13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Received: 10 November 2020 Revised: 6 February 2021 Accepted: 13 February 2021 DOI: 10.1111/bcp.14794 ORIGINAL ARTICLE Monitoring of gene expression in tacrolimus-treated de novo renal allograft recipients facilitates individualized immunosuppression: Results of the IMAGEN study Claudia Sommerer1 | Mercè Brunet2 | Klemens Budde3 | Olga Millán2 | 4 3 5 1 Lluis Guirado Perich | Petra Glander | Stefan Meuer | Martin Zeier | Thomas Giese5 1 Department of Nephrology, University of Heidelberg, University Hospital Heidelberg, Aims: Calcineurin inhibitors (CNI) have a small therapeutic window, and drug Heidelberg, Germany monitoring is required. Pharmacokinetic monitoring does not correlate sufficiently 2 Pharmacology and Toxicology Laboratory, CDB, CIBERehd, IDIBAPS, Hospital Clinic of with clinical outcome. Therefore, the expression of nuclear factor of activated T cells Barcelona, University of Barcelona, Spain (NFAT)-regulated genes in the peripheral blood has been suggested as a potentially 3 Department of Nephrology, Charité useful immune monitoring tool to optimize CNI therapy. NFAT-regulated gene University Hospital Berlin, Berlin, Germany 4 Renal Transplant Unit, Nephrology expression (RGE) was evaluated in renal allograft recipients as predictive biomarker Department, Fundació Puigvert, Barcelona, to detect patients at risk of acute rejection or infections. Spain 5 Methods: NFAT-RGE (interleukin-2, interferon-γ, granular-macrophage colony- Institute of Immunology, University Hospital Heidelberg, Heidelberg, Germany 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 Correspondence Claudia Sommerer, MD, MSc, University 64 de novo renal allograft recipients from 3 European centres. Immunosuppression Hospital Heidelberg, Department of consisted of tacrolimus (Tac), mycophenolic acid, and corticosteroids. Nephrology, Im Neuenheimer Feld 162, D-69120, Heidelberg. Results: Tac concentrations (C0 and C1.5) correlated inversely with NFAT-RGE Email: claudia.sommerer@med.uni-heidelberg. (P < .01). NFAT-RGE showed a high interindividual variability (1–61%). Patients with de 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). Conclusions: NFAT-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. The authors confirm that Claudia Sommerer is the principal investigator for this paper and that she had direct clinical responsibility for patients. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. Brit Jnl Clinical Pharma. 2021;87:3851–3862. wileyonlinelibrary.com/journal/bcp 3851 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3852 SOMMERER ET AL. KEYWORDS biomarker, cytomegalovirus, pharmacodynamics, pharmacokinetics, rejection, renal transplantation, tacrolimus 1 | I N T RO DU CT I O N What is already known about this subject Optimal immunosuppression is necessary to achieve the best trans- plant function and patient survival after renal transplantation. Several • Pharmacokinetic monitoring does not correlate suffi- attempts have been undertaken to improve immunosuppressive ciently with clinical outcome. treatment. Most of the new promising agents failed before market • Pharmacodynamic monitoring provides the opportunity introduction. At this time, cell therapy to induce tolerance seems to to individualize calcineurin inhibitor therapy. be promising but is far from daily clinical use,1,2 while calcineurin • Monitoring of the residual expression of nuclear factor of inhibitors (CNIs) are still the most widely used immunosuppressive activated T cells regulated gene expression (NFAT-RGE) agents in transplantation.3,4 CNIs are critical dose drugs and have a reflects the biological effect of ciclosporin A treatment in large pharmacokinetic interpatient variability. Therapeutic drug moni- long-term renal allograft recipients. toring is necessary to balance efficacy and toxicity due to the narrow therapeutic window. One step to an optimized immunosuppression What this study adds might be an individualized CNI treatment based on adequate monitoring tools.5,6 Pharmacokinetic monitoring is well established in • Monitoring of NFAT-RGE reflects the biological effect of clinical practice but does not reflect the biological activity of the tacrolimus. drug. Sensitivity to CNIs varies widely between individuals due to • NFAT-RGE is confirmed as a noninvasive early predictive differences in intracellular drug accumulation but more importantly, marker in the post-transplant period for the risk of acute due to variable and difficult to predict genetic and epigenetic rejection and infections and allows individualized differences in the immune responsiveness. Therefore, the measure- tacrolimus treatment adjustments. ment of a CNI trough concentration in peripheral blood does not • NFAT-RGE can be used as biomarker in international necessarily predict the biological activity of the drug in immune cells multicentre