Bronchiolitis Obliterans Syndrome Patients Research Articles

Association Between FEV₁ Decline Rate and Mortality in Long-Term Follow-Up of a 21-Patient Pilot Clinical Trial of Inhaled Liposomal Cyclosporine Plus Standard-of-Care Versus Standard-of-Care Alone for Bronchiolitis Obliterans Syndrome After Lung Transplantation.

BACKGROUND The association between forced expiratory volume in 1 second (FEV1) trajectory and mortality in bronchiolitis obliterans syndrome (BOS) is not well defined. Using long-term data from a prior clinical trial of inhaled liposomal cyclosporine A (L-CsA-I) for lung transplant patients with BOS, this study examined the association between longitudinal FEV₁ change and mortality. MATERIAL AND METHODS We analyzed long-term data from a clinical trial which randomized 21 patients with BOS (³20% decrease in FEV1 from personal maximum) to receive L-CsA-I plus standard-of-care (n=11) or standard-of-care (SOC) alone (n=10) for 24 weeks. A joint statistical model, combining a linear mixed model for FEV₁ change and Cox regression for mortality, was utilized to examine the overall association between FEV₁ trajectory and mortality during follow-up. RESULTS The 21 trial participants (10 single, 11 double lung recipients) had a mean FEV₁ of 1.7±0.6 Liters at randomization. Median follow-up post-randomization was 35 months. In joint model analysis, 1 percent FEV₁ decline predicted 1.076-fold increased mortality risk (95% confidence interval: -0.998 to 1.160, p=0.058). FEV₁ decline was reduced by 2.6% per year in L-CsA-I patients compared to SOC (p=0.210), and overall survival at 1/3/5 years was 91%/64%/27% vs 90%/20%/0% for L-CsA-I versus SOC, respectively (p=0.164). CONCLUSIONS In BOS patients, greater longitudinal FEV₁ decline predicts increased mortality. Trends towards prolonged stabilization of FEV₁ and improved survival were observed with L-CsA-I receipt. Further analyses will aid in evaluating the utility of FEV₁ change as a survival predictor, having implications in BOS management and future trial design.

