Background/objectives: Kidney transplant recipients require lifelong immunosuppression and monitoring to prevent rejection, infection, and graft dysfunction. Current surveillance relies on tacrolimus therapeutic drug monitoring and, when needed, invasive biopsies. Dried blood spot (DBS) sampling provides a minimally invasive, patient-friendly option for remote follow-up. This study aims to develop and evaluate a DBS-based method for CXCL-10 quantification that, in combination with tacrolimus exposure monitoring, could help identify kidney recipients at risk of rejection and cytomegalovirus (CMV) infection and guide immunosuppression adjustment. Methods: The study included 81 selected kidney recipients for CXCL-10-DBS analysis by ELISA (12 T-cell mediated rejection; 10 antibody-mediated rejection; 6 CMV infection and 53 clinical event-free) and 10 healthy volunteers. A Tacrolimus-DBS LC-MS/MS method was developed and validated, and it was compared with the reference method on venous whole blood (WB) LC-MS/MS in a validation cohort (n = 160) and a clinical cohort (n = 36) using linear regression, Passing-Bablok and Bland-Altman analyses. Results: CXCL-10-DBS concentrations were significantly higher in rejectors (p < 0.001), with intermediate increases in CMV infection in comparison with event-free patients and healthy volunteers. ROC analysis demonstrated excellent diagnostic accuracy for rejection (AUC: 0.952; cutoff: 216.2 pg/mL; sensitivity: 100%; specificity: 79%; PPV: 88%; NPV: 100%). In contrast, tacrolimus trough concentrations did not differ significantly among the three clinical groups but showed strong correlation and agreement between DBS and venous WB with no systematic or proportional bias. Conclusions: This pilot study demonstrates the feasibility and diagnostic potential of DBS-based CXCL-10 measurement in adult kidney recipients. Integration of DBS-tacrolimus monitoring supports a minimally invasive pharmacokinetic-pharmacodynamic approach for personalized immunosuppression management.

The use of biomarkers in liver transplantation (LT) may be useful for the noninvasive assessment of T-cell-mediated rejection (TCMR). This study aimed to: (1) determine if changes in circulating microRNAs (miRNAs) in rejector patients reflect those at the tissue level; (2) assess a biomarker score (including miRNAs 155-5p, 181a-5p, and CXCL-10) in an independent cohort and (3) evaluate the score’s utility in detecting other LT-related complications. A prospective cohort of 151 LT recipients was followed during the first year after surgery. Patients with abnormal liver function tests (LFTs) underwent liver biopsy (LB) to rule out TCMR. Plasma samples were collected at predefined time points to analyse miRNAs and chemokines and their ability to predict and diagnose TCMR. To correlate plasma biomarkers with their tissue expression, 27 liver tissue samples (9 with normal LFTs and 18 with TCMR) were selected. Forty patients were diagnosed with TCMR. Circulating miRNAs (155-5p and 122-5p) reflected tissue-level changes in TCMR (p < 0.001, 3.3-fold increase for miR-155 and 2.3 for miR-122). Additionally assessed plasma miRNA 885-5p and chemokines CXCL-9 and CXCL-11, were also significantly elevated in TCMR but did not improve the performance of our previously biomarker score for TCMR (AUROC 0.94 for prediction, 0.99 for diagnosis). In conclusion, we confirmed in an independent cohort the performance of the score based on miR-155-5p, miR-181a-5p, and CXCL10. The correlation between circulating miRNAs and liver tissue expression underscores their value for predicting and diagnosing TCMR.

