The effects of inhaled corticosteroids on healthy airways.

BACKGROUND: The effects of inhaled corticosteroids (ICS) on healthy airways are poorly defined. OBJECTIVES: To delineate the effects of ICS on gene expression in healthy airways, without confounding caused by changes in disease-related genes and disease-related alterations in ICS responsiveness. METHODS: Randomized open-label bronchoscopy study of high-dose ICS therapy in 30 healthy adult volunteers randomized 2:1 to (i) fluticasone propionate 500 mcg bd daily or (ii) no treatment, for 4 weeks. Laboratory staff were blinded to allocation. Biopsies and brushings were analysed by immunohistochemistry, bulk RNA sequencing, DNA methylation array and metagenomics. RESULTS: ICS induced small between-group differences in blood and lamina propria eosinophil numbers, but not in other immunopathological features, blood neutrophils, FeNO, FEV1, microbiome or DNA methylation. ICS treatment upregulated 72 genes in brushings and 53 genes in biopsies, and downregulated 82 genes in brushings and 416 genes in biopsies. The most downregulated genes in both tissues were canonical markers of type-2 inflammation (FCER1A, CPA3, IL33, CLEC10A, SERPINB10 and CCR5), T cell-mediated adaptive immunity (TARP, TRBC1, TRBC2, PTPN22, TRAC, CD2, CD8A, HLA-DQB2, CD96, PTPN7), B-cell immunity (CD20, immunoglobulin heavy and light chains) and innate immunity, including CD48, Hobit, RANTES, Langerin and GFI1. An IL-17-dependent gene signature was not upregulated by ICS. CONCLUSIONS: In healthy airways, 4-week ICS exposure reduces gene expression related to both innate and adaptive immunity, and reduces markers of type-2 inflammation. This implies that homeostasis in health involves tonic type-2 signalling in the airway mucosa, which is exquisitely sensitive to ICS.

Visualization of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness.

Acute cardiorespiratory breathlessness accounts for one in eight of all emergency hospitalizations. Early, noninvasive diagnostic testing is a clinical priority that allows rapid triage and treatment. Here, we sought to find and replicate diagnostic breath volatile organic compound (VOC) biomarkers of acute cardiorespiratory disease and understand breath metabolite network enrichment in acute disease, with a view to gaining mechanistic insight of breath biochemical derangements. We collected and analyzed exhaled breath samples from 277 participants presenting acute cardiorespiratory exacerbations and aged-matched healthy volunteers. Topological data analysis phenotypes differentiated acute disease from health and acute cardiorespiratory exacerbation subtypes (acute heart failure, acute asthma, acute chronic obstructive pulmonary disease, and community-acquired pneumonia). A multibiomarker score (101 breath biomarkers) demonstrated good diagnostic sensitivity and specificity (≥80%) in both discovery and replication sets and was associated with all-cause mortality at 2 years. In addition, VOC biomarker scores differentiated metabolic subgroups of cardiorespiratory exacerbation. Louvain clustering of VOCs coupled with metabolite enrichment and similarity assessment revealed highly specific enrichment patterns in all acute disease subgroups, for example, selective enrichment of correlated C5-7 hydrocarbons and C3-5 carbonyls in heart failure and selective depletion of correlated aldehydes in acute asthma. This study identified breath VOCs that differentiate acute cardiorespiratory exacerbations and associated subtypes and metabolic clusters of disease-associated VOCs.

Airway remodelling rather than cellular infiltration characterizes both type2 cytokine biomarker-high and -low severe asthma.

