Mixed Rejection Research Articles

Donor-derived cell-free DNA versus left ventricular global longitudinal strain in noninvasive detection of heart allograft injury

Abstract Introduction Invasive endomyocardial biopsy (EMB) is still considered the gold standard method for monitoring heart transplant (HTx) rejection. However, it is associated with potentially serious complications, and its histopathologic interpretation has high interobserver variability. Thus, noninvasive methods for surveillance of the transplanted organ remains of great interest. The noninvasive donor-derived cell-free DNA is a marker of graft injury which can indicate acute rejection, even before the histopathological diagnosis of rejection detected by EMB. The assay has a high negative predictive value for acute rejection. Left ventricular global longitudinal strain (LV GLS) is an emerging marker of subclinical myocardial injury which plays a crucial role in the evaluation of several cardiac diseases; however, its role in HTx injury has so far been poorly investigated. Purpose We aimed to investigate the efficacy of a local laboratory-run dd-cfDNA assay-based heart transplant rejection surveillance programme, and the prognostic and discriminatory performance of LV GLS for subsequent heart allograft injury. Methods HTx recipients without a history of allograft rejection requiring intensification of immunosuppressive therapy were transitioned from our EMB-based surveillance protocol to dd-cfDNA-based rejection surveillance. We assessed the percentage of dd-cfDNA to total cell-free DNA. Injury cut-off was defined by a dd-cfDNA level ≥0.20%, while the severe injury threshold was at ≥0.35%. LV ejection fraction was calculated using 2D Simpson’s method. To derive LV GLS, we analyzed apical 4-chamber, 2-chamber, and long-axis images by 2D speckle tracking echocardiography. Results A total of 244 dd-cfDNA samples were analyzed from 46 patients in fifteen runs performed monthly between October 2022 and December 2023. The dd-cfDNA fraction remained below the injury cut-off in most patients (81%). There were no missed rejection episodes. 88% of routine EMBs that would have otherwise been performed were safely avoided. Eighteen for-cause EMBs were performed based on dd-cfDNA levels suggesting injury. One EMB verified moderate cellular rejection (ISHLT grade 2R) that required steroid pulse therapy. Two EMBs proved mild cellular rejection, one EMB confirmed mild antibody-mediated rejection, while one EMB proved mixed rejection. Thirteen EMBs found no rejection. Mean LVEF was preserved (57.6%±7.6%). Mean LV GLS was -15.1%±2.5% in the whole cohort, while -15.6%±4.7% in recipients with HTx rejection on indication EMB. There was no significant correlation between dd-cfDNA and LV GLS (n=159). Conclusion The noninvasive dd-cfDNA assay is effective in ruling out acute rejection and safely decreases the necessity of invasive surveillance EMBs after HTx. LV GLS is less sensitive in comparison with the dd-cfDNA assay for detection of myocardial injury in the early stages of graft rejection in HTx recipients at low risk for rejection.

Prevalence and Impact of Recurrent Rejection on Pediatric Heart Transplant Recipients: A PHTS Multi-Institutional Analysis

BackgroundStudies evaluating the prevalence and impact of recurrent rejection (RR) in pediatric heart transplant (HT) are sparse. ObjectivesThe purpose of this study was to describe prevalence and impact of RR on cardiac allograft vasculopathy (CAV) and graft loss after pediatric HT. MethodsData on HT from January 1, 2000, to June 30, 2020, in the Pediatric Heart Transplant Society database were included. Freedom from RR (≥2 rejection episodes) was compared by era (early: 2000-2009; current: 2010-2020). Outcomes for children experiencing RR were compared with those experiencing 0 or 1 rejection episodes and by type of RR (antibody-mediated rejection [AMR], acute cellular rejection [ACR], mixed [ACR/AMR]). ResultsOf 6,342 HT recipients, 1,035 (17%) experienced RR. In the current era, pediatric HT recipients were less likely to experience RR (P < 0.001). Freedom from CAV was similar for those experiencing RR to those experiencing 0 or 1 episode (96.6% vs 95.3% vs 96.6%); and similar regardless of the type of RR (AMR, ACR, or mixed) (65.5% vs 82.9% vs 100%) (P > 0.05). Freedom from graft loss was significantly lower for those experiencing RR to those experiencing 0 or 1 episode (56.3% vs 72.3% vs 82.3%) and lower for those experiencing recurrent mixed rejection or recurrent AMR compared with those experiencing recurrent ACR (65.3% vs 50% vs 81.8%). Black children experiencing RR subsequently had lower freedom from CAV and graft loss than White children (P < 0.05 for all). ConclusionsAlthough prevalence of RR has decreased, children experiencing RR are at greatly increased risk for losing their graft, particularly those who have recurrent mixed or antibody-mediated rejection.

306.6: Phenotype of interstitial inflammation and tubulitis in mixed rejection compared to T cell mediated rejection: A study using CODEX multiplex imaging and machine learning.

