental Digital Content is available in the text., Background. Higher Banff inflammation and chronicity scores on kidney transplant biopsies are associated with poorer graft survival, although histology alone has limitations in predicting outcomes. We investigated if integrating donor-derived cell-free DNA (dd-cfDNA, Allosure; CareDx, Inc.) with Banff biopsy scores into a predictive model for estimated glomerular filtration rate over time can improve prognostic assessment versus histology alone. Methods. We identified 180 kidney transplant patients with dd-cfDNA assessed within 1 mo of biopsy. Using linear mixed–effects models, a prediction model of Banff histology scores and dd-cfDNA on estimated glomerular filtration rate over time was derived. Nested models were compared using the likelihood-ratio test, Akaike Information Criterion, and Bayesian Information Criterion to assess if inclusion of dd-cfDNA into a model consisting of Banff biopsy scores would improve model fit. Results. Univariate models identified significant covariate-by-time interactions for cg = 3 versus

Background. Donor-derived cell-free DNA (dd-cfDNA) is increasingly recognized as a valuable biomarker for acute transplant injury, with possible indications in the detection of cellular or humoral rejection and the guidance of immunosuppressive therapy. There is an ongoing debate on whether relative or absolute quantification of dd-cfDNA is more reliable for the detection of acute transplant injury. Methods. We retrospectively reviewed all 22 kidney transplant recipients who underwent dd-cfDNA measurements (percentage and absolute) between April 2020 and April 2021 at our institution. Of these, 9 (41%) showed discrepancies between absolute (cutoff: 50 copies/mL) and relative (cutoff: 0.5%) quantification in at least 1 dd-cfDNA measurement. Results. We report on 9 of 22 cases with discrepancies in relative and absolute quantification of dd-cfDNA, which were predominantly late posttransplant patients. We found bacterial and viral infections, as well as low leukocyte count from chronic myeloid leukaemia treatment, to be reasons for variability in total cell-free DNA (cfDNA), leading to inter- and intraindividual variability in relative dd-cfDNA quantification. When correlating dd-cfDNA quantification and biopsy results, as well as clinical course, our data indicate that relying solely on relative dd-cfDNA can lead to false-negative and false-positive results. Conclusions. In summary, these cases argue that absolute quantification of dd-cfDNA is better suited in patients with underlying conditions affecting total cfDNA levels and suggest using both absolute and relative dd-cfDNA together for higher reliability and interindividual comparability in the clinical setting. Especially for patients with chronic active antibody–mediated rejection, further studies on the use of dd-cfDNA are desirable.

Background Donor-derived cell-free DNA (dd-cfDNA) has generated interest as a biomarker for kidney injury including transplant (KT) rejection. It is possible that the KT biopsy procedure can cause the release of dd-cfDNA, therefore affecting the reliability of this assay in the postbiopsy period. We evaluated the effect of KT biopsy on the kinetics of dd-cfDNA. Methods We conducted a single-arm prospective study. Samples were collected from 16 adult KT recipients undergoing KT biopsy. All participants had samples drawn within 8 h before the biopsy (prebiopsy), within 20 min (hour 0), 2 h (hour 2), and 24-48 h (hours 24-48) after the biopsy. We evaluated the change in dd-cfDNA from the prebiopsy time point to the following 3 time points after the biopsy. Results At hour 0 and hour 2, there was a significantly larger log dd-cfDNA mean score compared with the prebiopsy score (least square mean estimate 0.4 [0.17-0.63] and 0.39 [0.09-0.68], respectively). By 24-28 h postbiopsy, there was no significant difference in log dd-cfDNA mean score compared with the prebiopsy score (least square mean estimate -0.21 [-0.6 to 0.19]). Conclusions Mechanical injury from a KT biopsy can transiently increase circulating dd-cfDNA. The increase resolves by 24-48 h after the biopsy. Providers should wait 48 h postbiopsy to obtain dd-cfDNA levels to establish the correct baseline to be used for monitoring.

Background. Approximately 15% of kidney transplant recipients (KTRs) develop BK viremia (BKV), with 1%–10% developing BK virus-associated nephropathy (BKVAN), which histologically resembles rejection. The Diagnosing Acute Rejection in Kidney Transplant Recipients (DART) study showed that donor-derived cell-free DNA (dd-cfDNA) levels 10 000 copies/mL. Results. Of 102 participants with biopsies, 10 patients with BKV and BKVAN had paired dd-cfDNA, and viral loads available for analysis. Patients diagnosed with BKV and BKVAN had a median dd-cfDNA of 0.58% (IQR 0.43–1.15) and 3.38% (IQR 2.3–4.56, P = 0.001), respectively. dd-cfDNA titers correlated with BK PCR viral loads (R = 0.874, P = 0.01) and the presence of histologic evidence of BKVAN (100% sensitivity, 50% specificity). Five of 7 patients with BKVAN, but only 2 of 7 with BKV, had biopsies meeting Banff criteria for T-cell–mediated rejection. Median dd-cfDNA in nonrejection patients was 0.43% versus 2.84% in rejection patients (P = 0.001). Conclusion. Higher dd-cfDNA titers were associated with higher BK viral loads, biopsy-diagnosed BVAN, as well histologic changes meeting Banff criteria for as T-cell–mediated rejection. dd-cfDNA may be a useful noninvasive test to assess for progression of BKV to BKVAN.

Background. Elevated levels of donor-derived cell-free DNA (dd-cfDNA) in the plasma of renal allograft recipients indicates organ injury and an increased probability of active rejection. Donor-specific antibodies (DSA) to HLA antigens are associated with risk of antibody-mediated rejection (ABMR). This study assessed the combined use of dd-cfDNA and DSA testing to diagnose active ABMR. Methods. Donor-derived cell-free DNA was assayed in 90 blood samples with paired DSA and clinically indicated biopsies from 87 kidney transplant patients. Sixteen cases met criteria for active ABMR. Performance characteristics of dd-cfDNA for diagnosis of active ABMR were determined for samples with prior or current positive DSA (DSA+, n = 33). Results. The median level of dd-cfDNA (2.9%) in DSA+ patients with active ABMR was significantly higher than the median level (0.34%) in DSA+ patients without ABMR (P < 0.001). The median level of dd-cfDNA in DSA− patients was 0.29%. The positive predictive value of dd-cfDNA (at 1%) to detect active ABMR in DSA+ patients was 81%, whereas the negative predictive value was 83%. The positive predictive value for DSA+ alone was 48%. Conclusions. The combined use of dd-cfDNA and DSA testing may improve the noninvasive diagnosis of active ABMR in kidney transplant patients. Patients with dd-cfDNA+/ DSA+ results have a high probability of active ABMR.

Gastrointestinal neuroendocrine tumors (NET) are rare but the age-adjusted incidence in the United States has increased, possibly due to improved radiographic and endoscopic detection. In advanced NET, hepatic metastases are common. Orthotopic liver transplant (OLT) is currently considered an acceptable therapy for selected patients with limited hepatic disease or liver metastases where complete resection is thought to have curative intent. The development of NET of donor origin is very uncommon after organ transplant, and it is unclear if the same treatment strategies applied to hepatic NET would also be efficacious after OLT. Here, we describe a unique case of an OLT recipient with a donor-derived NET that was treated with redo OLT as the primary therapy. The donor-derived NET recurred in the recipient's second liver allograft suggesting an extrahepatic reservoir. This case describes the natural history of such a rare event. Here, we highlight the treatment options for hepatic NET and challenge the role of OLT for a donor-derived hepatic NET.