Background/Objectives: Tacrolimus is the most used immunosuppressive agent in solid organ transplantation due to its efficacy in preventing acute rejection, but it has a narrow therapeutic range, and overexposure often leads to toxicities, including neurological side effects like tremors. Tremor affects up to 54% of renal transplant patients under tacrolimus. Extended-release tacrolimus (LCPT) has demonstrated efficacy in reducing tremor severity, as evidenced by studies employing quality of life (QoL) questionnaires, the Fahn–Tolosa–Marin (FTM) scale, and Accelerometer devices. The objectives of this study were to evaluate the benefits of the conversion to LCPT formulation in kidney transplant recipients experiencing tremors on prolonged-release tacrolimus (PR-TAC) treatment and to validate the DyCare device, a wearable wireless sensor for tremors. Results: The DyCare device measured tremor frequencies of 8.74 ± 0.11 Hz and 1.36 ± 0.08° and 17.38 ± 1.16°, as root mean square (RMSx100 for accelerometer and Gyroscope, respectively) in PR-TAC patients. After switching ten patients to LCPT, tremor severity significantly decreased, as confirmed by DyCare and the QoL in the Essential Tremor Questionnaire (QUEST). Additionally, LCPT allowed a 34% reduction in tacrolimus dosage while maintaining therapeutic trough concentrations. Immunological and pharmacodynamic biomarkers (p-miR-210-3p, p-IL10, p-IL12p70, p-IFNγ uCXCL10, NFAT-regulated gene expression) confirmed stable immunosuppression post-conversion. Conclusions: The conversion to the LCPT formulation significantly reduced tremors in kidney transplant recipients without altering their immunological status, as confirmed through a panel of immunologic and pharmacodynamic biomarkers. The DyCare device enables a precise quantification of tremors in transplant recipients, allowing physicians to optimize treatment strategies.

Introduction:The identification of noninvasive biomarkers for monitoring liver transplant (LT) recipients is crucial for the early detection of graft dysfunction and rejection. Donor-derived cell-free DNA (dd-cfDNA) and microRNAs (miRNAs) have been identified as promising biomarkers for assessing graft integrity. While the levels of dd-cfDNA have been validated for this use in kidney and heart transplantation, there are limited data regarding its potential in liver graft monitoring. Similarly, the expression levels of miRNAs, key regulators of immune responses and liver injury, have potential utility in distinguishing between rejection and other causes of graft dysfunction. Methods:In this prospective, observational study, we monitored the levels of dd-cfDNA and miRNAs by analyzing 437 plasma samples from 64 LT recipients over a 12-month period, measuring the levels of dd-cfDNA and signature miRNAs at predefined time points and during episodes of graft dysfunction. The diagnostic performance of the levels of dd-cfDNA and signature miRNAs was assessed through receiver operating characteristic (ROC) curve analysis and logistic regression models. Results:dd-cfDNA levels were significantly elevated during acute rejection (AR) episodes, with a median 3.9-fold increase over those in stable patients. A diagnostic cut-off value of 9.88% yielded an area under the ROC curve (AUROC) of 0.812, a sensitivity of 100%, a specificity of 66.7%, a positive predictive value (PPV) of 17.5% and a negative predictive value (NPV) of 100%. Interestingly, patients with cholestasis also exhibited increased dd-cfDNA levels (3.0-fold vs. stable patients), suggesting that it could serve as a potential confounder in the diagnosis of transplant rejection. Plasma miRNA analysis demonstrated significant upregulation of the expression levels of miR-155-5p, miR-122-5p, and miR-181a-5p during rejection episodes, and the incorporation of these factors improved diagnostic accuracy when combined with the level of dd-cfDNA. Conclusions:dd-cfDNA and miRNA profiling represent promising noninvasive biomarkers for diagnosing liver graft rejection and dysfunction. The combined use of these biomarkers may result in increased diagnostic accuracy, reduce unnecessary biopsies, and allow personalized immunosuppressive management. Further studies in larger cohorts are needed to validate the clinical applicability of these compounds as diagnostic biomarkers.

ABSTRACT:The Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology established the second consensus report to guide Therapeutic Drug Monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice 7 years after the first version was published in 2016. This version provides information focused on new developments that have arisen in the last 7 years. For the general aspects of the pharmacology and TDM of EVR that have retained their relevance, readers can refer to the 2016 document. This edition includes new evidence from the literature, focusing on the topics updated during the last 7 years, including indirect pharmacological effects of EVR on the mammalian target of rapamycin complex 2 with the major mechanism of direct inhibition of the mammalian target of rapamycin complex 1. In addition, various concepts and technical options to monitor EVR concentrations, improve analytical performance, and increase the number of options available for immunochemical analytical methods have been included. Only limited new pharmacogenetic information regarding EVR has emerged; however, pharmacometrics and model-informed precision dosing have been constructed using physiological parameters as covariates, including pharmacogenetic information. In clinical settings, EVR is combined with a decreased dose of calcineurin inhibitors, such as tacrolimus and cyclosporine, instead of mycophenolic acid. The literature and recommendations for specific organ transplantations, such as that of the kidneys, liver, heart, and lungs, as well as for oncology and pediatrics have been updated. EVR TDM for pancreatic and islet transplantation has been added to this edition. The pharmacodynamic monitoring of EVR in organ transplantation has also been updated. These updates and additions, along with the previous version of this consensus document, will be helpful to clinicians and researchers treating patients receiving EVR.

