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Long-term efficacy of everolimus as de novo immunosuppressant on the cardiac allograft vasculopathy in heart transplant recipients

  • Hyo-In Choi
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, Seoul, 03181, South Korea
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  • Do-Yoon Kang
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Min-Seok Kim
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Sang Eun Lee
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Jung-Min Ahn
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Jong-Young Lee
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, Seoul, 03181, South Korea
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  • Yong-Hak Kim
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Duk-Woo Park
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Sung-Ho Jung
    Affiliations
    Department of Thoracic and Cardiovascular Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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  • Jae-Joong Kim
    Correspondence
    Corresponding author. Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, 05505, Republic of Korea.
    Affiliations
    Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea
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      Highlights

      • Data on cardiac allograft vasculopathy progression in everolimus-immunosuppressed heart recipients are scarce.
      • Everolimus is associated with reduced plaque progression and positive vascular remodeling.
      • De novo everolimus is an effective immunosuppressant therapy.

      Abstract

      Background and aims

      Data on the long-term effects of everolimus (EVL) on the de novo immunosuppression of heart transplant (HT) recipients with progressive cardiac allograft vasculopathy (CAV) and vascular remodeling are lacking. Hence, in this study, we aimed to determine the long-term safety and efficacy of EVL as a de novo immunosuppressant therapy for CAV progression and the clinical outcomes after HT.

      Methods

      We retrospectively reviewed the medical records of 144 HT recipients who survived for at least one year after HT. CAV progression was assessed via serial coronary intravascular ultrasonography (IVUS) in recipients who underwent at least two IVUS studies.

      Results

      A significant attenuation in the percentage of the atheroma volume progression was observed in those who took EVL (1.2%) compared with those who took cyclosporin (CSA; 7.3%; p = 0.005 vs. EVL) or tacrolimus (TAC; 6.6%; p = 0.0052 vs. EVL) at 1 year after HT. This trend persisted for the next 3 and 5 years after HT. Moreover, the remodeling index was greater in the EVL (1.08) group than in the CSA (0.23) or TAC (−0.25) groups 1 year after HT. The results of the Kaplan–Meier analysis over a median follow-up period of 8 years revealed that there was no statistical difference in the primary endpoint between the three groups.

      Conclusions

      De novo immunosuppression with EVL is associated with attenuated CAV progression for the first 5 years of follow-up via IVUS. Moreover, EVL has comparable long-term clinical outcomes to those of CSA- or TAC-based protocols.

      Graphical abstract

      Keywords

      1. Introduction

      Over the last 50 years, heart transplantation (HT) has become the treatment of choice for patients with advanced heart diseases. Nevertheless, HT is associated with complications and late morbidity and mortality. The primary cause of late morbidity and mortality in HT recipients is cardiac allograft vasculopathy (CAV), which accounts for one-third of all deaths at five years after HT [
      • Khush K.K.
      • et al.
      The international thoracic organ transplant registry of the international society for heart and Lung transplantation: 37th adult heart transplantation report-2020; focus on deceased donor characteristics.
      ]. CAVs are caused by immune and non-immune mechanisms that promote inflammation, endothelial damage, and smooth muscle cell hyperplasia in both the epicardial vessels and microvasculature [
      • Pober J.S.
      • et al.
      Cardiac allograft vasculopathy: current review and future research directions.
      ]. Endothelial injury and an exaggerated repair response cause diffuse intimal hyperplasia and luminal stenosis of the CAV. Because of the detrimental impacts of CAV on graft survival, prevention remains the best strategy for achieving the best long-term outcomes.
      Various medications have been used to prevent CAVs. Several studies showed that statins could improve patient survival while reducing the incidence and severity of CAV [
      • Mallah S.I.
      • et al.
      Evidence-based pharmacotherapy for prevention and management of cardiac allograft vasculopathy.
      ]. Moreover, mammalian target of rapamycin (mTOR) inhibitors are immunosuppressants that exert antiproliferative effects on fibroblasts and smooth muscle cells. As a result, they have the potential to reduce intimal hyperplasia in the coronary arteries, thereby preventing CAV progression [
      • Mallah S.I.
      • et al.
      Evidence-based pharmacotherapy for prevention and management of cardiac allograft vasculopathy.
      ]. The use of mTOR inhibitors in place of azathioprine or mycophenolate mofetil (MMF) as secondary immunosuppressive agents and in combination with lower doses of calcineurin inhibitors (CNIs) attenuated the progression of CAV [
      • Eisen H.J.
      • et al.
      Everolimus versus mycophenolate mofetil in heart transplantation: a randomized, multicenter trial.
      ,
      • Kobashigawa J.A.
      • et al.
      Cardiac allograft vasculopathy by intravascular ultrasound in heart transplant patients: substudy from the everolimus versus mycophenolate mofetil randomized, multicenter trial.
      ,
      • Arora S.
      • et al.
      Effect of everolimus initiation and calcineurin inhibitor elimination on cardiac allograft vasculopathy in de novo heart transplant recipients.
      ]. However, the outcomes of large randomized studies on intravascular ultrasonography (IVUS) were obtained over a short-to mid-term follow-up period only. Thus, long-term data on these patients are scarce [
      • Kobashigawa J.A.
      • et al.
      Cardiac allograft vasculopathy by intravascular ultrasound in heart transplant patients: substudy from the everolimus versus mycophenolate mofetil randomized, multicenter trial.
      ,
      • Eisen H.J.
      • et al.
      Everolimus versus mycophenolate mofetil in heart transplantation: a randomized, multicenter trial.
      ]. Furthermore, there is currently limited evidence regarding the de novo use of EVL for HT in routine clinical practice. To the best of our knowledge, there have been no studies regarding the effect and safety of de novo everolimus (EVL)-based protocols on Asian HT recipients.
      The purpose of this study was to investigate the long-term effect of EVL as a de novo immunosuppressant on CAV progression as assessed by serial IVUS examinations and to investigate the long-term safety and efficacy of an EVL-based regimen on clinical events compared with those on cyclosporine (CSA)- and tacrolimus (TAC)-based regimens.

