Restrictive Allograft Syndrome Patients Research Articles

Characterization of chronic lung allograft dysfunction phenotypes using spectral and intrabreath oscillometry.

Background: Chronic lung allograft dysfunction (CLAD) is the major cause of death beyond 2 years after lung transplantation and develops in 50% of all patients by 5 years post-transplant. CLAD is diagnosed on the basis of a sustained drop of 20% for at least 3 months in the forced expiratory volume (FEV1), compared to the best baseline value achieved post-transplant. CLAD presents as two main phenotypes: bronchiolitis obliterans syndrome (BOS) is more common and has better prognosis than restrictive allograft syndrome (RAS). Respiratory oscillometry is a different modality of lung function testing that is highly sensitive to lung mechanics. The current study investigated whether spectral and intrabreath oscillometry can differentiate between CLAD-free, BOS- and RAS-CLAD at CLAD onset, i.e., at the time of the initial 20% drop in the FEV1. Methods: A retrospective, cross-sectional analysis of 263 double lung transplant recipients who underwent paired testing with oscillometry and spirometry at the Toronto General Pulmonary Function Laboratory from 2017 to 2022 was conducted. All pulmonary function testing and CLAD diagnostics were performed following international guidelines. Statistical analysis was conducted using multiple comparisons. Findings: The RAS (n = 6) spectral oscillometry pattern differs from CLAD-free (n = 225) by right-ward shift of reactance curve similar to idiopathic pulmonary fibrosis whereas BOS (n = 32) has a pattern similar to obstructive lung disease. Significant differences were found in most spectral and intrabreath parameters between BOS, RAS, and time-matched CLAD-free patients. Post-hoc analysis revealed these differences were primarily driven by BOS instead of RAS. While no differences were found between CLAD-free and RAS patients with regards to spectral oscillometry, the intrabreath metric of reactance at end-inspiration (XeI) was significantly different (p < 0.05). BOS and RAS were differentiated by spectral oscillometry measure R5, and intrabreath resistance at end expiration, ReE (p < 0.05 for both). Conclusion: Both spectral and intrabreath oscillometry can differentiate BOS-CLAD from CLAD-free states while intrabreath oscillometry, specifically XeI, can uniquely distinguish RAS-CLAD from CLAD-free. Spectral and intrabreath oscillometry offer complementary information regarding lung mechanics in CLAD patients to help distinguish the two phenotypes and could prove useful in prognostication.

Restrictive Allograft Syndrome Patients Have Higher Cell-Free DNA Assessed Allograft Injury Prior to Diagnosis

