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Purpose Phenotyping chronic lung allograft dysfunction (CLAD) in single lung transplant (SLTX) recipients is challenging, due to the native lung contribution to pulmonary function tests (PFT). We aimed to assess the ISHLT CLAD classification and its prognostic performance in SLTX. Methods In this retrospective cohort of adult, first, SLTX from 2009 to 2017, patients with persistent drop in FEV1>20% were assessed by 2 independent reviewers to determine CLAD status and phenotype, specifically noting presence or absence of RAS-like opacities. Disagreements were resolved by a 3rd reviewer. Interobserver agreement (IOA) was calculated by Cohen's Kappa. Association of CLAD phenotypes with time to death/retransplant (ReTx), adjusted for age, sex, CMV mismatch and native lung condition, were assessed with Cox models. Results Out of 172 SLTX patients, 92 had a persistent drop in FEV1>20%, of whom 67 got a diagnosis of CLAD. We noted a moderate IOA for CLAD status (Kappa 0.69) and poor IOA for phenotype (Kappa 0.5). When applying the ISHLT criteria strictly (based on exact cut-offs for PFT, along with imaging), 34 patients had BOS (50.7%), 9 RAS/mixed (13.4%), 7 undefined (10.4%), and 17 unclassified (25.5%). We found no association of these strict phenotypes with death/ReTx (Fig A). When using adjudicated CLAD phenotypes, 31 patients had BOS (46.3%), 15 RAS/mixed (22.4%), 2 Undefined (3%), and 19 Unclassified (28.3%). Using these adjudicated phenotypes, RAS/mixed was significantly associated with higher risk of death/ReTx (HR 2.98, 95%CI [1.39-6.4]) (Fig B). Finally, the specific adjudication of RAS-like opacities had the best IOA (Kappa 0.73). Presence of RAS-like opacities was a strong predictor of death/ReTx (HR 2.3, 95%CI [1.2-4.5]) (Fig C). Conclusion PFT interpretation is challenging in SLTX. Using a classification relying more on imaging analysis that harboured good IOA, we obtained better prognostic performances than with a strict application of published physiologic cut-offs.

Definitions for chronic lung allograft dysfunction (CLAD) phenotypes were recently revised (2019 ISHLT consensus). Post-CLAD onset phenotype transition may occur as a result of change in obstruction, restriction, or RAS-like opacities (RLO). We aimed to assess the prevalence and prognostic implications of these transitions. This was a single-center, retrospective cohort study of bilateral lung transplants performed in 2009-2015. CLAD phenotypes were determined per ISHLT guidelines. CLAD phenotype transition was defined as a sustained change in obstruction, restriction or RLO. We specifically focused on phenotype changes based on RLO emergence. Association of RLO development with time to death or retransplant were assessed using Kaplan-Meier and Cox proportional hazards models. Among 211 patients with CLAD, 47 (22.2%) experienced a phenotype transition. Nineteen patients developed RLO. Development of RLO phenotype after CLAD onset was associated with a shorter time to death/retransplant when considering the entire CLAD patient cohort (HR = 4.00, CI 2.74-5.83, P

Ex vivo lung perfusion (EVLP) has being increasingly used for the pretransplant assessment of extended-criteria donor lungs. Mathematical models to predict lung acceptance during EVLP have not been reported so far. Thus, we hypothesized that predictors of lung acceptance could be identified and used to develop a mathematical model describing the clinical decision-making process used in our institution. Donor lungs characteristics and EVLP physiologic parameters included in our EVLP registry were examined (derivation cohort). Multivariable logistic regression analysis was performed to identify predictors independently associated with lung acceptance. A mathematical model (EX vivo lung PerfusIon pREdiction [EXPIRE] model) for each hour of EVLP was developed and validated using a new cohort (validation cohort). Two hundred eighty donor lungs were assessed with EVLP. Of these, 186 (66%) were accepted for transplantation. ΔPO2 and static compliance/total lung capacity were identified as independent predictors of lung acceptance and their respective cut-off values were determined. The EXPIRE model showed a low discriminative power at the first hour of EVLP assessment (AUC: 0.69 [95% CI: 0.62-0.77]), which progressively improved up to the fourth hour (AUC: 0.87 [95% CI: 0.83-0.92]). In a validation cohort, the EXPIRE model demonstrated good discriminative power, peaking at the fourth hour (AUC: 0.85 [95% CI: 0.76-0.94]). The EXPIRE model may help to standardize lung assessment in centers using the Toronto EVLP technique and improve overall transplant rates.

BACKGROUND Procuring a good quality transbronchial-biopsy sample is essential for diagnosing acute cellular rejection after lung transplantation (LT). Insufficient transbronchial-biopsy samples are graded "AX." We hypothesized that AX samples may be associated with a higher risk for chronic lung allograft dysfunction (CLAD) or death/retransplant, through a potential anatomic or physiologic underlying pulmonary process or because of undiagnosed acute cellular rejection episodes. METHODS We conducted a single-center, retrospective, cohort study drawn from all consecutive adult, first, bilateral LT between 1999 and 2015. We reviewed all biopsies obtained within the first year posttransplant and compared outcomes of patients with ≥1 AX to patients with no AX. Association of any AX or percent AX with time to CLAD or death/retransplant was assessed using Cox Proportional Hazards models. RESULTS The cohort consisted of 809 patients with a median of 6 (interquartile range 5-6) biopsies and 16.7% (interquartile range 0-25) AX samples within the first year posttransplant. Four hundred thirty-nine (54.3%) subjects had ≥1 AX sample obtained within the time period. Median time to CLAD or death/retransplant, from 1 year posttransplant, was 761 (320, 1587) and 1200 (662, 2308) days, respectively. In the multivariable analysis, there was no difference in risk for CLAD (hazard ratio = 1.05, 95% confidence interval, 0.87-1.28, P = 0.60), or death/retransplant (hazard ratio = 1.14, 95% confidence interval, 0.92-1.42, P = 0.24) between patients with ≥1 AX biopsy versus none. Among subjects with ≥1 AX, having >50% AX biopsies was not associated with outcome. CONCLUSIONS This is the first study to demonstrate that AX biopsies are not associated with an increased risk of CLAD or death/retransplant after LT and may not require to repeat the biopsy.