      2. Patients and methods

      2.1 Study population

      This study included patients aged ≥18 years who underwent HT at Asan Medical Center between 2005 and 2015 and survived for at least one year. Multi-organ transplant recipients were excluded from this study. Since 2005, 24 patients used an EVL-based protocol as the de novo immunosuppressant (EVL group). 48 and 72 patients not using EVL, representing two and three times the number of patients in the EVL group, respectively, were consecutively included from the patients using the CSA-based protocol (CSA group) and the TAC-based protocol (TAC group). Patients who survived at least 1 year after HT and had at least two IVUS examinations were included to determine CAV progression.
      Demographic, clinical follow-up, and laboratory data were collected from the patients' medical records. At each outpatient visit after HT, immunosuppressive medications were reviewed and recorded. The Asan Medical Center's Institutional Review Board (IRB) approved the study protocol (IRB No. 2019-0627), and the IRB waived the requirement for patient consent due to the retrospective nature of the study.

      2.2 Immunosuppression and management

      All HT recipients received interleukin-2 monoclonal antibody (basiliximab) induction therapy and a three-drug maintenance immunosuppressive regimen consisting of a CNI, an antimetabolite agent (MMF), and tapering doses of prednisone after HT as part of a standard immunosuppressive protocol. The decision to withdraw or continue steroids at a maintenance dose was made at the discretion of the attending physician. In patients with stable condition at least 2 weeks post-HT without wound problems or serious infection, MMF was replaced with EVL (trough level, 3–8 ng/mL). After 2007, TAC replaced CSA as the primary CNI. The CNI target trough concentrations in the first 3 months after HT were 300–350 ng/mL for CSA and 10–15 ng/mL for TAC. Twelve months post-HT, the CSA and TAC target trough concentrations were adjusted to 100–150 ng/mL and 5–10 ng/mL, respectively. High-performance liquid chromatography with tandem mass spectroscopy (XEVO TQ-S, Waters, Milford, USA) or microparticle enzyme immunoassay (Dimension® EXL™ 200 Integrated Chemistry System; Siemens, Munich, Germany) were used to measure the trough levels of CSA, TAC, and EVL.
      Until one year after HT, all patients underwent serial endomyocardial biopsies at regular intervals. Patients with grade 2R or a higher grade of acute cellular rejection on surveillance endomyocardial biopsy were treated with augmented immunosuppression and intravenous steroids [
      • Kirklin J.K.
      • et al.
      ISHLT Guidelines for the Care of Heart Transplant Recipients.
      ]. The grading system of acute cellular rejection was based on the International Society of Heart and Lung Transplantation (ISHLT) [
      • Mehra M.R.
      • et al.
      International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010.
      ].
      Except for those who experienced adverse effects from statin therapy, all post-HT recipients received statin therapy (initial daily dose: 10 mg of pravastatin) within 2 weeks after HT regardless of their cholesterol level [
      • Mallah S.I.
      • et al.
      Evidence-based pharmacotherapy for prevention and management of cardiac allograft vasculopathy.
      ]. If the patient's lipid profile worsened, either the dosage of pravastatin was increased, or the statin was changed to a stronger one. Intensive statin therapy was defined as 1) increasing statin dosage during the trial period and 2) switching from pravastatin to a stronger statin during the study period.
      Until one year after HT, a cytomegalovirus (CMV) antigenemia assay was performed weekly during hospitalization and at every outpatient clinic visit. Prophylactic gancyclovir and trimethoprim/sulfamethoxazole were administered to all patients for one year after HT.

