MD Paolo Malvezzi
email: pmalvezzi@chu-grenoble.fr

Clara Fischman

MD Guillaume Rigault

MD Marie Christine Jacob

MD Tatiana Raskovalova

MD Thomas Jouve

MD Bénédicte Janbon

MD Lionel Rostaing

MD, PhD Paolo Cravedi

Switching renal transplant recipients to belatacept therapy: results of a real-life gradual conversion protocol

Keywords: CXCL9, renal transplant, belatacept, tacrolimus, conversion

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The use of calcineurin inhibitor (CNI)-based maintenance immunosuppression has dramatically reduced the rates of acute cellular rejection after kidney transplantation [START_REF] Kasiske | KDIGO clinical practice guideline for the care of kidney transplant recipients[END_REF][START_REF] Kasiske | KDIGO clinical practice guideline for the care of kidney transplant recipients: a summary[END_REF], but long-term graft survival has not improved significantly over the past decades [START_REF] Lamb | Long-Term Renal Allograft Survival in the United States: A Critical Reappraisal[END_REF]. Several studies have correlated the long-term use of CNIs with renal interstitial fibrosis and tubular atrophy [START_REF] Nankivell | The natural history of chronic allograft nephropathy[END_REF][START_REF] Naesens | Calcineurin inhibitor nephrotoxicity[END_REF][START_REF] Gaston | Chronic calcineurin inhibitor nephrotoxicity: reflections on an evolving paradigm[END_REF] and have prompted attempts to transition patients to other immunosuppressive agents. Mammalian target of rapamycin inhibitor (mTORi)-based regimens resulted in more frequent rejection episodes than tacrolimus and comparable chronic arteriolar toxicity [START_REF] Naesens | Calcineurin Inhibitor Nephrotoxicity in the Era of Antibody-Mediated Rejection[END_REF]. Furthermore, CNI-free mTORibased regimens have been associated with an increased risk of de novo donor-specific antihuman leukocyte antibodies (DSA) [START_REF] Naesens | Calcineurin Inhibitor Nephrotoxicity in the Era of Antibody-Mediated Rejection[END_REF][START_REF] Ekberg | Reduced exposure to calcineurin inhibitors in renal transplantation[END_REF]. More recently, belatacept, a selective T-cell inhibitor of CD28-CD80/86 co-stimulation, in combination with mycophenolate mofetil (MMF) and steroids, preserved long-term graft function in de novo kidney-transplant recipients better than cyclosporine-receiving counterparts, and reduced the incidence of DSA development [START_REF] Durrbach | A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFIT-EXT study)[END_REF][START_REF] Vincenti | A phase III study of belatacept-based immunosuppression regimens versus cyclosporine in renal transplant recipients (BENEFIT study)[END_REF]. Despite these promising results, belatacept treated patients experienced significantly higher rates of acute rejection. Rejection events occurred mainly during the first-year post-transplantation, suggesting that selective co-stimulation blockade may not be sufficient to inhibit alloreactive memory T cells (less dependent on CD28-CD80/86 signaling for activation) in early phase posttransplantation when the alloimmune response is strongest [START_REF] Durrbach | Long-Term Outcomes in Belatacept-Versus Cyclosporine-Treated Recipients of Extended Criteria Donor Kidneys: Final Results From BENEFIT-EXT, a Phase III Randomized Study[END_REF][START_REF] Vincenti | Belatacept and Long-Term Outcomes in Kidney Transplantation[END_REF].

In an attempt to limit long-term nephrotoxicity while maintaining the beneficial peri-transplant immunosuppressive effects of CNIs, several groups have conducted trials in which kidneytransplant recipients were switched from CNIs to belatacept maintenance immunotherapy at 6 to 24 months post-transplantation [START_REF] Rostaing | Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study[END_REF][START_REF] De Graav | A Randomized Controlled Clinical Trial Comparing Belatacept With Tacrolimus After De Novo Kidney Transplantation[END_REF][START_REF] Darres | Conversion to belatacept in maintenance kidneytransplant patients: A retrospective multicenter European study[END_REF]. Although these studies demonstrated an improved or stable glomerular filtration rate (GFR) while receiving belatacept, acute-rejection episodes were common during conversion [START_REF] Rostaing | Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study[END_REF]. One potential explanation for the high rates of acute rejections may rely in the relatively fast conversion, that generally occurred in one month or less [START_REF] Rostaing | Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study[END_REF][START_REF] Darres | Conversion to belatacept in maintenance kidneytransplant patients: A retrospective multicenter European study[END_REF][START_REF] Grinyó | Safety and Efficacy Outcomes 3 Years After Switching to Belatacept From a Calcineurin Inhibitor in Kidney Transplant Recipients: Results From a Phase 2 Randomized Trial[END_REF]. A longer time period may be needed to allow the immune system to "adapt" to the different targets of the new immunosuppressive agent that often also imply changes in cell metabolism and epigenetic profile.

Moreover, the ability to identify patients at risk of acute rejection prior to graft damage during conversion to belatacept would improve the safety of this strategy. Urinary chemokine (C-X-C motif) ligand 9 (CXCL9), an interferon-γ-induced T-cell chemoattractant chemokine released by monocytes/macrophages, endothelial cells, and renal parenchymal cells [START_REF] Lazzeri | CXCR3-binding chemokines: novel multifunctional therapeutic targets[END_REF], can non-invasively detect clinical and subclinical acute cellular rejection with a high negative predictive value [17 -22]. Data from the Clinical Trial in Organ Transplantation 09 (CTOT-09), a study where kidney transplant recipients underwent tacrolimus withdrawal, show that urinary CXCL9 at a positivity threshold of ≥200 pg/ml can detect acute rejection 3 to 30 days before clinical presentation [START_REF] Hricik | Adverse Outcomes of Tacrolimus Withdrawal in Immune-Quiescent Kidney Transplant recipients[END_REF].

Data from a case-series indicate that urinary CXCL9 can also be used to monitor response to therapy for acute rejection [START_REF] Gandolfini | Rapid Biolayer Interferometry Measurements of Urinary CXCL9 to Detect Cellular Infiltrates Noninvasively After Kidney Transplantation[END_REF]. Therefore, serial measurements of urinary CXCL9 could allow to identify early patients at risk for acute cellular rejection during conversion from CNI to belatacept and to treat them promptly.

Based on this background, we developed a strategy of gradual (over a 2-month time period) conversion from CNI to belatacept to reduce the risk of acute rejection. In the present study, we tested the safety and feasibility of such approach in the real-life setting of a large transplant center and we tested the association between changes in urinary CXCL9 and acute rejection episodes during conversion. We also performed serial measurement of main circulating immune B and T cell subsets to monitor immune changes associated with conversion to belatacept therapy.

MATERIALS AND METHODS

Design

This was a retrospective study performed at the Grenoble University Hospital (Grenoble, France), where deceased or living kidney-transplant recipients on chronic CNI immunosuppressive therapy underwent conversion to belatacept immunosuppression. Inclusion criteria were: time from transplant > 3 months, signs of CNI nephrotoxicity (eGFR decline without donor specific antibodies and compatible allograft biopsy findings), CNI neurotoxicity, or toxicity related to any other immunosuppressive drug, eGFR < 35 mL/min/1.73 m 2 , or metabolic disorders (e.g. metabolic syndrome or new onset of diabetes after transplant). Patients with negative EBV serology were excluded. Urine samples were collected on the day of the first belatacept infusion (before infusion) and at 1, 3, 6, and 12 months thereafter. All patients provided their written informed consent for urine collection. All medical data were collected from the Grenoble University Hospital database [CNIL (French national committee for data protection) approval number 1987785v0].

Conversion to belatacept

All included patients started conversion by receiving 5 mg/kg of belatacept on day 1, 15, 28 and then monthly thereafter. During the first month following initiation of belatacept, CNI dose was unchanged; then, at month 2 after initiation of belatacept, CNI dose was halved and then stopped at the beginning of month 3. For one patient, CNI was stopped at 6 months after initiation of belatacept because of a prescription error. The doses of mycophenolate mofetil and/or corticosteroids remained unchanged in all the participants. The two subjects that were on combined tacrolimus and everolimus therapy had everolimus replaced by mycophenolic acid at conversion.