trials. of a particular patient. Several biomarkers have shown promising results predicting the rejection risk, graft dysfunction, and the personal response to immunosuppressive agents. Pharmacodynamic monitoring strategies have been used to directly measure the functional effects of CNIs.7–10 CNIs inhibit the phosphatase activity of calcineurin.11,12 The main The aim of the blinded, prospective study was to assess the clini- substrate of calcineurin in T cells is the nuclear factor of activated T cal utility and validate the biomarker residual NFAT-RGE in adult renal cells (NFAT). This transcription factor does not translocate into the allograft recipients on tacrolimus therapy early after transplantation in nucleus in its phosphorylated form; therefore, CNIs prevent the 3 large European transplant centres. transcription of genes regulated by NFAT. More than 100 genes encoding a variety of cytokines (such as interleukins [ILs]-2,13 4 and 17, interferon-γ [IFNγ],14 and tumour necrosis factor [TNF]-α15) as 2 | M A T E R I A L S A N D M ET H O D S well as several components of signal transduction pathways, cell cycle regulatory proteins, and apoptosis-controlling proteins in T cells 2.1 | Patient cohort seems to be regulated by this transcription factor.16 Therefore, the transcription of NFAT-regulated genes in the De novo renal allograft recipients from 3 European transplant centres peripheral blood has been suggested as a potentially useful immune (Barcelona, Berlin, Heidelberg) were recruited in this prospective 17–19 monitoring tool to individualize CNI therapy. Previous studies noninterventional trial (EudraCT number: 2013–001817-33). Inclusion have evaluated the residual expression of NFAT-regulated gene criteria were age ≥18 years, primary renal transplant patients without expression (RGE) in solid organ transplant recipients.20,21 Most of other transplanted organs from deceased or living donors, willing to these studies evaluated NFAT-RGE in long-term stable transplanted participate in the study, and from whom written informed consent has patients and clearly showed that strong inhibition of these genes is a been obtained.28 Exclusion criteria were recipients older than sign of oversuppression reflected by a higher incidence of infectious 70 years, positive for HCV, HBV or HIV panel reactive antibodies (mostly viral) and malignant complications.22–27 >10%, or AB0-incompatibility. 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SOMMERER ET AL. 3853 2.2 | Study design 0 with 500 mg of methylprednisolone, 250 mg on day 1, 125 mg on day 2, 80 mg on day 3, 60 mg on day 4, 40 mg on day 5, and progres- The study included a total of 6 visits: pretransplantation, post- sively decreasing the dose to reach 20 mg on day 14, and from day transplantation at day 7, and months 1, 2, 3 and 6. The patient cohort 15 to day 29, 15 mg of prednisone. Patients received 10 mg of predni- was divided into 2 groups according to the residual NFAT-RGE at day sone from day 30 to day 60 after transplantation. After the 60th day, 7. The threshold of NFAT-RGE was detected by receiver operator 5 mg/d of prednisone was administered. curve (ROC) analysis in a patient cohort with a maximum time after Within 6 months after transplantation all patients had transplantation of 6 months of an earlier evaluation in Tac treated Pneumocystis jirovecii prophylaxis with cotrimoxazole. Patients with renal allograft recipients (test cohort).29 CMV-positive donors had CMV prophylaxis with valganciclovir during At each visit safety parameters were assessed, including renal the first 3 months after transplantation. allograft function (serum creatinine, glomerular filtration rate), urinary protein, blood counts, and routine chemistry. In addition, regular monitoring of cytomegalovirus (CMV), polyomavirus (BKV) and 2.5 | Pharmacokinetic monitoring Epstein Barr virus replication was performed. Polyomavirus viraemia was defined as BKV or John Cunningham virus levels ≥10 000 DNA Tac trough concentration (C0), Tac peak concentration (C1.5), Tac copies mL−1 in whole blood and cytomegalovirus viraemia as CMV concentration–dose ratio (Tac C/D) at day 7, and months 1, 2, 3, and levels ≥1000 DNA copies mL−1. Renal allograft function was assessed 6 post-transplantation, and complete area under the curve (AUC; by calculating the glomerular filtration rate with the Modification of 0, 30 min, 1, 1.5, 2, 3, 4, 6, 8 and 12 h after drug administration) Diet in Renal Disease formula. at day 7 post-transplantation were analysed. Abbreviated Tac Apparent acute infections, e.g. urinary tract infections, infections AUC was calculated according to Miura et al.31 with the term of the upper or lower respiratory system, CMV infection and other AUC0–12 = 7.04C0 + 1.71C2 + 3.23C4 + 15.19 and Mathew et al.32 herpes virus infections, were recorded. Indication biopsies were with the term AUC0–12 = 19.16 + (6.75C0) + (3.33C1.5). obtained