The Toronto Lung Transplant Program has been using a peri-operative desensitization regimen of plasma exchange, intravenous immune globulin, and antithymocyte globulin in order to accept donor-specific antibody (DSA)-positive lung transplants safely since 2008. There are no long-term data on the impact of this practice on allograft survival or the development of chronic lung allograft dysfunction (CLAD). We extended our prior study to include long-term follow-up of 340 patients who received lung transplants between January 1, 2008 and December 31, 2011. We compared allograft survival and CLAD-free survival among patients in three cohorts: DSA-positive, panel reactive antibody (PRA)-positive/DSA-negative, and unsensitized at the time of transplant. The median follow-up time in this extension study was 6.7 years. Among DSA-positive, PRA-positive/DSA-negative, and unsensitized patients, the median allograft survival was 8.4, 7.9, and 5.8 years, respectively (p = .5908), and the median CLAD-free survival was 6.8, 7.3, and 5.7 years, respectively (p = .5448). This follow-up study confirms that long-term allograft survival and CLAD-free survival of patients who undergo DSA-positive lung transplants with the use of our protocol do not differ from other lung transplant recipients. Use of protocols such as ours, therefore, may improve access to transplant for sensitized candidates.

Purpose Gastroesophageal reflux disease is common in lung transplant recipients and is thought to cause lung graft injury through aspiration of gastric contents. Bile acids in bronchoalveolar lavage (BAL) are putative markers of gastric microaspiration and associated with inflammation and earlier chronic lung allograft dysfunction (CLAD). Microaspiration is thought to augment CLAD risk through potentiation of acute rejection (AR), although this has not been demonstrated. We hypothesized that BAL bile acid levels would be associated with AR presence and severity. Methods Among all adult, first, bilateral lung transplants, performed at two large lung transplant centers 2010-2015, transbronchial biopsies obtained within the first-year post-transplant were categorized as spirometrically-significant or stable based on the presence or absence of ≥10% concurrent drop in FEV1. AR diagnosis was based histology and biopsies with concurrent infection were excluded. BAL supernatant levels of bile acids (taurocholic acid (TCA), glycocholic acid (GCA), cholic acid), and inflammatory proteins (IL6, CXCL8, S100A8, CCL2, IL1β, CCL5, IL1α, RAGE, S100A12, IL12) were compared between patient groups. Association between BA levels and subsequent CLAD or death/retransplant was assessed using Cox Proportional Hazards models, adjusted for relevant peri-transplant clinical covariates and AR group. Endpoints We identified 42 patients with spirometrically-significant AR, 56 patients with stable AR, and 81 patients with stable no-AR with available BAL samples (one sample per patient). BAL at time of spirometrically-significant AR, compared to other groups, had elevated levels of TCA (ANOVA

Purpose Previous studies suggest Chronic Lung Allograft Dysfunction (CLAD) subtypes with RAS-like opacities (RLO) (i.e. persistent parenchymal or pleural fibrosis) at CLAD onset to have worse survival. However, the significance of transitions between CLAD subtypes remains obscure. Methods This was a single center, retrospective cohort study of consecutive adult, first bilateral lung transplants from 2010-2015. Patients with CLAD were classified into ISHLT phenotypes BOS, RAS, Mixed, undefined and some remained unclassified. We defined phenotype transition as a change of at least one of three determinants: obstruction, restriction or RLO. Association of phenotype transition with time to death or retransplant starting at CLAD onset or transition date was assessed using Cox PH models. Multivariable models included age and CMV mismatch. Results Of 173 patients with CLAD, 108 patients with no RLO and 28 with RLO at CLAD onset did not change phenotype. 37 patients (21.3%) had a phenotype transition at a median time of 329 days (IQR 166-591) post CLAD onset. 15 patients without RLO (12 BOS, 2 undefined, 1 unclassified) developed RLO and changed phenotype to RAS (1), mixed (7), or undefined (7). 22 patients had a phenotype transition owing to a change in the ventilatory defect pattern (10 with RLO and 12 without RLO). Patients who developed RLO after CLAD onset had a non-significant trend towards worse survival from CLAD onset (HR=1.29, p=0.7) and also from transition date (HR=3.45, p=0.1) compared to patients who transitioned to a phenotype without RLO (Fig 1A), probably due to small number of patients. Survival after RLO emergence did not differ between patients with RLO at CLAD onset and patients who developed RLO post CLAD onset (HR=1.69, p=0.17 (Fig 1B). Conclusion Our study shows that phenotype transition is a relatively common phenomenon, although a transition to RAS-like opacities occurred in only 9% of CLAD patients. The poor survival from emergence of RAS-like opacities appears to be similar regardless of timing relative to CLAD onset.