Purpose Restrictive Allograft Syndrome (RAS) and Bronchiolitis Obliterans Syndrome (BOS) are two forms of chronic lung allograft dysfunction (CLAD), an irreversible loss of allograft function and a major cause of death in lung transplant recipients (LTR). Clinical features and trends in allograft injury associated with RAS and BOS remain incompletely understood. This study examines the clinical characteristics and trends of allograft injury, as assessed by donor-derived cell free DNA (ddcfDNA), in RAS and BOS. Methods 369 LTR enrolled in two cohorts were examined for the development of BOS or RAS, as judged by adjudication committees using standardized definitions for CLAD. Demographic and clinical variables were collected prospectively for each patient. Serial plasma samples were collected for ddcfDNA quantification via shotgun sequencing. Results During the median 38 months follow-up (IQR 24.3-49.7), 116 patients (31.4%) developed CLAD, including 83 (71.5%) with BOS and 33 (28.4%) with RAS. There were no significant differences in demographic and baseline clinical factors between the two groups, including sex, race, BMI, pre-transplant diagnosis, or lung allocation score. RAS patients had a higher percentage of positive donor-specific antibodies (80.6%) compared to BOS (60.5%) (p=0.03). RAS patients had higher levels of ddcfDNA than BOS before CLAD diagnosis (p=0.02), with a rise in ddcfDNA levels preceding RAS diagnosis by 3 months (Fig 1a). RAS patients had significantly lower baseline forced expiratory volume in 1 second (FEV1) (p=0.007) and forced vital capacity (FVC) (p=0.001) six months prior to CLAD diagnosis (Fig 1b). Conclusion RAS patients showed more prevalent DSA and significantly lower baseline lung function compared to BOS. RAS patients also showed higher allograft injury measured via ddcfDNA than BOS patients, with a ddcfDNA rise preceding RAS diagnosis by 3 months. This study demonstrates differences in clinical variables and molecular injury between RAS and BOS. Restrictive Allograft Syndrome (RAS) and Bronchiolitis Obliterans Syndrome (BOS) are two forms of chronic lung allograft dysfunction (CLAD), an irreversible loss of allograft function and a major cause of death in lung transplant recipients (LTR). Clinical features and trends in allograft injury associated with RAS and BOS remain incompletely understood. This study examines the clinical characteristics and trends of allograft injury, as assessed by donor-derived cell free DNA (ddcfDNA), in RAS and BOS. 369 LTR enrolled in two cohorts were examined for the development of BOS or RAS, as judged by adjudication committees using standardized definitions for CLAD. Demographic and clinical variables were collected prospectively for each patient. Serial plasma samples were collected for ddcfDNA quantification via shotgun sequencing. During the median 38 months follow-up (IQR 24.3-49.7), 116 patients (31.4%) developed CLAD, including 83 (71.5%) with BOS and 33 (28.4%) with RAS. There were no significant differences in demographic and baseline clinical factors between the two groups, including sex, race, BMI, pre-transplant diagnosis, or lung allocation score. RAS patients had a higher percentage of positive donor-specific antibodies (80.6%) compared to BOS (60.5%) (p=0.03). RAS patients had higher levels of ddcfDNA than BOS before CLAD diagnosis (p=0.02), with a rise in ddcfDNA levels preceding RAS diagnosis by 3 months (Fig 1a). RAS patients had significantly lower baseline forced expiratory volume in 1 second (FEV1) (p=0.007) and forced vital capacity (FVC) (p=0.001) six months prior to CLAD diagnosis (Fig 1b). RAS patients showed more prevalent DSA and significantly lower baseline lung function compared to BOS. RAS patients also showed higher allograft injury measured via ddcfDNA than BOS patients, with a ddcfDNA rise preceding RAS diagnosis by 3 months. This study demonstrates differences in clinical variables and molecular injury between RAS and BOS.

Long-Term Pirfenidone for Restrictive Allograft Syndrome: A Case Series

<h3>Purpose</h3> Restrictive allograft syndrome (RAS) after lung transplantation (LTx) carries a bad prognosis, with generally a poor survival limited to 1-2 years after diagnosis. RAS is typically characterized by parenchymal fibrosis and distortion of lung architecture, a restrictive pulmonary function decline and persistent pleuroparenchymal abnormalities on computed tomography. There is no established treatment for RAS, although case series have reported on off-label treatment with antifibrotic drugs, such as pirfenidone (PFD), in RAS. However, reported treatment duration with PFD up to now was <2 years. We aim to report on RAS patients treated with PFD for at least 2 years in our center, in whom we evaluated evolution of pulmonary function during treatment with PFD. <h3>Methods</h3> Patients treated with off-label PFD for RAS were retrospectively assessed; and patients treated with PFD for at least 2 years were included for analysis. FVC, FEV1, TLC and TLCO at start of PFD and during the last visit on PFD were evaluated. GraphPad Prism 9.0 was used to compare parameters at different time points (start vs. last). <h3>Results</h3> In our cohort of 28 RAS patients treated with PFD since 2013 (median duration 177.5 (64.5-359.8) days), we identified 4 RAS patients who were treated with PFD for a mean (±SD) duration of 5.2 (±1.1) years (range 3.8-6.3 years). In these patients, total follow-up was 10.4±3.1 years (3/4 patients currently alive), RAS was diagnosed 4.6 (±1.7) years post-LTx (2/4 patients had prior BOS) and PFD was started 6.0 (±2.5) years post-LTx. FVC at start of PFD was 2.40 (±1.24) L vs. 1.85 (±0.91) L (p=0.091) at last measurement, with a monthly decline of 9.5 (±7.7) mL/month during PFD treatment. FEV1 at start was 1.69(±0.86) L vs. 1.28 (±0.55) L (p=0.147) at last measurement, with a monthly decline of 8.0 (±10.0) mL/month during PFD treatment. TLC at start was 4.00 (±1.58) L vs. 2.82 (±0.60) L (p=0.080) at last measurement, with a monthly decline of 11.3 (±8.6) mL/month during PFD treatment. Also, TLCO did not significantly differ over time (p=0.258). <h3>Conclusion</h3> A subset of RAS patients may demonstrate significantly better long-term survival, in these patients progressive fibrosing interstitial lung disease may possibly be attenuated by antifibrotic drugs, such as PFD, which requires further investigation.