when necessary. Acute rejection was diagnosed by clinical Blood samples were collected in EDTA-K3 tubes and processed and laboratory findings and confirmed by histological evaluation of immediately. Tac concentrations in whole blood were measured by graft biopsies by an independent pathologist according to Banff 2007 liquid chromatography/tandem mass spectrometry and MPA concen- criteria.30 trations were measured by high-performance liquid chromatography The study was approved by the institutional Ethical Review Board with ultraviolet spectrometry. (Ethics Committee University Hospital Heidelberg, Germany, refer- ence number ABmu-507/2013). The study was conducted following the ethical principles of the Declaration of Helsinki and in compliance 2.6 | Pharmacodynamic monitoring with the Good Clinical Practice Guidelines. All samples for the evaluation of the residual expression of NFAT-RGE were analysed at the Institute of Immunology, University 2.3 | Study objectives Hospital Heidelberg, Germany. The primary outcome was the combined endpoint consisting of biopsy-proven acute rejection (BPAR), renal allograft failure, and 2.6.1 | Sample preparation death. Secondary outcomes were efficacy results (treated BPAR) and safety data (renal function, infectious complications, malignancy, Residual NFAT-RGE was assessed in whole blood samples. cardiovascular risk factors, e.g. hypertension, hyperlipidaemia, diabe- Heparinized peripheral blood was stimulated with 1 mL of com- tes), and neurotoxic effects. plete RPMI 1640 containing 100 ng/mL PMA and 5 μg/mL ionomycin (Sigma-Aldrich Corp. St. Louis, MO, USA) for 3 hours at 37 C. After red cell lysis with ACK buffer (0.15 M NH4CI, 1.0 mM 2.4 | Immunosuppressive regimen KHCO3), leucocytes were lysed with 400 μL of MagNA-Pure lysis buffer supplemented with an additional 1% (w/v) of dithiothreitol All enrolled patients received an immunosuppressive regimen con- (RAS, Mannheim, Germany), and the sample was frozen at −70  C. sisting of tacrolimus (Tac; Prograf, Astellas Pharma), mycophenolic After thawing, mRNA was isolated with the MagNA-Pure-LC acid (MPA; Myfenax, Teva Pharmaceuticals), prednisone, and device using the mRNA standard protocol for cells. The elution basiliximab induction. The initial oral daily dose of Tac was 0.1 mg/kg volume was set to 50 μL. One aliquot of 8.2 μL RNA was reverse and administered twice daily. The Tac dose had to be subsequently transcribed in a thermocycler using avian myeloblastosis virus adjusted to achieve target trough concentrations of 7–10 and reverse transcriptase and oligo (dT) as a primer (First Strand cDNA 5–8 ng/mL during the first and third months, respectively. MPA was synthesis kit; Roche, Mannheim, Germany) according to the administered 1 g twice daily. Steroid treatment was initiated at day manufacturer's protocol. After the termination of the cDNA 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3854 SOMMERER ET AL. synthesis, the reaction mixture was diluted to a final volume of 2.7 | Statistical analysis 200 μL and stored at −20 C until polymerase chain reaction (PCR) analysis. Demographic data and the results of the prospective analysis were collected in a unified database. Statistical analysis was performed using SPSS software (version 26.0; Chicago, IL, USA). Data were pres- 2.6.2 | Quantitative analysis of gene expression ented as median ± standard deviation or percentages. ROC analyses were performed to assess how well NFAT-RGE dis- The NFAT-regulated genes IL-2, IFNγ and granular-macrophage criminated between patients at risk for the combined endpoint (death, colony-stimulating factor (GM-CSF)33 were selected for this assay transplant failure and acute rejection). Optimal thresholds were calcu- from previous studies.4,5 Gene expression was quantified using real- lated using simultaneous maximization of sensitivity and specificity. time (RT)-PCR with the LightCycler. Target sequences were amplified Frequency distributions were provided for categorical variables, using commercially available LightCycler Primer Sets (Search-LC, and treatment groups were compared with χ2 tests. Descriptive statis- Heidelberg, Germany) with the LightCyclerFastStart DNA Sybr Green tics were presented for continuous variables, and comparisons of both I Kit (Roche, Mannheim, Germany) according to the manufacturer's groups were performed with a nonparametric test (Mann–Whitney protocol. The transcript concentration for the measured genes was U test). Correlations between gene expression and Tac levels were calculated from a virtual standard curve, obtained by plotting a known analysed using Spearman correlation coefficient (r). All statistical tests input concentration of a plasmid to the PCR cycle number at which were 2-sided and used the .05 level of statistical significance (P < .05). the detected fluorescence intensity reaches a fixed value. mRNA input was normalized by a constant expression value of 2 housekeeping genes (β-actin and peptidyl propyl isomerase B). 