Single Cell Transcriptomics of Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS) is a manifestation of pulmonary chronic graft versus host disease (cGVHD), a serious complication of allogeneic stem cell transplantation (ASCT). BOS also occurs following bi-orthotopic lung transplant (BOLT), where it is termed chronic lung allograft dysfunction (CLAD)-BOS and is the leading cause of long-term allograft failure. In both contexts, fibrotic obliteration of the small airways leads to progressive respiratory failure. Since the molecular mechanisms leading to fibrotic airway remodeling in humans are poorly understood, we used single cell RNA-sequencing to map intercellular interactions between immune (CD45+ve) and non-immune (CD45-ve) cells of lung explants from patients undergoing first lung transplantation for cGVHD-BOS (n=1) or second lung transplantation for CLAD-BOS (n=2) compared to normal controls (NC) (n=3). BOS patients ranged in age from 44 to 61 years old; NC were similar in age and died of cerebrovascular accidents. Lungs were surgically removed at the time of BOLT (BOS samples) or at autopsy (NC). CD45+ve and CD45-ve cells were magnetically sorted and freshly processed. Single cell libraries were prepared using the 10X Genomics Chromium Controller and sequenced on an Illumina Novaseq 6000 instrument. Quality control filtering, transcript count normalization, integration, PCA, UMAP projection, and cell clustering were performed with Seurat in R v4.2.1. CD45+ve and CD45-ve cells were annotated in Azimuth using the Human Lung Cell Atlas and LungMAP Cell Cards reference data sets, respectively. Differences in gene expression between conditions were evaluated with the Seurat FindMarkers function. Inter-cellular communications were characterized with CellChat. Interaction strength was calculated as a function of ligand/receptor pair expression, and incoming and outgoing signaling was classified using network centrality analysis. We identified 45,932 CD45+ve cells (28,241 in BOS and 17,691 in NC) and 33,428 CD45-ve cells (23,390 in BOS and 10,038 in NC) including 17 distinct cell types in the CD45+ve fractionand 24 in the CD45-ve fraction. As samples came from patients with end-stage disease, we hypothesized that CD45+ve cells would be highly activated while CD45-ve cells would exhibit strongly fibrogenic programs. To test this hypothesis, we analyzed the incoming and outgoing signal strength for each cell type. Stromal cells such as type 1 (AF1) and type 2 alveolar fibroblasts (AF2), secondary crest myofibroblasts (SCMF), and pericytes had the strongest outgoing signals in BOS ( Figure 1). Except for AF2, incoming and outgoing signaling from stromal cells was stronger in BOS than in NC. These observations support stromal cells as important drivers of BOS pathogenesis. Next, we characterized signaling directionality between stromal cells and CD45+ve cells. In general, incoming signals to stromal cells from CD45+ve cells were weaker in BOS than in NC, while outgoing signals from stromal cells to CD45+ve cells were stronger. For example, signaling from stromal cells expressing THY1, which may attenuate fibrosis in fibrotic lung disease, to integrins on monocytes and macrophages was stronger in BOS. Likewise, BOS was enriched for signaling from collagens and fibronectin on stromal cells to CD44 on CD45+ve cells. As expected, genes implicated in fibrosis and wound healing were upregulated in stromal cells in BOS patients. In AF1 ( Figure 2), these included genes encoding collagens (COL6A3, COL14A1), actins (ACTA2), matricellular proteins (POSTN, TNC, SPARC), protease inhibitors (SERPINE1, TIMP1), hyaluronic acid synthetases (HAS2), proteoglycans (LUM, VCAN), and proteases (MMP19, ADAMTS9). Interestingly, we found evidence for active anti-fibrotic programs such as MMP19, PTGS2, and THY1. To our knowledge, this is the first study employing single cell transcriptomics of lung explants from patients with cGVHD-associated BOS. Robust signaling from stromal cells to CD45+ve cells may support their role in modifying the inflammatory infiltrate in BOS. We identified outgoing, stromal derived signaling associated with fibrotic (collagen and fibronectin) and anti-fibrotic programs (THY1). Our findings illustrate an interplay between pro- and anti-fibrotic programs even in end-stage BOS lungs, raising the tantalizing possibility to intervene therapeutically in this dyshomeostatic balance.

Clinical risk factors and prognostic model for patients with bronchiolitis obliterans syndrome after hematopoietic stem cell transplantation.

Bronchiolitis obliterans syndrome (BOS) is a common and potentially devastating noninfectious pulmonary complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, predictive tools for BOS are not available. We aimed to identify the clinical risk factors and establish a prognostic model for BOS in patients who undergo allo-HSCT. We retrospectively identified a cohort comprising 195 BOS patients from 6100 consecutive patients who were allografted between 2008 and 2022. The entire cohort was divided into a derivation cohort and a validation cohort based on the time of transplantation. Via multivariable Cox regression methods, declining forced expiratory volume at 1 s (FEV1) to <40%, pneumonia, cGVHD except lung, and respiratory failure were found to be independent risk factors for the 3-year mortality of BOS. A risk score called FACT was constructed based on the regression coefficients. The FACT model had an AUC of 0.863 (95% CI: 0.797-0.928) in internal validation and 0.749 (95% CI: 0.621-0.876) in external validation. The calibration curves showed good agreement between the FACT-predicted probabilities and actual observations. The FACT risk score will help to identify patients at high risk and facilitate future research on developing novel, effective interventions to personalize treatment.

EE539 Estimating the Lifetime Burden of Illness in Patients with Bronchiolitis Obliterans Syndrome (BOS)

Bronchiolitis Obliterans Syndrome (BOS) is a rare, life-threatening disease where the immune system attacks the airways of the lungs. BOS is estimated to affect approximately 25,000 individuals worldwide and can occur after hematopoietic stem cell transplantation (HSCT) or lung transplantation, with up to half of all lung transplant recipients developing BOS within 5 years of transplantation. BOS is rapidly progressive and irreversible, leading to lung failure after diagnosis, and making it the leading cause of death. The aim of this analysis was to estimate the lifetime economic burden of illness for BOS patients from the perspective of a US Payer.