Clinical and immunological follow-up

Participants were examined at baseline (before first belatacept administration) and before each subsequent belatacept infusion. We also measured systolic and diastolic blood pressure in a seated position after 10 minutes of rest using vital signs monitor (DYNAMAP PRO 300 V2, GE Healthcare). Patients had serum creatinine and proteinuria measured on the day of the first infusion of belatacept and at 1, 3, 6, and 12 months thereafter. GFR was estimated using the CKD EPI equation [START_REF] Levey | A new equation to estimate glomerular filtration rate[END_REF]. Complete blood count and total CD4 + and CD8 + T cells, B cells, NK cells and T and B cells subset were measured at the same time points.

For patients with diabetes, HbA1c was measured on the day of the first infusion of belatacept and at 3, 6, 12 months after starting conversion. For the others, HbA1c was assessed before conversion and 1-year later. Hemoglobin A1C (HbA1c) dosage was performed by High-Performance Liquid Chromatography (HPLC). Patients had total cholesterol, LDL-c, and triglycerides measured before conversion and 1-year after the conversion to belatacept. All these analyses were performed at the central lab of the Grenoble University Hospital.

Anti-donor human leukocyte antigen (HLA) antibodies were measured at the time of first belatacept infusion, at the end of the 12-month follow-up period, and at the time of any suspected rejection episode by Luminex (Immucor®). Identification of anti-HLA specificity was performed by single antigen assays if screening test was positive. Assessment of anti-HLA antibodies was performed at the Etablissement Français du Sang -EFSat Grenoble University Hospital.

Measurement of urinary CXCL9

Urinary samples for CXCL9 analyses were centrifuged at 2,000g for 30 min at 4°C within 4 h from collection. The supernatant was divided into aliquots and frozen at -80°C. Urinary CXCL9 was measured in batches at the end of the study. We strictly followed the protocol defined by a multicenter validation study for CXCL9 measurement [START_REF] Hricik | Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury[END_REF] and reported the data as pg/ml without creatinine correction accordingly. We used the same previously defined threshold for positivity of 200 pg/mL [START_REF] Hricik | Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury[END_REF]. Frozen aliquots of urine supernatants were diluted (1:1) in 0.05% Tween 20/0.4% bovine serum albumin in phosphate-buffered saline (pH 7.2-7.4) and tested by ELISA for CXCL9 (R&D Systems, Minneapolis, MN), as previously reported [START_REF] Hricik | Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury[END_REF]. .

Flow cytometric peripheral blood lymphocyte analysis

Cell staining was performed on whole blood samples using a direct immunofluorescence method with erythrocytes lysis and washing, then lymphocyte subsets were identified using a 3-laser, 8color BD FACSCanto-II TM flow cytometer and FACSDiva software (BD Biosciences, San José, CA). The absolute numbers of subsets were calculated by multiplying their percentage by the total lymphocyte number obtained from an ABX MICROS 60 device (HORIBA ABX SAS, Montpellier, France). CD3 + HLA-DR + evaluated activated T-cells. The second combination discriminated naive (CD45RA + CCR7 + ), central memory (CD45RA -CCR7 + ), and effector memory (CD45RA +/- CCR7 -) CD4 + and CD8 + T-cell subsets [START_REF] Saule | Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment[END_REF]. The last combination distinguished naive (CD19 + IgD + CD27 -), natural memory (CD19 + IgD + CD27 + ), post-germinal center (CD19 + IgD - CD27 + ) B-cell subsets [START_REF] Aspord | Remodeling of B-Cell Subsets in Blood during Pegylated IFNα-2a Therapy in Patients with Chronic Hepatitis B Infection[END_REF], and plasma cell precursors (CD19 + IgD -CD27 high CD38 high ).

Three combinations of anti-human antibodies

Statistical analyses

Baseline characteristics were compared by Wilcoxon rank sum, χ 2 , or Fisher's exact test as appropriate. eGFR slopes were calculated using a single slope linear model for each patient who had at least three eGFR values. eGFR slopes before vs. after belatacept initiation were compared using a paired t test, as appropriate. Statistical analyses were performed with GraphPad Prism 5.0 software using paired Wilcoxon rank tests to compare parameters with baseline values. Twosided significance tests were used throughout. P < 0.05 was considered statistically significant.

RESULTS

Patients

Between September 2016 and March 2017, the study included 35 kidney transplant recipients (Table 1). The median time between transplant and conversion to belatacept was 3.3 years (interquartile interval: 1.3-7.2). Four patients were recipients of a second transplant. Mean age at conversion was 55.5 ± 12.5 years. Seven (20%) subjects had circulating anti-HLA antibodies and two subjects had low level (MFI < 2000) class I DSA. No one had class II DSA. All patients received induction therapy (32 with antithymocyte globulins and 3 with basiliximab). Before belatacept conversion, 31 patients were receiving tacrolimus, 3 were on cyclosporine, and one was receiving azathioprine (this patient was switched from azathioprine to belatacept, due to recurrent squamous cell carcinomas [START_REF] Jiyad | Azathioprine and Risk of Skin Cancer in Organ Transplant Recipients: Systematic Review and Meta-Analysis[END_REF]. Thirty-one patients took mycophenolic acid in association with the CNIs, 2 patients received everolimus, and 11 patients received low doses of steroids. The reasons for the conversion are specified in Table 2.

No patient had BK virus nephropathy previous to or at the time of conversion. BKV viremia was performed only in those patients that had kidney graft dysfunction. None of these patients developed viremia during the first year following conversion. No patient developed CMV disease.

Clinical outcomes and safety

All patients withdrew CNI and were converted to belatacept over a 2-month time period except for one patient who stayed with halved CNI doses from month 2 to month 6 after study entry because of a prescription error (starting from month 3, his tacrolimus trough levels were below detection limit at our lab of 1 ng/ml).

Patient-and graft-survival rates at 1-year post-conversion were 100%. Kidney function measured by eGFR (Figure 1) and proteinuria was stable over the 12-month follow-up: mean delta between conversion and one year later was 0.11±10.5 ml/min/1.73m 2 (p=0.448) for eGFR and 0.02±0.3g/24h (p=0.987) for proteinuria. For patients with kidney transplantation performed more than one year before conversion, the 12-month slope of eGFR before conversion showed faster worsening of graft function the year before conversion compared to the one after conversion, but this difference did not reach statistical significance (median difference: -6.2 vs -1.8 ml/min/1.73m 2 for eGFR slope before vs. after conversion, respectively; p=0.213). When we stratified patients according to eGFR at study entry, we found that during the 12-month followup period, eGFR was stable across all levels of renal function (Supplemental Figure 1).

Changes in serum creatinine, eGFR, and proteinuria remained significant when we repeated the analyses after excluding the patient that continued CNI for 6 months.

Two patients underwent per-cause graft biopsy due to acute rise in serum creatinine following belatacept conversion (at 4.5 months and 3.5 months after first belatacept infusion). In one patient, the graft biopsy showed mild acute rejection (IA), while the biopsy of the second patient did not show any infiltrate reaching criteria for acute rejection. Both patients received steroid pulses followed by rapid dose reduction and creatinine values returned to baseline following treatment. After steroid pulses, both patients were maintained on belatacept.

Conversion was overall well tolerated, including belatacept infusions. Adverse events are listed in Table 3 and none of them was associated with belatacept therapy. No major infection requiring hospitalization occurred. No patient stopped belatacept during the study. Two patients were converted to belatacept for neuropathy: one patient felt a significant improvement after one year, while the second did not.

Blood pressure and metabolic profile

Mean systolic arterial blood pressure significantly decreased between baseline (before conversion) and 12 months post-conversion (146.3±18.8 vs. 137.5±16.1 mmHg; p=0.03), while mean diastolic blood pressure did not significantly change (86.5±12.3 at baseline vs, 84.1±13.5 at 12 months post-conversion; p=0.36) (Figure 2A). Twenty-three patients reduced or did not change the dosage of their antihypertensive drugs, while 12 patients had their doses increased. In patients with diabetes mellitus history (n=10), the mean HbA1c also significantly declined after conversion (7.1±1.2%, 6.4±1.4%, 6.3±1.1% at baseline, 6 and 12 months after first belatacept administration; p<0.05 for both time points vs. baseline) (Figure 2B). None of these patients had their antidiabetic drugs increased and in 4 patients their doses were reduced.

Total cholesterol, LDL cholesterol, and triglyceride levels did not significantly change between the baseline levels and those at the end of the study (not shown).

Circulating immune globulin and lymphocyte subsets

Concentrations of various immunoglobulin subclasses did not significantly change from baseline to 12 months post-conversion (IgG: 9.0±2.3 vs. 9.3±2.4 g/l; p=0.44, IgA: 2.1±1.1 vs. 2.0±1.0 g/l; p= 0.72, and IgM: 0.8±0.6 vs. 0.8 ±0.6 g/l; p=0.80). No patient developed de novo DSA during the follow-up period.