Imaging Mass Cytometry for Detailed Cellular and Spatial Characterization of Chronic Lung Allograft Dysfunction (CLAD)

<h3>Purpose</h3> CLAD, the main limitation to long-term survival after a lung transplantation (LTx), has two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). There is great need for better characterization of cells within the lung allograft tissue. We aimed to assess CLAD lung allografts using imaging mass cytometry (IMC), a high dimensional tissue imaging system allowing a comprehensive and multiparametric in situ exploration of tissue microenvironments at a single cell level, using heavy metal-tagged antibodies. <h3>Methods</h3> Explanted lung tissue was obtained at the time of retransplantation from 4 BOS patients, 4 RAS patients, and 4 control donor lungs. Paraffin-embedded lung tissue was sectioned and stained with 35 heavy-metal-tagged antibodies selected to identify structural and immune proteins of interest. For each sample, three 1 mm² regions of interest, centered on an airway, were ablated and data was analyzed using MCD viewer and HistoCAT software. <h3>Results</h3> To identify specific cell subsets identified by combinations of markers, we used tSNE to create a 2D representation of cells organised in 50 immune and non-immune clusters according to the similarity of their antibody-binding in 35-dimensional space (Figure 1A). We present several key examples of our findings: CLAD lungs had significantly reduced club cells (Figure 1B-D, H) and a trend towards reduced basal cells (Figure 1B-D, I). A Ki67-high basal cell population was mostly present in RAS samples (Figure 1B-D, I-J) and in close proximity to memory T cells (neighborhood analysis not shown, Figure 1L). Memory CD8+ T cells were more frequent in CLAD lungs (Figure 1E-G, K) with prominent regulatory T cells in RAS (Figure 1E-G). <h3>Conclusion</h3> IMC allowed us to identify distinct basal cells and their proximity to cytotoxic memory T cells in CLAD lungs. IMC is a powerful technology for detailed cellular analysis within intact organ structures that may shed further light on CLAD mechanisms.

Prediction of Chronic Lung Allograft Dysfunction by Monitoring Serum KL-6 Levels

<h3>Purpose</h3> Chronic lung allograft dysfunction (CLAD) is the main cause of long-term mortality in lung transplantation (LT). The main phenotypes of CLAD are bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and mixed. Biomarkers for early identification of CLAD are needed. KL-6, an antigen produced mainly by damaged type II pneumocytes, has been described as a RAS-biomarker. The aim of our study was to assess the utility of serum KL-6 levels as a CLAD biomarker. <h3>Methods</h3> This was a longitudinal study in which KL-6 levels were measured in 52 bilateral LT patients at 3, 6, 9, 12, 18 and 24 months after LT. At the same time-points, clinical features and pulmonary function were determined. CLAD diagnosis was assessed during a three years follow-up. <h3>Results</h3> Seventeen out of 52 patients developed CLAD (11 BOS and 6 patients RAS or mixed). No differences in demographics were found, but patients with RAS or mixed phenotypes had a higher incidence of positive Donor-Specific Antibodies (DSA). Also, serum KL-6 levels before CLAD diagnosis were higher in the patients who developed RAS-mixed (563 [439.7-752.8 U/ml]) compared to patients diagnosed with BOS (356.7 [265.7-492.2 U/ml]) or those without CLAD (372.8 [274.8-606.9 U/ml]) at 6 months after LT, although not statistically significant (p=0.187). However, a KL-6 cut-off of 520.4 U/ml at 6-month post-transplant was able to differentiate RAS-mixed from non- RAS-mixed patients with a sensitivity of 80% (IC95 28.36-99.49%) and specificity of 72.73% (IC95 57.21-85.04%). When analyzing serum KL-6 levels in all samples, even those obtained after CLAD diagnosis, RAS patients had higher levels which progressively increase along follow-up (see table). <h3>Conclusion</h3> Serum KL-6 could be a useful biomarker for RAS prediction.

Quantitative CT Correlates with Local Inflammation in Lung of Patients with Subtypes of Chronic Lung Allograft Dysfunction.

