Cell-free DNA for the detection of kidney allograft rejection.

Donor-derived cell-free DNA (dd-cfDNA) is an emerging noninvasive biomarker that has the potential to detect allograft injury. The capacity of dd-cfDNA to detect kidney allograft rejection and its added clinical value beyond standard of care patient monitoring is unclear. We enrolled 2,882 kidney allograft recipients from 14 transplantation centers in Europe and the United States in an observational population-based study. The primary analysis included 1,134 patients. Donor-derived cell-free DNA levels strongly correlated with allograft rejection, including antibody-mediated rejection (P < 0.0001), T cell-mediated rejection (P < 0.0001) and mixed rejection (P < 0.0001). In multivariable analysis, circulating dd-cfDNA was significantly associated with allograft rejection (odds ratio 2.275; 95% confidence interval (CI) 1.902-2.739; P < 0.0001) independently of standard of care patient monitoring parameters. The inclusion of dd-cfDNA to a standard of care prediction model showed improved discrimination (area under the curve 0.777 (95% CI 0.741-0.811) to 0.821 (95% CI 0.784-0.852); P = 0.0011) and calibration. These results were confirmed in the external validation cohorts (n = 1,748) including a cohort of African American patients (n = 439). Finally, dd-cfDNA showed high predictive value to detect subclinical rejection in stable patients. Our study provides insights on the potential value of assessing dd-cfDNA, in addition to standard of care monitoring, to improve the detection of allograft rejection. ClinicalTrials.gov registration: NCT05995379 .

Management and Outcomes of Kidney Transplant Candidates With Severe Pulmonary Hypertension: A Single-center Strategy and Experience.

Background: Severe pulmonary hypertension (PH) is associated with high mortality posttransplant and thus is considered a contraindication to kidney transplantation. In this study, we describe the pretransplant management and posttransplant outcomes in patients with severe PH using a multidisciplinary approach. Methods: Between 11 of 2013 and 8 of 2022, we identified all patients with severe PH on initial pretransplant workup who underwent ultrafiltration (UF) or medical therapy for PH before transplant. Posttransplant we evaluated the perioperative course, renal function, graft, and patient survival. We compared survival to those who remained waitlisted or were delisted. Results: Three-two patients (mean age = 55.03 ± 10.22 y) diagnosed with severe PH on pretransplant screening echocardiogram. Thirty patients (94%) were subjected to a median of 4 (range, 3-8) UF sessions with an average weight loss of 4.33 ± 2.6 kg. Repeat assessment of PH revealed a decline in mean pulmonary artery systolic pressure from 67 ± 12 mm Hg to 43 ± 13 mm Hg (P < 0.0001). Seventeen patients (53%) received a kidney transplant. The mean estimated Glomerular Filtration Rate at 3, 6, 9, and 12 mo was 72 ± 27, 72 ± 28, 75 ± 29, and 75 ± 29 mL/min/1.73 m2. Among, those who underwent transplantation both graft and patient survival was 100% at 1-y posttransplant. Overall, since the UF intervention, at a median follow-up of 88 ± 12 mo those transplanted had a patient survival of 88% while those who remained on dialysis had a survival of 53% (P = 0.0003). Conclusion: In this single-center study, we report postcapillary PH can be a significant contributor to elevations in pulmonary artery systolic pressure. Using a multidisciplinary approach, PH can improve with volume removal and phosphodiesterase 5 inhibitors therapy leading to a successful posttransplant outcome.

Right versus left fully robotic live donor nephrectomy and open kidney transplantation: Does the laterality of the donor kidney really matter?

