Small airway brush gene expression predicts chronic lung allograft dysfunction and mortality.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) limits survival following lung transplant, but substantial lung damage occurs before diagnosis by traditional methods. We hypothesized that small airway gene expression patterns could identify CLAD risk before spirometric diagnosis and predict subsequent graft failure. METHODS: Candidate genes from 4 rejection-associated transcript sets were assessed for associations with CLAD or graft failure in a derivation cohort of 156 small airway brushes from 45 CLAD cases and 37 time-matched controls with >1-year stable lung function. Candidate genes not associated with CLAD and time to graft failure were excluded, yielding the Airway Inflammation 2 (AI2) gene set. Area under the receiver operating curve (AUC) for CLAD and competing risks of death or graft failure were assessed in an independent validation cohort of 37 CLAD cases and 37 controls. RESULTS: Thirty-two candidate genes were associated with CLAD and graft failure, comprising the AI2 score, which clustered into 3 subcomponents. The AI2 score identified CLAD before its onset, in early and late post-CLAD brushes, as well as in the validation cohort (AUC 0.69-0.88). The AI2 score association with CLAD was independent of positive microbiology, CLAD stage, or CLAD subtype. However, transcripts most associated with CLAD evolved over time from CLAD onset. The AI2 score predicted time to graft failure and retransplant-free survival in both cohorts (p ≤ 0.03). CONCLUSIONS: This airway inflammation gene score is associated with CLAD development, graft failure, and death. Future studies defining the molecular heterogeneity of airway inflammation could lead to endotype-targeted therapies.

Macrophage and CD8 T cell discordance are associated with acute lung allograft dysfunction progression.

BACKGROUND: Acute lung allograft dysfunction (ALAD) is an imprecise syndrome denoting concern for the onset of chronic lung allograft dysfunction (CLAD). Mechanistic biomarkers are needed that stratify risk of ALAD progression to CLAD. We hypothesized that single cell investigation of bronchoalveolar lavage (BAL) cells at the time of ALAD would identify immune cells linked to progressive graft dysfunction. METHODS: We prospectively collected BAL from consenting lung transplant recipients for single cell RNA sequencing. ALAD was defined by a ≥10% decrease in FEV1 not caused by infection or acute rejection and samples were matched to BAL from recipients with stable lung function. We examined cell compositional and transcriptional differences across control, ALAD with decline, and ALAD with recovery groups. We also assessed cell-cell communication. RESULTS: BAL was assessed for 17 ALAD cases with subsequent decline (ALAD declined), 13 ALAD cases that resolved (ALAD recovered), and 15 cases with stable lung function. We observed broad differences in frequencies of the 26 unique cell populations across groups (p = 0.02). A CD8 T cell (p = 0.04) and a macrophage cluster (p = 0.01) best identified ALAD declined from the ALAD recovered and stable groups. This macrophage cluster was distinguished by an anti-inflammatory signature and the CD8 T cell cluster resembled a Tissue Resident Memory subset. Anti-inflammatory macrophages signaled to activated CD8 T cells via class I HLA, fibronectin, and galectin pathways (p < 0.05 for each). Recipients with discordance between these cells had a nearly 5-fold increased risk of severe graft dysfunction or death (HR 4.6, 95% CI 1.1-19.2, adjusted p = 0.03). We validated these key findings in 2 public lung transplant genomic datasets. CONCLUSIONS: BAL anti-inflammatory macrophages may protect against CLAD by suppressing CD8 T cells. These populations merit functional and longitudinal assessment in additional cohorts.

Decreased Lymphocytic Bronchitis Severity in the Era of Azithromycin Prophylaxis.

Large-airway lymphocytic inflammation (LB), assessed on endobronchial biopsies, has been associated with acute cellular rejection and chronic lung allograft dysfunction (CLAD). Azithromycin (AZI) prophylaxis has been used to prevent airway inflammation and subsequent CLAD, with inconsistent results. We hypothesized that AZI prophylaxis would be associated with reduced LB, changes in bronchoalveolar lavage (BAL) immune cell populations, and improved CLAD-free survival. Methods: We compared frequencies of LB from endobronchial biopsies before (N = 1856) and after (N = 975) protocolized initiation of AZI prophylaxis at our center. LB was classified as none, minimal, mild, or moderate by histopathologic analysis. LB grades were compared using ordinal mixed-model regression. Corresponding automated BAL leukocyte frequencies were compared using mixed-effects modeling. The effect of AZI prophylaxis on CLAD-free survival was assessed by a Cox proportional hazards model adjusted for age, sex, ethnicity, transplant indication, and cytomegalovirus serostatus. Results: Biopsies in the pre-AZI era had 2-fold increased odds (95% confidence interval, 1.5-2.7; P < 0.001) of higher LB grades. LB was associated with BAL neutrophilia in both eras. However, there was no difference in risk for CLAD or death between AZI eras (hazard ratio 1.3; 95% confidence interval, 0.7-2.0; P = 0.45). Conclusions: Decreased airway inflammation in the era of AZI prophylaxis may represent a direct effect of AZI therapy or reflect other practices or environmental changes. In this cohort, AZI prophylaxis was not associated with improved CLAD-free survival.