Chronic rejection of lung allografts has two major subtypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), which present radiologically either as air trapping with small airways disease or with persistent pleuroparenchymal opacities. Parametric response mapping (PRM), a computed tomography (CT) methodology, has been demonstrated as an objective readout of BOS and RAS and bears prognostic importance, but has yet to be correlated to biological measures. Using a topological technique, we evaluate the distribution and arrangement of PRM-derived classifications of pulmonary abnormalities from lung transplant recipients undergoing redo-transplantation for end-stage BOS (N = 6) or RAS (N = 6). Topological metrics were determined from each PRM classification and compared to structural and biological markers determined from microCT and histopathology of lung core samples. Whole-lung measurements of PRM-defined functional small airways disease (fSAD), which serves as a readout of BOS, were significantly elevated in BOS versus RAS patients (p = 0.01). At the core-level, PRM-defined parenchymal disease, a potential readout of RAS, was found to correlate to neutrophil and collagen I levels (p < 0.05). We demonstrate the relationship of structural and biological markers to the CT-based distribution and arrangement of PRM-derived readouts of BOS and RAS.

MiR-21 in Lung Transplant Recipients With Chronic Lung Allograft Dysfunction.

Background: Micro-RNA-21 (miR-21) is a post-translational regulator involved in epithelial-to-mesenchymal transition (EMT). Since EMT is thought to contribute to chronic lung allograft dysfunction (CLAD), we aimed to characterize miR-21 expression and distinct EMT markers in CLAD. Methods: Expression of miR-21, vimentin, Notch intracellular domain (NICD) and SMAD 2/3 was investigated in explanted CLAD lungs of patients who underwent retransplantation. Circulating miR-21 was determined in collected serum samples of CLAD and matched stable recipients. Results: The frequency of miR-21 expression was higher in restrictive allograft syndrome (RAS) than in bronchiolitis obliterans syndrome (BOS) specimens (86 vs 30%, p = 0.01); Vimentin, NICD and p-SMAD 2/3 were positive in 17 (100%), 12 (71%), and 7 (42%) BOS patients and in 7 (100%), 4 (57%) and 4 (57%) RAS cases, respectively. All four markers were negative in control tissue from donor lungs. RAS patients showed a significant increase in serum concentration of miR-21 over time as compared to stable recipients (p = 0.040). Conclusion: To the best of our knowledge this is the first study highlighting the role miR-21 in CLAD. Further studies are necessary to investigate the involvement of miR-21 in the pathogenesis of CLAD and its potential as a therapeutic target.

Connective Tissue Growth Factor Is Overexpressed in Explant Lung Tissue and Broncho-Alveolar Lavage in Transplant-Related Pulmonary Fibrosis.

BackgroundConnective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD).Materials and MethodsCTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients.ResultsqPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74).ConclusionsLung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD.

Potential novel biomarkers for chronic lung allograft dysfunction and azithromycin responsive allograft dysfunction

Chronic Lung Allograft Dysfunction (CLAD), manifesting as Bronchiolitis Obliterans Syndrome (BOS) or Restrictive Allograft Syndrome (RAS), is the main reason for adverse long-term outcome after Lung Transplantation (LTX). Until now, no specific biomarkers exist to differentiate between CLAD phenotypes. Therefore, we sought to find suitable cytokines to distinguish between BOS, RAS and Azithromycin Responsive Allograft Dysfunction (ARAD); and reveal potential similarities or differences to end-stage fibrotic diseases. We observed significantly increased Lipocalin-2 serum concentrations in RAS compared to BOS patients. In addition, in RAS patients immunohistochemistry revealed Lipocalin-2 expression in bronchial epithelium and alveolar walls. Patients with ARAD showed significantly lower Activin-A serum concentrations compared to Stable-LTX and BOS patients. Further, increased serum concentrations of Lipocalin-2 and Activin-A were predictors of worse freedom-from-CLAD in Stable-LTX patients. These biomarkers serve as promising serum biomarkers for CLAD prediction and seem suitable for implementation in clinical practice.

Connective Tissue Growth Factor in Chronic Lung Allograft Dysfunction: An Explorative Study