Pulmonary epithelial markers in phenotypes of chronic lung allograft dysfunction

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 fluid (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). CLAD status and phenotypes were retrospectively determined in a cohort of all consecutive adult, first, bilateral lung transplants performed 2010-2015, with available BAL samples. All patients with RAS-related phenotypes were included and 1:1 matched with BOS patients based on the time from transplant to CLAD-onset. Subjects who were CLAD-free for a minimum of 3 years post-transplant were 1:1 matched to CLAD patients and included 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 epithelial cell apoptosis and overall death, respectively), were measured in BAL obtained within 6-months post CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using 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 CLAD phenotype. Fifty-four CLAD (27 BOS, 11 RAS, 7 mixed, 9 others with RAS-like opacities) patients and 23 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. 88.7 U/L), MUC1 (6.8 vs. 3.2 pg/mL), and MUC16 (121.0 vs. 30.1 pg/mL). When comparing CLAD phenotypes, M30 was significantly higher in patients with RAS-related phenotypes than BOS (160.9 vs. 114.6 U/L). In multivariable models, higher M30 and MUC5B levels were associated with decreased allograft survival after CLAD onset independent of phenotype (P<0.05 for all). Airway epithelial mucins and cell death markers are enhanced in the BAL of patients with CLAD and can assist in differentiating between CLAD phenotypes and post-CLAD outcomes. Abnormal airway mucin expression and epithelial cell death may be involved in the pathogenesis of CLAD, and therefore their detection may aid in future selection of targeted therapies.

Interstitial lung diseases after hematopoietic stem cell transplantation: New pattern of lung chronic graft-versus-host disease?

Bronchiolitis obliterans syndrome (BOS) after allogeneic HSCT is the only formally recognized manifestation of lung chronic graft-versus-host disease (GVHD). Other lung complications were reported, including interstitial lung diseases (ILDs). Whether ILDs belong to the spectrum of lung cGVHD remains unknown. We compared characteristics and specific risk factors for both ILD and BOS. Data collected from consecutive patients diagnosed with ILD or BOS from 1981–2019 were analyzed. The strength of the association between patient characteristics and ILD occurrence was measured via odds ratios estimated from univariable logistic models. Multivariable models allowed us to handle potential confounding variables. Overall survival (OS) was estimated using the Kaplan-Meier method. 238 patients were included: 79 with ILD and 159 with BOS. At diagnosis, FEV1 was lower in patients with BOS compared to patients with ILD, while DLCO was lower in ILD. 84% of ILD patients received systemic corticosteroids, leading to improved CT scans and pulmonary function, whereas most BOS patients were treated by inhaled corticosteroids, with lung-function stabilization. In the multivariable analysis, prior thoracic irradiation and absence of prior treatment with prednisone were associated with ILD. OS was similar, even if hematological relapse was more frequent in the ILD group. Both complications occurred mainly in patients with GVHD history.

Transition from BOS to RAS impairs prognosis after lung transplantation—CLAD subtype analysis by CT volumetry

BackgroundChronic lung allograft dysfunction (CLAD), subclassified into bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS), limits survival after lung transplantation. Information concerning transition from BOS to RAS is limited. We aimed to characterize the lung volume change after BOS diagnosis by computed tomography (CT) volumetry and to determine the incidence, risk factors and clinical significance of BOS to RAS transition.MethodsCT volumetry measurements were performed from 63 patients with CLAD initially classified as BOS by CT volumetry. BOS patients with lung volume remaining >85% of baseline were classified as persistent BOS, whereas BOS patients whose lung volume permanently decreased to ≤85% of baseline were classified as BOS to RAS transition.ResultsDuring follow-up (median 9.8 years) eight patients (12.7%) were classified as BOS to RAS transition, which decreased recipient (p = 0.004) and graft survival (p = 0.020) in comparison to patients with persistent BOS. Opacities on chest imaging preceded BOS to RAS transition in 88% of patients. Opacities on chest imaging at BOS diagnosis and early CLAD diagnosis after transplantation were risk factors for transition.ConclusionBased on lung volume decrease measured by CT volumetry, a small proportion of BOS patients transitioned to RAS which had an adverse effect on recipient and graft survival.