Design and implementation of a digital health home spirometry intervention for remote monitoring of lung transplant function.

BACKGROUND: We developed an automated, chat-based, digital health intervention using Bluetooth-enabled home spirometers to monitor for complications of lung transplantation in a real-world application. METHODS: A chat-based application prompted patients to perform home spirometry, enter their forced expiratory volume in 1 second (FEV1), answer symptom queries, and provided patient education. The program alerted patients and providers to substantial FEV1 decreases and concerning symptoms. Data was integrated into the electronic health record (EHR) system and dashboards were developed for program monitoring. RESULT: Between May 2020 and December 2021, 544 patients were invited to enroll, of whom 427 were invited remotely and 117 were enrolled in-person. 371 (68%) participated by submitting ≥1 FEV1 values. Overall engagement was high, with an average of 197 unique patients submitting FEV1 data per month. In-person enrollees submitted an average of 4.6 FEV1 values per month and responded to 55% of scheduled chats. Home and laboratory FEV1 values correlated closely (rho = 0.93). There was an average of 133 ± 59 FEV1 decline alerts and 59 ± 23 symptom alerts per month. 72% of patients accessed education modules, and the program had a high net promoter score (53) amongst users. CONCLUSIONS: We demonstrate that a novel, automated, chat-based, and EHR-integrated home spirometry intervention is well accepted, generates reliable assessments of graft function, and can deliver automated feedback and education resulting in moderately-high adherence rates. We found that in-person onboarding yields better engagement and adherence. Future work will aim to demonstrate the impact of remote care monitoring on early detection of lung transplant complications.

Development of the Lung Transplant Frailty Scale (LT-FS).

BACKGROUND: Existing measures of frailty developed in community dwelling older adults may misclassify frailty in lung transplant candidates. We aimed to develop a novel frailty scale for lung transplantation with improved performance characteristics. METHODS: We measured the short physical performance battery (SPPB), fried frailty phenotype (FFP), Body Composition, and serum Biomarkers representative of putative frailty mechanisms. We applied a 4-step established approach (identify frailty domain variable bivariate associations with the outcome of waitlist delisting or death; build models sequentially incorporating variables from each frailty domain cluster; retain variables that improved model performance ability by c-statistic or AIC) to develop 3 candidate "Lung Transplant Frailty Scale (LT-FS)" measures: 1 incorporating readily available clinical data; 1 adding muscle mass, and 1 adding muscle mass and research-grade Biomarkers. We compared construct and predictive validity of LT-FS models to the SPPB and FFP by ANOVA, ANCOVA, and Cox proportional-hazard modeling. RESULTS: In 342 lung transplant candidates, LT-FS models exhibited superior construct and predictive validity compared to the SPPB and FFP. The addition of muscle mass and Biomarkers improved model performance. Frailty by all measures was associated with waitlist disability, poorer HRQL, and waitlist delisting/death. LT-FS models exhibited stronger associations with waitlist delisting/death than SPPB or FFP (C-statistic range: 0.73-0.78 vs. 0.57 and 0.55 for SPPB and FFP, respectively). Compared to SPPB and FFP, LT-FS models were generally more strongly associated with delisting/death and improved delisting/death net reclassification, with greater improvements with increasing LT-FS model complexity (range: 0.11-0.34). For example, LT-FS-Body Composition hazard ratio for delisting/death: 6.0 (95%CI: 2.5, 14.2), SPPB HR: 2.5 (95%CI: 1.1, 5.8), FFP HR: 4.3 (95%CI: 1.8, 10.1). Pre-transplant LT-FS frailty, but not SPPB or FFP, was associated with mortality after transplant. CONCLUSIONS: The LT-FS is a disease-specific physical frailty measure with face and construct validity that has superior predictive validity over established measures.

The Lung Allocation Score Remains Inequitable for Patients with Pulmonary Arterial Hypertension, Even after the 2015 Revision.