BACKGROUND Gastroesophageal reflux disease (GERD) is a risk factor for chronic lung allograft dysfunction. Bile acids—putative markers of gastric microaspiration—and inflammatory proteins in the bronchoalveolar lavage (BAL) have been associated with chronic lung allograft dysfunction, but their relationship with GERD remains unclear. Although GERD is thought to drive chronic microaspiration, the selection of patients for anti-reflux surgery lacks precision. This multicenter study aimed to test the association of BAL bile acids with GERD, lung inflammation, allograft function, and anti-reflux surgery. METHODS We analyzed BAL obtained during the first post-transplant year from a retrospective cohort of patients with and without GERD, as well as BAL obtained before and after Nissen fundoplication anti-reflux surgery from a separate cohort. Levels of taurocholic acid (TCA), glycocholic acid, and cholic acid were measured using mass spectrometry. Protein markers of inflammation and injury were measured using multiplex assay and enzyme-linked immunosorbent assay. RESULTS At 3 months after transplantation, TCA, IL-1β, IL-12p70, and CCL5 were higher in the BAL of patients with GERD than in that of no-GERD controls. Elevated TCA and glycocholic acid were associated with concurrent acute lung allograft dysfunction and inflammatory proteins. The BAL obtained after anti-reflux surgery contained reduced TCA and inflammatory proteins compared with that obtained before anti-reflux surgery. CONCLUSIONS Targeted monitoring of TCA and selected inflammatory proteins may be useful in lung transplant recipients with suspected reflux and microaspiration to support diagnosis and guide therapy. Patients with elevated biomarker levels may benefit most from anti-reflux surgery to reduce microaspiration and allograft inflammation.

BACKGROUND Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the new International Society for Heart and Lung Transplantation (ISHLT) CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. METHODS We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed from 2010 to 2015. Patients with CLAD were classified on the basis of the 2019 ISHLT consensus document. CLAD phenotypes and other potential predictors of survival after CLAD onset were assessed using Kaplan–Meier and Cox proportional hazards models. RESULTS Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed, and 19 (10.9%) undefined phenotype. A total of 26 patients (14.9%) did not match any of these 4 categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The 45 patients (26.8%) with undefined/unclassified phenotype were combined and recategorized on the basis of the presence or absence of characteristic RAS-like opacities on chest imaging; those with RAS-like opacities had significantly worse allograft survival than patients with BOS (hazard ratio, 2.14; 95% confidence interval, 1.17–3.93; p = 0.014) and similar survival to RAS or mixed phenotype. CONCLUSIONS The new ISHLT CLAD phenotype classification is informative with regards to post-CLAD outcomes. Chest imaging demonstrating persistent parenchymal or pleural fibrosis may be used for risk-stratification of patients who do not match the major CLAD phenotypes.

Lung transplantation has become an invaluable approach for the treatment of end-stage respiratory diseases. However, survival after lung transplant remains limited due to chronic lung allograft dysfunction (CLAD), a fibrotic process affecting the airway and/or parenchymal compartments of the lung graft leading to a significant and persistent deterioration in lung function [1, 2]. With our accelerating understanding of CLAD, it has become evident that CLAD is a heterogeneous process comprised of multiple patterns of physiological and radiological features, which have strong implications for post-CLAD survival [3]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Levy has nothing to disclose. Conflict of interest: Dr. Huszti has nothing to disclose. Conflict of interest: Dr. Berra has nothing to disclose. Conflict of interest: Dr. Renaud-Picard has nothing to disclose. Conflict of interest: Dr. Kawashima has nothing to disclose. Conflict of interest: Dr. Takahagi has nothing to disclose. Conflict of interest: Dr. Moshkelgosha has nothing to disclose. Conflict of interest: Dr. Ghany has nothing to disclose. Conflict of interest: Dr. Fuchs has nothing to disclose. Conflict of interest: Dr. Chow has nothing to disclose. Conflict of interest: Dr. Keshavjee has nothing to disclose. Conflict of interest: Dr. Singer has nothing to disclose. Conflict of interest: Dr. Tikkanen has nothing to disclose. Conflict of interest: Dr. Martinu has nothing to disclose.

RationaleThe diffusing capacity of the lung for carbon monoxide corrected for haemoglobin (DLCOcor) measures gas movement across the alveolar–capillary interface. We hypothesised that DLCOcor is a sensitive measure of injurious allograft processes disrupting this interface.ObjectivesTo determine the prognostic significance of the DLCOcor trajectory on chronic lung allograft dysfunction (CLAD) and survival.MethodsA retrospective analysis was conducted of all bilateral lung transplant recipients at a single centre, between January 1998 and January 2018, with one or more DLCOcor measurements. Low baseline DLCOcor was defined as the failure to achieve a DLCOcor >75% predicted. Drops in DLCOcor were defined as >15% below recent baseline.Results1259 out of 1492 lung transplant recipients were included. The median (range) time to peak DLCOcor was 354 (181–737) days and the mean±sdDLCOcor was 80.2±21.2% pred. Multivariable analysis demonstrated that low baseline DLCOcor was significantly associated with death (hazrd ratio (HR) 1.68, 95% CI 1.27–2.20; pDLCOcor was not independently associated with CLAD after adjustment for low baseline forced expiratory volume in 1 s or forced vital capacity. Any DLCOcor declines ≥15% were significantly associated with death, independent of concurrent spirometric decline. Lower percentage predicted DLCOcor values at CLAD onset were associated with shorter post-CLAD survival (HR 0.75 per 10%-unit change, pConclusionLow baseline DLCOcor and post-transplant declines in DLCOcor were significantly associated with survival, independent of spirometric measurements. We propose that DLCOcor testing may allow identification of a subphenotype of baseline and chronic allograft dysfunction not captured by spirometry. There may be benefit in routine monitoring of DLCOcor after lung transplantation to identify patients at risk of poor outcomes.