3 | RE SU LT S RGE after Tac intake was calculated as Cpeak/C0 × 100, where C0 is the adjusted number of transcripts at the Tac predose level, and 3.1 | Patients Cpeak is the number of transcripts 1.5 hours (C1.5) after drug intake. Mean RGE was calculated as mean RGE from the 3 measured In total, 64 patients were enrolled between April 2014 and June 2015 NFAT-regulated genes: IL-2, IFNγ and GM-CSF. in all 3 European transplant centres (Figure 1). Transplantation was FIGURE 1 Patient flow chart 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SOMMERER ET AL. 3855 cancelled in 2 patients due to medical reasons, 1 patient suffered a was shown at NFAT-RGE of 30% (sensitivity 90.1%, specificity 85%), heart attack and 1 other patient from a severe infection early after whereas the positive predictive value was 0.4 and the negative transplantation. Altogether, 60 de novo renal allograft recipients could predictive value (NPV) was 0.99. Hit rate was 85.6%. be evaluated. Consequently, we used NFAT-RGE 30% as the cut-off for further All, except for 3 patients with NFAT-RGE <30% and 5 patients discrimination between 20 patients with low inhibition (defined as with NFAT-RGE ≥30%, completed the study. Patient characteristics high NFAT-RGE ≥30%), and 40 patients with high inhibition defined were similar between groups (Table 1). as NFAT-RGE <30%. 3.2 | ROC analysis for group definition 3.3 | AUC ROC analyses for the ability of NFAT-RGE to detect patients at risk of Complete AUC0–12 at day 7 post-transplantation could be obtained in acute rejection episodes was performed in a group of 111 renal 36 of the patients. Mean TAC AUC0–12 was 176 ± 81 μg*h/L. TAC allograft recipients with a maximum time of 6 months after transplan- AUC0–12 correlated significantly with abbreviated AUC calculated tation of an earlier evaluation of NFAT-RGE as prognostic marker according to Miura (R = .972, P < .001) or Mathew (0.968, P < .001). in Tac treatment.26 Results showed a good diagnostic accuracy TAC AUC0–12 and RGE correlated inversely (IL-2 RGE, R = −.408, (P < .01; AUC = 0.874; 95% CI 0.759–0.989; Figure 2). Simultaneous P = .017; IFNy, R = −,459, P = .006; GM-CSF RGE, −0.499, P = .003; maximization of sensitivity and specificity obtained from ROC curves NFAT-RGE, −0.459, P = .006). TABLE 1 Demographics and clinical characteristics of de novo renal allograft recipients. Data of all enrolled patients and in both study groups are shown All* Low NFAT-RGE (<30%) High NFAT-RGE (≥30%) P-value Number of patients 64 40 20 - Recipient Age (y), mean ± SD 48.1 ± 12.3 47.7 ± 12.4 48.1 ± 12.7 .896 Male 41 (64.1) 24 (60%) 14 (70%) .449 Caucasian 57 (89.1) 36 (90%) 18 (90%) .472 BMI (kg/m2) 25.8 ± 5.1 25.7 ± 5.0 26.1 ± 5.6 .789 ESRD leading to transplantation .057 Glomerulonephritis 17 (26.6) 12 (30%) 3 (15%) ADPKD 14 (21.9) 10 (25%) 3 (15%) Diabetes 6 (9.4) 3 (7.5%) 3 (15%) Other 21 (32.8) 9 (22.5%) 11 (55%) Unknown 6 (9.4) 6 (15%) 0 Transplantation Pre-emptive transplantation 14 (21.9%) 12 (30%) 2 (10%) .084 Pretransplant dialysis (mo) 59.5 ± 46.7 51.2 ± 51.3 65.9 ± 42.4 .316 Cold ischaemia time (h) 9.5 ± 7.7 11.3 ± 4.6 13.6 ± 5.1 .241 Donor Age (y), mean ± SD 51.9 ± 12.9 51.1 ± 12.8 52.8 ± 12.3 .644 Male (%) 27 (42.2) 18 (45%) 8 (40%) .713 Living (%) 30 (46.9) 23 (57.5%) 7 (35%) .100 CMV- infection status donor (D) recipient (R) .342 D−/R- 4 (6.3%) 1 (2.5%) 3 (15%) D−/R+ 10 (15.6%) 6 (15%) 4 (20%) D+/R- 16 (25%) 11 (27.5%) 3 (15%) D+/R+ 34 (53.1%) 22 (55%) 10 (50%) *all enrolled patients. ADPKD, autosomal-dominant polycystic kidney disease; BMI, body mass index; D, donor; HLA, human leucocyte antigen; NFAT, nuclear factor of activated T cells; RE, residual expression; R, recipient, SD, standard deviation 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3856 SOMMERER ET AL. C, concentration; C0, trough level; C/D, concentration-dose ratio; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-γ, interferon-γ; IL-2, interleukin 2; NFAT, nuclear factor of activated T cells; p, .218 .090 .007 .744 .016 .010 .029 .015 P T A B L E 2 Pharmacokinetics and pharmacodynamics in de novo renal allograft recipients with low (<30%) and high (≥30%) residual expression of NFAT-regulated genes (NFAT-RGE) on High NFAT-RGE 1.89 ± 0.88 5.1 ± 2.5 7.8 ± 2.6 15.1 ± 8.7 54 ± 25 61 ± 24 51 ± 25 55 ± 24 N = 15 Low NFAT-RGE 25.1 ± 10.8 1.79 ± 0.88 6.2 ± 2.9 9.3 ± 2.6 32 ± 26 39 ± 24 31 ± 26 34 ± 25 At mo 6 N = 37 <.001 .312 .054 .001 .769 .075 .003 .011 P F I G U R E 2 Receiver operating curve on nuclear factor of High NFAT-RGE activated T cells-regulated gene expression (threshold 30%) to detect patients at risk of acute rejection episodes. Results showed a good 1.19 ± 0.83 10.7 ± 4.9 10.0 ± 2.9 15.5 ± 7.9 40 ± 28 48 ± 24 39 ± 28 42 ± 27 diagnostic accuracy (P < .01; area under the curve [AUC] = 0.874; N = 20 95% confidence interval [CI]0.759–0.989) 3.4 | Pharmacokinetic and pharmacodynamics monitoring Low NFAT-RGE Pharmacokinetic data of Tac treatment are summarized in Table 2. 