Rationale: The lung allocation score (LAS) was revised in 2015 to improve waiting list mortality and rate of transplant for patients with pulmonary arterial hypertension (PAH). Objectives: We sought to determine if the 2015 revision achieved its intended goals. Methods: Using the Standard Transplant Analysis and Research file, we assessed the impact of the 2015 LAS revision by comparing the pre- and postrevision eras. Registrants were divided into the LAS diagnostic categories: group A-chronic obstructive pulmonary disease; group B-pulmonary arterial hypertension; group C-cystic fibrosis; and group D-interstitial lung disease. Competing risk regressions were used to assess the two mutually exclusive competing risks of waiting list death and transplant. Cumulative incidence plots were created to visually inspect risks. Measurements and Main Results: The LAS at organ matching increased by 14.2 points for registrants with PAH after the 2015 LAS revision, the greatest increase among diagnostic categories (other LAS categories: Δ, -0.9 to +2.8 points). Before the revision, registrants with PAH had the highest risk of death and lowest likelihood of transplant. After the 2015 revision, registrants with PAH still had the highest risk of death, now similar to those with interstitial lung disease, and the lowest rate of transplant, now similar to those with chronic obstructive pulmonary disease. Conclusions: Although the 2015 LAS revision improved access to transplant and reduced the risk of waitlist death for patients with PAH, it did not go far enough. Significant differences in waitlist mortality and likelihood of transplant persist.

NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes.

Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

NCCN Guidelines® Insights: Lung Cancer Screening, Version 1.2022.

The NCCN Guidelines for Lung Cancer Screening recommend criteria for selecting individuals for screening and provide recommendations for evaluation and follow-up of lung nodules found during initial and subsequent screening. These NCCN Guidelines Insights focus on recent updates to the NCCN Guidelines for Lung Cancer Screening.

A survey of use of mTOR inhibitors in patients with lymphangioleiomyomatosis listed for lung transplant.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is an uncommon indication for lung transplantation. The use of mechanistic target of rapamycin (mTOR) inhibitors, which are the mainstay of treatment in progressive LAM, in patients awaiting lung transplant is controversial. We sought to examine worldwide practice patterns in use of mTOR inhibitors in LAM patients on the lung transplant waiting list. METHODS: We designed and disseminated an online survey about institution-specific practice patterns, particularly regarding listing LAM patients for lung transplant and use of mTOR inhibitors in those patients on the transplant waitlist. RESULTS: Of the 49 unique respondent programs, 83.6% had previously listed a LAM patient for lung transplant. Thirteen centers allowed patients to continue on mTOR inhibitor until time of lung transplant. None of those centers reported any complications or deaths attributable to mTOR inhibitor adverse effects. CONCLUSION: There exists significant variability in practice patterns concerning the use of mTOR inhibitors in LAM patients on the lung transplant waiting list. Our survey suggests favorable outcomes for those patients that did continue mTOR inhibitor up to time of transplant. Further data regarding the risk of anastomotic complication with use of mTOR inhibitors in the pre-transplant period would help provide clarity in this debate.

Depressive symptoms in lung transplant recipients: trajectory and association with mortality and allograft dysfunction.

OBJECTIVE: Most studies observing an association between depressive symptoms following lung transplantation and mortality are limited to depressive symptom measurement at a single time point, unrelated to allograft function. We aimed to test the association of depressive symptoms over multiple assessments with allograft dysfunction and with mortality. METHODS: We assessed depressive symptoms before and serially up to 3 years after lung transplantation in lung transplant recipients. We quantified depressive symptoms with the Geriatric Depression Scale (GDS; range 0-15; minimally important difference (MID): 2). We quantified changes in GDS using linear mixed effects models and tested the association with mortality using Cox proportional hazards models with GDS as a time-dependent predictor. To determine if worsening in GDS preceded declines in lung function, we tested the association of GDS as a time-dependent predictor with the lagged outcome of FEV1 at the following study visit. RESULTS: Among 266 participants, depressive symptoms improved early after transplantation. Worsening in post-transplant GDS by the MID was associated with mortality (HR 1.25, 95% CI 1.05 to 1.50), and in lagged outcome analyses with decreased per cent predicted FEV1 (Δ, -1.62%, 95% CI -2.49 to -0.76). Visual analyses of temporal changes in GDS demonstrated that worsening depressive symptoms could precede chronic lung allograft dysfunction. CONCLUSIONS: Depressive symptoms generally improve after lung transplantation. When they worsen, however, there is an association with declines in lung function and mortality. Depression is one of the few, potentially modifiable, risk factors for chronic lung allograft dysfunction and death.