306.6: Phenotype of interstitial inflammation and tubulitis in mixed rejection compared to T cell mediated rejection: A study using CODEX multiplex imaging and machine learning.

Clinical and molecular spectrum of v-lesion

Isolated v-lesion presents diagnostic stratification and clinical challenges. We characterized allograft outcomes for this entity based on posttransplant time (early: ≤1 month vs late: >1 month) and compared its molecular phenotype with other v+ rejection forms. Using the NanoString B-HOT panel, we analyzed 92 archival formalin-fixed paraffin-embedded tissue kidney biopsies from 3 centers: isolated v-lesion (n = 23), antibody-mediated rejection (ABMR) v+ (n = 26), T cell–mediated rejection (TCMR) v+ (n = 10), mixed rejection v+ (n = 23), and normal tissue (n = 10). Six gene sets (ABMR, DSAST, ENDAT, TCMR, early/acute injury, late injury) were assessed. Early isolated v-lesions had the poorest 1-year death-censored graft survival compared with late isolated v-lesions or other rejections (P = .034). Gene set analysis showed lower TCMR-related gene expression in isolated v+ groups than TCMR and mixed rejection (P < .001). Both early- and late isolated v-lesions had lower ABMR-related gene expression than ABMR, mixed rejection, and TCMR (P ≤ .022). Late isolated v-lesions showed reduced DSAST and ENDAT gene expression versus ABMR (P ≤ .046) and decreased early/acute injury gene expression than early isolated v+, ABMR, TCMR, and mixed rejection (P ≤ .026). In conclusion, isolated v-lesions exhibit distinct gene expression patterns versus other rejection v+ forms. Early isolated v+ is associated with poorer prognosis and increased early/acute injury gene expression than late isolated v+, suggesting distinct etiologies.

T-cell Mediated Rejection Associated Microvascular Inflammation in the Allograft Kidney: RNAseq Analysis Using the Banff Human Organ Transplant Gene Panel.

Microvascular inflammation (MVI) can occur in biopsies showing T-cell mediated rejection (TCMR), but it is not well established that T-cells can directly mediate microvascular injury (TCMR-MVI). This was a cross sectional RNAseq based Banff Human Organ Transplant (BHOT) gene expression (GE) analysis. The objective of this study was to probe the molecular signature of TCMR-MVI in comparison with C4d+, DSA+ antibody mediated rejection (ABMR), stable renal function (STA), and TCMR without MVI. Transcriptome analysis utilized CLC genomic workbench and R-studio software. No gene set was specific for any diagnostic category, and all were expressed at low levels in STA biopsies. BHOT gene set scores could differentiate ABMR from TCMR and TCMR-MVI, but not TCMR from TCMR-MVI. TCMR-MVI underexpressed several genes associated with ABMR including DSATs, ENDAT, immunoglobulin genes, ADAMDEC1, PECAM1 and NK cell transcripts (MYBL1, GNLY), but overexpressed C3, NKBBIZ, and LTF. On the other hand, there was no significant difference in the expression of these genes in TCMR-MVI versus TCMR. This indicates that the GE profile of TCMR MVI aligns more closely with TCMR than ABMR. The limitations of classifying biopsies using the binary ABMR-TCMR algorithm, and the occurrence of common pathogenesis mechanisms amongst different rejection phenotype was highlighted by the frequent presence of molecular mixed rejection. T-cell mediated mechanisms play a significant role in the pathogenesis of MVI. GE was broadly different between rejection phenotypes, but molecular scores varied substantially between biopsies with the same Banff grade. It was not always possible to achieve precise molecular score-based diagnostic categorization of individual patients.

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.

#1658 Association of blood dd-cfDNA levels with Banff scores and histopathological lesions in kidney allograft biopsies: results from an observational trial