Purpose Chronic Lung Allograft Dysfunction (CLAD) is a heterogeneous disease. Three major phenotypes: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and mixed phenotype, are defined based on combinations of lung physiology and chest imaging. Total lung capacity (TLC)≤90% of baseline is used to define RAS/Mixed; however, many centers do not routinely obtain TLC and use instead forced vital capacity (FVC)≤80%. The objective of the current study was to assess the diagnostic accuracy of FVC≤80% for determining RAS/Mixed in a large center that routinely monitors both TLC and FVC. Methods This was a retrospective cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with CLAD were included. Spirometry, lung volumes, and chest imaging were available. RAS/mixed were determined based on 2019 ISHLT definitions using TLC≤90% and the presence of RAS-like opacities. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic accuracy of FVC≤80% combined with RAS-like opacities for diagnosis of RAS/Mixed were computed. Results A total of 174 patients developed CLAD: 25(14.4%) met criteria for RAS/mixed by TLC. The sensitivity, specificity, PLR, NLR and diagnostic accuracy for FVC to determine RAS/Mixed was 84.0% (95%CI 63.9-95.5), 95.3% (95%CI 90.6-98.1), 17.9 (95%CI 8.5-37.6), 0.17 (95%CI 0.07-0.41) and 93.7% (95%CI 89.0-96.8). Conclusion Low FVC has an overall good diagnostic value for estimating RAS/mixed phenotype. Given the high specificity, it is possible to rule in RAS/mixed phenotype with spirometry. However, it cannot be ruled out confidently based on spirometry alone. In CLAD patients with a clinical suspicion of RAS/mixed phenotype and a preserved FVC, measurement of TLC is important for a more accurate diagnosis.

Purpose Chronic allograft dysfunction (CLAD) limits survival after lung transplantation (LT), affecting 50% by 5-years and 75% by 10 years. Long-term survival (≥10 years) is seen in 35-40% after LT. While 10-15% of patients survive ≥10 years without CLAD, the reasons remain unclear. Our aim was to determine clinical factors that differ between long-term survivors with and without CLAD Methods This was a single-center retrospective cohort study of all first LT performed between 1983 and 2010. Re-transplants and those without follow-up data were excluded. CLAD was adjudicated by ISHLT criteria in patients with ≥ 4 pulmonary function tests (PFTs). Associations of clinical variables with CLAD-free status were assessed using univariate logistic regression Results 1110 patients underwent primary LT in the study period. 1-year survival was 79.5 (882/1110). 32.2% (357/1110) survived ≥10 years, and of those with sufficient PFTs, 10.6% (118/348) were long-term CLAD-free survivors. 73.7% (87/357) of this cohort are still alive at a median follow-up of 12.49 (IQR 11.3-14.85) years. Among very long-term survivors (≥20 years; n=49) with sufficient PFTs, 83% were CLAD-free at a median of 23.3 (IQR 21.1-25.87) years. Shorter ischemic time, lower donor age, indication for transplant, higher baseline total lung capacity and baseline renal function, and absence of gastroparesis, major infections and treatment for acute rejection were all associated with ≥10-year CLAD-free survival. Compared to long-term survivors without CLAD, those with CLAD had higher mortality ( Conclusion We identified clinical variables associated with ≥10-year CLAD-free survival in our cohort. We will perform multiple regression and compare this cohort with those surviving 1 to 5 years post-LT using Cox proportional hazards model. We aim to optimize recipient-allograft matching leading to better long-term CLAD-free survival.

Purpose Bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS), and mixed are major phenotypes of CLAD. In previous, older studies, CMV viremia has been associated with reduced survival. We hypothesized that, in a more modern era of CMV prophylaxis, CMV viremia would remain associated with death, but also represent a risk factor for CLAD and specifically RAS/mixed phenotype. Methods This is a retrospective cohort study of all consecutive adult, first, bilateral-/single-lung transplants performed 2010-2016 who had ≥4 pulmonary function tests and ≥1 CMV-PCR measurement. CMV-PCR levels were divided into 3 categories: undetectable, Results Of 645 patients, 257 developed CLAD (143 BOS, 44 RAS/mixed, 66 undefined/unclassified) and 388 were CLAD-free at last follow-up. In univariate analysis, CMV-PCR≥1000 IU/mL was a significant risk factor for CLAD (HR 1.33, p=0.05). In multivariate analysis, CMV-PCR was not a significant risk factor for CLAD, whereas CMV serostatus mismatch was (Table). CMV-PCR was not a significant risk factor for RAS/mixed (CMV-PCR Conclusion CMV viremia was significantly associated with death/re-transplantation. Conversely, CMV viremia was not strongly associated with CLAD or RAS/mixed, while CMV serostatus mismatch was. CMV serostatus mismatch may have an impact on CLAD through mechanisms independent of viremia. In light of these paradoxical results, we are further assessing the relationship between immunosuppression, immune function, and CMV.

Purpose Microaspiration, primarily driven by gastroesophageal reflux, is deleterious to the lung allograft and contributes to poor clinical outcomes in lung transplant (LTx) recipients. Although recent studies have identified bile acids as promising biomarkers, early detection and mitigation of microaspiration remain a clinical challenge. Compared to bronchoalveolar lavage (BAL), bronchial wash (BW) has not gained widespread use at LTx centers but likely better samples the proximal airways, where gastric refluxate first enters the lung allograft. We hypothesized that BW is more useful than BAL to diagnose microaspiration and prognosticate poor clinical outcome in LTx recipients. Methods BW and BAL (as defined per ISHLT consensus statement, Nov 2020) were concurrently collected during surveillance bronchoscopies at 3 months post-LTx from 61 patients. Three bile acids, taurocholic acid (TCA), glycocholic acid (GCA) and cholic acid, were measured by mass spectrometry. Eleven protein biomarkers of inflammation or epithelial injury were measured by immunoassays. Results TCA and GCA levels were higher in BW than in BAL (Figure A). BW TCA and GCA were associated with concurrent decline in lung function (TCA OR=2.2; p=0.03; GCA OR=2.1; p=0.02). TCA, GCA, and most inflammatory proteins in BW, but not in BAL, were associated with decreased overall survival in multivariate Cox proportional hazard models adjusted for recipient age at transplant, sex, primary disease, days post-LTx to bronchoscopy, and concurrent acute rejection (Figure B). Exploratory derivation of BW-based biomarker signatures identified both bile acids and proteins as key parameters in predicting CLAD-free survival and overall survival. Conclusion BW may be more useful than BAL as a source of biomarkers to aid in the diagnosis and prognosis of clinically-significant microaspiration after LTx. The potential utility of BW needs to be tested in a larger, independent, prospective validation cohort.