Inflammation on bronchoalveolar lavage cytology is associated with decreased chronic lung allograft dysfunction-free survival.

BACKGROUND: Lung transplant recipients undergo bronchoalveolar lavage (BAL) to detect antecedents of chronic lung allograft dysfunction (CLAD), but routine assessment of BAL cytology is controversial. We hypothesized that inflammation on BAL cytology would predict CLAD-free survival. METHODS: In a single-center retrospective cohort, associations between cytology results and clinical characteristics were compared using generalized-estimating equation-adjusted regression. The association between BAL inflammation and CLAD or death risk was assessed using time-dependent Cox models. RESULTS: In 3365 cytology reports from 451 subjects, inflammation was the most common finding (6.2%, 210 cases), followed by fungal forms (5.3%, 178 cases, including 24 cases of suspected Aspergillus). Inflammation on BAL cytology was more common in procedures for symptoms (8.5%) versus surveillance (3.2%, p < .001). Inflammation on cytology was associated with automated neutrophil and lymphocyte counts, acute cellular rejection, infection, and portended a 2.2-fold hazard ratio (CI 1.2-4.0, p = .007) for CLAD or death. However, inflammation by cytology did not inform CLAD-free survival risk beyond automated BAL cell counts (p = .57). CONCLUSIONS: Inflammation on BAL cytology is clinically significant, suggesting acute rejection or infection and increased risk of CLAD or death. However, other indicators of allograft inflammation can substitute for much of the information provided by BAL cytology.

Consensus document for the selection of lung transplant candidates: An update from the International Society for Heart and Lung Transplantation.

Tens of thousands of patients with advanced lung diseases may be eligible to be considered as potential candidates for lung transplant around the world each year. The timing of referral, evaluation, determination of candidacy, and listing of candidates continues to pose challenges and even ethical dilemmas. To address these challenges, the International Society for Heart and Lung Transplantation appointed an international group of members to review the literature, to consider recent advances in the management of advanced lung diseases, and to update prior consensus documents on the selection of lung transplant candidates. The purpose of this updated consensus document is to assist providers throughout the world who are caring for patients with pulmonary disease to identify potential candidates for lung transplant, to optimize the timing of the referral of these patients to lung transplant centers, and to provide transplant centers with a framework for evaluating and selecting candidates. In addition to addressing general considerations and providing disease specific recommendations for referral and listing, this updated consensus document includes an ethical framework, a recognition of the variability in acceptance of risk between transplant centers, and establishes a system to account for how a combination of risk factors may be taken into consideration in candidate selection for lung transplantation.

Construct and Predictive Validity of Sarcopenia in Lung Transplant Candidates.

Rationale: Sarcopenia is associated with disability and death. The optimal definition and clinical relevance of sarcopenia in lung transplantation remain unknown. Objectives: To assess the construct and predictive validity of sarcopenia definitions in lung transplant candidates. Methods: In a multicenter prospective cohort of 424 lung transplant candidates, we evaluated limited (muscle mass only) and expanded (muscle mass and quality) sarcopenia definitions from the European Working Group on Sarcopenia in Older People 2 (EWGSOP2), the Foundation for the National Institutes of Health (FNIH), and a cohort-specific distribution-based lowest quartile definition. We assessed construct validity using associations with conceptually related factors. We evaluated the relationship between sarcopenia and frailty using generalized additive models. We also evaluated associations between sarcopenia definitions and key pretransplant outcomes, including disability (quantified by the Lung Transplant Valued Life Activities scale [range, 0-3; higher scores = worse disability; minimally important difference, 0.3]) and waitlist delisting/death, by multivariate linear and Cox regression, respectively. Results: Sarcopenia prevalence ranged from 6% to13% by definition used. The limited EWGSOP2 definition demonstrated the highest construct validity, followed by the expanded EWGSOP2 definition and both limited and expanded FNIH and lowest quartile definitions. Sarcopenia exhibited a linear association with the risk of frailty. The EWGSOP2 and expanded lowest quartile definitions were associated with disability, ranging from 0.20 to 0.25 higher Lung Transplant Valued Life Activities scores. Sarcopenia was associated with increased risk of waitlist delisting or death by the limited and expanded lowest quartile definitions (hazard ratio [HR], 3.8; 95% confidence interval [CI], 1.4-9.9 and HR, 3.5; 95% CI, 1.1-11.0, respectively) and the EWGSOP2 limited definition (HR, 2.8; 95% CI, 0.9-8.6) but not with the three other candidate definitions. Conclusions: The prevalence and validity of sarcopenia vary by definition; the EWGSOP2 limited definition exhibited the broadest validity in lung transplant candidates. The linear relationship between low muscle mass and frailty highlights sarcopenia's contribution to frailty and also questions the clinical utility of a sarcopenia cut-point in advanced lung disease. The associations between sarcopenia and important pretransplant outcomes support further investigation into using body composition for candidate risk stratification.