      2.3 CAV and IVUS assessment

      Since 2004, most HT recipients underwent coronary angiography with 3-dimensional (3D) IVUS as part of the surveillance for CAV progression. After intracoronary administration of 0.2 mg of nitroglycerin, grayscale IVUS imaging was performed using motorized transducer pullback (0.5 mm/s) and a commercial scanner (Boston Scientific/SCIMED, Minneapolis, MN) consisting of a rotating 40 MHz transducer within a 3.2 F imaging sheath. The inter- and intraobserver agreements of IVUS measurement was excellent, with intraclass correlation coefficient values of 0.986 (95% Confidence Interval [CI]: 0.953 to 0.995; p < 0.001) and 0.978 (95% CI: 0.945 to 0.991; p < 0.001), respectively [
      • Park S.J.
      • et al.
      Intravascular ultrasound-derived minimal lumen area criteria for functionally significant left main coronary artery stenosis.
      ]. Baseline coronary angiography was performed approximately 1 month after HT unless patients had contraindications to coronary angiography. At 1, 3, 5, 8, and 10 years after HT or with any change in clinical status, follow-up coronary angiography was performed as routine surveillance. CAVs were classified according to the ISHLT criteria [
      • Mehra M.R.
      • et al.
      International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010.
      ]. By manual planimetry in images spaced 1-mm apart and where there were no artifacts obscuring >90° of the contiguous outer vascular wall, core laboratory analysts at the Asan Medical Center defined the leading edges of the lumen and the outer vessel wall with reproducibility, as previous reported [
      • Nicholls S.J.
      • et al.
      ,
      • Nissen S.E.
      • et al.
      Effect of very high-intensity statin therapy on regression of coronary atherosclerosis: the asteroid trial.
      ]. The analysts were blinded to the treatment status and imaging study sequencing (baseline vs. follow-up). The percentage of the atheroma volume (PAV) was calculated as follows:
      Σ(EEMareaLumenarea)ΣEEMarea×100


      where EEMarea was the cross-sectional area of the external elastic membrane and Lumenarea was the cross-sectional area of the lumen. The normalized total atheroma volume (TAV) was calculated as follows to compensate for the differences in the examined vessel to the examined segment length:
      Σ(EEMareaLumenarea)NumberofImagesinPullback×median number of images in a group.


      Vessel volume (VV) and lumen volume (LV) were normalized. CAV progression was assessed by calculating the changes in PAV, TAV, VV, and LV between baseline and 1, 3, and 5 years of follow-up IVUS examinations for each patient.
      The remodeling index was defined as the ratio of the change in the vascular area at the lesion site to the change in the intimal area, which was a quantitative assessment of the extent and direction of remodeling [
      • Zhang P.
      • et al.
      IVUS plus multivariate analysis for evaluating the stability of coronary artery plaque in coronary heart disease.
      ]. Positive remodeling that adequately compensated (=1) or overcompensated (>1) for intimal growth was indicated by an index of ≥1, while positive remodeling that was inadequate for intimal growth was indicated by an index of >0 and < 1. Negative remodeling with no compensation (=0) or shrinkage (<0) was indicated by an index ≤0.

      2.4 Outcome endpoints

      The primary clinical outcome endpoint was treatment failure, which was defined as the composite of all-cause death, graft failure, re-transplantation, and treatment requiring rejection at the median follow-up. Treatment requiring rejection was defined as biopsy-proven rejection of at least grade 2R or any episode of rejection associated with hemodynamic compromise.
      Post-transplant diabetes mellitus was diagnosed using the American Diabetes Association criteria [
      2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2021.
      ]. Post-transplant hypertension was defined as a blood pressure of >140/90 mmHg or intake of antihypertensive medication. Hyperlipidemic patients were defined as those receiving intensive statin therapy. CMV anemia was defined as having >50 CMV-positive cells per 200,000 leukocytes. CMV treatment with ganciclovir was started when >100 CMV-positive cells per 200,000 and/or CMV disease was diagnosed.

      2.5 Statistical analysis

      Group sample sizes of 144 achieve 84.135% power to reject the null hypothesis of equal means when the population mean difference is μ1 - μ2 = 5.3–7.6 = −2.3 with standard deviations of 2.8 for group 1 and 5.9 for group 2 [
      • Arora S.
      • et al.
      Effect of everolimus initiation and calcineurin inhibitor elimination on cardiac allograft vasculopathy in de novo heart transplant recipients.
      ], and with a significance level (alpha) of 0.050 using a two-sided two-sample unequal-variance t-test. Quantitative data is reported as mean ± standard deviation or as median (interquartile range [IQR]). Categorical data are expressed as numbers and percentages. Student's t-test or the Chi-square test was used to compare baseline variables, as applicable. Wilcoxon signed-rank test or Mann-Whitney was used to compare non-parametric data. We compared the survival function with the occurrence of clinical events using a log-rank test and Kaplan–Meier survival curve estimations. Logistic regression analysis was used to evaluate adverse events. A linear mixed model was used to assess the absolute changes in PAV, TAV, VV, and LV between the treatment groups. Two-sided p-values <0.05 were considered significant. All analyses were performed using the SAS version 9.4 (SAS Institute, Cary, NC, USA).