Purpose CLAD occurs in 50-70% of LTx patients by year 5 and is the major obstacle to longterm survival. Multiple factors contribute to CLAD, of which acute rejection (AR) is the most significant. Routine monitoring with spirometry aims to identify and treat AR early. We reported that the oscillometry (Osc) metrics of small airway obstruction and ventilatory inhomogeneity, AX, X5 and R5-19, are more sensitive than spirometry in detecting AR associated graft injury. Hypothesis Osc detects AR associated graft injury and predicts risk of CLAD. Methods All double LTx recipients were enrolled from Dec 2017 onwards for Osc with each routine spirometry. By Mar 13, 2020, 238/287 enrolled patients had follow-up of ≥3 months while 170 had ≥6 months, of whom 32 developed CLAD. We excluded 6 CLAD cases due to insufficient Osc measurements by CLAD-onset date. Cox regression factored in the initial, baseline (the average of 2 best achieved post LTx) and intra-subject variance of AX, X5 and R5-19; demographics, primary diagnosis, CMV match status, HLA status and biopsy A-score, a cumulative index of biopsy-proven ARs. Results Cox analysis in 170 patients with ≥ 6 months follow up found higher A-scores to increase the risk of CLAD (HR 1.60, p Conclusion We provide evidence that 1) Osc metrics of lung function reflect graft injury associated with AR and 2) intra-subject variance in Osc measurements is a marker of ongoing graft injury that provides a risk assessment of subsequent CLAD. As such, post LTx graft monitoring with Osc can improve early detection of CLAD.

Purpose Airway epithelial injury is thought to be a key event in the pathogenesis of Chronic lung allograft dysfunction (CLAD). We investigated whether markers of epithelial activity and injury in bronchoalveolar lavage (BAL) correlate with CLAD diagnosis and major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS)-related phenotypes (including RAS, mixed phenotype, and all other patients with RAS-like opacities). Methods CLAD status and phenotypes were retrospectively determined in a cohort of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with RAS-related phenotypes were included and 1:1 matched with BOS patients based on time from transplant to CLAD-onset. CLAD-free subjects were used as controls. Proteins that maintain the barrier function of the airway epithelial mucosa (club cell secretory protein (CCSP), surfactant protein-D (SP-D) and epithelial mucins: MUC1, MUC5AC, MUC5B, MUC16), as well as epithelial cell death markers (M30&M65 representing apoptosis and overall death, respectively), were measured in BAL obtained within 6-months from CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using Kruskal-Wallis and Mann-Whitney-U-test. Association between protein levels and graft survival was assessed using Cox proportional hazards models, adjusted for CMV serology mismatch status and phenotype. Results Fifty-four CLAD (27 BOS, 11 RAS, 7 mixed, 9 others with RAS-like opacities) patients and 26 CLAD-free controls were included. Median BAL levels were significantly higher in patients with CLAD compared to CLAD-free controls for M30 (124.5 vs. 88.9), MUC1 (6.8 vs. 3.28) and MUC16 (121.0 vs. 31.1). When comparing CLAD phenotypes, M30 was significantly higher in patients with RAS-related phenotypes compared to BOS (160.9 vs. 114.6). In multivariable models, levels of M30 and MUC5B were associated with allograft survival after CLAD onset independent of phenotype (P Conclusion Airway epithelial mucin and cell death markers are enhanced in the BAL fluid of patients with CLAD and can assist in differentiating between CLAD phenotypes. Abnormal airway mucin expression and epithelial cell death may be involved in the airway inflammatory response of CLAD and thus aid in future selection of targeted therapies.

Acute cellular rejection (ACR) is a significant risk factor for chronic lung allograft dysfunction (CLAD). Although clinically manifest and higher grade (≥A2) ACR is generally treated with augmented immunosuppression, management of minimal (grade A1) ACR remains controversial. In our program, patients with subclinical and spirometrically stable A1 rejection (StA1R) are routinely not treated with augmented immunosuppression. We hypothesized that an untreated first StA1R does not increase the risk of CLAD or death compared to episodes of spirometrically stable no ACR (StNAR). The cohort was drawn from all consecutive adult, first, bilateral lung transplantations performed between 1999 and 2017. Biopsies obtained in the first-year posttransplant were paired with (forced expiratory volume in 1 second FEV1 ). The first occurrence of StA1R was compared to a time-matched StNAR. The risk of CLAD or death was assessed using univariable and multivariable Cox proportional hazards models. The analyses demonstrated no significant difference in risk of CLAD or death in patients with a first StA1R compared to StNAR. This largest study to date shows that, in clinically stable patients, an untreated first A1 ACR in the first-year posttransplant is not significantly associated with an increased risk for CLAD or death. Watchful-waiting approach may be an acceptable tactic for stable A1 episodes in lung transplant recipients.

