Soufian Meziyerh

Introduction: Timely diagnosis of kidney allograft rejection is critical for post-transplant management, yet treatment decisions may need to be made empirically while awaiting the final pathology report. We previously demonstrated that biopsy transport medium (BTM) contains molecular signals reflective of allograft rejection. Building on these findings, we developed a rapid BTM–qPCR workflow using a targeted gene panel for early molecular assessment of rejection. Methods: Kidney transplant biopsies assessed at Leiden University Medical Center from April 2023 to November 2024 were eligible. A predefined gene panel was quantified in BTM by qPCR, while matched formalin-fixed paraffin-embedded tissues were profiled using Tempo-Seq. Elastic Net, Random Forest, and support vector machine models were developed to classify rejection and predict continuous histological indices directly from the BTM. Results: A total of 134 paired BTM samples and biopsy tissues were analyzed. The best-performing BTM gene-panel models demonstrated good discrimination for rejection, with a median AUC of 0.80, and good calibration (median slope, 0.92; median intercept, 0.08). For continuous histological indices, the best-performing BTM gene-panel models achieved R² values of 0.33 for Activity, 0.31 for TCMR/TI, and 0.27 for AMR/MVI. Corresponding biopsy gene-panel models showed similar patterns across diagnostic outcomes. Conclusions: The BTM–qPCR workflow captures molecular features of kidney allograft rejection and enables prediction of rejection status along the spectrum of continuous activity indices. By providing same-day molecular information while definitive biopsy interpretation is pending, this rapid approach has the potential to complement conventional histopathological diagnostics and support early clinical decision-making. Lay Summary: Kidney transplantation is the optimal treatment for end-stage kidney disease, and effective post-transplant management is critical for long-term survival of the transplanted kidney. Therefore, prompt treatment decisions and timely interventions are essential. However, a complete and final pathological assessment of a kidney biopsy can sometimes take several days. A molecular diagnostic approach using qPCR on biopsy transport medium (BTM), the liquid used to transport biopsy samples, has the potential to complement conventional pathological assessment by providing rapid information to support clinical decision-making. Our study included 134 kidney transplant biopsy samples and measured the expression of a panel of 19 genes in BTM. These gene expression measurements were able to distinguish rejection from non-rejection and predict continuous histological indices. Our findings suggest that, while pathological assessment is pending, the BTM–qPCR approach could provide same-day molecular information to support earlier clinical decision-making.

Background: Histopathological diagnoses in kidney transplant biopsies, as described by the Banff classification, suffer from substantial inter-observer variability and misinterpretation, making them prone to diagnostic errors. Continuous indices based on BanffNET automated lesion scores are proposed to quantify the continuous phenotypic spectrum of kidney transplant biopsies, complementary to pathologist assessment of biopsies. Methods: The BanffNET model was used to obtain continuous lesion scoring for a training cohort of 2544 biopsies, a large validation cohort of 3863 biopsies, and a multi-reader cohort of 36 biopsies scored by 67 pathologists. A penalized regression approach was used to condense 17 automated lesion scores into four indices, representing the most important dimensions of graft injury: a TCMR/TI Index, an AMR/MVI Index, an Activity Index and a Chronicity Index. The BanffNET Indices were evaluated against Banff diagnoses and histological indices in the training cohort and validation cohort. Since pathologist-assigned Banff lesions and diagnoses suffer from inter-observer variability, associations were also assessed with diagnoses in a multi-reader study, with time to kidney graft failure in the training and validation cohort and with biopsy-based molecular signatures in the validation cohort. Results: The BanffNET TCMR/TI Index, AMR/MVI Index and Activity Index showed excellent discrimination of Banff TCMR, Banff MVI and Banff Any Diagnosis respectively, with validation AUCs ranging from 0.80 to 0.92. In addition, the BanffNET Indices explained large amounts of variability in multi-observer reference standards, time to graft failure and molecular biopsy-based diagnostics. In the large majority of cases, variability explained by BanffNET Indices matched or exceeded the variability explained by pathologist-assigned diagnoses and histological indices. Conclusions: BanffNET Indices are reproducible descriptors of whole slide images that could complement and augment kidney transplant biopsy evaluation by pathologists. Associations of BanffNET Indices with multi-observer diagnoses and related outcomes suggest that they capture additional information on tissue injury type and severity on top of pathologist-assigned Banff diagnoses and histological indices.