      3. Results

      3.1 Patient characteristics

      The baseline demographics, medications, and laboratory results of the three groups are shown in Table 1. The mean recipient age at HT was 48 years, and most of the patients were men. The median follow-up duration were 8.3 years. At the time of HT, the baseline characteristics were not significantly different among the three groups. The TAC group showed a slight difference from the CSA group, with fewer female-to-male transplants and less use of statins. In the TAC group, 80% of patients used statins for 5 years of follow-up, which was significantly lower than that in the CSA group. Only one patient in the EVL group used TAC as a conjunctive CNI, while the remaining patients used CSA. The average trough levels of CSA and TAC during the follow-up period were significantly lower in the EVL group compared with those in the CSA and TAC groups (Supplement Table 1). Throughout the study, the EVL trough levels were within the target range of 3–8 ng/mL. At 5 years, the mean dose of EVL was 0.9 ± 0.3 mg/day (Supplementary Table 2).
      Table 1Baseline characteristics of the patients according to their immunosuppressant regimen.
      CharacteristicsEverolimus (N = 24)Cyclosporine (N = 48)Tacrolimus (N = 72)
      Recipient characteristics
      Age, years48.9 ± 13.047.0 ± 8.9348.4 ± 13.2
      Male (no. %)20 (83.3%)40 (83.3%)45 (62.5%)
      BMI, kg/m222.6 ± 4.3022.2 ± 3.2222.5 ± 3.60
      Comorbidities
      CMV IgG-positive24 (100%)48 (100%)71 (98.6%)
      Diabetes4 (16.7%)4 (8.33%)8 (11.1%)
      Diagnosis
      DCMP17 (70.8%)25 (52.1%)44 (61.1%)
      ICMP1 (4.17%)11 (22.9%)14 (19.4%)
        Other6 (25.0%)12 (25.0%)14 (19.4%)
      Preoperative MCS0 (0.00%)1 (2.08%)7 (9.72%)
      Laboratory data
      Creatinine, mg/dl1.03 ± 0.421.30 ± 0.501.39 ± 1.50
      Bilirubin, mg/dl1.17 ± 0.751.72 ± 1.011.73 ± 1.36
      Donor characteristics
      Age of donor, years34.7 ± 9.5631.4 ± 9.3134.4 ± 11.0
      Donor male18 (75.0%)39 (81.2%)57 (79.2%)
      Female to Male2 (8.33%)7 (14.6%)1 (1.39%)
      p = 0.02 for the comparison with the cyclosporine group.
      Weight difference−9.09 ± 19.2−7.78 ± 19.6−10.83 ± 20.9
      Total ischemic time, minutes169 ± 72.3163 ± 59.4155 ± 53.1
      Medications at 5 years after HT
      Statin22 (91.7%)47 (97.9%)58 (80.6%)
      p = 0.03 for the comparison with the cyclosporine group.
      Aspirin14 (58.3%)37 (77.1%)42 (58.3%)
      Steroid8 (33.3%)11 (22.9%)15 (21.1%)
      BMI, body mass index; CMV, cytomegalovirus; IgG, immunoglobulin G; DCMP, dilated cardiomyopathy; ICMP, ischemic cardiomyopathy; MCS, mechanical circulatory support; HT, heart transplantation.
      a p = 0.02 for the comparison with the cyclosporine group.
      b p = 0.03 for the comparison with the cyclosporine group.