ABSTRACT:The use of noninvasive biomarkers may reduce the need for biopsy and guide immunosuppression adjustments during transplantation. The scientific community in solid organ transplantation currently considers that chemokines, T- and B-cell immunophenotypes, and gene expression, among other molecular biomarkers, have great potential as diagnostic and predictive biomarkers for graft evolution; however, in clinical practice, few valid early biomarkers have emerged. This review focuses on the most relevant scientific advances in this field in the last 5 years regarding the role of 3 biomarkers: miRNAs, chemokines, and ddcf-DNA, in both adult and pediatric populations. An update was provided on the scores based on the combination of these biomarkers. The most-featured articles were identified through a literature search of the PubMed database. This review provides a comprehensive analysis of the potential clinical applications of these biomarkers in the diagnosis and prediction of graft outcomes and discusses the reasons why none have been implemented in clinical practice to date. Translating these biomarkers into routine clinical practice and combining them with pharmacogenetics and pharmacokinetic monitoring is challenging; however, it is the key to present/future individualized immunosuppressive therapies. It is essential that they be shown to be applicable and robust in real-life patient conditions and properly evaluate their added value when combined with the standard-of-care factor monitoring for graft clinical assessment. Partnership strategies among scientists, academic institutions, consortia, including expert working groups and scientific societies, and pharmaceutical and/or biotechnology companies should promote the development of prospective, randomized, multicenter intervention studies for adequate clinical validation of these biomarkers and their monitoring frequency, and their commercialization to make them available to transplant physicians.

IntroductionThe use of noninvasive biomarkers may avoid the need for liver biopsy (LB) and could guide immunosuppression adjustment in liver transplantation (LT). The aims of this study were: to confirm the predictive and diagnostic capacity of plasmatic expression of miR-155-5p, miR-181a-5p, miR-122-5p and CXCL-10 for assessing T-cell mediated rejection (TCMR) risk; to develop a score based on a panel of noninvasive biomarkers to predict graft rejection risk and to validate this score in a separate cohort. MethodsA prospective, observational study was conducted with a cohort of 79 patients followed during the first year after LT. Plasma samples were collected at predetermined time points for the analysis of miRNAs and the CXCL-10. Patients with LFTs abnormalities were submitted to a LB to rule out rejection, assessing previous and concurrent expression of the biomarkers to evaluate their predictive and diagnostic ability. Information from 86 patients included in a previous study was collected and used as a validation cohort. ResultsTwenty-four rejection episodes were diagnosed in 22 patients. Plasmatic CXCL-10 concentration and the expression of the three miRNAs were significantly elevated prior to and at the moment of the diagnosis of rejection. We developed a logistic model for rejection prediction and diagnosis, which included CXCL-10, miR-155-5p and miR-181a-5p. The area under the ROC curve (AUROC) for rejection prediction was 0.975 (79.6% sensitivity, 99.1% specificity, 90,7% PPV; 97.7% NPV; 97.1% correctly classified) and 0.99 for diagnosis (87.5% sensitivity, 99.5% specificity, 91.3% PPV; 99.3% NPV; 98.9% correctly classified). In the validation cohort (n=86; 14 rejections), the same cut-off points were used obtaining AUROCs for rejection prediction and diagnosis of 0.89 and 0.92 respectively. In patients with graft dysfunction in both cohorts the score could discriminate those with rejection regarding other causes with an AUROC of 0.98 (97.3% sensitivity, 94.1%specificity). ConclusionThese results suggest that the clinical implementation of the monitoring of this noninvasive plasmatic score may allow the prediction and diagnosis of rejection and identify patients with graft dysfunction due to rejection, helping with a more efficient guide for immunosuppressive therapy adjustment. This finding warrants the development of prospective biomarker-guided clinical trials.