Absolute numbers and percentages of circulating total leukocytes, T cells, B cells, and NK cells

were stable across the study period. Percentages of central memory CD4 + and effector CD8 + T cells and memory and post-germinative B cells significantly declined between the day of first belatacept infusion and 12 months thereafter (Figure 3 and Table 4). The other subsets did not vary significantly.

Urinary CXCL9

Urinary CXCL9 levels were below the positivity threshold of 200 pg/mL for all but 3 patients, who showed elevated values at the time of first belatacept administration (n=1) or during followup (n=2) (Figure 4A).

The subject with high baseline CXCL9 value was on tacrolimus and everolimus at the time of first belatacept administration. Tacrolimus was stopped at month 2 after belatacept initiation, but two months later (4 months after starting conversion) the creatinine-level rose, and a graft biopsy showed acute cellular rejection (grade IA). Following steroid pulses, serum creatinine returned to baseline values (Figure 4B).

In a second patient, CXCL9 was normal at baseline and increased to > 250 pg/mL at 3 months after conversion. This increase happened 3 weeks before the rise in serum creatinine, which prompted a kidney graft biopsy. The histology showed interstitial inflammation, although it did not meet the Banff criteria for acute rejection. The patient received steroid pulses that lowered serum creatinine and urinary CXCL9 levels (Figure 4C).

In the third patient, urinary CXCL9 was normal at baseline and at 1-month post-conversion, but became positivity threshold and then remained elevated. Creatinine level fluctuated but remained within ± 40 mol/L from baseline, therefore no biopsy was performed (Figure 4D). At 12 helper cell differentiation, B cell clonal expansion, germinal center reaction and DSA formation months after conversion, the patient's serum creatinine level was lower than baseline (131µmol/l vs. 119µmol/l).

None of the other patients with stable negative urinary CXCL9 values experienced rises in serum creatinine that could be interpreted as clinical rejection episodes.

DISCUSSION

We demonstrated that conversion of kidney transplant recipients from CNI to belatacept can be performed safely in the real-life setting of a large transplant center, provided that it is performed gradually and more than 3 months post-transplant. Conversion was associated with improved blood pressure control, better metabolic profile, and stable graft function. Circulating T and B cells with effector or memory phenotype significantly declined after conversion to belatacept, which may suggest a lower level of immune activation. Serial urinary CXCL9 measurements were able to detect the few patients who developed intragraft infiltrates or graft dysfunction during conversion prior to an increase in serum creatinine, providing an important tool to monitor safety of the maneuver.

In contrast to the high rate of acute rejection reported by others, i.e. 8.2% to 20% [START_REF] Rostaing | Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study[END_REF][START_REF] Darres | Conversion to belatacept in maintenance kidneytransplant patients: A retrospective multicenter European study[END_REF][START_REF] Schulte | Late conversion from tacrolimus to a belataceptbased immuno-suppression regime in kidney transplant recipients improves renal function, acid-base derangement and mineral-bone metabolism[END_REF][START_REF] Grinyó | Safety and Efficacy Outcomes 3 Years After Switching to Belatacept From a Calcineurin Inhibitor in Kidney Transplant Recipients: Results From a Phase 2 Randomized Trial[END_REF], only one patient developed biopsy proven acute rejection (BPAR) in our series. This lowrate of acute rejections could be explained by the CNI weaning strategy that we adopted: CNIs and belatacept were overlapped for one month and then CNIs were fully tapered off over a 2month period, which exceeds the 1-month conversion time reported in most of the previous studies [START_REF] Darres | Conversion to belatacept in maintenance kidneytransplant patients: A retrospective multicenter European study[END_REF][START_REF] Grinyó | Safety and Efficacy Outcomes 3 Years After Switching to Belatacept From a Calcineurin Inhibitor in Kidney Transplant Recipients: Results From a Phase 2 Randomized Trial[END_REF]. Importantly, since previous studies showed that most BPAR events develop within 1 year of belatacept initiation, the extent of our follow-up period was enough to reasonably exclude that our conversion strategy simply delayed their occurrence and that we did not capture them.

No patient developed de novo DSA in our study, which is consistent with previous studies showing that de novo DSA is a rare event in belatacept-treated patients [START_REF] Bray | De novo donor-specific antibodies in belatacepttreated vs cyclosporine-treated kidney-transplant recipients: Post hoc analyses of the randomized phase III BENEFIT and BENEFIT-EXT studies[END_REF][START_REF] Everly | Comparison of de novo IgM and IgG anti-HLA DSAs between belatacept-and calcineurin-treated patients: An analysis of the BENEFIT and BENEFIT-EXT trial cohorts[END_REF]. This result is supported by experimental evidence indicating that costimulatory blockade inhibits T follicular [START_REF] Gallon | Differential Effects of Calcineurin and Mammalian Target of Rapamycin Inhibitors on Alloreactive Th1, Th17, and Regulatory T Cells[END_REF][START_REF] Wekerle | Mechanisms of transplant tolerance induction using costimulatory blockade[END_REF][START_REF] Kim | Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection[END_REF]. Consistently, our data indicate a significant reduction in circulating CD4 + and CD8 + effector T cells and post-germinal center and memory B cells after conversion to belatacept. Albeit small, these significant changes in peripheral cell subsets may actually have a biological effect, as previously described by others in other kidney transplant recipient cohorts [START_REF] Schaier | End-stage renal disease, dialysis kidney transplantation and their impact CD4 + T-cell differentiation[END_REF][START_REF] Schlößer | Prospective analyses of circulating B cell subsets in ABO-compatible and ABO-incompatible kidney transplant recipients[END_REF].

Belatacept conversion showed clinical benefit. Patients' kidney function did not deteriorate and, although not statistically significant, the rate of GFR decline was reduced after conversion to belatacept. CNI interruption also resulted in a significant lowering of HbA1c in diabetic patients despite no change in sugar-lowering drugs. This may infer that tacrolimus, which is a wellknown diabetogenic drug, may enable hyperglycemia late after its introduction. [START_REF] Talawila | Does belatacept improve outcomes for kidney transplant recipients? A systematic review[END_REF] Importantly, conversion to belatacept improved blood pressure and metabolic profile and stabilized graft function. The patient on azathioprine had numerous recurrences of squamous cell carcinomas and has been free from recurrence since conversion to belatacept. One of the two patients with CNI-induced neuropathy reported significant improvement of the symptoms.

Following conversion, all patients reported satisfaction with the new intravenous regimen and none asked to return to CNI.

Our data also support the favorable safety profile of belatacept. Belatacept infusions were well tolerated, the adverse events were limited in number, and in no case were any adverse events attributable to belatacept. All included patients were EBV positive, which reduced the risk for post-transplant lymphoproliferative disorder (PTLD) previously associated with belatacept therapy [START_REF] Martin | Risk of posttransplant lymphoproliferative disorder associated with use of belatacept[END_REF].

In the present study, urinary CXCL9 levels were within the normal range in most of the measurements. The three patients with elevated CXCL9 levels were the only ones who eventually developed kidney dysfunction, possibly mediated by an immunological reaction.

Because the CXCL9 measurements were done post hoc, this information did not bias the clinical actions of the treating physicians, thus explaining why patients did not receive a kidney-graft biopsy despite the high CXCL9 levels. However, these data provide rationale for future interventional studies testing the hypothesis that serial measurements of urinary CXCL9 can lead to early identification of patients at risk for acute rejection, for whom CNI tapering should be avoided or performed cautiously to improve safety. On the other hand, low CXCL9 levels could reassure transplant physicians and lead to less frequent surveillance graft biopsies. Because histological diagnoses in kidney transplantation are subject to sampling bias [START_REF] John | Determinants of long-term graft outcome in transplant glomerulopathy[END_REF][START_REF] Kretzler | Repuncturing the renal biopsy: strategies for molecular diagnosis in nephrology[END_REF] and interreader variation [START_REF] Furness | Convergence of European Renal Transplant Pathology Assessment Procedures (CERTPAP) Project. International variation in the interpretation of renal transplant biopsies: report of the CERTPAP Project[END_REF], and serum creatinine is an insensitive measurement of graft function [START_REF] Rush | Protocol transplant biopsies: an underutilized tool in kidney transplantation[END_REF], the present data support the testable hypothesis that elevated urinary CXCL9 measurements are better indicators of ongoing pathological inflammation than serum creatinine or histological evaluation.