Abstract Background and Aims Donor-derived cell-free DNA (dd-cfDNA) is a novel blood biomarker of kidney allograft injury. While primarily studied to discriminate rejection from no rejection, knowledge about its dynamics in other graft pathologies is scarce and merits further investigation. Previous research has highlighted its superior diagnostic performance in detecting ABMR compared to TCMR and borderline changes, however, recent studies have suggested that its increase is not limited to damage due to alloimmune injury. Therefore, we aimed to assess the association between dd-cfDNA release and diverse histopathological patterns as well as its correlation with Banff lesion scores in renal allograft biopsies. Method For this analysis, we identified all dd-cfDNA-paired allograft biopsies from a prospective observational trial of kidney transplant recipients undergoing an indication biopsy with deterioration in graft function, persistence of anti-HLA donor-specific antibodies (DSA), or proteinuria. Dd-cfDNA was collected at the time of allograft biopsy and measured using droplet-digital PCR. Both relative and absolute quantification of dd-cfDNA, with cutoffs of 0.5% and 50cp/ml, respectively, were available for clinical interpretation. All biopsy specimens were examined by a central pathologist blinded to the dd-cfDNA results. Histopathological diagnoses were assigned according to the Banff 2019 classification. Results In total, we examined 125 dd-cfDNA paired biopsies from 110 patients performed at our center between April 2020 and October 2023. Absolute levels of dd-cfDNA were significantly higher in mixed rejection (median 156 cp/mL, IQR 138-176), antibody-mediated rejection (ABMR) (median 63cp/mL, IQR 48-81), DSA-negative microvascular inflammation (DSAnegMVI) (median 74 cp/mL, IQR 35-134), and BK-virus associated nephropathy (BKVAN) (median 168cp/mL, IQR 54-311) in comparison to glomerulonephritis (median 12cp/mL, IQR 8-19; p &amp;lt; 0.001, &amp;lt;0.001, 0.02, 0.01, respectively) as well as in comparison CNI-toxicity (median 10, IQR 6-15; p &amp;lt;0.001, &amp;lt;0.001, 0.004, 0.003, respectively). Additionally, patients with mixed rejection, ABMR, and BKVAN had higher absolute dd-cfDNA levels than patients with IFTA (median 13, IQR 8-16; p 0.004, 0.004, and 0.04, respectively), as well as normal histology (median 7, IQR 7-11, p 0.004, 0.008, 0.03, respectively). Finally, absolute dd-cfDNA levels were higher in patients with mixed rejection than in those with UTI (median 14, IQR 12-16, p = 0.02) (Fig. 1). In the univariable analysis, absolute dd-cfDNA levels were significantly correlated with Banff lesion scores for glomerulitis (g), peritubular capillaritis (ptc), and tubulitis (t). In the multivariable analysis, ptc and t were independently correlated with absolute dd-cfDNA levels. Conclusion Increased donor-derived cell-free DNA is not specific for rejection, but also occurs in alternative injury patterns showing microvascular inflammation and severe tubulointerstitial inflammation, such as DSAnegMVI and BKVAN (BK-virus associated nephropathy). Interestingly, dd-cfDNA levels remain low in the setting of de novo or recurrent glomerulonephritis and CNI-toxicity, which are important differential diagnoses in patients with clinical indication biopsy.

#2566 Clinicopathological and molecular characteristics of plasma cell rich rejection in renal transplant biopsies

Abstract Background and Aims Plasma cell rich rejection (PCRR) is an uncommon, ill-defined type of renal allograft rejection in the literature considered as a subtype of T cell-mediated rejection (TCMR). PCRR has poorer clinical outcome and is often refractory to classic immunosuppressive therapy. Our study analyzed In this study, we analyzed PCRR in a large cohort of patients who underwent renal transplantation at the Amsterdam UMC. Our objective was to study clinical, histological and molecular signatures of PCRR in comparison to other types of rejection, especially late presentations of TCMR and ABMR. We hypothesized PCRR could be a specific subtype of allograft rejection with different molecular and cellular characteristics compared to (late) TCMR and ABMR with differences in clinical outcome. Method We retrospectively scored and reclassified the last known biopsy of 263 renal transplant recipients, morphologically classified as rejection according to the 2019 Banff classification. mRNA expression analysis was performed using the Nanostring B-HOT panel on a subset of cases. PCRR was compared to (late) TCMR, ABMR and mixed rejection for renal function follow-up and graft survival. Results mRNA analysis revealed uniquely expressed genes in PCRR including LOX, CPA3, IL4, IL17F, and MMP12. PCRR is enriched for genes related to mast cells, memory B- and T-cells and transcripts involved in NK cells and allograft fibrosis with heterogeneity in gene expression in biopsies with PCRR. PCRR might be a late event compared to late TCMR and ABMR, with a higher degree of total inflammation and fibrosis. Graft survival and kidney function was similar to late TCMR and ABMR during a 5-year follow-up period after renal biopsy Conclusion PCRR represents a distinct late-onset stage of inflammation displaying diverse gene expression patterns, with presence of mainly mast cells, NK cells and transcripts involved in renal allograft fibrosis. Such insights could enable a better and more tailored treatment for these patients and ultimately enhance long-term graft survival for patients affected by PCRR. Clinical outcomes in patients with PCRR appeared similar to late TCMR and ABMR.