Previous results from our group and others have shown that urinary pellet expression of miR155-5p and urinary CXCL-10 production could play a key role in the prognosis and diagnosis of acute rejection (AR) in kidney transplantation patients. Here, a logistic regression model was developed using NONMEM to quantify the relationships of miR155-5p urinary expression, CXCL-10 urinary concentration and tacrolimus and mycophenolic acid (MPA) exposure with the probability of AR in adult kidney transplant patients during the early post-transplant period. Owing to the contribution of therapeutic drug monitoring to achieving target exposure, neither tacrolimus nor MPA cumulative exposure was identified as a predictor of AR in the studied population. Even though CXCL-10 urinary concentration showed a trend, its effect on AR was not significant. In contrast, urinary miR155-5p expression was prognostic of clinical outcome. Monitoring miR155-5p urinary pellet expression together with immunosuppressive drug exposure could be very useful during routine clinical practice to identify patients with a potential high risk of rejection at the early stages of the post-transplant period. This early risk assessment would allow for the optimization of treatment and improved prevention of AR.

INTRODUCTION:Actually, immunosuppression selection isn't based on individual immune alloreactivity, and immunosuppressive drug dosing is mainly based on the development of toxicity and the achievement of specific target concentrations. Since a successful outcome requires optimal patient risk stratification and treatment, several groups have evaluated candidate biomarkers that have shown promise in the assessment of individual immune responses, the prediction of personal pharmacodynamic effects of immunosuppressive drugs and the prognosis and diagnosis of graft outcomes..AREAS COVERED:This review includes biomarkers that the Scientific Community in Solid Organ Transplantation currently considers to have potential as diagnostic and prognostic biomarkers of graft evolution. We have focused on recent scientific advances and expert recommendations regarding the role of specific and non-specific pharmacodynamic biomarkers that are mainly involved in the T-cell-mediated response.EXPERT OPINION:Integral pharmacologic monitoring that combines pharmacokinetics, pharmacogenetics and predictive pharmacodynamic biomarkers may provide crucial information and allow personal adjustment of immunosuppressive drugs at an early stage before severe adverse events ensue. Multicentre, randomized, prospective and interventional trials are needed to fine tune the established cut-off values for each biomarker and the optimal monitoring frequency for each biomarker and to accurately evaluate possible clinical confounding factors to enable correct clinical qualification.

AimsCalcineurin inhibitors (CNI) have a small therapeutic window, and drug monitoring is required. Pharmacokinetic monitoring does not correlate sufficiently with clinical outcome. Therefore, the expression of nuclear factor of activated T cells (NFAT)‐regulated genes in the peripheral blood has been suggested as a potentially useful immune monitoring tool to optimize CNI therapy. NFAT‐regulated gene expression (RGE) was evaluated in renal allograft recipients as predictive biomarker to detect patients at risk of acute rejection or infections.MethodsNFAT‐RGE (interleukin‐2, interferon‐γ, granular‐macrophage colony‐stimulating factor) was evaluated by quantitative real‐time polymerase chain reaction in whole blood samples at day 7, day 14, month 1, 3, and 6 after transplantation in 64 de novo renal allograft recipients from 3 European centres. Immunosuppression consisted of tacrolimus (Tac), mycophenolic acid, and corticosteroids.ResultsTac concentrations (C0 and C1.5) correlated inversely with NFAT‐RGE (P < .01). NFAT‐RGE showed a high interindividual variability (1–61%). Patients with high residual gene expression (NFAT‐RGE ≥30%) were at the increased risk of acute rejection in the following months (35 vs. 5%, P = .02), whereas patients with low residual gene expression (NFAT‐RGE <30%) showed a higher incidence of viral complications, especially cytomegalovirus and BK virus replication (52.5 vs. 10%, P = .01).ConclusionsNFAT‐RGE was confirmed as a potential noninvasive early predictive biomarker in the immediate post‐transplant period to detect patients at risk of acute rejection and infectious complications in Tac‐treated renal allograft recipients. Monitoring of NFAT‐RGE may provide additional useful information for physicians to achieve individualized Tac treatment.