Previous studies showed that urinary tract infections and BKV increase urinary CXCL9 [START_REF] Jackson | Urinary chemokines CXCL9 and CXCL10 are noninvasive markers of renal allograft rejection and BK viral infection[END_REF].

While no patient in our study had either of these conditions when CXCL9 was tested in the urine, routine BKV monitoring would be important to increase specificity of positive CXCL9 results for acute rejection.

Herein, urinary CXCL9 was measured by ELISA. The 12-to 24-hour requisite turnaround time for ELISAs is short but not ideal for "real-time" implementation of therapeutic changes based on assay results. However, urinary CXCL9 can be also detected by biolayer interferometry [START_REF] Gandolfini | Rapid Biolayer Interferometry Measurements of Urinary CXCL9 to Detect Cellular Infiltrates Noninvasively After Kidney Transplantation[END_REF], an assay that requires less than 1 hour, making it potentially implementable in clinical transplantation practice.

Our study has limitations, including the single-center, retrospective design. However, the fact that these data were generated in the "real-life" setting of a large transplant center underlines the potential of using this strategy of conversion together with urinary CXCL9 to minimize the risk of rejection and early treat the subjects that develop such complication. Lack of a control group is another limitation. However, with the limitation of juxtapositions across different studies, our results compare well with previous conversion trials from CNI to belatacept in terms of acute rejection rate and metabolic improvement [START_REF] Rostaing | Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study[END_REF][START_REF] Darres | Conversion to belatacept in maintenance kidneytransplant patients: A retrospective multicenter European study[END_REF][START_REF] Schulte | Late conversion from tacrolimus to a belataceptbased immuno-suppression regime in kidney transplant recipients improves renal function, acid-base derangement and mineral-bone metabolism[END_REF][START_REF] Grinyó | Safety and Efficacy Outcomes 3 Years After Switching to Belatacept From a Calcineurin Inhibitor in Kidney Transplant Recipients: Results From a Phase 2 Randomized Trial[END_REF].

Overall, our data support the use of our strategy of conversion of kidney transplant recipients from CNI to belatacept and provide the rationale for a randomized control trial testing the hypothesis that CXCL9 driven management of patients improves the safety/efficacy profile of this procedure over standard serum creatinine monitoring. AUTHORSHIP PM, LR, PC: participated in study design, interpretation of data and writing of the manuscript. CF, PC: performed urinary CXCL9 measurements, GR: participated in collecting the data, MCJ, TR: performed the flow cytometric peripheral blood lymphocyte analysis, PM, TJ, LR, and BJ: were in charge of clinical follow-up of study participants. All the authors reviewed the manuscript.

Disclosure:

The authors declare no conflicts of interest.

Funding: nothing to disclose. Supplemental Figure 1. 12-month follow-up eGFR according to entry level eGFR.

HIGHLIGHTS

 Late CNI to belatacept conversion is feasible and safe when performed with a two-month CNI overlap.

 CNI to belatacept conversion improves blood pressure and metabolic parameters such as HbA1c.

 Urinary CXCL9 monitoring may allow early identification of acute rejection.

  were analyzed i) FITC-CD3 (Sk7 clone, BD), PE-CD16 (B73.1 clone, BD), PE-CD56 (NCAM16.2 clone, BD), PerCP-CD45 (2D1 HLE-1, BD), PE-Cy7-CD4 (SK3 clone, BD), APC-CD19 (SJ25C1 clone, BD), APC-Cy7-CD8 (SK1 clone, BD), BV450-HLA-DR (L243 clone, BD), ii) PE-CD8 (B9.11 clone, BD), PerCP-Cy5.5-CD4 (SK3 clone, BD), PE-Cy7-CD3 (UCHT1 clone, BD), APC-CD45RA (HI100 clone, BD), BV421-CD197 (CCR7) (150503 clone, BD) and iii) FITC-IgD (F0189 clone, AGILENT (DAKO)), PE-CD10 (ALB1 clone, Beckman Coulter (BC)), PerCP-Cy5.5-CD38 (HIT2, BD), PE-Cy7-CD27 (1A4CD27 clone, BC), APC-IgM (G20-127 clone, BD), and APC-H7-CD19 (SJ2SC1 clone, BD). The first combination allowed the identification of whole B-cells (CD19 + ), NK-cells (CD16/CD56 + CD3 -) and T-cells (CD3 + ), as well as CD4 + and CD8 + T-cell subsets.

Figure 1 .

 1 Figure 1. eGFR during the 12-month follow-up period. Each black line represents a single patient. Red line represents the median values.

Figure 2 .

 2 Figure 2. Blood pressure and glycemic control. A) Systolic (sBP) and diastolic (dBP) blood pressure at baseline (before conversion, blue) and at the end of the 12-month follow-up period (red). B) Glycosylated hemoglobin (HbA1c) at baseline (month 0), 3, 6 and 12 months after belatacept initiation in subjects with diabetes.

Figure 3 .

 3 Figure 3. Changes in circulating T and B lymphocyte subsets. Here are shown the subsets that showed significant changes between baseline and one year after, expressed as percentages of total circulating B cells, CD4 + , or CD8 + T cells.

Figure 4 .

 4 Figure 4. Urinary CXCL9. A) Urinary CXCL9 before and at various time points after belatacept initiation in the whole study cohort. Dotted line represents the positivity threshold of 200 pg/ml. In red are depicted the positive values. B-D) Changes in serum creatinine, and urinary CXCL9 in the three patients who displayed at least one CXCL9 value above the positivity threshold.

Figure 3 Figure 4A Figure 4B

 34A4B Figure 3

Figure 5

 5 Figure 5

Table 1 . Patient characteristics.

 1

	Patients

Table 2 . Reasons for conversion from CNI to belatacept.

 2

	Chronic allograft dysfunction	23 (65.7)
	Chronic allograft dysfunction + diabetes	1 (2.9)
	Nephroprotection without allograft dysfunction	2 (5.7)
	Peripheral neuropathy	2 (5.7)
	Diabetes	2 (5.7)
	Dyslipidemia	1 (2.9)
	CNI intolerance	2 (5.7)
	Non adherence	1 (2.9)
	Recurrent squamous cell carcinoma	1 (2.9)

Data are number (percentage).

Table 3 . Patients with adverse events.

 3

	Serious
	Cardiovascular
	Cerebral Stroke
	Myocardial infarction
	Arteriovenous fistula thrombosis
	Infectious
	Viral pneumonia
	Pyelonephritis
	Non serious
	Cardiovascular
	Superficial thrombophlebitis
	Infectious
	Cystitis

Table 4 . Changes in circulating lymphocyte subsets.

 4

	Month 0	Month 12	p

Month 0 represents the time of conversion. Data are expressed as mean ± SD of absolute numbers or percentages.

ACKNOWLEDGEMENTS

Some of the data in this study were presented at the American Transplant Congress, Seattle, WA, June 2-6, 2018.


Franck-Nicolas Bardou

Olivier Guillaud

Domitille Erard-Poinsot

Christine Chambon-Augoyard

Elsa Thimonier

Mélanie Vallin

Olivier Boillot

Jérôme Dumortier
email: jerome.dumortier@chu-lyon.fr

Tacrolimus exposure after liver transplantation for alcohol-related liver disease: Impact on complications

Keywords: ALD: Alcohol-related liver disease, LT: Liver transplantation, Liver transplantation, alcohol-related cirrhosis, tacrolimus, outcome

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Since the mid 80's, since cyclosporine became available, liver transplantation (LT) became the effective treatment of end-stage liver diseases, including alcohol-related liver disease (ALD) [START_REF] Mathurin | Trends in the management and burden of alcoholic liver disease[END_REF].

Over the years, the number of LT increased considerably and nowadays there are more than 10,000 recipients of a liver graft in France. During the past 30 years, several improvements were made in surgical techniques and postoperative management, allowing a better survival of both grafts and patients. This includes the development of new immunosuppressive drugs and strategies. In particular, tacrolimus (Tac), a calcineurin inhibitor (CNI), replaced cyclosporine in the late 90's, as the cornerstone of immunosuppressive regimen for LT recipients, because of its higher ability to prevent acute cellular rejection (ACR) [START_REF] Mcalister | Cyclosporin versus tacrolimus as primary immunosuppressant after liver transplantation: a meta-analysis[END_REF]. Nevertheless, the major drawback of long-term use of Tac is the risk for developing dose-dependent adverse effects, including malignancies, renal failure, hyperlipidemia, diabetes mellitus, arterial hypertension and cardiovascular diseases [START_REF] Karie-Guigues | Long-term renal function in liver transplant recipients and impact of immunosuppressive regimens (calcineurin inhibitors alone or in combination with mycophenolate mofetil): the TRY study[END_REF][START_REF] Rodríguez-Perálvarez | Tacrolimus trough levels, rejection and renal impairment in liver transplantation: a systematic review and meta-analysis[END_REF][START_REF] Peters | Tacrolimus. A review of its pharmacology, and therapeutic potential in hepatic and renal transplantation[END_REF][START_REF] Carenco | Solid, non-skin, post-liver transplant tumors: Key role of lifestyle and immunosuppression management[END_REF][START_REF] Sérée | Longterm risk of solid organ de novo malignancies after liver transplantation: a French national study on 11,226 patients[END_REF].