#2859 Clinical and morphological characteristics of biopsy-proven kidney graft rejection in Latvia

Abstract Background and Aims Kidney transplantation is a treatment for many patients with end stage renal disease leading to increased quality of life. In last decades results in kidney transplantation have been improving, but allograft rejection remains an important clinical problem. Graft biopsy remains the gold standard to assess the cause of kidney transplant status. This study aimed to determine clinical and histopathological characteristics, treatment and short-term outcomes of biopsy-proved kidney graft rejection in Latvia. Method In this retrospective observational study all kidney allograft biopsies performed in Pauls Stradins Clinical University Hospital from January 2014 till December 2022 was reviewed. Biopsies with rejection, ≥7 glomeruli, patients ≥18 years old were included. Acute rejection was diagnosed according to clinical, laboratory and biopsy-proven acute rejection (BPAR) histological criteria. BPAR was classified and subdivided using Banff criteria. All recipients universally received either depleting or non-depleting induction therapy. Most of the patient (82%) for maintenance immunosuppression (MI) received triple therapy with calcineurin inhibitor, mycophenolate mofetil, and prednisone, others—in different combinations with above and mammalian target of rapamycin (mTOR), azathioprine. Graft function was evaluated according to glomerular filtration rate (GFR), calculated with the CKD-EPI Creatinine, 2021 equation. Treatment strategies, laboratory data at the time of biopsy and after 1 year follow-up were analysed. Repeated-control biopsies after receiving therapy were excluded. Data were analysed using descriptive statistics and T-test. Results From all 523 biopsies, further included and analysed were 153 (total 146 patients). 58% were males, average patient age was 45.5 years. Most 84% of the grafts were from deceased, 8%—living donors. Mean time from transplantation to biopsy was 32 months. Acute antibody-mediated rejection (AAMR) was diagnosed in 41% cases, chronic-active antibody mediated (CAAMR)—25%, followed by mixed 16%, acute cellular (ACR) 10% and borderline 8% rejection. Besides augmented MI for almost all patients, pulse steroids (GCS) were the most commonly 70% administered treatment, followed by plasma exchange 59% (PEX), rituximab (RTX) 50%, immunoglobulins (IG) 24% and anti-thymocyte globulin (ATG) 10% in different, mostly non-homogenous combinations. Non adherence, advanced fibrotic changes in biopsy and serious comorbidities were the main reasons patients did not receive specific treatment besides intensified MI. The most favourable outcomes were in ACR where mean allograft GFR increased +20.7 ml/min after one year, compared to the worst outcome CAAMR group where GFR decreased −4.7 ml/min (p = 0.03). In ACR group 92% graft function stayed the same or improved, moreover 54% graft function increased ≥20% from baseline; similarly in mixed and AAMR group GFR increase ≥20% were for 45% and 48%, compared to CAAMR where it was only for 6% of patient. Furthermore, the CAAMR group was with the most patients (48%) who returned to dialysis or died. In AAMR and mixed rejection groups mean GFR changes were close +13.4 ml/min and +12.9 ml/min (p = 0.95). There were no statistically significant associations between treatment outcomes in PEX+RTX+GCS vs PEX+RTX+GCS+IG in the AAMR group (p = 0.382). Overall, 3.9% of patients died. Conclusion Overall BPAR prevalence between kidney graft biopsies was high (29%). Antibody mediated rejection was diagnosed most often with least favourable short-term outcomes in CAAMR group. Despite various improvements in immunosuppression, CAAMR remains a serious cause of transplant dysfunction or loss.

#1134 Molecular rejection phenotype patterns differentiate between probable AMR, DSA-negative microvascular inflammation, and AMR

Abstract Background and Aims Biopsy-based transcripts associated with antibody-mediated rejection (AMR) hold promise as substitutes for C4d positivity, according to the latest Banff Meeting Report of 2022. However, patterns and strength of rejection phenotype scores remain inadequately studied within the new subgroups along the AMR continuum. Method We analyzed 365 kidney allograft biopsies by histology and the Molecular Microscope Diagnostic System (MMDx) at the University Hospital of Zurich from July 2021 to November 2023. Histologic findings were classified according to Banff 2022 into (1) probable AMR (n = 32), (2) donor-specific antibodies (DSA)-negative C4d-negative microvascular inflammation (MVI, n = 32), and (3) AMR (n = 60). A control group of 118 cases without MVI was used for comparison. Results 10 out of 32 cases (31%) with probable AMR, 20 of 32 cases (63%) with DSA-negative C4d-negative MVI, and 31 of 60 cases (52%) with AMR showed molecular AMR. Among cases with molecular AMR the sum of the TCMR-related (R2) and late-stage AMR-related (R6) phenotype scores was significantly higher in probable AMR and DSA-negative MVI cases compared to AMR cases (p = 0.029). In contrast, the sum of the mixed rejection (R3), early-stage AMR (R4), fully-developed AMR (R5) phenotype scores was higher in AMR compared to probable AMR (p = 0.063). Among cases without molecular AMR the all AMR phenotype score was significantly higher in AMR and DSA-negative MVI compared to probable AMR and the control group without MVI (p = 0.012, p &amp;lt; 0.001). Conclusion Our results identify different molecular rejection phenotype patterns between cases with probable AMR, DSA-negative MVI, and AMR. Sub-threshold findings appear relevant along the AMR continuum.