Purpose DLCO measures gas movement at the alveolar-capillary interface. The DLCO at CLAD onset predicts post-CLAD survival. We hypothesized that DLCO is a sensitive measure of allograft fibrosis which would be reflected in the CLAD phenotype. Our aim was to describe the trajectory of DLCO measurements after lung transplantation based on CLAD phenotype. Methods A retrospective cohort analysis was conducted of 506 adult, first, bilateral lung transplants in a single center from 2010-2015. DLCO values were corrected for the nearest hemoglobin (DLCOcor). % Predicted (%-Pred) values were determined using the GLFI equations. Baseline DLCOcor was defined as the best achieved DLCOcor. CLAD phenotypes were determined with longitudinal volumetric and radiologic monitoring using the 2019 ISHLT Consensus Criteria. Results 174 patients with complete CLAD phenotyping and DLCO data were included: BOS in 104 (60%), Mixed in 9 (5%), RAS in 16 (9%), Undefined in 14 (8%) and 31 remained Unclassified (18%). There was a clinically significant difference in the post-transplant %-Pred DLCOcor trajectory based on CLAD phenotype, with RAS and Mixed showing greater decline than BOS and unclassified patterns [Figure 1, undefined not pictured]. DLCOcor was measured within 4 weeks of CLAD onset in 38/174 (21.8%) recipients with the phenotypes of: BOS 20/38 (53%), RAS/Mixed 9/38 (24%), Other 9/38 (24%). The %-Pred DLCOcor (median, IQR) at CLAD onset was significantly lower in the RAS/Mixed (45%, 40-67) groups compared with BOS (68%, 54-85) and Other (55%, 41-69) p=0.047. The %-Baseline DLCOcor (median, IQR) at CLAD onset was significantly lower in the RAS (66%, 53-77) group compared with BOS (85%, 78-98) and Other (70%, 61-81), p Conclusion CLAD phenotypes characterized by extensive parenchymal fibrosis showed significantly reduced DLCO. The DLCO may be a useful pulmonary function test to assist in the differentiation of CLAD phenotypes, especially when volumetric or radiologic measurements are unavailable.

Purpose Allograft infection after lung transplant (LTx) has a significant impact on outcome and has been associated with chronic lung allograft dysfunction (CLAD). Commensal bacteria (CB) are commonly isolated from bronchoalveolar lavages (BAL) in LTx recipients but the clinical significance is unknown. Our objective was to evaluate the association of high growth of CB (≥106 CFU/L) in BAL cultures with subsequent infection or CLAD. Methods We conducted a single-center, retrospective, cohort study of consecutive adult, first bilateral LTx performed 2010-2017. Patients who had no evidence of BAL infection and were alive and CLAD-free throughout the first 6-months post-transplant were included. Infection was defined as the presence of treatment-requiring pathogens in BAL. Outcomes of patients with ≥1CB within the first 6-months post-transplant were compared to patients with no CB isolation episodes within the same time interval. Association of CB with subsequent clinically significant BAL infections using logistic regression models and with time to CLAD was assessed using Cox Proportional Hazards models. Results The study cohort consisted of 202 patients with a median of 7 (IQR 7, 8) bronchoscopies per patient. Seventy of the 202 (34.7%) had subsequent BAL infection at some point post-transplant. Median time to CLAD was 497 days (IQR 131, 1043). Fifty-four (26.7%) patients had at least one episode of CB within the first 6-months post-transplant. In a multivariable analysis accounting for age, sex, primary lung disease, acute rejection score and CMV-mismatch, there was no difference in risk for subsequent infections (OR=0.83, 95% CI 0.40-1.69, P=0.60) or CLAD (HR=0.97, 95% CI 0.50-1.87, P=0.92) between patients with ≥1CB episode versus none. Conclusion We found that in the absence of specific infectious pathogens, isolation of commensal bacteria from BAL of LTx recipients during the first 6-months is not associated with an increased risk of subsequent infections or development of CLAD.

Purpose Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the 2019 ISHLT CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. Methods We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed 2010-2015. Classification of CLAD phenotypes was based on the 2019 ISHLT consensus document. Survival after CLAD onset was assessed using Kaplan-Meier and Cox proportional hazards models. Results Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed and 19 (10.9%) undefined phenotype. Twenty-six patients (14.9%) did not match any of these four categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The remaining 45 (25.9%) patients were combined and re-categorized based on the presence or absence of characteristic RAS-like opacities on chest imaging: those with RAS-like opacities at CLAD onset had significantly worse allograft survival compared to patients with BOS (HR=2.14, 95% CI 1.17-3.93, P=0.014) and similar survival to RAS or mixed phenotype. Conclusion The new ISHLT CLAD phenotype classification is informative with regards to post-CLAD outcomes. Chest imaging demonstrating persistent parenchymal or pleural fibrosis may be used for the risk-stratification of patients who do not match the major CLAD phenotypes.

Purpose Surveillance bronchoscopies with bronchoalveolar lavage (BAL) and transbronchial biopsies (TBB) are used in the evaluation of patients after lung transplant to detect acute cellular rejection (ACR) or infection. We have previously shown that a focused BAL protein signature early post-transplant may predict CLAD or death in a subset of clinically-stable patients with A1-grade (minimal) ACR. The aim of the current study was to determine whether similar BAL markers would have similar predictive characteristics irrespective of ACR grade. Methods The cohort consisted of all adult, first, bilateral lung transplants performed 2010-2017. Clinical status was categorized as unstable or stable based on presence or absence of ≥10% concurrent drop in FEV1. Clinically stable patients with grade AX TBB (inadequate biopsies, which are not routinely treated at our center, thus avoiding the confounding effect of ACR treatment) during the first-year post-transplant, not preceded by ACR (grade A≥1 or grade B≥1) were included. IL6, S100A8, IL10, TNF-receptor1, IL-1α, pentraxin3 and CXCL10, previously shown to be associated with worse outcomes, were measured in the BAL using a multiplex bead assay. Association with subsequent CLAD or death was assessed using Cox proportional hazards models adjusted for age, sex, native lung disease, and CMV-mismatch. Results 107 patients matched the inclusion criteria with a stable AX occurring at a median time of 188 days (IQR 96-279) post-transplant. Median time from AX TBB to CLAD or death was 462 (IQR 330, 928) and 582 days (IQR 251-1410), respectively. In multivariable models, levels of CXCL10 and IL10 were associated with both CLAD development and death while IL6, S100A8 and pentraxin3 were only associated with death (P Conclusion A focused BAL protein signature in clinically stable patients with ungradable TBB, early post-transplant, may be informative in determining increased risk for worse outcome and may benefit from a more aggressive management strategy.