Objective: Robotic-assisted live donor nephrectomy (LDN) is being gradually adopted across transplant centers. The left donor kidney is preferred over right due to anatomical factors and ease of procurement. We aimed to study donor and recipient outcomes after robotic procurement and subsequent open implantation of right and left kidneys. Methods: All fully robotic LDNs and their corresponding open kidney transplants performed at our center between February 2016 and December 2021 were retrospectively analyzed. Results: Out of 196 robotic LDN (49 [right] vs. 147 [left]), 10 (5.1%) donors had intra-operative events (6.1% [right] vs. 4.8% [left], p=0.71). None of the LDN required conversion to open surgery. The operative times were comparable for the two groups. Nausea (13.3%) was the most common post-operative complication. There was no mortality in either LDN group. Herein, we report our outcomes on 156 recipients (39 right and 117 left allografts) excluding robotic implants, exports, and pediatric recipients. There were no significant differences between right and left kidney recipients with respect to 1-year post-transplant patient survival (100.0% vs. 98.1%, p=0.45) or graft survival (93.9% vs. 97.1%, p=0.11), or delayed graft function (7.7% vs. 5.1%, p=0.55). Conclusion: Non-hand-assisted robotic live donor nephrectomies can be safely performed with excellent outcomes. Right LDN was not associated with higher incidence of complications compared to left LDN. Open implantation of robotically procured right renal allografts was not associated with higher risk of recipient complications.

Correlation of Donor-derived Cell-free DNA With Histology and Molecular Diagnoses of Kidney Transplant Biopsies.

BACKGROUND: Circulating donor-derived cell-free DNA (cfDNA), a minimally invasive diagnostic tool for kidney transplant rejection, was validated using traditional histology. The molecular microscope diagnostic system (MMDx) tissue gene expression platform may provide increased precision to traditional histology. METHODS: In this single-center prospective study of 208 biopsies (median = 5.8 mo) posttransplant, we report on the calibration of cfDNA with simultaneous biopsy assessments using MMDx and histology by area under the curve (AUC) analyses for optimal criterion, as well as for, previously published cfDNA cutoffs ≤ 0.21% to "rule-out" rejection and ≥1% to "rule-in" rejection. RESULTS: There were significant discrepancies between histology and MMDx, with MMDx identifying more antibody-mediated rejection (65; 31%) than histology (43; 21%); the opposite was true for T cell-mediated rejection [TCMR; histology: 27 (13%) versus MMDx: 13 (6%)]. Most of the TCMR discrepancies were seen for histologic borderline/1A TCMR. AUC for cfDNA and prediction of rejection were slightly better with MMDx (AUC = 0.80; 95% CI: 0.74-0.86) versus histology (AUC = 0.75; 95% CI: 0.69-0.81). A cfDNA ≤ 0.21% had similar sensitivity (~91%) to "rule-out" rejection by histology and MMDx. Specificity was slightly higher with MMDx (92%) compared with histology (85%) to "rule-in" rejection using cfDNA criterion ≥1%. Strong positive quantitative correlations were observed between cfDNA scores and molecular acute kidney injury for both "rejection" and "nonrejection" biopsies. CONCLUSIONS: Molecular diagnostics using tissue gene expression and blood-based donor-derived cell-free DNA may add precision to some cases of traditional histology. The positive correlation of cfDNA with molecular acute kidney injury suggests a dose-dependent association with tissue injury irrespective of rejection characteristics.

Non-Invasive Assessment of Liver Fibrosis and Steatosis in End-Stage Renal Disease Patients Undergoing Renal Transplant Evaluation.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has an increased prevalence in end-stage renal disease (ESRD) due to similar risk factors. The aim of this study was to assess non-invasive testing including transient elastography (TE) for liver stiffness (LS), controlled attenuated parameter (CAP) for steatosis, Fibrosis-4 (FIB-4) score, aspartate aminotransferase (AST) to platelet ratio index (APRI) and NAFLD fibrosis score (NFS), for evaluation of NAFLD along with advanced fibrosis (AF) in patients with ESRD undergoing renal transplant evaluation. METHODS: Data were retrospectively collected within 12 weeks of TE. Primary outcomes were AF, defined by LS ≥ 9 kPa compared to APRI > 1.5, FIB-4 > 2.67, and NFS of 0.675, and ≥ 5% steatosis by CAP ≥ 263 dB/m compared to liver histology when available. RESULTS: A total of 171 patients were evaluated: mean age 56, 65% male, 36% obese, 47% had diabetes, 96% hypertension, and 56% dyslipidemia. Mean LS was 6.5 kPa with 21% having AF. Mean CAP was 232 dB/m, with 25% having steatosis. Those with AF were older with higher NFS. Those with steatosis were obese and had diabetes without higher LS or fibrosis scores. Only NFS was associated with LS ≥ 9 kPa. In those with liver histology, AF was associated with LS ≥ 9 kPa but not with APRI, FIB-4, or NFS. CONCLUSIONS: Despite normal liver enzymes, non-invasive assessment via TE in ESRD patients exhibited high prevalence of AF and steatosis not detected by APRI or FIB-4 scores. This high prevalence was secondary to the common risk factors such as obesity and diabetes, among patients with NAFLD and ESRD.