31.9 ± 15.6 1.14 ± 0.44 12.0 ± 4.1 12.4 ± 4.8 22 ± 34 24 ± 25 18 ± 26 21 ± 28 There was no significant difference in Tac dose, Tac C0 or Tac C/D At mo 1 N = 39 between patients with low (RGE <30%) and high (RGE ≥30%) residual expression of NFAT-regulated genes throughout the study period. In contrast, Tac C1.5 was significantly higher at all time points in the <.001 <.001 <.001 <.001 .124 .204 .347 .001 P patient group with residual NFAT-RGE below 30%. NFAT-RGE and expression of all 3 genes (IL-2, IFNγ and GM-CSF) correlated inversely to the Tac levels with the highest inhibi- High NFAT-RGE tion of gene expression at the time of the peak Tac level at 1.5 hours after drug intake (day 7; NFAT-RGE 21 ± 16%, IL-2 RGE 20 ± 17%, 21 ± 14.7 0.84 ± 0.89 13.8 ± 5.5 8.8 ± 5.0 28 ± 22 35 ± 22 26 ± 20 30 ± 21 IFNγ RGE 25 ± 18%, GM-CSF RGE 18 ± 16%). NFAT-RGE showed N = 20 significance; RGE, residual gene expression; Tac, tacrolimus high interindividual variability, from 1 to 61% (Figure 3a). The pharma- codynamic marker NFAT-RGE increased from 21 ± 16% at day 7 to 34 ± 27% at month 2 in the whole patient cohort. After that, residual NFAT-RGE was stable (31 ± 25% at month 3, 39 ± 26% at month 6), Low NFAT-RGE but with persisting high interindividual variability ranging from 6 to 37.5 ± 16.1 0.69 ± 0.28 95% (Figure 3b). 15.8 ± 4.5 10.1 ± 3.2 7±7 13 ± 9 7±6 9±7 N = 40 Patients with an early low residual expression of NFAT-regulated At d 7 genes showed significantly lower expression of all 3 genes throughout the study period (Table 2). tacrolimus treatment GM-CSF RGE, % Tac dose, mg/d NFAT-RGE, % Tac C1.5, μg/L IFN-γ RGE, % Tac C0, μg/L 3.5 | Efficacy IL-2 RGE, % Laboratory parameter Tac C/D The combined endpoint (death, graft failure and BPAR) was reached in 9 patients with a significantly higher incidence in patients with 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SOMMERER ET AL. 3857 F I G U R E 3 (A) Residual nuclear factor of activated T cells-regulated gene expression (NFAT-RGE) in tacrolimus treated renal allograft recipients early after transplantation (day 7), and b) during the 6-month study period (median, 5 and 95% percentiles are shown) T A B L E 3 Adverse events in de novo renal allograft recipients with low (<30%) and high (≥30%) residual NFAT-regulated gene expression (NFAT-RGE) Low NFAT-RGE (<30%) High NFAT-RGE (≥30%) P-value Combined endpoint 2 (5%) 7 (35%) .002 Death 0 1 (5%) .154 Transplant failure 0 1 (5%) .154 Treated BPAR 2 (5%) 7 (35%) .002 GFR >60 mL/min 11 (27.5%) 6 (30%) .694 GFR <30 mL/min 4 (10%) 5 (25%) .092 Any infection 28 (70%) 9 (45%) .060 All viral 22 (55%) 5 (25%) .028 CMV 13 (32.5%) 1 (5%) .018 BKV 8 (20%) 1 (5%) .125 CMV and/or BKV 21 (52.5%) 2 (10%) .001 Bacterial 10 (25%) 5 (25%) 1.00 Fungal 1 (2.5%) 2 (10%) .209 Malignancy 0 0 - De novo/worsening hypertension 2 (5%) 2 (10%) .464 De novo diabetes 2 (5%) 5 (25%) .023 De novo hyperlipidaemia 1 (2.5%) 2 (10%) .209 Neurotoxic signs (tremor/trembling) 5 (12.5%) 5 (25%) .221 Other events 4 (2.4%) 3 (10.5%) .570 BPAR, biopsy-proven acute rejection; BKV, polyoma (BK) virus; CMV, cytomegalovirus; GFR, glomerular filtration rate; NFAT, nuclear factor of activated T cells; RGE, residual gene expression NFAT-RGE ≥30% (combined endpoint: 35 vs. 5%, P = .002; Table 3). specificity 80.4%), whereas the positive predictive value was 0.35 and There was 1 death and 1 graft failure in patients with low residual the NPV was 0.95. expression of NFAT-regulated genes. This 2 patients also showed BPAR occurred in 9 cases, 7 patients with NFAT-RGE ≥30% and BPAR.ROC analyses for the ability of NFAT-RGE to discriminate 2 in patients with NFAT-RGE <30% (35 vs. 5%, P = .002; Figure 5). patients at risk for the combined endpoint (death, transplant failure Histopathological classifications of these 2 patients demonstrated a and acute rejection) from stable patients showed a good diagnostic subclinical or borderline acute rejection. accuracy (P = .005; AUC = 0.797; 95% CI 0.607–0.988; Figure 4). Patients with a BPAR demonstrated a high residual expression of Simultaneous maximization of sensitivity and specificity obtained NFAT-regulated genes early after renal transplantation (35 ± 23 vs. from ROC curves was shown at NFAT-RGE of 30% (sensitivity 77.8%, 12 ± 13%, P < .001; Figure 6A). 