Tac pharmacokinetics is characterized by a high inter-and intra-patient variability; therefore, therapeutic drug monitoring is used to adjust the dosage in order to provide an effective immunosuppression and prevent toxicity [START_REF] Venkataramanan | Clinical utility of monitoring tacrolimus blood concentrations in liver transplant patients[END_REF]. New strategies have emerged to reduce doses of Tac ("minimizing tacrolimus strategy") [START_REF] Boudjema | Reduced-dose tacrolimus with mycophenolate mofetil vs. standard-dose tacrolimus in liver transplantation: a randomized study[END_REF][START_REF] Jia | Minimizing tacrolimus'' strategy and long-term survival after liver transplantation[END_REF][START_REF] Neuberger | Delayed introduction of reduced-dose tacrolimus, and renal function in liver transplantation: the 'ReSpECT' study[END_REF]. The exposure to Tac can be evaluated from different ways in transplant recipients. In daily practice, trough blood concentration is used to adapt Tac doses. Therefore, overall mean blood concentration of Tac (for a defined period of time) can be used as an estimation of Tac exposure, and it has been suggested that this could have a significant impact on renal impairment and risk of de novo malignancies [START_REF] Carenco | Tacrolimus and the risk of solid cancers after liver transplant: a dose effect relationship[END_REF]. Recently, Tac metabolism rate has been defined as the Tac blood concentration (C) divided by the daily dose of Tac (D). This C/D ratio leads to separate Tac fast-and low-metabolizers, and it has been suggested that fast-metabolizers could develop more frequently renal impairment than slowmetabolizers [START_REF] Thölking | The Tacrolimus Metabolism Rate Influences Renal Function after Kidney Transplantation[END_REF][START_REF] Thölking | Tacrolimus Concentration/Dose Ratio is Associated with Renal Function After Liver Transplantation[END_REF]. The impact on other Tac-related adverse events has not been investigated yet.

In France, ALD (with or without hepatocellular carcinoma (HCC)) is the main indication for LT [START_REF] Sérée | Longterm risk of solid organ de novo malignancies after liver transplantation: a French national study on 11,226 patients[END_REF].

In this specific population, past or persistent tobacco/alcohol intake has been linked with a dramatic higher risk of developing several malignancies [START_REF] Sérée | Longterm risk of solid organ de novo malignancies after liver transplantation: a French national study on 11,226 patients[END_REF][START_REF] Dumortier | Negative impact of de novo malignancies rather than alcohol relapse on survival after liver transplantation for alcoholic cirrhosis: a retrospective analysis of 305 patients in a single center[END_REF], especially cancers of the upper aero-digestive tract. Moreover chronic alcohol consumption is associated in higher risk of developing cardiovascular [START_REF] Toma | Alcohol and Cardiovascular Disease: How Much is Too Much?[END_REF][START_REF] Gardner | Alcohol effects on cardiac function[END_REF] and infectious diseases [START_REF] Zhang | Alcohol abuse, immunosuppression, and pulmonary infection[END_REF]. The aim of the present retrospective study was to assess the impact of Tac exposure during the first year after LT, on short-and long-term complications after LT for ALD.

METHODS

Study population

Since October 1990, we set up a prospective database in our medical center with the aim of generating detailed survival data. The present retrospective study included all adult patients who were transplanted for ALD (with or without HCC), from October 1990 to September 2010 at Edouard Herriot Hospital, Lyon, France, and who received Tac (twice-daily), beginning during the first 10 days following LT and maintained for more than one year. Steroids, azathioprine, mycophenolate mofetil (MMF), or everolimus (EVR) were either administrated as a part of initial triple immunosuppressive regimen or introduced during follow-up as maintenance immunosuppressive agents. Patients receiving combined kidney, heart or lung transplantation were not included.

Four hundred and eight patients were transplanted for ALD. We excluded 41 patients because they did not receive Tac as initial immunosuppressive therapy, 3 patients who received multi-organ transplant, 22 patients who died early (less than a year after LT), and 28 patients because alcohol was associated with another cause of liver disease (mainly HCV infection). On the 314 patients treated with Tac, 24 patients were exposed less than one year and 32 did not received Tac during the first month after LT. Finally, we excluded 7 patients who were lost to follow up and the study population consisted in 251 patients.

The aim of the study was to assess the impact of Tac exposure, during the first year after LT, on short-and long-term complications after LT for ALD.

Clinical and biological data collection

Data collection was based on written medical records and digital information, thereby providing a record of medical history of the patients. Recorded clinical data included: gender, age at LT, body mass index (BMI) at LT, Child-Pugh class at LT, Model for End Stage Liver Disease (MELD) score (retrospectively calculated from our data base for all the patients since 1997) at LT, presence of HCC at LT, initial immunosuppressive regimen after LT, smoking (defined as never, current, or past -in case of cessation of smoking before LT), alcohol relapse, occurrence of severe infections (requiring hospital stay and including cytomegalovirus (CMV) infections, and classified as early post-operative infection during the first month after LT, late post-operative infection between one and 6 months after LT or late infection after 6 months after LT), de novo post-LT malignancies (cases reported in the first 6 months following LT were excluded because it can be strongly assumed that the malignancies were probably present at the time of transplantation and undiagnosed), HCC recurrence, diabetes, dyslipidemia, hypertension, and cardiovascular events (CVE). Severe hypertension was defined as hypertension needing 3 or more antihypertensive drugs to be controlled. Cardiovascular event included were stroke, peripheral arterial disease (usually obliterative arterial disease of the lower limb or carotid stenosis) and coronary heart disease.

We separated cancer in several categories: all de novo malignancies, upper aerodigestive tract cancers, HCC recurrence, non-melanoma skin cancers, and post-transplant lymphoproliferative disorders (PTLD).

Renal function was assessed at time of LT, and at 1 month, 6 months, 12 months, 5 years and 10 years after LT. It was estimated by calculating the glomerular functional rate (eGFR) using the MDRD (modification of diet in renal disease) formula [START_REF] Levey | A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group[END_REF].

[186 x (creatinine (µmol/l) x 0.0113) -1.154 x age -0.203 ] x0.742 if a woman Patients were categorized depending on the eGFR as follows:

• Mild renal impairment: eGFR= 60-90 mL/min

• Moderate renal impairment: eGFR= 30-60 mL/min

• Severe renal impairment: eGFR<30 mL/min

Exposure to tacrolimus

After LT, Tac was routinely monitored using the Tac trough blood concentration (ng/mL). Tac concentration measurements were performed using liquid chromatography coupled with tandem mass spectrometry. Dose adaptations were made depending on several criteria including occurrence of rejection episodes, renal failure, infections or depending on the comorbidities of patients.

We collected Tac trough blood concentrations at different time points: day 3 (D3), D7, D14, D30, then at month 2 (M2), M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M15, M18, and then every year following LT.

We also estimated the mean exposition by calculating the overall mean concentration of Tac during the 1 st , 3 rd , 6 th and 12 th months after LT for each patient, using the trapezoidal method as described by Vivarelli [START_REF] Vivarelli | Liver transplantation for hepatocellular carcinoma under calcineurin inhibitors: reassessment of risk factors for tumor recurrence[END_REF].

Daily doses of Tac were recorded at 1, 3 and 6 months, and at the end of the first year after LT.

Tac metabolism rate was determined by dividing the Tac blood trough concentration (C) by the corresponding daily Tac dose (D), at M1, M3, M6 and M12 [START_REF] Thölking | The Tacrolimus Metabolism Rate Influences Renal Function after Kidney Transplantation[END_REF].

/

( / × 1/ ) = blood Tac trough concentration (ng/mL) daily Tac dose (mg)

Statistical analysis

The main clinical parameters of the patient were determined using mean and median for quantitative variable (for age, time from LT to diagnosis cancer or cardiovascular event or severe infectious episodes, eGFR, Tac blood concentration, weight) and frequencies and percentages for qualitative variable (for the incidence of diabetes, hypertension, dyslipidemia, alcohol consumption, tobacco consumption , severe infectious events and such).