Purpose Connective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD) by analyzing CTGF expression in broncho-alveolar lavage (BAL) fluid, plasma and lung tissue. Methods Sixty matched lung transplant patients were included (stable, n=20; bronchiolitis obliterans syndrome (BOS), n=20, restrictive allograft syndrome (RAS), n=20). CTGF expression was analyzed in BAL 3m post-transplant, 1y post-transplant, and at CLAD diagnosis or 2y post-transplant for stable patients. Explant lung tissue of CLAD (BOS, n=20; RAS, n=20), pulmonary GHVD (n=9), and discarded donor lungs (n=20) was immunohistochemically stained for CTGF. Results BAL CTGF protein expression was significantly higher at 3m post-transplant in patients who later developed RAS compared to stable or BOS patients (ANOVA p=0.028); while CTGF expression was similar at 1y post-transplant (ANOVA p=0.20). At CLAD diagnosis, CTGF expression was significantly increased in RAS compared to stable (p=0.0007) and BOS (p=0.042). Serial analysis revealed no difference in CTGF values between time points for stable and BOS patients (ANOVA p=0.84, p=0.92), whereas CTGF levels were significantly higher in RAS at CLAD diagnosis compared to 1y post-transplant (ANOVA p=0.029). CTGF plasma values were similar between BOS, RAS, and stable (ANOVA p=0.74). Immunohistochemistry revealed a higher percentage and intensity of CTGF positive respiratory epithelial cells in BOS and RAS lungs compared to controls (ANOVA p Conclusion Higher CTGF expression is present in BAL from RAS patients at CLAD diagnosis. Lung tissue CTGF expression is increased in end-stage RAS, BOS and pulmonary GVHD. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD, which warrants further investigation of CTGF and its potential therapeutic modulation in these conditions.

Clinical Characteristics of Patients with Chronic Lung Allograft Dysfunction Listed for Lung Re-Transplant

<h3>Purpose</h3> Chronic lung allograft dysfunction(CLAD) is the barrier to improve long term outcome of lung transplantation. This study aims to examine prevalence and characteristics of patients with CLAD who were listed for lung re-transplant. <h3>Methods</h3> This is a retrospective observational study. Adult patients (>18 YO) listed for lung re-transplant due to bronchiolitis obliterans syndrome(BOS) or restrictive allograft syndrome(RAS) were identified from SRTR database. Numerical data were reported in mean and standard deviation. Categorical data were reported in count and percent. T-test and Chi-square test were performed to compare 2 groups. <h3>Results</h3> 1546 adult CLAD patients were included. 85.51% of these patients have BOS phenotype. Mean age was 42.79. There was no significant difference in age, mPAP, history of smoking, previous malignancy, or pan-resistant infection between BOS and RAS groups. Oxygen requirement at listing time was significantly higher in RAS group (5.64±6.33 vs 2.93±3.31, p < 0.01). Proportion of patients who were on ECMO at listing time is significantly higher in RAS group (4.02% vs 0.91%, p<0.01). LAS of RAS group was significantly higher(52.07±18.71 vs 43.60±17.71, p < 0.01). 67.86% of RAS group received re-transplant compared to 59.15% of BOS group (p = 0.014). <h3>Conclusion</h3> RAS patients have worse clinical condition at listing time compared to BOS patients. They required higher FO2 supplement and have higher LAS. More proportion of patients with RAS received re-transplant compared to patients with BOS which reflects the above findings.

Lung Ultrasound to Phenotype Chronic Lung Allograft Dysfunction in Lung Transplant Recipients. A Prospective Observational Study.

Background: Bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) are two distinct phenotypes of chronic lung allograft dysfunction (CLAD) in lung transplant (LTx) recipients. Contrary to BOS, RAS can radiologically present with a pleuroparenchymal fibroelastosis (PPFE) pattern. This study investigates lung ultrasound (LUS) to identify potential surrogate markers of PPFE in order to distinguish CLAD phenotype RAS from BOS. Methods: A prospective cohort study performed at a National Lung Transplantation Center during June 2016 to December 2017. Patients were examined with LUS and high-resolution computed tomography of the thorax (HRCT). Results: Twenty-five CLAD patients (72% males, median age of 54 years) were included, corresponding to 19/6 BOS/RAS patients. LUS-identified pleural thickening was more pronounced in RAS vs. BOS patients (5.6 vs. 2.9 mm) compatible with PPFE on HRCT. LUS-identified pleural thickening as an indicator of PPFE in RAS patients’ upper lobes showed a sensitivity of 100% (95% CI; 54–100%), specificity of 100% (95% CI; 82–100%), PPV of 100% (95% CI; 54–100%), and NPV of 100% (95% CI; 82–100%). Conclusion: Apical pleural thickening detected by LUS and compatible with PPFE on HRCT separates RAS from BOS in patients with CLAD. We propose LUS as a supplementary tool for initial CLAD phenotyping.

Twelve-month effects of everolimus on renal and lung function in lung transplantation: differences in chronic lung allograft dysfunction phenotypes.