Primary graft dysfunction attenuates improvements in health-related quality of life after lung transplantation, but not disability or depression.

Disability, depressive symptoms, and impaired health-related quality of life (HRQL) are common among patients with life-threatening respiratory compromise. We sought to determine if primary graft dysfunction (PGD), a syndrome of acute lung injury, attenuates improvements in patient-reported outcomes after transplantation. In a single-center prospective cohort, we assessed disability, depressive symptoms, and HRQL before and at 3- to 6-month intervals after lung transplantation. We estimated the magnitude of change in disability, depressive symptoms, and HRQL with hierarchical segmented linear mixed-effects models. Among 251 lung transplant recipients, 50 developed PGD Grade 3. Regardless of PGD severity, participants had improvements in disability and depressive symptoms, as well as generic-physical, generic-mental, respiratory-specific, and health-utility HRQL, exceeding 1- to 4-fold the minimally clinically important difference across all instruments. Participants with PGD Grade 3 had a lower magnitude of improvement in generic-physical HRQL and health-utility than in all other participants. Among participants with PGD Grade 3, prolonged mechanical ventilation was associated with greater attenuation of improvements. PGD remains a threat to the 2 primary aims of lung transplantation, extending survival and improving HRQL. Attenuation of improvement persists long after hospital discharge. Future studies should assess if interventions can mitigate the impact of PGD on patient-reported outcomes.

Chronic lung allograft dysfunction small airways reveal a lymphocytic inflammation gene signature.

Chronic lung allograft dysfunction (CLAD) is the major barrier to long-term survival following lung transplantation, and new mechanistic biomarkers are needed. Lymphocytic bronchitis (LB) precedes CLAD and has a defined molecular signature. We hypothesized that this LB molecular signature would be associated with CLAD in small airway brushings independent of infection. We quantified RNA expression from small airway brushings and transbronchial biopsies, using RNAseq and digital RNA counting, respectively, for 22 CLAD cases and 27 matched controls. LB metagene scores were compared across CLAD strata by Wilcoxon rank sum test. We performed unbiased host transcriptome pathway and microbial metagenome analysis in airway brushes and compared machine-learning classifiers between the two tissue types. This LB metagene score was increased in CLAD airway brushes (p = .002) and improved prediction of graft failure (p = .02). Gene expression classifiers based on airway brushes outperformed those using transbronchial biopsies. While infection was associated with decreased microbial alpha-diversity (p ≤ .04), neither infection nor alpha-diversity was associated with LB gene expression. In summary, CLAD was associated with small airway gene expression changes not apparent in transbronchial biopsies in this cohort. Molecular analysis of airway brushings for diagnosing CLAD merits further examination in multicenter cohorts.

Type-1 immunity and endogenous immune regulators predominate in the airway transcriptome during chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long-term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush-derived transcriptomic signatures in CLAD (n = 24) versus non-CLAD (n = 21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type-1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO-1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type-1 profile with detection of IFN-γ, TNF-α, and IL-1ß as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.

Cystic Fibrosis Lung Transplant Recipients Have Suppressed Airway Interferon Responses during Pseudomonas Infection.

Lung transplantation can be lifesaving in end-stage cystic fibrosis (CF), but long-term survival is limited by chronic lung allograft dysfunction (CLAD). Persistent upper airway Pseudomonas aeruginosa (PsA) colonization can seed the allograft. While de novo PsA infection is associated with CLAD in non-CF recipients, this association is less clear for CF recipients experiencing PsA recolonization. Here, we evaluate host and pathogen contributions to this phenomenon. In the context of PsA infection, brushings from the airways of CF recipients demonstrate type 1 interferon gene suppression. Airway epithelial cell (AEC) cultures demonstrate similar findings in the absence of pathogens or immune cells, contrasting with the pre-transplant CF AEC phenotype. Type 1 interferon promoters are relatively hypermethylated in CF AECs. CF subjects in this cohort have more mucoid PsA, while non-CF PsA subjects have decreased microbiome α diversity. Peri-transplant protocols may benefit from consideration of this host and microbiome equilibrium.