Shapiro-Wilk test was used to assess whether the distribution of each data was normal. If so, Student's t-test was used to compare the data and if not, we used the Mann-Whitney U test. To evaluate the independence between 2 variables, chi-square was used. All the analyses were performed using the SPSS software, version 13.0 (IBM, Armonk, NY) and p-values under 0.05 were considered significant.

RESULTS

Study population and post-LT complications

Characteristics of the 251 patients included in the study are summarized in Table 1. There was a majority of males (76.9%) and almost all patients were Caucasian (248/251, 98.8%). The median age at LT was 53.5 years and the median follow-up after LT was 12.0 years. Most of patients (71.3%) were smoker before LT and severe alcohol relapse occurred in 23.9% of the patients after LT. During follow up, 44.6% of patients experienced a severe infectious event, 41.4% a malignancy, and 15.5% a cardiovascular event. In addition, 47.0% of patient had dyslipidemia after LT, 37.8% had diabetes and 69.3% had arterial hypertension.

After LT, patients were usually treated with double immunosuppressive regimen using Tac with mycophenolate mofetil (MMF). During follow-up, immunosuppressive therapy was modified. From the initial cohort of 251 patients, Tac was stopped in 107 (42.6%); Table 2 summarizes immunosuppressive drugs used after LT.

Figure 1 describes the evolution of eGFR at and after LT and the evolution of stages of chronic kidney disease before and after LT: the mean eGFR before LT was 91.2 mL/min/1.73m², and declined to 61.6+/-25.9 the first month after LT, and 63.2+/-24.7mL/min/1.73m² 10 years after LT. Table 3 and 4 describes the association between the mean Tac trough blood concentrations or overall mean Tac concentrations and the occurrence of clinical complications after LT.

Exposure to tacrolimus and association with post-LT complications

Tac trough blood concentration

Patients with moderate and severe renal impairment (eGFR under 60 mL/min) at 6 months had significantly lower overall mean Tac concentrations and Tac trough blood concentrations at 1, 3 and 6 months. Patients with moderate and severe renal impairment at 1 and 5 year had also at 6 and 12 months a lower overall mean Tac concentrations and Tac trough blood concentrations. Tac trough blood concentration at 3 months was significantly higher in patients who developed skin cancer, cardiovascular event, or de novo hypertension. Patients who presented CMV infection or HCC recurrence had significantly higher Tac trough blood concentration at 6 months.

The Tac trough blood concentrations and the overall mean Tac concentrations were not significantly different according to survival and according to the occurrence of de novo dyslipidemia, de novo diabetes (or diabetes treated with insulin), de novo hypertension (or severe hypertension), severe infectious events (or CMV infection), cardiovascular events, and de novo malignancies.

Finally, there was a trend between recurrence of HCC and a higher overall mean Tac concentrations at 6 and 12 months. Using fast/slow metabolizer status based on the median rate of C/D ratio for each time point, we evaluate the stability of that status with time: 126 patients (50.2%) kept the same fast/slow metabolizers status all along the 1 st year after LT. Between the 1 st and 3 rd month, 186 (74.1%) patients kept the same status, between the 1 st and 6 th month, 153 (61.0%) patients, between the 3 rd and 6th month, 192 (76.5%) patients, and between the 6 th month and 12 th month, 189 (75.3%) patients were classified in the same group. We then evaluated the stability of the fast/slow metabolizer status if the median at 6 or 12 months was used. If C/D ratio of 1.6 was taken as the cut-off for fast/slow metabolizer, 193 (76.9%) kept the same status between 6 and 12 months vs.

Tac metabolism and C/D ratio

190 (75.7%) with a C/D ratio of 1.8. We also evaluated the 1.09 cut-off of C/D separating fast and slow metabolizers as previously defined by Thölking and coll. [START_REF] Thölking | The Tacrolimus Metabolism Rate Influences Renal Function after Kidney Transplantation[END_REF][START_REF] Thölking | Tacrolimus Concentration/Dose Ratio is Associated with Renal Function After Liver Transplantation[END_REF][START_REF] Schütte-Nütgen | Fast Tac Metabolizers at Risk (-) It is Time for a C/D Ratio Calculation[END_REF][START_REF] Thölking | Influence of tacrolimus metabolism rate on BKV infection after kidney transplantation[END_REF]. Using this cut-off, C/D was not stable over time: between one month and one year after LT, 147 (58.6%) patients kept the same fast/slow metabolizer status. Between the 1 st and 3 rd month, 199 (79.3%) patients kept the same status, between the 1 st and 6 th month 162 (64.5%) patients, between the 3 rd and 6th month 197 (78.5%) patients and between the 6 th month and 12 th month, 214 (85.3%) patients were classified in the same group.

Table 5 describes the association between fast/slow metabolizer status (depending on 3 different cut-offs of C/D ratio: 1.6, 1.8 and 1.09) and the occurrence of complications after LT. Table 6 describes the mean eGFR depending of fast/slow metabolizer status. Using the 1.6 cut-off of C/D, at 6 and 12 months after LT, fast metabolizers at 6 months had a significantly lower eGFR at 1, 5

and 10 year and had more moderate and severe renal impairment at 5 year and more severe renal impairment at 10 year than slow metabolizer. There was no other significant association between fast/slow metabolizer status defined with C/D ratio<1.6 at 6 or 12 months. Using the 1.8 cut-off of C/D, at 6 and 12 months after LT, fast metabolizers at 6 months had a significantly lower eGFR at 1, 5 and 10 year and had more moderate and severe renal impairment at 5 year, more severe renal impairment at 10 year and developed also more cardiovascular events after LT. Fast metabolizers at 12 months developed more PTLD, de novo dyslipidemia, de novo hypertension, and insulin-treated diabetes than slow metabolizers.

Finally, using the 1.09 cut-off of C/D from Thölking, there was no significant association between fast/slow metabolizer status with the mean eGFR at 1, 5 and 10 year after LT. There was a trend for fast metabolizers, defined at M6, to have lower eGFR 5 years after LT.

DISCUSSION

Due to significant improvement in both surgical techniques and immunosuppressive therapy during the past decades, LT recipients are expected to live longer. Nevertheless, with time, complications of immunosuppression and immunosuppressive drugs themselves are frequent and can impact survival of patients. Tac became the cornerstone of immunosuppression after LT in the mid 90's and close Tac monitoring is needed because of a high intra-and inter-variability of its metabolism.

In addition to general complications of immunosuppression, mainly infections and malignancies, side-effects of Tac include hypertension, diarrhea, hyperglycemia, hyperlipemia, neurological symptom such as tremor, and renal impairment [START_REF] Karie-Guigues | Long-term renal function in liver transplant recipients and impact of immunosuppressive regimens (calcineurin inhibitors alone or in combination with mycophenolate mofetil): the TRY study[END_REF][START_REF] Rodríguez-Perálvarez | Tacrolimus trough levels, rejection and renal impairment in liver transplantation: a systematic review and meta-analysis[END_REF][START_REF] Peters | Tacrolimus. A review of its pharmacology, and therapeutic potential in hepatic and renal transplantation[END_REF][START_REF] Carenco | Solid, non-skin, post-liver transplant tumors: Key role of lifestyle and immunosuppression management[END_REF]. Tac nephrotoxicity could manifest either as acute kidney injury, which is a dose-dependent and reversible, or as a chronic kidney disease (usually defined as GFR<60mL/min/1.73m²) that appears usually after 6 months use of Tac and is irreversible (ranging from 4 to 73% depending on the length of follow up) [START_REF] Duvoux | Immunosuppression in liver transplant recipients with renal impairment[END_REF]. Tac use can also lead to diabetes (up to 15.9%), arterial hypertension (up to 70%) and hyperlipidemia (up to 69%), well known cardiovascular risk factors [START_REF] Pfitzmann | Long-term results after liver transplantation[END_REF][START_REF] Barnard | Medical Management of Metabolic Complications of Liver Transplant Recipients[END_REF], and cardiovascular events range from 9 to 25% in LT recipients [START_REF] D'avola | Cardiovascular morbidity and mortality after liver transplantation: The protective role of mycophenolate mofetil[END_REF][START_REF] Desai | Cardiovascular risk factors following orthotopic liver transplantation: predisposing factors, incidence and management[END_REF]. Therefore, the observed rates of all these complications of Tac in our cohort with long follow-up were as expected, and we report that the occurrence of a significant part of complications of Tac-based immunosuppression is associated with Tac exposure (trough blood concentrations and overall mean Tac concentrations) and Tac metabolism (C/D ratio).