Background:Chronic lung allograft dysfunction (CLAD), a complication affecting the survival of lung transplanted patients, includes two clinical phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Everolimus is used in CLAD because of its antiproliferative mechanism. In lung transplant patients treated with everolimus, the clinical course of renal and lung function has not yet been assessed systematically in CLAD, BOS and RAS patients for more than 6 months.Methods:We retrospectively evaluated the 12-month follow-up of renal and lung function of lung-transplanted patients switched to everolimus and evaluated the reduction in immunosuppressant dosage (ISD) and mortality. Subgroups were based on indication for everolimus treatment: CLAD and non-CLAD patients, BOS and RAS among CLAD patients.Results:We included 26 patients, 17 with CLAD (10 BOS, seven RAS). After 1 year from the everolimus switch, we observed renal function improvement (serum creatinine −17%, estimated glomerular filtration rate +24%) and stable pulmonary function [forced expiratory volume in the first second (FEV1) −0.5%, forced vital capacity (FVC) +0.05%]. RAS patients had progressive functional loss, whereas BOS patients had FEV1 improvement and FVC stability. All-cause mortality was higher in the CLAD versus non-CLAD group (41% versus 11%), without differences between BOS and RAS patients (p > 0.05). All patients had significant and persistent ISD reduction.Conclusion:Lung transplant patients treated with everolimus had improvements in renal function and reduced ISD. We observed sustained improvements in lung function for CLAD related to BOS subgroup results, whereas RAS confirmed the 1-year worsening functional trend. Data seem to suggest one more piece of the puzzle in CLAD phenotyping.

Transition from BOS to RAS Impairs Prognosis after Lung Transplantation - CLAD Subtype Analysis by CT-Volumetry

Chronic lung allograft dysfunction (CLAD) is the most important reason for poor patient survival after lung transplantation. Of the two CLAD subtypes, restrictive allograft syndrome (RAS) has worse prognosis than bronchiolitis obliterans syndrome (BOS), but only limited information is available on the incidence and clinical effect of CLAD subtype change after CLAD onset. We set out to characterize CLAD subtype development by computed tomography (CT) volumetry after CLAD onset. 167 adult patients who underwent de novo lung transplantation in a nationwide transplant centre between 2003-2015 were retrospectively analyzed. CLAD subtype was determined by CT volumetry at CLAD onset. Lung volume decrease of <15% from baseline resulted in BOS and >15% in RAS classification. Patients were re-classified to BOS->RAS or RAS->BOS if persistent lung volume change was detected, and no other specific reasons for lung volume change were present. Radiologic findings of air trapping and fibrosis were linked to lung volume change. Seventy-one (43%) patients were diagnosed with CLAD which was classified as BOS in 63 (89%) and RAS in 8 (11%) patients. After CLAD onset, 6 (9.5%) of BOS patients were re-classified as BOS->RAS, and one (12.5%) RAS patient was re-classified as RAS->BOS whereas other patients remained in their original CLAD subtype. Class change from BOS to RAS occurred at a median of 515.5 days after CLAD onset. Recipient (Figure A, p= 0.002) and graft survival (Figure B, p= 0.003) after CLAD diagnosis was impaired in BOS->RAS group compared to patients remaining in the BOS category. Radiological evidence of both air trapping and fibrosis was seen in 83% of BOS->RAS patients, and fibrotic changes preceded the subtype change in 67% of patients. A significant proportion of BOS patients transform to RAS after CLAD diagnosis, which has an adverse effect on recipient and graft survival.

Use of serum KL-6 level for detecting patients with restrictive allograft syndrome after lung transplantation.

KL-6 is an antigen produced mainly by damaged type II pneumocytes that is involved in interstitial lung disease. Chronic lung allograft dysfunction (CLAD) after lung transplantation (LT) is a major concern for LT clinicians, especially in patients with restrictive allograft syndrome (RAS). We investigated KL-6 levels in serum and bronchoalveolar lavage fluid (BALF) as a potential biomarker of the RAS phenotype. Levels of KL-6 in serum and BALF were measured in 73 bilateral LT recipients, and patients were categorized into 4 groups: stable (ST), infection (LTI), bronchiolitis obliterans syndrome (BOS), and RAS. We also studied a healthy cohort to determine reference values for serum KL-6. The highest levels of KL-6 were found in the serum of patients with RAS (918 [487.8–1638] U/mL). No differences were found for levels of KL-6 in BALF. Using a cut-off value of 465 U/mL serum KL-6 levels was able to differentiate RAS patients from BOS patients with a sensitivity of 100% and a specificity of 75%. Furthermore, higher serum KL-6 levels were associated with a decline in Forced Vital Capacity (FVC) at 6 months after sample collection. Therefore, KL-6 in serum may well be a potential biomarker for differentiating between the BOS and RAS phenotypes of CLAD in LT recipients.