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.

Reprogramming alveolar macrophage responses to TGF-β reveals CCR2+ monocyte activity that promotes bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients, but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-β bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-β and reduce TGF-β bioavailability through secretion of the TGF-β antagonist decorin. In untreated recipients, high airway TGF-β activity stimulated AMs to express CCL2, leading to CCR2+ monocyte-driven BOS development. Moreover, we found TGF-β receptor 2–dependent differentiation of CCR2+ monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8+ T cells that inflicted airway injury through Blimp-1–mediated granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-β–dependent network that couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS.

Epidemiology of Lower Respiratory Tract Infections and Community-Acquired Respiratory Viruses in Patients with Bronchiolitis Obliterans Syndrome after Hematopoietic Cell Transplantation: A Retrospective Cohort Study

Bronchiolitis obliterans syndrome (BOS)-chronic graft-versus-host disease (cGVHD) affecting the lungs-is an uncommon complication of allogeneic hematopoietic cell transplant (HCT). The epidemiology and complications of lower respiratory tract infections (LRTIs) and community-acquired respiratory viruses (CARVs) in these patients are poorly understood. We aim to characterize the epidemiology of LRTIs in patients with BOS complicating HCT. We also aim to explore the association of LRTIs and CARV detection on lung function in BOS patients. Adult patients with BOS at Stanford Health Care between January 2010 and December 2019 were included in this retrospective cohort study. LRTI diagnosis was based on combined clinical, microbiologic, and radiographic criteria, using consensus criteria where available. Fifty-five patients with BOS were included. BOS was diagnosed at a median of 19.2 (IQR 12.5-24.7) months after HCT, and patients were followed for a median of 29.3 (IQR 9.9-53.2) months from BOS diagnosis. Twenty-two (40%) patients died after BOS diagnosis; 17 patients died from complications of cGVHD (including respiratory failure and infection) and 5 died from relapsed disease. Thirty-four (61.8%) patients developed at least one LRTI. Viral LRTIs were most common, occurring in 29 (52.7%) patients, primarily due to rhinovirus. Bacterial LRTIs-excluding Nocardia and non-tuberculous mycobacteria (NTM)-were the second most common, occurring in 21 (38.2%) patients, mostly due to Pseudomonas aeruginosa. Fungal LRTIs, NTM, and nocardiosis occurred in 14 (25.5%), 10 (18.2%), and 4 (7.3%) patients, respectively. Median time to development of the first LRTI after BOS diagnosis was 15.3 (4.7-44.7) months. Twenty-six (76.5%) of the 34 patients who developed LRTIs had infections due to more than one type of organism-fungi, viruses, Nocardia, NTM, and other bacteria-over the observation period. Patients with at least one LRTI had significantly lower forced expiratory volume in one second percent predicted (FEV1%) (37% vs. 53%, p = 0.0096) and diffusing capacity of carbon monoxide (DLCO) (45.5% predicted vs. 69% predicted, p = 0.0001). Patients with at least one LRTI trended toward lower overall survival (OS) (p = 0.0899) and higher non-relapse mortality (NRM) (p = 0.2707). Patients with a CARV detected or LRTI diagnosed after BOS-compared to those without any CARV detected or LRTI diagnosed-were more likely to have a sustained drop in FEV1% from baseline of at least 10% (21 [61.8%] versus 7 [33.3%]) and a sustained drop in FEV1% of at least 30% (12 [36.4%] versus 2 [9.5%]). LRTIs are common in BOS and associated with lower FEV1%, lower DLCO, and a trend toward decreased OS and higher NRM. Patients with LRTIs or CARVs (even absent lower respiratory tract involvement) were more likely to have substantial declines in FEV1% over time than those without. The array of organisms-including P. aeruginosa, mold, Nocardia, NTM, and CARVs-seen in BOS reflects the unique pathophysiology of this form of cGVHD, involving both systemic immunodeficiency and structural lung disease. These patterns of LRTIs and their outcomes can be used to guide clinical decisions and inform future research.