Key Points: Repeated measurements of plasma tacrolimus concentrations were obtained retrospectively from 295 kidney transplant recipients. Plasma tacrolimus concentration was negatively associated with later eGFR, whereas whole blood concentration was not. Plasma tacrolimus concentration was associated with arteriolar hyalinosis in protocol biopsies, whereas whole blood concentration was not. Background: Tacrolimus is the cornerstone of immunosuppressive treatment after kidney transplantation. Its application requires therapeutic drug monitoring, which is routinely performed by assessment of whole blood tacrolimus concentrations (WBTC) that largely comprises a pharmacologically inactive fraction. Monitoring tacrolimus exposure using the plasma tacrolimus concentration (PTC) may better reflect the pharmacologic active fraction. We hypothesized that PTC better reflects effectivity and toxicity than WBTC. Methods: The Amsterdam, LEiden, GROningen trial randomized 295 kidney transplant recipients (KTR) to standard triple immunosuppression, lower tacrolimus exposure, or withdrawal of prednisolone. PTC was measured using liquid chromatography–tandem mass spectrometry. Associations between tacrolimus concentrations and eGFR were analyzed using linear regression, with bootstrap resampling used to compare PTC and WBTC. Associations with biopsy results and rejection were evaluated using logistic regression, and infection risk was assessed with joint modeling. Results: The mean age was 56.5±13.2 years, and 95 (32%) were female. The mean PTC during the first month post-transplantation (0.31±0.17 ng/ml) was negatively associated with eGFR at 4 weeks (st. β =−0.17 [−0.29 to −0.05], P = 0.006), whereas mean WBTC was not (st. β =0.09 [−0.03 to 0.22], P = 0.15), and the difference between these associations was statistically significant ( P = 0.004). Late mean PTC (6.5–24 months; 0.15±0.07 ng/ml) was negatively associated with eGFR at 2 years (st. β =−0.19 [−0.35 to −0.03], P = 0.02), whereas mean WBTC was not (st. β =0.04 [−0.13 to 0.20], P = 0.66), and this difference was also statistically significant ( P = 0.02). At 2 years, 10 (14%) of the 71 protocol biopsies showed moderate-to-severe arteriolar hyalinosis. Although in a limited number of events, late mean PTC was significantly associated with a risk of arteriolar hyalinosis (odds ratio=1.96 [1.07–3.80], P = 0.03), WBTC was not (odds ratio=1.65 [0.83–3.53], P = 0.17). Neither PTC nor WBTC was significantly associated with a risk of infection in univariable analysis. Twenty-two KTR (7%) experienced biopsy-proven rejection; neither PTC nor WBTC was associated with rejection. Conclusion: Larger studies are needed to determine whether late PTC is a better marker than WBTC to detect potential tacrolimus-related nephrotoxicity in KTR. Clinical Trial registry name and registration number: NCT01560572.

Background Pre-implantation biopsy utility for deceased-donor kidney acceptance is controversial due to processing limitations and interpretive variability. We evaluated whether systematic histological assessment, including expert evaluation and quantification with the BanffNET automated deep learning system (17 lesions), predicts post-transplant failure under optimal laboratory conditions. Methods Biopsies from deceased donor kidneys (N=733) underwent optimized laboratory processing, whole slide imaging, expert Banff 2024 pathologic assessment, and BanffNET lesion quantification. We assessed their incremental predictive value beyond clinical characteristics for early graft dysfunction, longitudinal eGFR/UPCR trajectories, need for indication biopsies, and death-censored graft failure. Results Histopathology improved clinical model discrimination by 1-4% and increased explained variance for longitudinal trajectories by 1-2%. Continuous bivariate surface analysis, however, revealed that while histology adds marginal overall predictive accuracy, mapping continuous composite and individual BanffNET scores against the Kidney Donor Risk Index (KDRI) uncovers distinct topological risk gradients for early graft dysfunction and long-term graft failure. Conclusion While pre-implantation biopsies add minimal overall prognostic value, continuous bivariate mapping of KDRI and BanffNET scores uncovers distinct synergistic risk gradients for graft failure. These continuous clinical-histological surfaces could optimize organ salvage by precisely identifying viable high-KDRI kidneys lacking critical compounded damage and vice versa.