Relationship between donor-derived cell-free DNA and tissue-based rejection-related transcripts in heart transplantation

IntroductionEndomyocardial biopsy (EMB)-based traditional microscopy remains the gold standard for the detection of cardiac allograft rejection, despite its limitation of inherent subjectivity leading to inter-reader variability. Alternative techniques now exist to surveil for allograft injury and classify rejection. Donor-derived cell-free DNA (dd-cfDNA) testing is now a validated blood-based assay used to surveil for allograft injury. The molecular microscope diagnostic system (MMDx) utilizes intragraft rejection-associated transcripts (RATs) to classify allograft rejection and identify injury. The use of dd-cfDNA and MMDx together provides objective molecular insight into allograft injury and rejection. The aim of this study was to measure the diagnostic agreement between dd-cfDNA and MMDx and assess the relationship between dd-cfDNA and MMDx-derived RATs which may provide further insight into the pathophysiology of allograft rejection and injury. MethodsThis is a retrospective observational study of 156 endomyocardial biopsy (EMB) evaluated with traditional microscopy and MMDx. All samples were paired with dd-cfDNA from peripheral blood prior to EMB (up to 9 days). Diagnostic agreement between traditional histopathology, MMDx, and dd-cfDNA (threshold of 0.20%) were compared for assessment of allograft injury. In addition, the relationship between dd-cfDNA and individual RAT expression levels from MMDx was evaluated. ResultsMMDx characterized allograft tissue as no rejection (NR) (62.8%), antibody-mediated rejection (ABMR) (26.9%), T-cell-mediated rejection (TCMR) (5.8%), and mixed ABMR/ TCMR (4.5%). For the diagnosis of any type of rejection (TCMR, ABMR, and mixed rejection), there was substantial agreement between MMDx and dd-cfDNA (76.3% agreement). All transcript clusters (group of gene sets designated by MMDx) and individual transcripts considered abnormal from MMDx had significantly elevated dd-cfDNA. In addition, a positive correlation between dd-cfDNA levels and certain MMDx-derived RATs was observed. Tissue transcript clusters correlated with dd-cfDNA scores, including DSAST, GRIT, HT1, QCMAT and S4. For individual transcripts, tissue ROBO4 was significantly correlated with dd-cfDNA in both non-rejection and rejection as assessed by MMDx. ConclusionCollectively, we have shown substantial diagnostic agreement between dd-cfDNA and MMDx. Furthermore, based on the findings presented, we postulate a common pathway between the release of dd-cfDNA and expression of ROBO4 (a vascular endothelial-specific gene that stabilizes the vasculature) in the setting of antibody-mediated rejection (AMR), which may provide a mechanistic rationale for observed elevations in dd-cfDNA in AMR, compared to acute cellular rejection (ACR).

Development and Validation of a Multiclass Model Defining Molecular Archetypes of Kidney Transplant Rejection: A Large Cohort Study of the Banff Human Organ Transplant Gene Expression Panel

Gene expression profiling from formalin-fixed paraffin-embedded (FFPE) renal allograft biopsies is a promising approach for feasibly providing a molecular diagnosis of rejection. However, large-scale studies evaluating the performance of models using NanoString platform data to define molecular archetypes of rejection are lacking. We tested a diverse retrospective cohort of over 1400 FFPE biopsy specimens, rescored according to Banff 2019 criteria and representing 10 of 11 United Network of Organ Sharing regions, using the Banff Human Organ Transplant panel from NanoString and developed a multiclass model from the gene expression data to assign relative probabilities of 4 molecular archetypes: No Rejection, Antibody-Mediated Rejection, T Cell–Mediated Rejection, and Mixed Rejection. Using Least Absolute Shrinkage and Selection Operator regularized regression with 10-fold cross-validation fitted to 1050 biopsies in the discovery cohort and technically validated on an additional 345 biopsies, our model achieved overall accuracy of 85% in the discovery cohort and 80% in the validation cohort, with ≥75% positive predictive value for each class, except for the Mixed Rejection class in the validation cohort (positive predictive value, 53%). This study represents the technical validation of the first model built from a large and diverse sample of diagnostic FFPE biopsy specimens to define and classify molecular archetypes of histologically defined diagnoses as derived from Banff Human Organ Transplant panel gene expression profiling data.

Report of the 2022 Banff Heart Concurrent: Focus on non-human leukocyte antigen antibodies in rejection and the pathology of “mixed” rejection

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to• Determine current knowledge gaps in heart transplant pathology• Identify limitations of current pathology classification systems• Discuss next steps in addressing gaps and refining classification system

Utilization of donor-derived Cell-Free DNA in pediatric kidney transplant recipients: A single center study.