      3.2 Effect of immunosuppression on plaque progression and vascular geometry

      We identified 107 recipients having a baseline and at least one IVUS examination after HT to assess volumetric changes and CAV progression (23 patients in the EVL group, 29 patients in the CSA group, and 55 patients in the TAC group). We excluded poor-quality images or images that could not be analyzed using IVUS. Finally, 376 coronary IVUS examinations (median: 4 per patient; range: 3.4 to 4.6 per patient) were analyzed. Table 2 summarizes the volumetric measurements of plaque progression via three-dimensional IVUS at baseline and at 1-, 3-, and 5-year follow-up time points. At baseline, the left anterior descending artery-normalized TAV, VV, PV, LV, and PAV were not significantly different among the groups. During follow-up, PAV was significantly increased in all of the three groups. However, the plaque volume change between 1-, 3-, and 5-year IVUS and the baseline measurement was significantly lower in the EVL group than in the CSA or TAC group. A significant attenuation in PAV progression was observed in the EVL group (+1.2%) compared with that in the CSA (+7.3%; p = 0.005) and TAC (+6.6%; p = 0.0052) groups at 1 year after HT. This trend remained unchanged for 3 years (+4.7% vs. +12.4% vs. +12.5% for EVL vs. CSA vs. TAC respectively, p = 0.006) and 5 years (+7.9% vs. +14.9% vs. +14.9% for EVL vs. CSA vs. TAC respectively, p = 0.02) after HT. Interestingly, after 1 year of treatment, EVL decreased TAV (−3.8 mm³); however, statistical significance was not observed. Conversely, use of CSA (+24.2 mm³; p = 0.005) and TAC (+19.3 mm³; p = 0.007) resulted in a statistically significant increase in TAV. Approximately 3 and 5 years after HT, the TAV in the EVL group was also increased, and the level of increase was statistically less than those in the other two groups. The efficacy of EVL in attenuating the progression of CAV was maintained at 5 years, as shown by the changes in PAV (Fig. 1A) and TAV (Fig. 1B) compared with those in the CSA and TAC groups.
      Table 2Assessment of the progression of cardiac allograft vasculopathy via IVUS during follow-up.
      EverolimusCyclosporineTacrolimusOverall pp between EVL-CSAp between EVL-TACp between CSA-TAC
      % atheroma volume change from baseline
       Baseline23.8 ± 1.621.9 ± 1.422.1 ± 1.10.620.390.370.92
       1 year after HT25 ± 2.229.4 ± 1.928.7 ± 1.40.270.130.160.76
       1 year change from baseline1.2 ± 1.67.3 ± 1.46.6 ± 10.0080.0050.0050.68
      p value0.45<0.001<0.001
       3 years after HT28.5 ± 2.634.6 ± 2.434.6 ± 1.70.130.090.050.99
       3 year change from baseline4.7 ± 2.112.4 ± 1.912.5 ± 1.40.0060.010.0030.97
      p value0.03<0.001<0.001
       5 years after HT31.7 ± 2.737.1 ± 2.437.1 ± 1.70.220.140.10020.95
       5 year change from baseline7.9 ± 2.214.9 ± 1.914.9 ± 1.30.0180.020.00720.99
      p value<0.001<0.001<0.001
      Total atheroma volume change from baseline
       Baseline123.9 ± 10.4125.1 ± 9.3127.8 ± 6.70.940.940.75680.81
       1 year after HT120.1 ± 12.4149.4 ± 11.1147.3 ± 80.140.080.0680.88
       1 year change from baseline−3.8 ± 7.324.2 ± 6.519.3 ± 4.70.010.0050.00880.55
      p value0.60<0.001<0.001
       3 years after HT143.7 ± 14.1180.5 ± 12.7176.3 ± 9.20.100.060.050.794
       3 year change from baseline19.8 ± 9.555.1 ± 8.548.3 ± 6.20.020.0070.010.52
      p value0.04<0.001<0.001
       5 years after HT151.8 ± 15.8192.3 ± 13.7184.5 ± 9.80.130.050.080.64
       5 year change from baseline26.9 ± 11.467 ± 9.556.3 ± 6.80.020.010.030.36
      p value0.01<0.001<0.001
      Total vessel volume change from baseline
       Baseline518.6 ± 26.1576.3 ± 23.2582.3 ± 16.90.110.100.040.84
       1 year after HT471.9 ± 24.5515 ± 22.1518.5 ± 15.90.260.190.110.89
       1 year change from baseline−46.7 ± 19.9−59.8 ± 18.0−64.1 ± 12.90.770.630.470.84
      p value0.020.001<.0001
       3 years after HT506.8 ± 23.7519.9 ± 21.7523.7 ± 15.50.840.680.550.89
       3 year change from baseline−11.9 ± 22.1−55.0 ± 20.1−58.7 ± 14.50.190.150.080.88
      p value0.590.006<0.001
       5 years after HT471.2 ± 26.7517 ± 22.7506.6 ± 16.20.400.190.260.71
       5 year change from baseline−39.7 ± 26.5−59.5 ± 22.4−75.2 ± 16.00.510.570.260.57
      p value0.070.009<0.001
      Total lumen volume change from baseline
       Baseline394.7 ± 23.1451.3 ± 20.6454.5 ± 150.080.070.030.90
       1 year after HT351.8 ± 22.1365.8 ± 20371.5 ± 14.30.760.640.460.82
       1 year change from baseline−42.9 ± 21.3−83.7 ± 19.3−83.5 ± 13.80.250.160.110.99
      p value0.05<0.001<0.001
       3 years after HT363.1 ± 22.9339.5 ± 21347 ± 150.740.450.560.77
       3 year change from baseline−31.6 ± 23.5−110.4 ± 21.3−107.0 ± 15.40.020.010.0080.90
      p value0.18<0.001<0.001
       5 years after HT319.9 ± 22.9326.8 ± 19.6322.9 ± 140.970.820.910.87
       5 year change from baseline−64.3 ± 25.3−127.4 ± 21.7−131.6 ± 15.50.070.060.020.87
      p value0.003<0.001<0.001
      EVL, everolimus; CSA, cyclosporine; TAC, tacrolimus; HT, heart transplantation.
      Fig. 1
      Fig. 1Changes in PAV (A) and TAV (B) as assessed by serial IVUS examinations during follow-up, stratified by type of immunosuppressive regimen.
      Values are expressed as mean ± SEM in each treatment group. PAV, percentage of the atheroma volume.
      The remodeling patterns observed after 1-, 3- and 5-years follow-up are shown in Fig. 2. The remodeling index was greater in the EVL (1.08) group than in the CSA (0.23) or TAC (−0.25) groups, indicating adequate remodeling in the EVL group and inadequate (CSA) or negative remodeling (TAC) in the other groups. In the EVL group, positive correlations between changes in vessel and atheroma volumes (adaptive vessel response) were observed throughout the study period. In the CSA group, inadequate vessel remodeling was observed at 1 and 3 years, while the TAC group showed negative vessel remodeling throughout the study period. At 1-year post-HT, negative vessel remodeling, defined as an intimal increase with negative vessel remodeling, was observed in 30.4%, 63.0% and 46.3% for the EVL, CSA, and TAC groups respectively.
      Fig. 2
      Fig. 2Remodeling patterns, such as vessel changes in response to intimal changes, during follow-up according to immunosuppressant regimen.
      Upper panel: 1-year change from baseline in EVL (A), CSA (B), TAC (C) group. Middle panel: 3-year change from baseline in EVL (D), CSA (E), TAC (F) group. Lower panel: 5-year change from baseline in EVL (G), CSA (H), TAC (I) group. EVL, everolimus; CSA, cyclosporine; TAC, tacrolimus.