Ruxolitinib as an Effective and Steroid-Sparing First-Line Treatment in Newly Diagnosed BOS Patients After Hematopoietic Stem Cell Transplantation.

Bronchiolitis obliterans syndrome (BOS) is a life-threatening pulmonary complication of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we retrospectively identified seven patients newly diagnosed with BOS post HSCT and analyzed the outcomes in those patients treated with ruxolitinib as a first-line treatment. All seven patients achieved symptom responses within 2 weeks after ruxolitinib administration. Three months after treatment, five patients (71.43%) achieved a CR, and two (28.57%) achieved a PR. The overall response rate (ORR) was 100%. In addition, the steroid therapy was determined within 2 months after ruxolitinib treatment, indicating ruxolitinib as a steroid-sparing agent. We also found that ruxolitinib was well-tolerated and safe in treating newly diagnosed BOS. According to our results, ruxolitinib would be a promising and safe option in newly diagnosed BOS post HSCT.

Corticotropin releasing hormone as an identifier of bronchiolitis obliterans syndrome

Lung transplantion (LTx) recipients have low long-term survival and a high incidence of bronchiolitis obliterans syndrome (BOS), an inflammation of the small airways in chronic rejection of a lung allograft. There is great clinical need for a minimally invasive biomarker of BOS. Here, 644 different proteins were analyzed to detect biomarkers that distinguish BOS grade 0 from grades 1–3. The plasma of 46 double lung transplant patients was analyzed for proteins using a high-component, multiplex immunoassay that enables analysis of protein biomarkers. Proximity Extension Assay (PEA) consists of antibody probe pairs which bind to targets. The resulting polymerase chain reaction (PCR) reporter sequence can be quantified by real-time PCR. Samples were collected at baseline and 1-year post transplantation. Enzyme-linked immunosorbent assay (ELISA) was used to validate the findings of the PEA analysis across both time points and microarray datasets from other lung transplantation centers demonstrated the same findings. Significant decreases in the plasma protein levels of CRH, FERC2, IL-20RA, TNFB, and IGSF3 and an increase in MMP-9 and CTSL1 were seen in patients who developed BOS compared to those who did not. In this study, CRH is presented as a novel potential biomarker in the progression of disease because of its decreased levels in patients across all BOS grades. Additionally, biomarkers involving the remodeling of the extracellular matrix (ECM), such as MMP-9 and CTSL1, were increased in BOS patients.

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.

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.

Mechanisms of Action of Extracorporeal Photopheresis in the Control of Bronchiolitis Obliterans Syndrome (BOS): Involvement of Circulating miRNAs.

Clinical evidence suggests an improvement or stabilization of lung function in a fraction of patients with bronchiolitis obliterans syndrome (BOS) treated by extracorporeal photopheresis (ECP); however, few studies have explored the epigenetic and molecular regulation of this therapy. The aim of present study was to evaluate whether a specific set of miRNAs were significantly regulated by ECP. Total RNA was isolated from serum of patients with established BOS grade 1–2 prior to the start and after 6 months of ECP treatment. We observed a significant downregulation of circulating hsa-miR-155-5p, hsa-miR-146a-5p and hsa-miR-31-5p in BOS patients at the start of ECP when compared to healthy subjects. In responders, increased miR-155-5p and decreased miR-23b-3p expression levels at 6 months were found. SMAD4 mRNA was found to be a common target of these two miRNAs in prediction pathways analysis, and a significant downregulation was found at 6 months in PBMCs of a subgroup of ECP-treated patients. According to previous evidence, the upregulation of miR-155 might be correlated with a pro-tolerogenic modulation of the immune system. Our analysis also suggests that SMAD4 might be a possible target for miR-155-5p. Further longitudinal studies are needed to address the possible role of miR-155 and its downstream targets.