Forty-two pediatric kidney transplant patients were enrolled between February 2020 and August 2021. Dd-cfDNA was tested before and after biopsy/rejection treatment. There was a total of 61 allograft biopsies (44 for-cause, 17 surveillance). Graft rejection was found in 35/61 biopsies. Rejection was more common in basiliximab induction compared to rATG (77.1% vs. 22.9%, p = .0121). Median dd-cfDNA was higher in those with rejection (1.2% [0.34-3.12] vs. 0.24% [0.08-0.78], p < .0001). Dd-cfDNA was highest in biopsies with AMR and mixed AMR/TCMR. In addition, dd-cfDNA in basiliximab induction was higher compared to rATG (0.92% [0.27-1.8] vs. 0.26% [0.08-2], p = .0437). Median change in dd-cfDNA after rejection treatment was -0.57% (-1.67 to 0.05). Median time to dd-cfDNA <1% post-rejection treatment was 8.5 days (3.0-19.5). Dd-cfDNA in AMR was higher compared to TCMR or mixed rejection, and levels remained higher in AMR after treatment. In surveillance biopsies, 4/17 had rejection. Median dd-cfDNA was not different in those with versus without rejection (0.48% vs. 0.28%, p = .2342). Those without rejection all had dd-cfDNA <1%. In those with rejection, only one patient had dd-cfDNA >1%, and all had TCMR. Our findings support dd-cfDNA as a useful indicator of graft rejection and response to treatment. Additional studies are needed to determine the role of dd-cfDNA in graft health surveillance.

A modified perioperative regimen for deceased donor kidney transplantation in presensitized recipients without prior desensitization therapy.

Renal transplantation in HLA-presensitized recipients entails an increased risk of antibody-mediated rejection (AMR) and graft loss. There is currently no accepted standard treatment protocol that can help transplant surgeons safely perform deceased donor (DD) kidney transplantation in presensitized patients without pretransplant desensitization. Fifty-one panel-reactive antibody (PRA)-positive recipients and 62 PRA-negative retransplant recipients (control) who received DD renal transplantation were included. Patients in the presensitized group (donor-specific antibody [DSA]-positive, n=25; DSA-negative, n=26) without desensitization received a modified perioperative treatment starting on day 0 or -1 with rituximab, thymoglobulin, and low daily doses of intravenous immunoglobulin (IVIG, 10-20 g/d, for 14 days). Plasmapheresis was performed once before surgery in DSA-positive recipients. The median follow-up time was 51 months in the presensitized group and 41 months in the control group. The incidence of early acute rejection (AR) and AMR (including mixed rejection) was 35.3% and 13.7% in the presensitized group, respectively, significantly higher than in the control group (14.5% and 1.6%, respectively). Within the presensitized group, the DSA-positive subgroup had more AMR than the DSA-negative subgroup (24.0% vs. 3.8%), but the incidence of T cell-mediated rejection was comparable (20.0% vs. 23.4%). In the presensitized group, all rejections were successfully reversed, and graft function remained stable during follow-up. The 1-year and 3-year survival rates of the grafts and recipients in this group were 98.0%. With a modified IVIG-based perioperative regimen, excellent intermediate-term graft and recipient survival outcomes can be achieved in presensitized patients who received DD kidney transplantation without prior desensitization.

Therapy in the Course of Kidney Graft Rejection-Implications for the Cardiovascular System-A Systematic Review.

Kidney graft failure is not a homogenous disease and the Banff classification distinguishes several types of graft rejection. The maintenance of a transplant and the treatment of its failure require specific medications and differ due to the underlying molecular mechanism. As a consequence, patients suffering from different rejection types will experience distinct side-effects upon therapy. The review is focused on comparing treatment regimens as well as presenting the latest insights into innovative therapeutic approaches in patients with an ongoing active ABMR, chronic active ABMR, chronic ABMR, acute TCMR, chronic active TCMR, borderline and mixed rejection. Furthermore, the profile of cardiovascular adverse effects in relation to the applied therapy was subjected to scrutiny. Lastly, a detailed assessment and comparison of different approaches were conducted in order to identify those that are the most and least detrimental for patients suffering from kidney graft failure.