A 2-fold Approach to Polyoma Virus (BK) Nephropathy in Kidney Transplants: Distinguishing Direct Virus Effects From Cognate T Cell-mediated Inflammation.

BACKGROUND: BK nephropathy (BKN) in kidney transplants diagnosed by histology is challenging because it involves damage from both virus activity and cognate T cell-mediated inflammation, directed against alloantigens (rejection) or viral antigens. The present study of indication biopsies from the Integrated Diagnostic System in the International Collaborative Microarray Study Extension study measured major capsid viral protein 2 (VP2) mRNA to assess virus activity and a T cell-mediated rejection (TCMR) classifier to assess cognate T cell-mediated inflammation. METHODS: Biopsies were assessed by local standard-of-care histology and by genome-wide microarrays and Molecular Microscope Diagnostic System (MMDx) algorithms to detect rejection and injury. In a subset of 102 biopsies (50 BKN and 52 BKN-negative biopsies with various abnormalities), we measured VP2 transcripts by real-time polymerase chain reaction. RESULTS: BKN was diagnosed in 55 of 1679 biopsies; 30 had cognate T cell-mediated activity assessed by by MMDx and TCMR lesions, but only 3 of 30 were histologically diagnosed as TCMR. We developed a BKN probability classifier that predicted histologic BKN (area under the curve = 0.82). Virus activity (VP2 expression) was highly selective for BKN (area under the curve = 0.94) and correlated with acute injury, atrophy-fibrosis, macrophage activation, and the BKN classifier, but not with the TCMR classifier. BKN with molecular TCMR had more tubulitis and inflammation than BKN without molecular TCMR. In 5 BKN cases with second biopsies, VP2 mRNA decreased in second biopsies, whereas in 4 of 5 TCMR classifiers, scores increased. Genes and pathways associated with BKN and VP2 mRNA were similar, reflecting injury, inflammation, and macrophage activation but none was selective for BKN. CONCLUSIONS: Risk-benefit decisions in BKN may be assisted by quantitative assessment of the 2 major pathologic processes, virus activity and cognate T cell-mediated inflammation.

Clinical Rationale for a Routine Testing Schedule Using Donor-Derived Cell-Free DNA After Kidney Transplantation.

Kidney transplant recipients require meticulous clinical and laboratory surveillance to monitor allograft health. Conventional biomarkers, including serum creatinine and proteinuria, are lagging indicators of allograft injury, often rising only after significant and potentially irreversible damage has occurred. Immunosuppressive medication levels can be followed, but their utility is largely limited to guiding dosing changes or assessing adherence. Kidney biopsy, the criterion standard for the diagnosis and characterization of injury, is invasive and thus poorly suited for frequent surveillance. Donor-derived cell-free DNA (dd-cfDNA) is a sensitive, noninvasive, leading indicator of allograft injury, which offers the opportunity for expedited intervention and can improve long-term allograft outcomes. This article describes the clinical rationale for a routine testing schedule utilizing dd-cfDNA surveillance at months 1, 2, 3, 4, 6, 9, and 12 during the first year following kidney transplantation and quarterly thereafter. These time points coincide with major immunologic transition points after transplantation and provide clinicians with molecular information to help inform decision making.