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3858 SOMMERER ET AL. Infectious complications resulting in a hospitalization occurred in 70% of patients with NFAT-RGE <30% compared to 45% in patients with NFAT-RGE ≥30% (P = .060; Table 3). In particular, viral infec- tions were significantly higher in the patient group with NFAT-RGE <30% compared to patients with NFAT-RGE ≥30% (55 vs. 25%, P = .028; Figure 7). More than half of the patients with NFAT-RGE <30% experienced CMV or BKV replication (52.5 vs. 10%, P = .001). Incidences of bacterial or fungal complications were compa- rable in both groups. Altogether patients with a viral complication, in particular CMV or BKV infection, showed a significantly lower residual expression of NFAT-regulated genes (10 ± 7 vs. 19 ± 19%, P = .010). In contrast, median values for Tac C0 and Tac C1.5 in both groups were compara- ble (Figure 6B). No malignancy occurred in either of patient groups. The incidence of drug-related adverse events (neurotoxic, e.g. trembling, de novo or worsening hypertension, or hyperlipidaemia) were comparable (Table 3). New-onset diabetes occurred more often in the group with NFAT-RGE ≥30%, i.e. with low inhibition of NFAT-RGE, showing the F I G U R E 4 Receiver operating curve on nuclear factor of effect of steroids on the development of diabetes in the early post- activated T cells-regulated gene expression (threshold 30%) de novo transplantation period (Table 3). renal allograft recipients at risk for the combined endpoint (death, transplant failure, and acute rejection; area under the curve [AUC] 0.797; 95% confidence interval [CI] 0.607–0.988, P = .005) 4 | DI SCU SSION Optimized immunosuppression might be possible by individualized CNI treatment monitored by pharmacodynamic tools that determine the immune response.6,7 The ultimate goal would be a balance between the optimal suppression of alloresponses while preserving maximal immunocompetence towards viral infections, reactivation, and virus-induced malignancy. Although both responses share the same mechanistic principle, the different level of effector responses allows fine-tuned therapeutic intervention. Also, treating patients with a minimal required dose of a CNI might reduce off-target side effect. Considering the proposed mode of action of CNIs, the transcriptional activity of NFAT-regulated genes might be a useful immune monitor- ing tool to individualize CNI therapy.19 In the present European multicentre study in renal allograft recipi- ents on Tac treatment, NFAT-RGE was monitored in addition to stan- dard Tac treatment. Patients on Tac treatment showed a high F I G U R E 5 Incidence of biopsy-proven acute rejection in renal interindividual variability of NFAT-RGE. Patients with low inhibition allograft recipients with low (<30%) and high (≥30%) residual of IL-2, IFNγ and GM-CSF gene expression resulting in a high expression of nuclear factor of activated T cells-regulated genes (NFAT-RGE) in the early post-transplant period NFAT-RGE could be identified; Tac doses and Tac C0 levels, which represent the standard monitoring tool in transplantation, were com- parable in this high NFAT-RGE group to patients with RGE <30%. These latter patients experienced the combined endpoint of BPAR, 3.6 | Safety graft loss, and death with a significantly higher incidence. Acute rejec- tion episodes occurred more often and earlier after transplantation if Renal allograft function was comparable in both patient cohorts at NFAT-RGE was ≥30% compared to patients with stronger inhibition month 6 with an S-creatinine of 124 ± 53 vs. 142 ± 59 μmol/L, of NFAT-RGE. An NPV of 0.95 at NFAT 30% led to better identifica- P = .247 (estimated glomerular filtration rate 56 ± 27 vs. 52 ± 18 mL/ tion of patients without rejection risk. By contrast, patients with low min, P = .525). Proteinuria was comparable at month 6 with NFAT-RGE were at increased risk of infectious complications, espe- 0.167 ± 0.192 vs. 0.152 ± 0.106 g/L, P = .869. cially viral infections. In patients with CMV or BKV infection or 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SOMMERER ET AL. 3859 F I G U R E 6 Pharmacokinetic and pharmacodynamics results in patients with (1) and without (0) (A) biopsy-proven acute rejection, and (B) cytomegalovirus (CMV) and/or polyoma virus (BKV) infection. NFAT-RGE, nuclear factor of activated T cells-regulated gene expression F I G U R E 7 Incidence of (A) any infection, (B) viral infection and (C) cytomegalovirus (CMV) infection in renal allograft recipients with low (30%) and high (≥30%) residual expression of nuclear factor of activated T cells-regulated genes (NFAT-RGE) replication within the first 6 months after transplantation, NFAT-RGE technique provides a rapid, highly reproducible and sensitive tool for was low (<10%). the quantitative analysis of gene expression.18 This assay can be The present study represents the first prospective multicentre semi-automated, standardized and performed in laboratories using study on standard Tac treatment monitored by the immune monitor- reverse transcription–PCR.34 Although overall gene expression is ing tool residual NFAT-RGE immune monitoring tool.The RT-PCR reduced within 24 hours upon shipment, the relative degree of NFAT 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3860 SOMMERER ET AL. inhibition remains stable during this period. NFAT-RGE has