#6352 CLINICAL AND MOLECULAR SPECTRUM OF V-LESION

Abstract Background and Aims Isolated v-lesion is an increasingly recognized but clinically challenging entity. Some studies suggest that isolated v+ early post-transplant may be caused by non-alloimmune etiologies such as ischemic injury. We aimed to characterize the allograft outcomes of isolated v-lesions according to post-transplant time (early: ≤1 month vs. late: &amp;gt;1 month) and further understand the significance of this lesion by characterizing its molecular phenotype in comparison with other forms of rejection v+. Method The NanoString B-HOT panel (770 genes) was used to measure the expression of six literature-derived gene sets in 92 archival FFPE kidney biopsies from two centers, including transplant biopsies with isolated v-lesion (n = 23), antibody-mediated rejection (ABMR) with v+ (n = 26), pure T-cell mediated rejection (TCMR) with v+ (n = 10), mixed rejection v+ (n = 23), and normal implant biopsies (Normal, n = 10). The evaluated gene sets included transcripts previously associated with ABMR, DSA (DSAST), endothelial injury (ENDAT), TCMR, early injury, and late injury. Gene expression was compared between groups using principal component and class comparison analyses. Death-censored graft survival according to diagnosis and time after KT was assessed. Results Isolated v+ early conferred the worst death-censored graft survival one year after the biopsy (40%) when compared to isolated v+ late (100%) or other forms of rejection (≥82%, p = 0.034). The principal component analysis demonstrated significant molecular overlap between sample groups (PC1: 29.4%, PC2: 8.1%). However, gene set analysis showed lower expression of TCMR-related genes in isolated v+ groups compared to TCMR and mixed rejection (p&amp;lt;0.001). Both Isolated v+ early and late had lower ABMR-related genes than ABMR, mixed rejection, and TCMR groups (p ≤ 0.022). Moreover, isolated v+ late showed lower DSAST and ENDAT gene set expression than ABMR (p ≤ 0.046); and lower early injury gene set expression than isolated v+ early, ABMR, TCMR, and mixed rejection (p ≤ 0.026). Late injury gene set expression was highest in TCMR and mixed rejection compared to the other groups (p ≥ 0.034). Conclusion These results suggest that early and late isolated v+ lesions display lower expression of TCMR-related genes than TCMR and mixed rejection and lower expression of ABMR-related genes than ABMR. Isolated v+ early confers a bad prognosis and is associated with higher expression of early injury genes compared to isolated v+ late, suggesting a different ethology.

#5811 URINARY CXCL9 AND CXCL10 CONCENTRATIONS AND KIDNEY TRANSPLANT INJURY

Abstract Background and Aims Kidney transplant rejection is diagnosed by the kidney biopsy which is an invasive diagnostic method, however, non-invasive biomarkers, distinguishing normal kidney transplant from transplant rejection would be prefered but are lacking. CXC chemokine ligand 9 (CXCL9) and CXC chemokine ligand 10 (CXCL10) are gamma interferon inducible small cytokines. The aim of this study was to evaluate urinary CXCL9 and CXCL10 as potential biomarkers of allograft rejection. Method 117 kidney transplant recipients undergoing kidney transplant biopsy (surveillance or indication) were included into this study. Spot urine samples were collected before kidney biopsy for urinary creatinine, CXCL9 and CXCL10 measurement. The ratios of CXCL9 over urinary creatinine and CXCL10 over urinary creatinine were calculated. Kidney biopsies were analyzed according to Banff criteria by an experienced pathologist. Results 26 (22.2%) of kidney biopsies were defined as normal histology, 24 (20.5%) – antibody mediated rejection, 9 (7.7%) – T cell mediated rejection, 10 (8.6%) mixed rejection, 5 (4.3%) BK virus nephopathy and 43 (36.8%) were defined as other histological lesions. Average CXCL9/creatinine was 0.39 ng/mmol in normal histology group and 12.1 ng/mmol in allograft rejection groups (p&amp;lt;0.01). Average CXCL10/creatinine was 0.26 ng/mmol in normal histology and 4.55 ng/mmol in allograft rejection group (p&amp;lt;0.01). CXCL9/creatinine and CXCL10 moderately correlated to proteinuria at the time of kidney biopsy (accordingly r = 0.595, p&amp;lt;0.01 and r = 0.408, p&amp;lt;0.01). ROC curve for CXCL9/creatinine detecting antibody mediated rejection was 0.79, CI 0.67 to 0.90, p&amp;lt;0.01. ROC curve for CXCL10/creatinine detecting antibody mediated rejection was 0.79, CI 0.64 to 0.94, p&amp;lt;0.01. Conclusion Urinary biomarkers CXCL9/creatinine and CXCL10/creatinine has a potential to distinguish normal renal histology from allograft rejection and correlates to proteinuria at the time of the biopsy.