      3.3 Effect of immunosuppressive regimens on clinical outcomes

      Over a median follow-up period of 8 years, the primary endpoint did not occur in the EVL group. However, it occurred in 10 (21.8%) and 14 (20.6%) patients in the CSA and TAC groups, respectively (Supplementary Table 3). There was no significant difference in the event-free survival among the three groups according to the results of the Kaplan–Meier analysis (Fig. 3). The incidence of treatment requiring rejection among patients treated with EVL-based protocol and those treated with CSA-based or TAC-based protocols during follow-up was lower in the EVL group, but the difference was not statistically significant (0% vs. 19.9% vs 19.4 in EVL, CSA, and TAC groups, respectively; p = 0.1). In the CSA group, nine patients (19.7%) had moderate-to-severe (ISHLT grade 2R or 3R) cellular rejection. In the TAC group, seven patients (11.1%) had moderate-to-severe cellular rejection. No patients in the EVL group had moderate-to-severe cellular rejection. Patients who received intensive statin therapy was numerically higher in the EVL group compared to those in the CSA or TAC groups (87.5% vs. 69.9% vs. 54.8% in EVL, CSA, and TAC groups, respectively). Nevertheless, the difference was not statistically significant. The incidence of CMV infections was lower with EVL-based protocol treatment (0%) than treatment with either CSA-based protocol (9 [18.8%], p = 0.27) or TAC-based protocol (13 [18.8%], p = 0.16). The incidence of non-CMV infections did not differ between groups. Patients treated with EVL had less development of moderate-toss-severe CAV compared to the other two groups (0 [0%] vs. 5 (9.5%) vs. 3 (4.2%) for the EVL, CSA, and TAC groups, respectively). Changes in serum creatinine levels did not differ significantly over the 5 years of follow-up between the groups.
      Fig. 3
      Fig. 3Event-free survival in heart transplant recipients according to their immunosuppressive regimen.