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.

Beyond Bronchiolitis Obliterans: In-Depth Histopathologic Characterization of Bronchiolitis Obliterans Syndrome after Lung Transplantation.

Bronchiolitis obliterans syndrome (BOS) is considered an airway-centered disease, with bronchiolitis obliterans (BO) as pathologic hallmark. However, the histologic spectrum of pure clinical BOS remains poorly characterized. We provide the first in-depth histopathologic description of well-characterized BOS patients and patients without chronic lung allograft dysfunction (CLAD), defined according to the recent consensus guidelines. Explant lung tissue from 52 clinically-defined BOS and 26 non-CLAD patients (collected 1993–2018) was analyzed for histologic parameters, including but not limited to airway lesions, vasculopathy and fibrosis. In BOS, BO lesions were evident in 38 (73%) patients and varied from concentric sub-epithelial fibrotic BO to inflammatory BO, while 10/14 patients without BO displayed ‘vanishing airways’, defined by a discordance between arteries and airways. Chronic vascular abnormalities were detected in 22 (42%) patients. Ashcroft fibrosis scores revealed a median of 43% (IQR: 23–69) of normal lung parenchyma per patient; 26% (IQR: 18–37) of minimal alveolar fibrous thickening; and 11% (IQR: 4–18) of moderate alveolar thickening without architectural damage. Patchy areas of definite fibrotic damage to the lung structure (i.e., Ashcroft score ≥5) were present in 28 (54%) patients. Fibrosis was classified as bronchocentric (n = 21/28, 75%), paraseptal (n = 17/28, 61%) and subpleural (n = 15/28, 54%). In non-CLAD patients, BO lesions were absent, chronic vascular abnormalities present in 1 (4%) patient and mean Ashcroft scores were significantly lower compared to BOS (p = 0.0038) with 78% (IQR: 64–88) normally preserved lung parenchyma. BOS explant lungs revealed evidence of various histopathologic findings, including vasculopathy and fibrotic changes, which may contribute to the pathophysiology of BOS.

Retrospective Analysis of Bronchiolitis Obliterans Syndrome after Allogeneic Hematopoietic Stem Cell Transplantation

Objective: To explore the incidence, clinical features,risk factors and survival prognosis of bronchiolitis obliterans syndrome (BOS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by using large samples form a single center.Provide a prerequisite for improving the early diagnosis of BOS after allo-HSCT.Methods: We retrospectively investigated the case data of 802 patients who underwent allo-HSCT in our institute in petiod of January 1, 2015 to December 31, 2019 and survived more than 100 days. Adopt a multidisciplinary discussion method in the Department of Hematology, Respiratory and Radiology,according to the 2014 National Institutes of Health (NIH) ,diagnose and assess the severity of patiencts with BOS.Describe the clinical characteristics of BOS, and then analyze the risk factors and survival prognosis.Result: Among the 802 patients，46 patients（5.74%), 26males and 20 females with a median age of 32 years was diagnosed BOS.The median time to first appearance of respiratory symptoms was 13.5 months after transplantation, and the median time to diagnosis was 14.4 months.The cumulative incidence rate for 1- year, 2- year, and 5 -year after allo-HSCT is 3.7%, 6.5%, and 8.4%,while the cumulative incidence rate for patients already diagnosed with cGVHD is 7.5%、12.4% and 15.0% . Univariate analysis showed several risk factors associated with the onset of BOS,including the presence of preoperative or postoperative pneumonia,free of ATG ,use of DLI,peripheral blood stem cell transplantation, occurrence of GVHD and the severity and number of these episodes .And multivariate analysis denotes :free of ATG ,use of DLI,and number of organs affected by cGVHD were the variables correlated with increased incidences of BOS. At the onset of BOS, FEV1, FEV1%pred, FEV1/FVC ,FEV1/FVC %pred were significantly lower than those before transplantation. Compare before and after onset, the average decrease rate of FEV1 is 0.17L/month, while FEV1%pred is 5.15%/month.We also recorded the last follow-up lung function to evaluate the treatment efficacy : after treatment, the average FEV1 was 2.46L, the average of FEV1%pred was 36.61%, the average of FEV1/FVC was 45.80%, and the average of FEV1/FVC%pred was 56.36%, before and after treatment., FEV1%pred has no statistical difference (P=0.455).Not only that, there was no statistical difference in the treatment effect among different severity of BOS.The median follow-up time after the diagnosis of BOS was 19 months, and there was no statistical difference in the survival rate between the BOS and non-BOS groups at the end of the follow-up. The analysis result shows that early-onset BOS is a independent risk factors for poor survival time of BOS.Compare BOS with other non-infectious lung complications,the severity of BOS and cGVHD involving extrapulmonary organs are statistically different.Conclusion: BOS is a serious refractory complication after allo-HSCT.Early detection of potential patients with BOS is very difficult , and because most of the BOS patients did not respond to the therapy well, eventually lead to the irreversibility of deterioration of PFT. Compared with other non-infectious lung complications, strong association with severe cGVHD and deterioration of lung function characterized by obstructive ventilation disorder，etc.It suggests that the clinical need to deepen the recognition of BOS, continue to explore the early diagnosis method of BOS , more importantly , strive to give effective treatment at the early stage to improve the prognosis.KEYWORDS Bronchiolitis obliterans Allogeneic hematopoietic stem cell transplantation Risk factors Non-infectious pulmonary complications DisclosuresNo relevant conflicts of interest to declare.