Outcomes of short-duration antiviral prophylaxis for hepatitis C positive donor kidney transplants.

Trials describing 4- to 12-week courses of direct-acting antiviral drugs (DAAs) to treat hepatitis C virus (HCV) transmission from infected donors to uninfected kidney transplant recipients (D+/R- transplants) may be limited in "real-world" application by costs and delayed access to DAAs. We previously reported HCV transmission of 13% among D+/R- transplants with 2- to 4-day pangenotypic sofosbuvir/velpatasvir (SOF/VEL) perioperative prophylaxis, where one patient with HCV transmission was a nonresponder to first-line full-course DAA. Here, we report new data with a 7-day prophylaxis protocol (N = 50), as well as cumulative treatment and outcome data on all HCV D+/R- transplants (N = 102). Overall, nine patients (9/102; 9%; 95% CI: 5%-16%) developed HCV transmission, with a significant decline noted in the 7-day group (2/50; 4%; 95% CI: 0%-13%) compared with 2- to 4-day prophylaxis (7/52; 13%; 95% CI: 5%-25%). All patients with HCV transmission achieved sustained virologic response post full-course therapy (including one nonresponder from initial trial). A 1:1 matched analysis (N = 102) with contemporary HCV D-/R- transplants (controls) showed that although the pretransplant wait time was significantly shorter for D+/R- compared with D-/R- (mean: 1.8 vs. 4.4 years; p < .001), there were no differences in infections, rejection, development of de novo donor-specific antibody, or transplant outcomes up to 6 months of transplant.

Early initiation of sodium-glucose linked transporter inhibitors (SGLT-2i) and associated metabolic and electrolyte outcomes in diabetic kidney transplant recipients.

There is a paucity of data on the use of SGLT2 inhibitors on outcomes in kidney transplant recipients. There may be concern in initiating these agents, especially within the first year post-transplant when renal function is more labile and immunosuppression more intense, due to a presumed high risk of urinary infections and acute kidney injury. This is a retrospective study on 50 kidney transplant recipients, half of whom were started on therapy within the first year of transplant. Over a follow-up period of 6 months, overall patients had a statistically significant improvement in weight by -2.95 kg [SD 3.54, P = <.0001 (CI: 3.53, 1.50)] as well as hypomagnesemia 0.13 [SD 1.73, P = .0004 (CI: 0.06, 0.20)]. Overall insulin usage declined by -3.7 units [SD 22.8, P = .17]. 14% of patients had at least one urinary tract infection although this rate is not different (~20%) than that reported historically in this high-risk population.

Donor-derived Cell-free DNA in Solid-organ Transplant Diagnostics: Indications, Limitations, and Future Directions.

The last few years have seen an explosion in clinical research focusing on the use of donor-derived cell-free DNA (dd-cfDNA) in solid-organ transplants (SOT). Although most of the literature published so far focuses on kidney transplants, there are several recent as well as ongoing research studies on heart, lung, pancreas, and liver transplants. Though initially studied as a noninvasive means of identifying subclinical or acute rejection in SOT, it is rapidly becoming clear that instead of being a specific marker for allograft rejection, dd-cfDNA is more appropriately described as a marker of severe injury, although the most common cause of this injury is allograft rejection. Multiple studies in kidney transplants have shown that although sensitivity for the diagnosis of antibody-mediated rejection is excellent, it is less so for T-cell-mediated rejection. It is possible that combining dd-cfDNA with other novel urine- or blood-based biomarkers may increase the sensitivity for the diagnosis of rejection. Irrespective of the cause, though, elevated dd-cfDNA seems to portend adverse allograft prognosis and formation of de novo donor-specific antibody. Although current data do not lend themselves to a clear conclusion, ongoing studies may reveal the utility of serial surveillance for the management of SOT as following levels of dd-cfDNA over time may provide windows of opportunity to intervene early and before irreversible allograft injury. Finally, cost-effectiveness studies will be needed to guide the ideal incorporation of dd-cfDNA into routine clinical practice.