shown pharmacodynamic tool for additional monitoring of Tac therapy low analytical variability in repeated measurements (<10%). Whereas for renal allograft recipients, especially in the context of over- interpatient variability is high, an earlier evaluation demonstrated low immunosuppression and viraemia.45 Both independent studies con- intraindividual variability in patients on stable CNI doses and stable firmed our previous finding that a considerable proportion of immunosuppressive comedication.21 Tac-treated patients show a relatively high residual expression of In earlier studies, the linearity, precision and limit of quantifica- NFAT-regulated genes without clinical evidence of inadequate immu- 18,22,34,35 tion, as well as sample stability, were investigated. For inter- nosuppression.29 Both Bremer et al.45 and Sommerer et al.29 found a laboratory comparison, samples were analysed in 2 experienced small difference between trough and peak drug levels as a technical laboratories. This analysis demonstrated that this assay could be set explanation for this phenomenon. Since the absolute expression of up with satisfactory analytical performance in a routine molecular NFAT-regulated genes in these patients is likewise relatively high, biological laboratory with results comparable between laboratories. additional regulatory pathways of suppressing alloresponses have to The reproducibility of the NFAT-RGE assay across laboratories be postulated. So far, previously published additional effects of Tac facilitates the implementation of this assay in different centres for on other activating pathways and T-cell metabolism46 could not be pharmacodynamic monitoring of CNI. confirmed in a clinically relevant physiologic setting. While this tool has already been well evaluated in cross-sectional, In an earlier noninterventional trial, NFAT-RGE was assessed in prospective observational and interventional studies of CsA-treated the early post-transplant period in renal allograft recipients on CsA adult and paediatric patients with renal, liver, heart or lung transplan- treatment.47 High residual NFAT-RGE was related to acute rejection tations, there are only a few studies in Tac treated patients.22–24,36–39 episodes and low residual expression with infectious complications. The inverse relationship between Tac levels and gene expression Recently, NFAT-RGE was evaluated as an early noninvasive predictive has already been demonstrated in an earlier single-centre study.29 biomarker for the risk of acute rejection and infection in de novo liver The highest gene inhibition could be detected at the time of Tac peak transplant recipients on CsA or Tac treatment.39 A significant increase levels showing the biological effect of Tac on the 3 evaluated in NFAT-RGE was observed in patients with T-cell mediated rejection NFAT-regulated genes IL-2, IFNγ and GM-CSF. High interindividual compared to patients without rejection or infection. However, the variability of NFAT-regulated genes was observed in CsA- and present study confirmed the previously identified threshold of Tac-treated patients. Combined with a low intraindividual variability, NFAT-RGE 30% for patients on Tac-based immunosuppression at risk this immune assay may be the optimal tool for monitoring of the of acute rejection.29 biological effect of CNIs. In conclusion, the residual expression of the 3 NFAT-regulated CNI dose adjustments using NFAT-RGE assists in the individuali- genes (IL-2, IFNγ and GM-CSF) measured by RT-PCR in whole blood zation of CNI treatment.40 NFAT-RGE monitoring in addition to PK samples directly show the biological effect of Tac. The present monitoring expands the possibility and improves individualization of European multicentre study confirmed this predictive immune marker CNI therapy. in de novo renal allograft recipients on Tac treatment. While the The feasibility of individualised CNI immunosuppression by phar- standard tools for pharmacokinetic monitoring could not detect macodynamic monitoring was assessed in the prospective randomized patients at risk of acute rejection or viral infections, the immune Calcineurin-Inhibitor Sparing trial, and the opportunity to reduce marker NFAT-RGE clearly shows this possibility. This pharmacody- cardiovascular risk and other CNI-induced adverse events were evalu- namic monitoring tool could be added to the standard monitoring, ated.41 NFAT-RGE proved efficacy and safety in individualizing CsA especially in patients at risk of adverse events. This promising treatment as a translational immune monitoring tool that can reduce new pharmacodynamic biomarker provides physicians with useful cardiovascular risk and improve long-term renal allograft function.42 information to specify individualized adjustments of treatment and This pharmacodynamic analysis supports the identification of prevent serious clinical events. patients at risk of side effects and