Purpose Esophageal dysmotility is very common in patients with end-stage lung disease and post lung transplant and is thought to predispose to allograft injury through higher risk of aspiration. We routinely test lung transplant recipients for esophageal dysmotility with High Resolution Manometry (HRM) at 3 months post-transplant. We hypothesize that lung transplant recipients (LTRs) with significant esophageal dysmotility are more likely to develop lung injury leading to CLAD and death. Methods Among 1753 LTRs transplanted at our center between July 2000 and June 2018, we retrospectively analyzed 426 patients who had HRM with a 24 hour-pH impedance study available post-transplantation. Esophageal hypomotility was defined as >= 30% of failed and/or simultaneous esophageal contractions, while esophageal hypermotility was defined as evidence of higher amplitude on HRM. Cox proportional hazards models adjusted for age, sex, native lung disease, CMV matching, and transplant type (single vs double) were used to determine the association between esophageal dysmotility and death/retransplant or CLAD. Results Esophageal hypomotility and hypermotility was found in 120 (28%) and 22 (5%) patients, respectively. Mild, moderate, and severe hypomotility was found in 58 (48%), 14 (12%), and 48 (40%) patients, respectively. In multivariate analyses, esophageal hypermotility or hypomotility was not associated with CLAD (HR 0.59 [95% CI 0.21-1.60]; HR 0.79 [95% CI 0.52-1.19]). or death (HR 0.53 [95% CI 0.16-1.68]; HR 0.62 [95% CI 0.37-1.03]). The risk of CLAD or death did not vary by severity of hypomotility. Conclusion In a large single-center lung transplant cohort study, we have determined that esophageal dysmotility is not associated with CLAD or death. Concurrent GI co-morbidities, such as reflux and gastroparesis, may play a role in modifying this risk. Additionally, center-specific approaches to patient selection and/or treatment may be related to outcomes and will be further assessed in future studies.

Purpose Microaspiration induces lung inflammation which contributes to chronic lung allograft dysfunction (CLAD) and death in lung transplant (LTx) recipients. Early detection and mitigation of microaspiration may therefore improve outcomes. Bronchial wash (BW) has been underused compared to bronchoalveolar lavage (BAL) at most LTx centers. As BW likely better reflects the proximal airways, we hypothesized that the BW is a useful sample to analyze biomarkers of microaspiration and inflammation which may predict CLAD and death. Methods Large airway BW and BAL were collected concurrently during recipient routine bronchoscopies at 3 months post-LTx (n=61, 28% of whom had gastroesophageal reflux). Mass spectrometry was performed to measure 3 bile acids, taurocholic acid (TCA), glycocholic acid (GCA), and cholic acid, as putative biomarkers of microaspiration. Ten inflammatory proteins were measured by multiplex assay. Spearman's correlation was used to assess relationships between biomarkers. Cox proportional hazards models were used for survival analysis, adjusted for age, sex, primary disease, days from LTx to BW, and acute rejection. Results BW bile acid levels were higher than their BAL equivalents, and correlated with inflammatory markers in both samples (Fig A). BW bile acids showed no association with CLAD, though high IL-1α and IL-6 were predictors of early CLAD (Fig B). Notably, high BW TCA and GCA, as well as IL-1β, IL-6, IL-8, IL-12, CCL2, and CCL5 were all associated with early death (Fig C). In BAL, high IL-6 predicted early CLAD, but no other biomarker was associated with CLAD or death. Conclusion Bile acid and inflammatory marker levels in BW at 3 months post-LTx are significant prognostic factors for CLAD or death in LTx recipients and may be more useful predictive biomarkers than BAL levels in the context of microaspiration. Collection and analysis of BW for these biomarkers may allow for better evaluation of microaspiration and its inflammatory sequalae.

Purpose Acute cellular rejection (ACR) remains the most significant risk factor for chronic lung allograft dysfunction (CLAD). While clinically-unstable and higher-grade ≥A2 ACR is generally treated with augmented immunosuppression (aIS), management of clinically-stable A1 ACR (CSA1R) remains controversial. At our center, patients with CSA1R are routinely not treated with aIS and constitute a unique population that enabled us to identify sub-phenotypes with differential risks. Our aim was to determine if bronchoalveolar lavage (BAL) biomarkers at the time of first CSA1R (fCSA1R) are predictive of subsequent CLAD or death. Methods Among all adult, first, bilateral lung transplants, performed 2010-2016, post-transplant transbronchial biopsies obtained within the first year, without concurrent infection, were categorized as clinically unstable or stable based on ≥10% concurrent drop in FEV1. fCSA1R episodes were defined as stable biopsy-FEV1 pairs not preceded by another ACR. 21 proteins previously associated with ACR or CLAD were measured in the BAL using a multiplex bead assay. Association between protein levels and subsequent CLAD or death was assessed using Cox PH models, adjusted for relevant peri-transplant clinical covariates. Results We identified 75 patients with fCSA1R at a median time of 98 days (range 48.5-197) post-transplant. Median time from transplant to CLAD or death was 1247 (756.5-1921.5) and 1641 days (1024.5-2326.5), respectively. In multivariable models, levels of IL6, S100A8, IL10, TNF-receptor 1, IL-1α and pentraxin 3 (PTX3) were associated with both CLAD development and death (P Conclusion Our data indicate that a focused BAL protein signature, with PTX3 having the strongest association, may be useful in determining a subset of CSA1R patients who are at increased risk and may benefit from a more aggressive management strategy.