      4. Discussion

      The main findings of this study can be summarized as follows: 1) de novo use of EVL as secondary immunosuppression combined with reduced-dose CNI was associated with attenuated progression of CAV to 5 years after HT; 2) EVL was not associated with significantly different all-cause mortality, graft failure, retransplantation, and treatment requiring rejection for 8 years of follow-up, although a trend of clinical benefit appeared in the EVL group; and 3) when compared to standard CNI-based regimens, early adoption of EVL-based immunosuppressive therapy was safe and well-tolerated. To the best of our knowledge, this is the first study to demonstrate the long-term IVUS results of de novo EVL use, efficacy, and safety in the Asian population.
      In our study, EVL was associated with a reduced progression of CAV, and these effects were sustained over a five-year period following HT. Several clinical trials using IVUS showed that EVL reduced intimal thickening. We found that at the 5-year follow-up, the CSA and TAC groups had significant increases in plaque volume and vessel shrinkage, whereas the EVL group had fewer worsening changes, which corresponded to an attenuation in CAV progression, which was determined by IVUS. mTOR inhibitors, such as sirolimus (SRL) [
      • Asleh R.
      • et al.
      Long-term sirolimus for primary immunosuppression in heart transplant recipients.
      ,
      • Asleh R.
      • et al.
      Hypercholesterolemia after conversion to sirolimus as primary immunosuppression and cardiac allograft vasculopathy in heart transplant recipients.
      ] and its derivative EVL [
      • Kobashigawa J.A.
      • et al.
      Cardiac allograft vasculopathy by intravascular ultrasound in heart transplant patients: substudy from the everolimus versus mycophenolate mofetil randomized, multicenter trial.
      ,
      • Howard J. Eisen
      • Murat Tuzcu E.
      • Dorent Richard
      • Kobashigawa Jon
      • Mancini Donna
      • Valantine-von Kaeppler Hannah A.
      • Starling Randall C.
      • Sørensen Keld
      • Hummel Manfred
      • Lind Joan M.
      • Abeywickrama Kamal H.
      • Bernhardt Peter
      for the RAD B253 Study Group
      Everolimus for the prevention of allograft rejection and vasculopathy in CardiacTransplant recipients.
      ], resulted in the attenuation of CAV progression in de novo HT patients taking full- or reduced-dose CNI compared to those taking azathioprine or MMF. The IVUS substudy of A2310 demonstrated that the maximal intimal thickness and incidence of CAV were significantly lower in the EVL and reduced-dose CSA groups than in the MMF and standard-dose CSA groups 12 months after HT [
      • Kobashigawa J.A.
      • et al.
      Cardiac allograft vasculopathy by intravascular ultrasound in heart transplant patients: substudy from the everolimus versus mycophenolate mofetil randomized, multicenter trial.
      ]. Plaque volume progression (SRL: 2.8 ± 2.3; CNI: 0.46 ± 1.8; p < 0.0001) and plaque index (SRL: 12.2 ± 9.6%; CNI: 1.1 ± 7.9%; p < 0.0001) were significantly reduced in patients who had primary immunosuppression with SRL and withdrawal of CNI compared to those in the CNI group [
      • Asleh R.
      • et al.
      Long-term sirolimus for primary immunosuppression in heart transplant recipients.
      ]. Interesting findings of our study include a decrease in TAV at 1 year in follow-up IVUS, and positive remodeling with EVL-based immunosuppression. CAV attenuation by mTOR inhibitors may be mediated by their antiproliferative and antimigratory effects on VSMCs, as demonstrated by in vitro and in vivo studies that explored beyond their immunosuppressive properties [
      • Mehra M.R.
      • et al.
      International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010.
      ,
      • Chaves Á.J.
      • et al.
      ]. Reduced extracellular matrix accumulation and fibrosis [
      • Anthony C.
      • et al.
      Everolimus for the prevention of calcineurin-inhibitor-induced left ventricular hypertrophy after heart transplantation (RADTAC study).
      ], combined with increased nitric oxide production [
      • Trøseid M.
      • et al.
      The carnitine-butyrobetaine-TMAO pathway after cardiac transplant: impact on cardiac allograft vasculopathy and acute rejection.
      ], resulted in improved vascular remodeling and reduced coronary lumen obliteration. Both resulted in positive vascular remodeling and less obliteration of the coronary artery lumen. It appeared that the vascular responses of the transplanted heart were distinct from those of the native coronary arteries regarding the remodeling of the coronary artery [
      • Pober J.S.
      • et al.
      Cardiac allograft vasculopathy: current review and future research directions.
      ]. The remodeling index at 5 years in the CSA group showed significant positive remodeling; however, this result should be interpreted carefully because patients with IVUS images who developed CAV or received PCI were excluded from the analysis.
      In our study, the primary composite endpoint of all-cause death, retransplantation, and treatments requiring rejection occurred much less frequently in EVL-based protocols than in CSA or TAC-based protocols. The individual variables responsible for the benefit were reduction in all-cause mortality and treatment-requiring rejection, although statistical significance was not reached. This may be attributed to the small sample size. Previous randomized trials on mTOR inhibitors showed favorable rejection and efficacy results in EVL combined with standard- or reduced-dose CNIs [
      • Howard J. Eisen
      • Murat Tuzcu E.
      • Dorent Richard
      • Kobashigawa Jon
      • Mancini Donna
      • Valantine-von Kaeppler Hannah A.
      • Starling Randall C.
      • Sørensen Keld
      • Hummel Manfred
      • Lind Joan M.
      • Abeywickrama Kamal H.
      • Bernhardt Peter
      for the RAD B253 Study Group
      Everolimus for the prevention of allograft rejection and vasculopathy in CardiacTransplant recipients.
      ,
      • Zuckermann A.
      • et al.
      Efficacy and Safety of Low-Dose Cyclosporine with Everolimus and Steroids in de novo Heart Transplant Patients: a Multicentre, Randomized Trial.
      ,
      • Lehmkuhl H.B.
      • et al.
      Everolimus with reduced cyclosporine versus MMF with standard cyclosporine in de novo heart transplant recipients.
      ]. Two randomized trials comparing EVL with reduced-dose CSA to MMF with standard-dose CSA in de novo HT populations showed that EVL with reduced-dose CSA was as effective as standard-dose CSA [
      • Eisen H.J.
      • et al.
      Everolimus versus mycophenolate mofetil in heart transplantation: a randomized, multicenter trial.
      ,
      • Lehmkuhl H.B.
      • et al.
      Everolimus with reduced cyclosporine versus MMF with standard cyclosporine in de novo heart transplant recipients.
      ]. Using an EVL target range of 3–8 ng/mL, the primary composite efficacy endpoint and incidence of biopsy-proven acute rejection were comparable in the EVL treatment group at 12 months post-transplantation in each of the study.
      There was no difference in serum creatinine levels among the three groups. Recent evidence relating the renal benefit of EVL with reduced CNI in HT recipients was less convincing. During the study period, the trough level of CNI was maintained at approximately half of that of the other groups. A recent study [
      • Asleh R.
      • et al.
      Long-term sirolimus for primary immunosuppression in heart transplant recipients.
      ] showed that an mTOR inhibitor, SRL, did not cause nephrotoxicity compared with CNIs. These results suggest that the level of CNI used in combination with EVL mainly influences renal function rather than EVL influencing renal function by itself. Furthermore, evidence shows that nephrotoxicity can be prevented by reducing the CNI trough level. CMV infection increases the risk of acute rejection [
      • Stern M.
      • et al.
      Cytomegalovirus serology and replication remain associated with solid organ graft rejection and graft loss in the era of prophylactic treatment.
      ], accelerated development of CAVs [
      • Klimczak-Tomaniak D.
      • et al.
      The association between cytomegalovirus infection and cardiac allograft vasculopathy in the era of antiviral valganciclovir prophylaxis.
      ] and risk of secondary infections [
      • Snydman D.R.
      • et al.
      Update and review: state-of-the-art management of cytomegalovirus infection and disease following thoracic organ transplantation.
      ]. mTOR inhibitors appear to block the phosphatidylinositol 3-kinase pathway, which is important for viral signaling and replication [
      • Ozaki K.S.
      • et al.
      Decreased Cytomegalovirus infection after antilymphocyte therapy in sirolimus-treated renal transplant patients.
      ,
      • Marty F.M.
      • et al.
      ]. Three randomized studies on de novo EVL therapy with standard CNI [
      • Howard J. Eisen
      • Murat Tuzcu E.
      • Dorent Richard
      • Kobashigawa Jon
      • Mancini Donna
      • Valantine-von Kaeppler Hannah A.
      • Starling Randall C.
      • Sørensen Keld
      • Hummel Manfred
      • Lind Joan M.
      • Abeywickrama Kamal H.
      • Bernhardt Peter
      for the RAD B253 Study Group
      Everolimus for the prevention of allograft rejection and vasculopathy in CardiacTransplant recipients.
      ], reduced-dose CNI [
      • Eisen H.J.
      • et al.
      Everolimus versus mycophenolate mofetil in heart transplantation: a randomized, multicenter trial.
      ,
      • Lehmkuhl H.B.
      • et al.
      Everolimus with reduced cyclosporine versus MMF with standard cyclosporine in de novo heart transplant recipients.
      ], or reduced CNI with early CNI withdrawal [
      • Andreassen A.K.
      • et al.
      Everolimus initiation and early calcineurin inhibitor withdrawal in heart transplant recipients: a randomized trial.
      ] provided a robust data on the effect of EVL on CMV infection and CMV-related events after HT. In the present study, the incidence of CMV infection was lower in the EVL-based protocol treatment group than in either CSA-based or TAC-based protocol groups.