We first focused our attention on trough blood concentrations and overall mean Tac concentrations, which are used in daily practice. It has already been demonstrated that high Tac blood concentration, especially during the first year after LT leads to renal impairment [START_REF] Karie-Guigues | Long-term renal function in liver transplant recipients and impact of immunosuppressive regimens (calcineurin inhibitors alone or in combination with mycophenolate mofetil): the TRY study[END_REF][START_REF] Rodríguez-Perálvarez | Tacrolimus trough levels, rejection and renal impairment in liver transplantation: a systematic review and meta-analysis[END_REF]. In our study, the most of renal function loss occurred during the first month after LT confirming that longterm renal impairment might be determined by early exposition of Tac. Several studies reported that the use of lower dose of Tac during the first month after LT could therefore decrease the number of patient with eGFR<60mL/min/1.73m² [START_REF] Rodríguez-Perálvarez | Tacrolimus trough levels, rejection and renal impairment in liver transplantation: a systematic review and meta-analysis[END_REF][START_REF] Neuberger | Delayed introduction of reduced-dose tacrolimus, and renal function in liver transplantation: the 'ReSpECT' study[END_REF]. In our population patients with moderate and severe renal impairment (eGFR under 60mL/min/1.73m²) at 6 months had significantly lower Tac concentrations during the first year after LT; it can be hypothesized that clinicians adapted (lowered) Tac doses in patients with impaired renal function in the early post-operative period.

Although different factor contributes as well such as history of smoking or genetic predisposition, Tac is also a well-known factor of cancer progression [START_REF] Maluccio | Tacrolimus enhances transforming growth factor-beta1 expression and promotes tumor progression[END_REF]. The risk of solid cancer after LT is two to three times higher compared to the general population, and 10 to 30 times higher for skin cancer (16% to 22.5% of patients) and PTLD (reported up to 2.8% of patients) [START_REF] Sérée | Longterm risk of solid organ de novo malignancies after liver transplantation: a French national study on 11,226 patients[END_REF][START_REF] Carenco | Incidence of solid organ cancers after liver transplantation: comparison with regional cancer incidence rates and risk factors[END_REF]. We initially chose a LT population of patients with ALD because of the high risk of malignancies. Carenco and coll.

reported [START_REF] Carenco | Solid, non-skin, post-liver transplant tumors: Key role of lifestyle and immunosuppression management[END_REF] that the mean Tac concentration during the first year after LT was significantly higher in patients who developed non-skin solid tumors (10.3 and 7.9 ng/mL, respectively, p<0.0001). In our population, we failed to find such association; except for non-melanoma skin cancers and HCC recurrence (patients had a higher Tac trough blood concentration at 3 or 6 months). One possible explanation is that the mean blood concentration of Tac in our study was lower during the first 6 months following LT compared to the population of the Carenco and coll. study [START_REF] Carenco | Tacrolimus and the risk of solid cancers after liver transplant: a dose effect relationship[END_REF]. For example, the mean Tac trough blood concentration at 3 months was 8.2 in our population vs. 9.2 in Carenco study, and the overall mean Tac concentration at 3 months for patients with de novo malignancy was in our population 8.8 (+/-2.4) vs. 8.6 (+/-2.6) for patient without de novo cancer (10.8 vs. 8.9 in Carenco study). Regarding HCC recurrence, our results confirm that a reduced exposure to CNIs (for instance by using sirolimus) within the first months after LT could be associated with decreased risk of HCC recurrence [START_REF] Rodríguez-Perálvarez | Reduced exposure to calcineurin inhibitors early after liver transplantation prevents recurrence of hepatocellular carcinoma[END_REF][START_REF] Geissler | Sirolimus Use in Liver Transplant Recipients With Hepatocellular Carcinoma: A Randomized, Multicenter, Open-Label Phase 3 Trial[END_REF]. Similarly, conversion from CNI to sirolimus is able to prevent squamous-cell carcinoma recurrence in kidney transplant recipients [START_REF] Euvrard | Sirolimus and Secondary Skin-Cancer Prevention in Kidney Transplantation[END_REF].

Finally, we report here that patients who presented CMV infection had significantly higher Tac trough blood concentration at 6 months. Since the impact of Tac exposure on CMV infection has not been previously reported, this probably needs further evaluation.

Until now, only Tac trough blood concentration is used to adjust the dosage of Tac, and other tools, easy to use in daily practice, to determine patient at risks to developed long-term complications related to Tac therapy are needed. Several factors (outside patients' adherence) are known to influence and explained the high intra-and inter-variability of Tac metabolism. Genetic polymorphisms of cytochrome P450 enzymes such as CYP3A4 or CYP3A5 and P-glycoprotein, of both recipient and donor are involved, but this cannot be used routinely [START_REF] Hendijani | Effect of CYP3A5*1 expression on tacrolimus required dose after liver transplantation: A systematic review and meta-analysis[END_REF][START_REF] Ji | Combinational effect of intestinal and hepatic CYP3A5 genotypes on tacrolimus pharmacokinetics in recipients of living donor liver transplantation[END_REF]. Some drugs, such as corticosteroids or antibiotics, or food, are able to modify the activity of those enzymes, leading to modification in Tac bioavailability [START_REF] Iwahori | Pharmacokinetic differences between morning and evening administration of cyclosporine and tacrolimus therapy[END_REF]. Some studies found contradictory results as to the influence of age or sex on Tac metabolism: some reported that females required higher dosage of Tac compared to male [START_REF] Rancic | Tacrolimus concentration/dose ratio as a therapeutic drug monitoring strategy: The influence of gender and comedication[END_REF]. Tac metabolism rate, defined as C/D [START_REF] Thölking | The Tacrolimus Metabolism Rate Influences Renal Function after Kidney Transplantation[END_REF][START_REF] Thölking | Tacrolimus Concentration/Dose Ratio is Associated with Renal Function After Liver Transplantation[END_REF][START_REF] Thölking | Influence of tacrolimus metabolism rate on BKV infection after kidney transplantation[END_REF] ratio, has been recently proposed as an easy tool to predict patient at risk to develop complications after liver or kidney transplantation. C/D ratio is usually calculated at 6 months, in order to prevent the interaction of corticosteroids or antibiotics with Tac metabolism. The C/D ratio are calculated whether directly the 6 th month or using the mean C/D ratio at 6 months (using the data at 1, 3 and 6 months). Interestingly, we demonstrated here that C/D ratio progressively increases with time during the first year after LT, as previously reported after kidney transplantation [START_REF] Rostaing | Novel once-daily extended-release tacrolimus versus twice-Daily tacrolimus in de novo kidney transplant recipients: two-year results of phase 3, double-blind, randomized trial[END_REF].

Using the median calculated C/D ratio, patients can be separated in 2 groups, patients with a C/D ratio over the median (slow-metabolizers) and those with a C/D ratio under the median (fastmetabolizers). Fast-metabolizers are thought to be exposed to higher risk of Tac adverse effect because of the need of higher doses of Tac to achieve target, increasing thus the risk of higher (and deleterious) Tac peak. After kidney transplantation, fast-metabolizers could have more BK viremia and significantly lower eGFR [START_REF] Rostaing | Novel once-daily extended-release tacrolimus versus twice-Daily tacrolimus in de novo kidney transplant recipients: two-year results of phase 3, double-blind, randomized trial[END_REF]. In the study of Thölking and coll. [START_REF] Thölking | Tacrolimus Concentration/Dose Ratio is Associated with Renal Function After Liver Transplantation[END_REF], LT recipients considered as fast-metabolizers (defined with a C/D ratio <1.09), had significantly lower eGFR than slow-metabolizers. We did find similar result in our study, using different cut-offs because of significant differences in C/D ratio (and median) calculated in our study, which were much higher (for instance, 1.6 vs. 1.09 at 6 months). This difference of C/D ratio might reside in the difference in the populations: for example, there were less women in our study than in Thölking (23.1% vs.

49.4%) and gender had already been associated with difference in Tac metabolism (women probably require higher doses of Tac). In addition, our study focused only on patients with ALD whereas Thölking included all indications for LT: there might exist differences between Tac metabolism depending on the clinical profile of LT recipients.