Letermovir prophylaxis in solid organ transplant-Assessing CMV breakthrough and tacrolimus drug interaction.

BACKGROUND: Valganciclovir is the preferred drug for cytomegalovirus (CMV) prophylaxis in solid organ transplantation. A limitation to its use is profound myelosuppression. Letermovir is a new agent approved for CMV prophylaxis in hematopoietic stem cell transplantation and is associated with less toxicity. This study aims to assess the effectiveness and safety of letermovir in solid organ transplantation. METHODS: A single-center, matched cohort study was performed on 31 transplant recipients who were converted from valganciclovir to letermovir from November 2017 to June 2020. The primary outcome was the rate of CMV breakthrough infections while on prophylaxis. Secondary outcomes included rate of leukopenia, doses of immunosuppression, rejection, non-CMV infection, and renal function. Statistical analyses of continuous variables included the student's t-test, ANOVA test, and Wilcoxon Signed Rank test. Categorical data were analyzed with chi-square test and Fisher's Exact test. RESULTS: There was no difference in the rate of CMV breakthrough between patients on letermovir (8.7%) and valganciclovir (13.5%), (P = .7097). After conversion to letermovir, patients required lower tacrolimus doses at -3.34 mg (SD-1.3, P = .0273), between conversion and day 7. Transplant Infectious Disease The median difference in tacrolimus trough concentrations from conversion to day seven was 9.1 ng/ml [4.9, 16.95] (P = .0002). Leukopenia improved by 1.8 109/L [1.08, 4.85] (P < .0001). CONCLUSIONS: Patients converted from valganciclovir to letermovir did not show an increased rate of CMV breakthrough compared to a historical, matched cohort of patients remaining on valganciclovir. A significant drug interaction was noted with tacrolimus, leading to a recommendation to reduce the dose by 40-50% upon initiation of letermovir.

Impact of Belatacept Conversion on Renal Function, Histology, and Gene Expression in Kidney Transplant Patients With Chronic Active Antibody-mediated Rejection.

BACKGROUND: Here, we present our initial experience with a prospective protocol of belatacept conversion in patients with chronic active antibody-mediated rejection (caAMR) and a high degree of chronicity at the time of diagnosis. METHODS: We converted 19 patients (mean age, 45 ± 12 y) with biopsy-proven caAMR from tacrolimus to belatacept at a median of 44 months post-kidney transplant. RESULTS: At a median of 29 months (interquartile range, 16-46 mo) postconversion, death-censored graft and patient survivals were 89% and 95%, respectively. When compared to a 1:2 propensity-matched control cohort from the INSERM U970 registry maintained on calcineurin inhibitor, the belatacept group had progressive improvement (P = 0.02) in estimated glomerular filtration rate from a mean of 33.9 ± 10 at baseline to 37.8 ± 13 at 6 months and 38.5 ± 12 mL/min/1.73 m2 at 12 months postconversion, as compared to a steady decline noted in the controls (36.2 [baseline] → 33.1 [6 mo] → 32.7 mL/min/1.73 m2 [12 mo] of follow-up). A paired histologic comparison of preconversion and postconversion (performed at median 9.5 mo postconversion) biopsies showed no worsening in microvascular inflammation or chronicity. The paired tissue gene expression analysis showed improved mean total rejection score (0.68 ± 0.26-0.56 ± 0.33; P = 0.02) and a trend toward improved antibody-mediated rejection score (0.64 ± 0.34-0.56 ± 0.39; P = 0.06). CONCLUSIONS: Here, we report that in patients diagnosed with caAMR who were not subjected to intensive salvage immunosuppressive therapies, isolated belatacept conversion alone was associated with stabilization in renal function. These results are bolstered by molecular evidence of improved inflammation.

Coronavirus Disease 2019 Viremia, Serologies, and Clinical Course in a Case Series of Transplant Recipients.