      4.1 Limitations

      The relatively small sample size and retrospective observational design without randomization were the main limitations of this study. However, to overcome this limitation, we included patients in the control groups consecutively. Only stable patients who did not have wound problems or serious infections were converted to EVL, which might have resulted in biased results. In addition, patients who were unable to convert to EVL due to its side effects were excluded. Patients who did not undergo serial 3D IVUS were excluded from IVUS analysis. Patients with rapidly progressing CAVs, who could not undergo serial IVUS examinations, were also excluded. Despite these limitations, this study provides evidence of the underlying mechanism of CAV progression by evaluating both the vascular response and plaque change in de novo EVL immunosuppression.

      4.2 Conclusion

      De novo immunosuppression with EVL is associated with attenuated CAV progression during the first 5 years of follow-up. Moreover, de novo immunosuppression with EVL resulted in long-term clinical outcomes comparable to those of CSA- or TAC-based protocols. This 3D-IVUS study demonstrated an association between the EVL and the suppression of vascular negative remodeling as well as a reduction in plaque progression. Early introduction of EVL can provide adequate and safe immunosuppression.

      Funding

      This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

      CRediT authorship contribution statement

      Hyo-In Choi: Formal analysis, Investigation, Methodology, Writing – original draft. Do-Yoon Kang: Conceptualization, Methodology, Validation, Writing – review & editing. Min-Seok Kim: Conceptualization, Methodology, Validation, Writing – review & editing. Sang Eun Lee: Conceptualization, Validation, Writing – review & editing. Jung-Min Ahn: Conceptualization, Methodology, Validation, Writing – review & editing. Jong-Young Lee: Conceptualization, Validation, Writing – review & editing. Yong-Hak Kim: Conceptualization, Methodology, Validation, Writing – review & editing. Duk-Woo Park: Conceptualization, Methodology, Validation, Writing – review & editing. Sung-Ho Jung: Conceptualization, Methodology, Validation, Writing – review & editing. Jae-Joong Kim: Conceptualization, Supervision, Writing – review & editing.

      Declaration of interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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