overimmunosuppression and might serve as a reliable assay to lower CNI dosing. In a previous observa- tional single-centre study, NFAT-RGE was low in stable renal allograft 4.1 | Nomenclature of targets and ligands 43 recipients with CMV infections. In the present prospective multicentre study, these results were also confirmed in the early post- Key protein targets and ligands in this article are hyperlinked to transplant period. corresponding entries in http://www.guidetopharmacology.org, and An independent investigator group studied the feasibility to are permanently archived in the Concise Guide to PHARMACOLOGY adjust Tac dosing according to the residual activity of NFAT-regulated 2019/20 (Alexander et al., 2019 a,b). genes in renal transplantation in a single-centre clinical study.44 They suggested that quantitative analysis of the NFAT-RGE might serve as AC KNOWLEDG EME NT S a reliable assay to lower Tac dosing in patients who seem to be TEVA Pharmaceuticals supported partly the core study IMAGEN. oversuppressed. However, the assay could not identify patients with insufficient immunosuppression.Another small study from Oslo, COMPETING INTER ESTS Norway supported the potential of NFAT-RGE measurements as a There are no competing interests to declare. 13652125, 2021, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.14794 by Readcube (Labtiva Inc.), Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License SOMMERER ET AL. 3861 CONT R IBUT ORS 15. Alexander SPH, Kelly E, Mathie A, et al. The concise guide to Research idea and study design: C.S., K.B., M.B.; data acquisition and PHARMACOLOGY 2019/20: Introduction and Other Protein Targets. Br J Pharmacol. 2019;176(Suppl 1):S291. study performance: C.S., K.B., M.B., O.M., L.G., P.G.; data analysis/ 16. Kel A, Kel-Margoulis O, Babenko V, Wingender E. Recognition of statistics: C.S., T.G.; supervision and mentorship: M.Z., S.M. Each NFATp/AP-1 composite elements within genes induced upon the author contributed valuable intellectual content during manuscript activation of immune cells. J Mol Biol. 1999;288(3):353-376. drafting, accepts personal accountability for the author's contribu- 17. Giese T, Zeier M, Meuer S. Analysis of NFAT-regulated gene expression in vivo: a novel perspective for optimal individualized tions, and agrees to ensure that questions on the accuracy or integ- doses of calcineurin inhibitors. Nephrol Dial Transplant. 2004;19: rity of any portion of the work are appropriately investigated and iv55-iv60. resolved. 18. Giese T, Zeier M, Schemmer P, et al. Monitoring of NFAT-regulated gene expression in the peripheral blood of allograft recipients: a novel perspective toward individually optimized drug doses of cyclosporine DATA AVAI LAB ILITY S TATEMENT A. Transplantation. 2004;77(3):339-344. Research data cannot be shared due to restriction by law. 19. Sommerer C, Giese T. Nuclear factor of activated T cells-regulated gene expression as predictive biomarker of personal response to cal- ORCID cineurin inhibitors. Ther Drug Monit. 2016;38(Supplement 1):S50-S56. Claudia Sommerer https://orcid.org/0000-0001-5080-0979 20. Sommerer C, Giese T, Meuer S, et al. Pharmacodynamic monitoring Olga Millán https://orcid.org/0000-0002-6861-5832 of calcineurin inhibitor therapy: is there a clinical benefit? Nephrol Dial Transplant. 2009;24:21-27. 21. Sommerer C, Meuer S, Zeier M, Giese T. Calcineurin inhibitors and RE FE R ENC E S NFAT-regulated gene expression. Clin Chim Acta. 2012;413(17-18): 1. Webber AB, Vincenti F. An Update on Calcineurin Inhibitor-Free 1379-1386. Regimens: The Need Persists, but the Landscape has Changed. 22. Sommerer C, Konstandin M, Dengler T, et al. 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Pediatr Transplant. 2010;14(7):844-851. 39. Millán O, Ruiz P, Fortuna V, Navasa M, Brunet M. Nuclear factor of activated T cells as potential pharmacodynamic biomarker for the risk How to cite this article: Sommerer C, Brunet M, Budde K, of acute and subclinical rejection in de novo liver recipients. Liver Int. et al. Monitoring of gene expression in tacrolimus-treated de 2019;40:931-946. 40. Sommerer C, Giese T, Schmidt J, Meuer S, Zeier M. Ciclosporin A novo renal allograft recipients facilitates individualized tapering monitored by NFAT-regulated gene expression: a new immunosuppression: Results of the IMAGEN study. Brit Jnl concept of individual immunosuppression. Transplantation. 2008;85 Clinical Pharma. 2021;87(10):3851–3862. https://doi.org/10. (1):15-21. 1111/bcp.14794 41. Sommerer C, Schaier M, Morath C, et al. The Calcineurin Inhibitor- Sparing (CIS) Trial - individualised calcineurin-inhibitor treatment by immunomonitoring in renal allograft recipients: protocol for a ran- domised controlled trial. Trials. 2014;15(1):489. 42. Sommerer C, Brocke J, Bruckner T, et al. improved pulse wave veloc- ity and renal function in individualized calcineurin inhibitor treatment