When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

This study evaluate the potential of plasmatic CXCL-10 (pCXCL-10) as a pre&post transplantation prognostic and diagnostic biomarker of T-cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR) and subclinical rejection (SCR) risk in adult kidney recipients considering BKV and CMV infections as possible clinical confounder factors. Twenty-eight of 100 patients included experienced rejection (TCMR:14; ABMR:14); 8 SCR; 13 and 16 were diagnosed with BKV and CMV infection, respectively. Pre-transplantation pCXCL-10 was significantly increased in TCMR and ABMR and post-transplantation in TCMR, ABMR and SCR compared with nonrejectors. All CMV+ patients showed pCXCL-10 levels above the cutoff values established for rejection whereas the 80% of BKV+ patients showed pCXCL-10 concentration < 100 pg/mL. pCXCL-10 could improve pre-transplantation patient stratification and immunosuppressive treatment selection according to rejection risk; and after kidney transplantation could be a potential early prognostic biomarker for rejection. Clinical confounding factor in BKV+ and particularly in CMV+ patients must be discarded.

MicroRNAs (miRNAs) are small noncoding RNAs that can be detected in plasma and whose expression is associated with pathological processes. The role of miRNAs in the noninvasive diagnosis of T cell–mediated rejection (TCMR) after liver transplantation (LT) is unclear. Thus, we aimed to assess the effectiveness of a panel of 4 miRNAs (155‐5p, 122‐5p, 181a‐5p, and 148‐3p) in diagnosing TCMR in LT recipients with graft dysfunction (GD), and we compared its accuracy with previously published tests for diagnosing TCMR based on routine laboratory parameters. From a prospective cohort of 145 patients followed during the first year after transplant, 49 developed GD and underwent a liver biopsy and plasma collection for miRNA analysis using quantitative real‐time polymerase chain reaction. Patients with GD due to TCMR (n = 21) exhibited significantly higher (P < 0.001) expression of miRNA 155‐5p (2.05 versus 0.07), 122‐5p (19.36 versus 1.66), and 181a‐5p (1.33 versus 0.37) compared with those with GD from other causes (n = 28). The area under the receiver operating characteristic curve of miRNAs 155‐5p, 122‐5p, and 181a‐5p for the diagnosis of TCMR was 0.87, 0.91, and 0.89, respectively, significantly higher than those of the other noninvasive tests (P < 0.001). Furthermore, miRNA 155‐5p identified all patients who presented TCMR during the first 2 weeks after transplant. miRNA plasmatic expression differentiates TCMR from other causes of GD in patients who have undergone LT and may be a useful tool in clinical practice.

BACKGROUND & AIMS:Sorafenib and lenvatinib are the first-line treatments approved in hepatocellular carcinoma (HCC), but information is lacking about the relationships between their pharmacokinetics, patients pharmacogenetic profiles, adverse events (AE) and overall survival. We aimed to elucidate these relationships of tyrosine Kinase Inhibitors, such as sorafenib, in order to improve the design of trials testing it in combination with checkpoint inhibitors.METHODS:We assessed the pharmacokinetics of sorafenib and its N-oxide metabolite at day-0, day-7, day-30, day-60, day-90, day-120, day-150 and day-180 and nine single-nucleotide polymorphisms (SNP) in five genes related to sorafenib metabolism/transport to identify the best point for starting the combination between tyrosine kinases and checkpoint inhibitors.RESULTS:We prospectively included 49 patients (96% cirrhotic, 37% hepatitis-C, 82% Child-Pugh-A and 59% BCLC-C). Pharmacokinetic values peaked at day-7 and progressively declined until day-60. In the 16 patients without further dose modifications after day-60, pharmacokinetic values remained stable through day-180 (sorafenib P = .90; N-oxide P = .93). Pharmacokinetic values were higher in patients with early dermatological adverse events and lower in patients with early diarrhoea. Sorafenib and N-oxide pharmacokinetic values varied linearly with different alleles of MRP2*3972.CONCLUSIONS:Sorafenib's pharmacokinetics is heterogeneous across HCC patients. This heterogeneity affects adverse events development and must be taken into account in setting the dose and timing of its combination with checkpoint inhibitors.