Impact of mir21 Expression in Chronic Lung Allograft Dysfunction after Lung Transplantation

Purpose Long-term survival after lung transplantation is still limited by the development of chronic lung allograft dysfunction (CLAD), presenting either as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Epithelial-to-mesenchymal transition (EMT) is recognized as one of the pathogenic processes contributing to CLAD. MiR-21 is a post-translational regulator of several pathways of fibrosis and has recently been related to EMT. In this study we aimed to characterize miR-21 expression and other markers of EMT in lung tissue of BOS and RAS patients. Methods Formalin-fixed paraffin-embedded tissue of 17 BOS patients and 7 RAS patients undergoing a lung retransplantation due to CLAD and 5 normal lung tissue samples (control) were evaluated for the expression following markers: vimentin, notch intracellular domain (NICD), p-SMAD 2/3, β-catenin, and E-cadherin. Slides were additionally stained for miR-21 by in situ hybridization (ISH). Expression of respective markers were graded in a semi-quantified manner by two blinded pathologists. Results Vimentin, NICD, p-SMAD 2/3 were positive in 13 (77%), 12 (71%), and 7 (42%) BOS patients, and in 5 (71%), 4 (57%) and 4 (57%) RAS patients, respectively. In contrast, β-catenin and E-cadherin staining were consistently negative in both groups. In comparision, no significant difference was found between BOS and RAS regarding these markers. MiR-21 expression was positive in 5 (30%) BOS cases and 6 (86%) RAS cases. The rate of miR-21 expression was significantly higher in RAS than in BOS (p=0.01). MiR-21 expression was associated with significant shorter freedom to BOS (11.5 vs 46.0 months, p=0.001, Figure 1), but had no impact on the development of RAS. Conclusion This first preliminary study could identify miR-21 as a possible biomarker and therapeutic target of CLAD and mirR-21 expression was related to worse outcomes in BOS cases. Further research is necessary to validate these findings in a larger cohort of patients.

Diagnosis of Restrictive Allograft Syndrome (RAS) in Single-Lung Transplantation Using PFTs and CT-Scan Volumetry and Score

Purpose Restrictive allograft syndrome (RAS) has been identified as a chronic lung allograft dysfunction (CLAD) with the worst prognosis after lung transplantation (LTx). Diagnostic criteria of RAS have been proposed in double-LTx (DLTx), but how to apply these criteria in single (S)-LTx remain debatable. Hence, there is a need to validate and refine these criteria in SLTx. Our aim was to investigate functional, allosensitization, and CT-scan abnormalities patterns in SLTx recipients with RAS, as compared to single LTx with BOS or in stable condition. Methods We retrospectively reviewed data from all SLTx recipients during the 2009-2015 period at Bichat hospital. Among them, RAS phenotype in SLTx was identified using criteria defined in BLTx, including both PFTs (FVC ≤ 80% of baseline or FEV1 ≤ 80% of baseline with FEV1/FVC > 0.7) and CT-scan with persistent opacities suggestive of RAS. These SLTx with RAS were compared with SLTx with BOS and stable patients. We compared outcome of PFTs (FVC, FEV1, FEV1/FVC), repeated CT-scan (both CT-volumetry and CT parenchymal score), and DSA between the 3 groups at the following 4 time points with available CT-scan: (1) at the date of best post-Tx functional status, (2) at the date of CLAD onset, defined at first irreversible decline of PFTs or graft CT-volume, or first onset of pleuroparenchymal abnormalities reflected by RAS parenchymal score; (3) at last available CT-scan; (4) at an average time between 2 and 3. Results 17 SLTx were identified with RAS diagnosis, and compared to 17 SLTx with BOS and 17 stable SLTx. In RAS patients, outcome of FVC, FEV1, CT-volume, and CT-score showed all a significant decrease from time-point of CT baseline to last CT-scan (p Conclusion RAS diagnosis remains challenging in SLTx. We observed that the increase of a CT score ≥3 may lead to earlier identification of RAS patients in this population, as compared to standard criteria of RAS initially defined in DLTx.