In the present study, we evaluated 2 cut-offs of C/D, derived from median at 6-and 12 months (1.6 and 1.8). We consider 1.8 as the most efficient cut-off rather than 1.6, because it was more powerful and more associated with long term complications. Defining fast metabolizers patient with C/D ratio under 1.8, we found similar results than Thölking and coll.: fast-metabolizers had lower eGFR and more renal impairment (eGFR<60mL/min/1.73m²). Moreover, in our population, C/D ratio was more efficient than trough blood concentrations to identify patients with renal failure. In addition, we report here for the first time that fast-metabolizers also developed more de novo dyslipidemia, more de novo hypertension, more diabetes treated with insulin and presented more cardiovascular events while Tac trough blood concentration was only associated with the onset of de novo hypertension and cardiovascular events. Interestingly, it has been recently reported that kidney transplant fast-metabolizers develop more cardiovascular events [START_REF] Schütte-Nütgen | Fast Tac Metabolizers at Risk (-) It is Time for a C/D Ratio Calculation[END_REF]. Finally, we report that fast-metabolizers had a higher risk of developing PTLD, and this also needs further evaluation.

In conclusion, our results support that, in addition to blood trough concentrations, Tac metabolism, as estimated by the simple C/D ratio, could be an efficient parameter in daily practice to identify LT patients at risk to develop long term general complications of Tac, such as renal impairment, but also de novo dyslipidemia, diabetes and hypertension. Limitations of our study include its retrospective and monocentric nature, the characteristics of the French study population (Caucasian men (both ethnicity and gender influence genetic polymorphism on Tac metabolism), ALD LT recipients), variation of immunosuppressive regimen all along follow-up (long-term Tac withdrawal in almost half of our patients). Further studies are therefore needed to evaluate our findings in larger cohorts, including non-ALD LT recipients, and with a specific focus on other than Tac immunosuppressants, especially mTOR inhibitors which emerged in the recent years.

LEGENDS FOR FIGURES

Figure 2

 2 Figure 2 describe the patients' exposure to Tac using Tac trough blood concentrations and overall mean Tac concentrations. The mean Tac trough blood concentration was 9.1+/-3.7 (median at 8.7) 1 month after LT and decreased to 7.2+/-3.0 (median at 6.8) one year after LT. The overall mean Tac concentration was 9.6+/-3.1 during the first month and 8.0+/-2.1 during the first year.

Figures 3 and 4

 4 Figures 3 and 4 describe the time course of the median C/D ratio after LT and the distribution of C/D ratio after LT. The median C/D ratio increased with time, from 1.3 at one month to 1.8 at one year after LT. We therefore defined fast and slow metabolizers in our cohort, depending on the median C/D ratio, at each time point. Thus, fast metabolizers were patients with C/D ratio under 1.3, 1.4, 1.6 and 1.8 at 1, 3, 6 and 12 months after LT, respectively.

Figure 1 :Figure 2 :

 12 Figure 1: Renal function after LTA: Evolution of eGFR after LT Before LT mean eGFR was 91.2+/-28.1 mL/min/1.73m² and then decrease to 61.6+/-25.9 the first month after LT. After there was a small improvement of eGFR: At 6 months, mean eGFR was 67.1+/-20.5, then 67.0+/-21.4 at 1 year, 65.9+/-21.9 at 5 years and 63.2+/-24.7 at 10 years after LT.B: Evolution of stages of chronic kidney failure after LT

Figure 3 : 3 (

 33 Figure 3: Evolution of Tac C/D ratio after LT At 1 month, mean+/-SD (cross) Tac C/D ratio was at 1.7+/-1.4, then 1.6+/-1.0 at 3 months, 2.0+/-1.4 at 6 months and 2.2+/-1.8 at 12 months. Median Tac C/D ratio (straight line) was 1.3 (IQR=1.1), 1.4 (IQR=1.0), 1.6 (IQR=1.4) and 1.8 (IQR=1.2) at 1, 3, 6 and 12 months respectively.

Figure 4 : 2 At 1

 421 Figure 4: Distribution of Tac C/D ratio after LT At 1 month the median Tac C/D ratio (dotted line) was 1.3. Tac C/D ratio range from 0.3 to 12.9 At 3 month the median Tac C/D ratio (dotted line) was 1.5. Tac C/D ratio range from 0.2 to 7.3

Table 1 . Characteristics of the study population (n=251)

 1

	Characteristics

Table 2 . Immunosuppressive drugs used after LT in the study population (n=251)

 2

	cyclosporine azathioprine	MMF	EVR	Tac

Table 5 : Fast/slow metabolizers status and complications after LT

 5

			C/D<1.6 at M6		C/D<1.6 at M12		C/D <1.8 at M6		C/D <1.8 at M12
		Yes	No	p	Yes	No	p	Yes	No	p	Yes	No	p
	Cardiovascular event	16.9%	11.8%	0.1	17.6%	14.1%	0.45	19.7%	17.2%	0.046	17.2%	14.0%	0.48
		(21/124)	(15/127)		(18/102)	(21/149)		(27/137)	(12/114)		(21/122)	(18/129)
	Infections
	Severe infectious	48.4%	40.9%	0.26	43.1%	45.6%	0.66	49.6%	44.3%	0.09	44.3%	45.0%	0.87
	event	(60/124)	(52/127)		(44/102)	(68/149)		(68/137)	(44/114)		(54/122)	(58/129)
	CMV infection	10.5%	16.5%	0.16	8.8%	16.8%	0.07	12.4%	9.8%	0.56	9.8%	17.1%	0.1
		(13/124)	(21/127)		(9/102)	(25/149)		(17/137)	(17/114)		(12/122)	(22/129)
	Metabolic disorder
	Dyslipidemia	50.0%	44.1%	0.35	50.0%	45.0%	0.43	51.1%	52.5%	0.16	52.5%	41.9%	0.09
		(62/124)	(56/127)		(51/102)	(67/149)		(70/137)	(48/114)		(64/122)	(54/129)
	De novo dyslipidemia	49.2%	39.4%	0.12	49.0%	40.9%	0.21	49.6%	51.6%	0.06	51.6%	37.2%	0.02
		(61/124)	(50/127)		(50/102)	(61/149)		(68/137)	(43/114)		(63/122)	(48/129)
	Hypertension	74.2%	64.6%	0.1	71.6%	67.8%	0.52	74.5%	73.0%	0.05	73.0%	65.9%	0.23
		(92/124)	(82/127)		(73/102)	(101/149)		(102/137)	(72/114)		(89/122)	(85/129)
	De novo hypertension	54.0% (67/124)	44.9% (57/127)	0.12	68.6% (59/86)	58.6% (65/111)	0.15	52.6% (72/137)	56.6% (52/114)	0.27	56.6% (69/122)	42.6% (55/129)	0.03
	Severe hypertension	22.6% (28/124)	17.3% (22/127)	0.3	17.6% (18/102)	21.5% (32/149)	0.46	24.1% (33/137)	22.1% (17/114)	0.07	22.1% (27/122)	17.8% (23/129)	0.39
	Diabetes	36.3% (45/124)	39.4% (50/127)	0.62	36.3% (37/102)	38.9% (58/149)	0.69	38.7% (53/137)	39.3% (42/114)	0.76	39.3% (48/122)	36.4% (47/129)	0.64
	De novo diabetes	17.7% (22/124)	19.7% (25/127)	0.69	17.6% (18/102)	19.5% (29/149)	0.72	18.2% (25/137)	19.7% (22/114)	0.83	19.7% (24/122)	17.8% (23/129)	0.71
	Diabetes treated with insulin	21.0% (26/124)	21.3% (27/127)	0.93	24.5% (25/102)	18.8% (28/149)	0.29	21.9% (30/137)	26.2% (23/114)	0.77	26.2% (32/122)	16.3% (21/129)	0.049
	De novo diabetes treated with Insulin	12.9% (16/124)	13.4% (17/127)	0.91	14.7% (15/102)	12.1% (18/149)	0.55	13.9% (19/137)	16.4% (14/114)	0.71	16.4% (20/122)	10.1% (13/129)	0.14
	Malignancies
	De novo malignancy or HCC recurrence	45.2% (56/124)	37.8% (48/127)	0.24	38.2% (39/102)	43.6% (65/149)	0.4	44.5% (61/137)	38.5% (43/114)	0.28	38.5% (47/122)	44.2% (57/129)	0.36
	De novo malignancy	43.5% (54/124)	34.6% (44/127)	0.15	37.3% (38/102)	40.3% (60/149)	0.63	43.1% (59/137)	37.7% (39/114)	0.15	37.7% (46/122)	40.3% (52/129)	0.67

This study was not funded from any grant.