#6147 BELATACEPT OUTCOMES IN PEDIATRIC KIDNEY TRANSPLANTATION: AN INTERNATIONAL MULTICENTER STUDY

Abstract Background and Aims Belatacept is a co-stimulation blocker associated with better long-term outcomes in adult patients compared to CNI based regimens. Data on its use in older children and young adults are lacking. We are the 1st to report outcomes for 45 pediatric kidney transplant recipients converted to belatacept. Method 45 patients were included from 4 centers (USA and France) between 05/2018 and 12/2021. Patients received an induction with basiliximab (n = 39) or ATG (n = 6). Maintenance immunosuppression included CNI, MMF +/− steroids. Patients’ viral status (EBV, CMV) were monitored monthly and allograft biopsies were performed prior and ∼6 months after starting belatacept. The first 5 belatacept injections were administered at 5 mg/kg/dose (10 mg/kg/dose if early conversion) every 2 weeks, then monthly. CNI were progressively reduced and stopped. MMF doses were also increased at CNI withdrawal. Results Median age at conversion was 17.7 y (range 10.3-20.6). 7/45 patients received an early conversion (median: 1 month post-transplant, IQR 0.5-1.2): 6 patients because of delayed graft function and 1 to avoid CNI toxicity (post-transplant diabetes). 38/45 patients were converted after a median of 4.1 years post-transplant (IQR 1.7-6.0). Conversion indication was based on the need for long term CNI avoidance: either because of toxicity (histology, post-transplant diabetes, tremors; n = 13) or sub-optimal creatinine (n = 12) or to improve adherence (e.g. monthly IV-treatment, n = 13). CNI were withdrawn in 42/45 patients by a median of 2.4 months (IQR 1.4-6.0). GFR was stable or improved over a median follow-up time of 1.6 years (IQR 1.1-2.4), Fig 1 A. Rejection episodes were observed in 10/45 patients (22%) after a median of 10.2 months (IQR 6.1-15.8) and included 7 TCMR, 2 ABMR and 1 mixed rejection. None of these patients were converted early (&amp;lt;3 m), 5 had been converted for non-adherence, 4 had pre-existing DSA and 4 had prior history of rejection. Evolution of GFR in rejectors is shown in Fig 1 B. CNI were reintroduced for 6/10 and belatacept stopped for 3/10. Regarding viral complications, 1 severe BKv nephropathy required the discontinuation of belatacept. All patients were EBV+ at conversion (4 were EBV- at the time of transplant). No EBV replication was observed. Conclusion Selected pediatric kidney recipients benefit from long-term CNI toxicity avoidance, but selection criteria need to be refined to avoid rejection under costimulation blockade.

#2683 NEXT GENERATION SEQUENCING: FROM BANFF SCORE MOLECULAR SIGNATURES AND CLASSIFIERS TO PREDICTED HISTOLOGICAL ARCHETYPES OF KIDNEY BIOPSIES

Abstract Background and Aims To improve risk stratification in kidney transplantation, molecular diagnostic tools are increasingly investigated for their feasibility and gain in diagnostic power. Nonetheless, previously published studies focused on sequencing technologies and gene panels with limited vision of the transcriptome (pathogenesis-based transcripts [PBTs], Banff Human Organ Transplant [BHOT] panel). The EU-TRAIN consortium was built to discover new predictive and informative biomarkers for kidney transplant histology and rejection diagnoses, leveraging a next generation sequencing technology. Method EU-TRAIN (NCT03652402) is a prospective multicentric study including unselected kidney transplant cohorts from 11 centres from 4 countries (France, Spain, Germany, Switzerland). We performed a bulk RNA sequencing on the polyadenylated probes of 770 kidney biopsies (n = 540 kidney recipients) collected between 2018 and 2021. Differential gene expression analyses were computed to obtain a molecular signature for all Banff score lesions. We then derived three different feature selections by either i) training an ElasticNet model on all differentially-expressed genes (DEGs), or by taking the top 30 ii) overall DEGs or iii) the top 30 DEGs focusing on transcripts included in the BHOT gene panel. From them, we trained four machine learning (ML) classifiers through a 10-times repeated 3-fold cross-validation. Models’ performances were assessed on a hold-out test set accounting for 30% of the total samples. Finally, we derived prototypic histological profiles using an archetypal analysis on the samples’ predicted Banff score probabilities from the best classifier. Results The ElasticNet feature selection lowered the number of DEGs to be included from a range of [859; 10,839] to [52; 867]. These selections were composed of a mixture of PBTs [55.2%; 84.2%], BHOT genes [2.2%;13.5%] and new transcripts [12.4%; 37.2%]. Based on these findings, four ML classifiers (Naïve Bayes, Extreme Gradient Boosting, Linear Support Vector Machine and K-Nearest Neighbours) were trained on the three different feature selections and their performances in predicting Banff score lesions were compared using the precision-recall area under the curve (PRAUC). In all settings, the ElasticNet feature selection outperformed the other two methods with a minimal/maximal increase in PRAUC of 0.068 (t) / 0.726 (cg). Excluding the ah score, the best discriminations were obtained with the Linear Support Vector Machine with a PRAUC in the interval [0.708; 0.980] (t/ptc). Excluding cv and ah, all models calibrated properly (Hosmer and Lemeshow goodness of fit p-value &amp;gt; 0.05). The archetypal analysis resulted in 8 profiles: acute and chronic antibody-mediated rejection (presence of circulating donor-specific antibodies, C4d deposition, g, ptc and cg lesions), acute T-cell mediated rejection (i, t and ti lesions), chronic TCMR (i, t, ti, ci, ct and i-IFTA lesions), mixed rejection (g, ptc, i, t and ti lesions), vascular injuries (cv and ah), fibrosis (ci and ct lesions, older donors with history of hypertension), minimal fibrotic change (ci and ct) and minor changes (no lesions). Conclusion From new transcripts, we managed to develop models that predict accurately the Banff score lesions and 8 profiles were identified among these predictions. External validation and archetypes’ association with graft loss will be addressed in the future.