Identification and Characterization of Patients With a Mixed Phenotype of CLAD

Purpose Bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), have been identified as main phenotypes of chronic lung allograft dysfunction (CLAD), however patients can change phenotype. We aimed to describe incidence, prognosis, radiology, pulmonary function and pathology in this mixed group of CLAD. Methods We retrospectively assessed the chest CT-scans of all double lung patients transplanted between 2001 and 2015 with a FEV1 decline >20% of baseline. The diagnosis of RAS was made using a decline in TLC>10% compared to baseline or a decline in FVC>20% compared to baseline. A mixed group of CLAD was defined as patients developing a TLC or FVC decline with persistent CT opacities after initial CLAD diagnosis (without TLC or FVC drop) or vice versa. Results 44% of the patients developed CLAD (268/608) of which 77 developed persistent CT opacities (29%). 45 patients were immediately diagnosed with RAS. We found twenty-seven patients with a mixed phenotype of CLAD (25 BOS to RAS, 2 RAS to BOS). In 5 patients persistent pleuroparenchymal opacities were found without an additional decrease in FVC or TLC. The median time between CLAD and mixed diagnosis was 2Y (IQR 0.9-4.8Y). Median survival after CLAD diagnosis was longer in the mixed group compared to the RAS group (4.3Y vs 1.17, p=0.0052), however survival after the appearance of opacities was similar in both groups (p=0.39). We found significantly more emphysema patients in the mixed group (p=0.0062), with a lower FEV1 (p Conclusion We demonstrated that 35% of patients showed a mixed phenotype of CLAD, which showed difference in survival, demographics, pulmonary function, radiology and pathology compared to RAS patients.

The pleural mesothelium and transforming growth factor-β1 pathways in restrictive allograft syndrome: A pre-clinical investigation.

Chronic lung allograft dysfunction (CLAD) hampers long-term survival after lung transplantation. Common fibrosis-related mechanisms in idiopathic pulmonary fibrosis and CLAD instigated the consideration of investigating the differential regulation of pleural mesothelium and transforming growth factor-β1 (TGF-β1) in restrictive allograft syndrome (RAS). TGF-β1 was assessed in bronchoalveolar lavage (BAL) fluid using enzyme-linked immunoassay and via immune staining of explant biopsies. To assess the role of the pleura, explanted bronchiolitis obliterans syndrome (BOS) and RAS lungs were compared using computed tomography scans, calretinin stainings, Western blot, and quantititative real-time PCR. Last, a pleural mesothelial cell line was used to assess mesothelial-to-mesenchymal transition and its inhibition. TGF-β1 was increased in BAL of RAS patients (p = 0.035), and was present in the (sub)pleural area of biopsies. Explanted RAS lungs demonstrated an increased volume fraction of pleura (p = 0.0004), a higher proportion of calretinin-positive stainings (p = 0.0032), and decreased E-cadherin (p = 0.019) and increased α-smooth muscle actin (p = 0.0089) mRNA expression and protein levels in isolated pleural tissue. Moreover, TGF-β1 stimulation of pleural mesothelial cells led to a phenotypical switch to mesenchymal cells, accompanied with an increased migratory capacity. Interleukin-1α was able to accentuate TGF-β1‒induced mesothelial-to-mesenchymal transition. None of the tested drugs could inhibit mesothelial-to-mesenchymal transition at the used concentrations. Our results support an interplay between TGF-β1 and the pleural mesothelium in the pathophysiology of RAS.

BALF cytokines in different phenotypes of chronic lung allograft dysfunction in lung transplant patients.

The long-term success of lung transplantation (LT) is limited by chronic lung allograft dysfunction (CLAD). Different phenotypes of CLAD have been described, such as bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). The purpose of this study was to investigate the levels of cytokines and chemokines in bronchoalveolar lavage fluid (BALF) as markers of these CLAD phenotypes. BALF was collected from 51 recipients who underwent (bilateral and unilateral) LT. The study population was divided into three groups: stable (ST), BOS, and RAS. Levels of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured using the multiplex technology. BALF neutrophilia medians were higher in BOS (38%) and RAS (30%) than in ST (8%) (P=.008; P=.012). Regarding BALF cytokines, BOS and RAS patients showed higher levels of INF-γ than ST (P=.02; P=.008). Only IL-5 presented significant differences between BOS and RAS (P=.001). BALF neutrophilia is as a marker for both CLAD phenotypes, BOS and RAS, and IL-5 seems to be a potential biomarker for the RAS phenotype.