Background and Objective Tacrolimus therapeutic drug monitoring after kidney transplantation is primarily guided by the pre-dose concentration (C₀), assuming it reflects overall drug exposure. At the same time, the tacrolimus concentration-to-dose ratio (C₀/dose) has emerged as a marker associated with clinical outcomes after transplantation, suggesting that differences in peak (maximum concentration [C_max]) and total tacrolimus exposure (area under the concentration–time curve from 0 to 24 h [AUC_0–24h]) may not be fully captured by C₀ alone. Methods In 534 kidney transplant recipients followed for up to 13 months post-transplantation, 635 tacrolimus pharmacokinetic profiles with corresponding C₀ values were analyzed. The C₀/dose category was defined using the C₀/dose ratio and classified as fast (1·mg⁻¹, n = 344), intermediate (1.05–1.53, n = 127), or slow (≥1.54, n = 164). Analyses were restricted to measurements with C₀ between 5.0 and 6.0 ng/mL to compare C_max and AUC_0–24h across C₀/dose categories and tacrolimus formulations (immediate release vs extended release) using linear mixed-effects models. Sensitivity analyses evaluated C₀-adjusted C_max and AUC_0–24h in the full cohort (4271 pharmacokinetic profiles) and assessed consistency when C_max was predicted using population pharmacokinetic models. Results Despite comparable C₀ levels, the fast C₀/dose category was associated with substantially higher tacrolimus doses and had higher peak concentration and total exposure. Under immediate-release tacrolimus, C_max and AUC_0–24h were 30 and 18% higher, respectively, compared with the slow category. Under extended-release tacrolimus, differences were more pronounced, with 86% higher C_max (estimate: 1.86, 95% confidence interval 1.61–2.13) and 46% higher AUC_0–24h (estimate: 1.45, 95% confidence interval 1.35–1.55). The intermediate category showed a smaller but consistent increase in exposure. Higher C_max and AUC_0–24h relative to C₀ in fast metabolizers remained consistent in the full cohort and when C_max was model predicted. Conclusions Tacrolimus C₀ alone incompletely reflects the full pharmacokinetic profile and may miss clinically relevant peak-related toxicity. The C₀/dose ratio may help identify patients with disproportionate exposure, in whom more individualized therapeutic drug monitoring beyond C₀ monitoring is recommended.

Background: The presence of lymphoid tissues and their impact on long-term kidney graft outcome remains unclear. This study investigates the occurrence, molecular and phenotypic characteristics of lymphoid aggregates (LA) in protocol biopsies of clinically stable kidney transplant recipients, and their correlation with long-term clinical outcome. Methods: We retrospectively analyzed 414 protocol biopsies from a multicenter cohort of 278 stable kidney transplant recipients taken at either 6-, 12- or 24-months post-transplant. On a subset of 16 biopsies at 6 months post-transplant, mRNA gene expression analysis was performed using Nanostring B-HOT panel alongside phenotypic analysis with immunostaining for developmental staging of LA. Finally, the presence of LA was correlated to various clinical kidney allograft outcomes. Results: Stage I LA were characterized by upregulation of genes linked to immune regulation (LAG3, CTLA4, FOXP3), T-cell signaling, and chemotaxis pathways, with enrichment of regulatory T cells (Tregs), memory T cells, T follicular helper cells (Tfh), and dendritic cells. Immunohistochemistry confirmed higher numbers of FOXP3+ Tregs and dendritic cells in stage I LA, while high endothelial venules were absent. Over follow-up, participants with stage I LA showed slightly higher eGFR values and a trend toward better graft survival compared with those without LA or with stage II LA, although most differences did not reach statistical significance. Conclusions: In stable kidney transplant recipients, stage I LA displayed molecular and phenotypic features consistent with a regulated immune environment. The molecular and immunohistochemical analysis of stage I LA suggests a more regulatory molecular phenotype and composition, which may contribute to better long-term graft survival observed in this cohort of functionally stable transplant recipients. Observed trends toward more favorable outcomes suggest that early LA may not be deleterious and could represent local immune regulation, though larger prospective studies are needed to confirm these findings.