Here we report a single-center cohort of 6 patients (4 kidney only, and 2 simultaneous liver/kidney transplants) diagnosed with COVID-19 at a median of 1.9 years (range = 0.2-9.3 years) post transplant. Five (of 6) patients required inpatient admission, 2 patients (mortality = 33%) died. Among those with mortality, an increased concentration of inflammatory biomarkers (interleukin-6 and C-reactive protein) was noted with a lack of response to interleukin-6 blockade, remdesivir, and/or convalescent plasma. None of the kidney-only transplants (4/6; 67%) had elevation in plasma donor-derived cell-free DNA above the previously published cut-off of 1%, suggesting absence of significant allo-immune injury. Four (of 5) admitted patients had detectable SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) in blood on samples obtained at/during hospitalization. Of the 4 discharged patients, 2 patients with undetectable virus on repeat nasopharyngeal swabs had seroconversion with positive SARS-CoV-2 IgG formation at 30 to 48 days post infection. One patient had prolonged shedding of virus on nasopharyngeal swab at 28 days post discharge despite lack of symptoms. In this preliminary report, we find that immunocompromised transplant patients had higher rates of RNAemia (67%) than reported in the general population (15%), seeming absence of allo-immune injury despite systemic inflammation, and formation of IgG overtime after recovery from infection.

Impact of belatacept conversion on kidney transplant function, histology, and gene expression - a single-center study.

Prior studies on belatacept conversion from calcineurin inhibitor (CNI) have been limited by an absence of postconversion surveillance biopsies that could underestimate subclinical rejection, or a case-controlled design. A total of 53 adult patients with allograft dysfunction underwent belatacept conversion (median: 6 months) post-transplant. At a median follow-up = 2.5 years, patient survival was 94% with a death-censored graft survival of 85%. Seven (13%) patients had acute rejection (including 3 subclinical) at median 6 months postconversion. Overall, eGFR improved (P = <0.001) from baseline = 31±15 to 40.2 ± 17.6 ml/min/1.73m2 by 6 months postconversion, but then stayed stable. This improvement was also observed (P < 0.001) in comparison with a propensity matched control cohort on CNI, where eGFR stayed stable (mean ~ 32ml/min/1.72m2 ) over 2-year follow-up. Patients converted < 6 months post-transplant were more likely to have a long-term improvement in kidney function. Paired gene expression analysis of 30 (of 53) consecutive pre- and postconversion surveillance biopsies did not reveal changes in inflammation/acute injury; although atrophy-fibrosis score worsened (mean = 0.28 to 0.44; P = 0.005). Thus, improvement in renal function with belatacept conversion occurred early and then sustained in comparison with controls where renal function remained unchanged overtime. We were unable to show molecular signals that could be related to CNI administration and regressed after withdrawal.

High levels of dd-cfDNA identify patients with TCMR 1A and borderline allograft rejection at elevated risk of graft injury.

The clinical importance of subclinical, early T cell-mediated rejection (Banff TCMR 1A and borderline lesions) remains unclear, due, in part to the fact that histologic lesions used to characterize early TCMR can be nonspecific. Donor-derived cell-free DNA (dd-cfDNA) is an important molecular marker of active graft injury. Over a study period from June 2017 to May 2019, we assessed clinical outcomes in 79 patients diagnosed with TCMR 1A/borderline rejection across 11 US centers with a simultaneous measurement of dd-cfDNA. Forty-two patients had elevated dd-cfDNA (≥0.5%) and 37 patients had low levels (<0.5%). Elevated levels of dd-cfDNA predicted adverse clinical outcomes: among patients with elevated cfDNA, estimated glomerular filtration rate declined by 8.5% (interquartile rate [IQR] -16.22% to -1.39%) (-3.50 mL/min/1.73 m2 IQR -8.00 to -1.00) vs 0% (-4.92%, 4.76%) in low dd-cfDNA patients (P = .004), de novo donor-specific antibody formation was seen in 40% (17/42) vs 2.7% (P < .0001), and future or persistent rejection occurred in 9 of 42 patients (21.4%) vs 0% (P = .003). The use of dd-cfDNA may complement the Banff classification and to risk stratify patients with borderline/TCMR 1A identified on biopsy.