Clinical Analysis of Air-Leak Syndrome Following Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients

Background: Air-leak syndrome (ALS) occurs when there is leakage of gas from the alveoli, which results in clinical symptoms including cough and sputum, dyspnea, and hypoxemia. ALS is rare but potentially life-threatening in patients who have received an allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is an independent prognosis factor of patients undergoing HSCT. However, the clinical features and risk factors for post-transplant ALS among pediatric patients have rarely been explored.Aims: This study analyzed the clinical characteristics, risk factors, clinical treatment options and prognosis to provide a scientific basis for ALS prevention and treatment for pediatric patients.Methods: We retrospectively reviewed patients who were diagnosed with ALS following allo-HSCT between January 2013 and December 2019 at the Hebei Yanda Lu Daopei Hospital, and analyzed the role of ALS in the prognosis of the HSCT.Results: A total of 2026 pediatric patients received an HSCT between January 2013 and December 2019. The ALS incidence rate was 1.4% (28 of 2,206 patients) with a survival rate of 64.3% (10 of 28 patients). The median overall survival (OS) time was 429 days (range: 55-1614 days). Sixteen patients were males and 12 were female. The median patients age was 12 years old (range: 1-16 years), and the median follow-up time was 871 days (range: 55-2973 days).We divided ALS into two categories: 15 cases of bronchiolitis obliterans syndrome (BOS) and 13 cases of idiopathic pneumonia syndrome (IPS). There was a significant difference in OS between the twogroups (80% among BOS patients versus 46% among IPS patients; P=0.037). Logical regression analysis showed that gender, an unmatched transplant donor and recipients (P=0.049), time to first occurrence of graft-versus-host disease after transplantation (P=0.021), and dosage of methylprednisolone >0.5mg/kg at the onset of ALS (P=0.049), were independent risk factors for poor prognosis in ALS. In addition, we found fluticasone, azithromycin, and montelukast (FAM) could significantly improve the prognosis following ALS (P=0.005). Compared with IPS, our results showed that some patients with BOS may benefit from imatinib (P=0.055), ruxolitinib (P=0.009), or pirfenidone (P=0.044).Conclusion: ALS is a rare manifestation of pulmonary complications following HSCT among pediatric patients. Our analysis demonstrates that early diagnosis and FAM treatment may improve the survival rate of ALS following HSCT among pediatric patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.