A bstract: Background: Banff histological diagnoses do not capture how a graft’s injury has evolved over time. Repeated injury is common, but the consequences of repeated histological injury are currently not known, especially when observed in the absence of clinical signs. Methods: In a single-center observational cohort of 1818 kidney transplantations performed between 2004 and 2021, with 5218 post-transplant biopsies of which 82% were pre-planned protocol biopsies, transitions between six Banff histological diagnoses were described as recurrent events, with landmark Aalen-Johansen estimates and unadjusted Andersen-Gill rate ratios. A multistate model, with states for a first and a second diagnosis of rejection-related injury (RRI, comprising TCMR, AMR and MVI _DSA-/C4d- ), estimated cause-specific hazards of death-censored graft failure, in all biopsies and in protocol biopsies only. Follow-up ran from day 90 to five years after transplantation. Results: Each diagnosis was more frequent after an earlier episode of the same diagnosis, and several diagnoses also made a different diagnosis more or less frequent: TCMR was more frequent after AMR, MVI _DSA-/C4d- and BKPyVAN, and AMR less frequent after MVI _DSA-/C4d- . The hazard of graft failure was increased after a first RRI diagnosis (HR 2.18, 95% CI 1.35–3.51) and substantially further after a second (HR 9.31, 95% CI 5.84–14.85). The association persisted when both diagnoses were made on protocol biopsy, in the absence of clinical signs (HR 1.76, 95% CI 1.09–2.84 and HR 2.58, 95% CI 1.31–5.08), although the difference between a second and a first diagnosis was not statistically significant. Conclusions: A prior Banff diagnosis strongly predicts that same diagnosis recurring, and in several cases also changes the expected incidence of a different diagnosis. A second diagnosis of rejection-related injury identifies a markedly higher-risk graft than a first, independently of baseline risk factors, and subclinical episodes detected on protocol biopsy also mark increased risk. This defines both an unmet therapeutic need and a high-risk population for trial enrichment. K ey learning points: What was known: A Banff diagnosis is rendered from a single biopsy, yet alloimmune injury often persists, recurs or changes over time. Repeated T-cell mediated rejection adds risk of graft failure, but evidence comes almost entirely from indication biopsies. How other diagnoses recur, and whether one predicts another, was largely unknown. This study adds: In 1818 transplants with 5218 biopsies (82% protocol), a prior diagnosis strongly predicted its recurrence and altered the incidence of others. A second rejection-related episode carried a significantly higher hazard of death-censored graft failure than a first (9.31 versus 2.18). Subclinical protocol-biopsy episodes also carried risk (2.58 versus 1.76). Potential impact: A first episode marks a graft at elevated risk, not a resolved problem. Biopsies are best read alongside the graft’s diagnostic history. Protocol biopsies detect prognostically relevant injury in apparently well recipients. Recipients with a second episode form a high-risk group suited to trials of more durable immune modulation.