Purpose Gastroesophageal reflux disease (GERD) is thought to expose the lungs to refluxed gastric contents leading to injury and chronic lung allograft dysfunction (CLAD) in lung transplant recipients (LTRs). Prior small studies showed correlations between lung bile acids and CLAD/survival. The goal of this study was to determine whether bile acid levels in the bronchoalveolar lavage (BAL) correlate with GERD, concurrent lung allograft inflammation, or development of CLAD. Methods The cohort consisted of 25 GERD-positive and 50 GERD-negative adult sequential LTRs who underwent 24h pH/impedance reflux testing and BAL collection within 6 months post-transplant. Bile acids - taurocholic acid (TCA), glycocholic acid (GCA), and cholic acid (CA) - were measured using LC-MS/MS mass spectrometry; 10 cytokines were quantified using a multiplex assay. Levels were compared between groups using generalized linear models. Spearman correlations between biomarkers were assessed. The relationship between biomarkers and time to CLAD was examined using Cox proportional hazards models. Results TCA was significantly higher in BALs of GERD-positive patients compared to GERD-negative (0.4 vs. 0.1, p=0.04), with no significant differences in GCA, CA, or cytokines. Importantly, there were correlations (adjusted for 10 multiple comparisons) between TCA and GCA and inflammatory markers IL-6, CXCL8, CCL2, IL-1β, CCL5, IL-1α, and IL-12p70 (Fig 1A). Additionally, TCA (HR 1.37, p=0.04), IL-6 (HR 1.59, p=0.012), S100A8 (HR 1.39, p=0.033), and CCL2 (HR 1.41, p=0.03) at the time of the BAL were significantly associated with time to CLAD, independent of GERD status (Fig 1B shows additional Kaplan-Meier analysis). Conclusion BAL TCA correlates with GERD, concurrent innate immune inflammation, as well as risk of CLAD. We propose that BAL TCA levels, in conjunction with inflammatory marker measurements, may be useful in identifying LTRs with chronic aspiration of enteric contents that have a higher risk of subsequent adverse outcomes.

Purpose The DLCO measures the capacity of the lungs to exchange gas across the alveolar-capillary interface. We hypothesized that DLCO is a sensitive measure of injurious allograft processes that can disrupt this interface. Our goals were to describe the trajectory of DLCO measurements after lung transplantation, and to determine the prognostic significance of DLCO on chronic lung allograft dysfunction (CLAD) and survival. Methods A retrospective cohort analysis was conducted of all bilateral lung transplant recipients at a single center, between Jan-1998 and Jan-2018, with ≥2 DLCO measurements. DLCO values were corrected for the nearest haemoglobin (DLCOcor). Low baseline DLCO was defined as the failure to ever attain a best achieved DLCO >75% predicted. Drops in DLCOcor were defined as >15% below best achieved. Multivariate Cox proportional hazard models were used for time-to-event analyses. Results 1158/1571 patients were included. The median (IQR) number of DLCOcor measurements per patient was 7 (5). The median (IQR) time to peak DLCOcor was 360 (555) days and the mean (SD) %-predicted DLCO was 81.2% (20.7). 433 patients (37.4%) had low baseline DLCO. Multivariate analysis demonstrated that low baseline DLCO was significantly associated with a shorter time to CLAD (HR 1.50 p Conclusion Low baseline DLCO was significantly associated with both CLAD and survival. Significant drops in DLCOcor at 18 months and the % predicted DLCO at CLAD onset were significantly and independently associated with reduced survival. There may be benefit in routine monitoring of DLCO after lung transplantation to identify patients at risk of poor outcomes.

Purpose Gastroparesis and GERD are very common conditions in patients with end-stage lung disease and can be exacerbated after lung transplantation (LTx). At our center, we routinely test lung transplant recipients (LTRs) for gastric emptying abnormalities and for GERD at 3 months post-transplant. We hypothesized that LTRs with significant gastroparesis and/or GERD are more likely to develop lung injury leading to subsequent CLAD and death. Methods Among 2812 LTRs transplanted at our center between May 1998 and April 2018, we retrospectively analyzed 1071 patients who had a solid gastric emptying study available within seven months post-transplantation. Delayed gastric emptying (DGE) was defined as > 10% gastric retention at four hours and classified based on severity: mild (10-15%), moderate (16 - 35%), and severe (>35%). We obtained routine 24h pH/impedance testing in LTRs from 2004-2015, where GERD was defined as > 48 episodes of acid or non-acid reflux. 295 patients had both gastric emptying and reflux testing available. Cox proportional hazards models were used to determine the association between DGE, GERD, and combined DGE(moderate/severe)+GERD with time to CLAD or death. Results Mild, moderate, and severe DGE was found in 128 (12%), 163 (15.2%), and 302 (28.2%) patients, respectively. DGE alone was not associated with CLAD or death (HR 0.87 [95% CI 0.64-1.19]; HR 1.06 [95% CI 0.81-1.39]; HR 1.07 [95% CI 0.86-1.35]) for mild, moderate, severe, respectively (Figure 1A). Similarly, evidence of GERD alone was not associated with CLAD or death (abstract ISHLT 2017). However, the combination of moderate-severe DGE and GERD was significantly associated with death (HR: 1.81 [95% CI 1.07-3.05]). Conclusion In a single-center lung transplant cohort study , we observed that while DGE or GERD alone are not individually associated with CLAD or death, patients with both moderate-severe DGE and significant GERD had significantly worse survival.