Background: Clinical workplace learning was severely disrupted during the COVID‐19 pandemic because of social distancing measures. In response, we developed a Virtual Clinic using head‐mounted cameras to preserve authentic patient contact and clinical team interaction for medical students. Our aim was to create virtual teaching activities that allowed participation in real‐time patient care and facilitated peer learning and feedback. Approach: The Virtual Clinic was established in a separate space with live video connections to the hospital ward. Students rotated between wearing head‐mounted cameras during rounds, teaching visits and consultations, whereas peers observed remotely in small groups. Educational design was informed by principles of authentic and active learning strategies. Remote students contributed through discussion, documentation tasks and peer feedback. Evaluation: Twenty‐seven medical students participated in the Virtual Clinic during their internal medicine clerkship. Data were collected through 37 Likert‐scale items and 13 open‐ended questions. Students reported that Virtual Patient Rounds and Virtual Teaching Visits were both engaging and intellectually stimulating. Despite remote engagement, students reported a strong sense of integration within the medical team. Limitations included occasional connectivity issues. Implications: This innovation supported continued clinical education during pandemic restrictions while fostering student autonomy and team integration. The approach is transferable to other disciplines and contexts, such as interprofessional or global health education. Key lessons include the potential for virtual bedside learning beyond COVID‐19.

Pharmacogenomic research has historically focused on individuals of European ancestry, leading to the underrepresentation of genetic variants common in non‐European populations. This bias is exemplified by CYP3A5 *6, a functionally consequential variant common in individuals of African ancestry (MAF: 11–19%) but virtually absent in Europeans (MAF: 0.15%). We conducted a retrospective, longitudinal cohort study using real‐world data from 1,461 adult kidney transplant recipients across 67 countries, analyzing 4,293 dose‐normalized 24‐hour area‐under‐the‐curve (AUC _0‐24 ) measurements of tacrolimus. Patients with CYP3A5* 1/*1 were excluded. Linear mixed‐effects models (LME) were used to assess the association between CYP3A5* 6 carriage and tacrolimus exposure, adjusting for clinical factors and ancestry using both HLA‐based principal components and country of birth. CYP3A5 *6 carriers had a 17% lower dose‐normalized AUC _0‐24 than CYP3A5 *3 carriers ( P = 0.015). Sensitivity analyses using dose‐normalized trough concentrations (C ₀ ) confirmed these findings, with a 20% lower exposure in CYP3A5 *6 carriers ( P = 0.011). An interval‐based analysis demonstrated persistently lower tacrolimus exposure across the first post‐transplant year. All CYP3A5 *6‐containing genotypes showed significantly lower dose‐normalized AUC _0‐24 compared to CYP3A5 *3/*3, the most common genotype in European populations, with the largest reductions observed in CYP3A5 *1/*6 (−39%; P < 0.001) and CYP3A5 *3/*6 (−18%; P = 0.006). African origin, defined by country of birth, was independently associated with a 23% higher AUC _0‐24 ( P < 0.001). This is the first study to demonstrate a differential effect on tacrolimus exposure between the CYP3A5 *6 and CYP3A5 *3 loss‐of‐function alleles. Our results may help bridge the ethnicity gap, advance the applicability of pharmacogenomic findings, and promote health equity.

Current alloimmune risk assessment in kidney transplantation relies on HLA antigen mismatches, lacking precision for individualized care. In contrast, solvent-accessible HLA amino acid (saAA) mismatches, empirically defined across all HLA loci, offer a stable and objective alternative. We conducted a multicenter longitudinal study evaluating saAA mismatches, calculated using HLA-EMMA, in relation to key transplant outcomes. Among 2,473 kidney transplant recipients (median follow-up 6.9 years), 36% reached a hierarchical composite endpoint of death-censored graft failure(DCGF), biopsy-proven acute rejection(BPAR), or de novo donor-specific antibody(dnDSA). Multivariable analyses showed total, class I, class II, and locus-specific saAA mismatch scores were significantly associated with the composite outcome, as well as DCGF, BPAR and dnDSA individually (all P<.001). These findings suggest that saAA mismatch load provides a more granular and reproducible measure of alloimmune risk compared to traditional antigen-level mismatches. Incorporating saAA mismatch analysis could enable personalized immunosuppression strategies and optimized transplant monitoring in clinical practice.