BACKGROUND: The most recognizable phenotype of severe asthma comprises people who are blood eosinophil and FeNO-high, driven by type 2 (T2) cytokine biology, which responds to targeted biological therapies. However, in many people with severe asthma, these T2 biomarkers are suppressed but poorly controlled asthma persists. The mechanisms driving asthma in the absence of T2 biology are poorly understood. OBJECTIVES: To explore airway pathology in T2 biomarker-high and -low severe asthma. METHODS: T2 biomarker-high severe asthma (T2-high, n = 17) was compared with biomarker-intermediate (T2-intermediate, n = 21) and biomarker-low (T2-low, n = 20) severe asthma and healthy controls (n = 28). Bronchoscopy samples were processed for immunohistochemistry, and sputum for cytokines, PGD2 and LTE4 measurements. RESULTS: Tissue eosinophil, neutrophil and mast cell counts were similar across severe asthma phenotypes and not increased when compared to healthy controls. In contrast, the remodelling features of airway smooth muscle mass and MUC5AC expression were increased in all asthma groups compared with health, but similar across asthma subgroups. Submucosal glands were increased in T2-intermediate and T2-low asthma. In spite of similar tissue cellular inflammation, sputum IL-4, IL-5 and CCL26 were increased in T2-high versus T2-low asthma, and several further T2-associated cytokines, PGD2 and LTE4 , were increased in T2-high and T2-intermediate asthma compared with healthy controls. CONCLUSIONS: Eosinophilic tissue inflammation within proximal airways is suppressed in T2 biomarker-high and T2-low severe asthma, but inflammatory and structural cell activation is present, with sputum T2-associated cytokines highest in T2 biomarker-high patients. Airway remodelling persists and may be important for residual disease expression beyond eosinophilic exacerbations. Registered at ClincialTrials.gov: NCT02883530.

Diagnosis of COVID-19 by exhaled breath analysis using gas chromatography-mass spectrometry.

BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic has claimed over two and a half million lives worldwide so far. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is perceived to be seasonally recurrent, and a rapid noninvasive biomarker to accurately diagnose patients early on in their disease course will be necessary to meet the operational demands for COVID-19 control in the coming years. OBJECTIVE: The aim of this study was to evaluate the role of exhaled breath volatile biomarkers in identifying patients with suspected or confirmed COVID-19 infection, based on their underlying PCR status and clinical probability. METHODS: A prospective, real-world, observational study was carried out, recruiting adult patients with suspected or confirmed COVID-19 infection. Breath samples were collected using a standard breath collection bag, modified with appropriate filters to comply with local infection control recommendations, and samples were analysed using gas chromatography-mass spectrometry (TD-GC-MS). RESULTS: 81 patients were recruited between April 29 and July 10, 2020, of whom 52 out of 81 (64%) tested positive for COVID-19 by reverse transcription-polymerase chain reaction (RT-PCR). A regression analysis identified a set of seven exhaled breath features (benzaldehyde, 1-propanol, 3,6-methylundecane, camphene, beta-cubebene, iodobenzene and an unidentified compound) that separated PCR-positive patients with an area under the curve (AUC): 0.836, sensitivity: 68%, specificity: 85%. CONCLUSIONS: GC-MS-detected exhaled breath biomarkers were able to identify PCR-positive COVID-19 patients. External replication of these compounds is warranted to validate these results.

Volatile organic compounds in a headspace sampling system and asthmatics sputum samples.

The headspace of a biological sample contains exogenous volatile organic compounds (VOCs) present within the sampling environment which represent the background signal. This study aimed to characterise the background signal generated from a headspace sampling system in a clinical site, to evaluate intra- and inter-day variation of background VOC and to understand the impact of a sample itself upon commonly reported background VOC using sputum headspace samples from severe asthmatics. The headspace, in absence of a biological sample, was collected hourly from 11am to 3pm within a day (time of clinical samples acquisition), and from Monday to Friday in a week, and analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Chemometric analysis identified 1120 features, 37 of which were present in at least the 80% of all the samples. The analyses of intra- and inter-day background variations were performed on 13 of the most abundant features, ubiquitously present in headspace samples. The concentration ratios relative to background were reported for the selected abundant VOC in 36 asthmatic sputum samples, acquired from 36 stable severe asthma patients recruited at Glenfield Hospital, Leicester, UK. The results identified no significant intra- or inter-day variations in compounds levels and no systematic bias ofz-scores, with the exclusion of benzothiazole, whose abundance increased linearly between 11am and 3pm with a maximal intra-day fold change of 2.13. Many of the identified background features are reported in literature as components of headspace of biological samples and are considered potential biomarkers for several diseases. The selected background features were identified in headspace of all severe asthma sputum samples, albeit with varying levels of enrichment relative to background. Our observations support the need to consider the background signal derived from the headspace sampling system when developing and validating headspace biomarker signatures using clinical samples.

A Feasibility Study of a Randomized Controlled Trial of Asthma-Tailored Pulmonary Rehabilitation Compared with Usual Care in Adults with Severe Asthma.

BACKGROUND: Currently, the acceptability and efficacy of pulmonary rehabilitation for adults with severe asthma is unknown. OBJECTIVE: To investigate the feasibility of performing a randomized controlled trial of asthma-tailored pulmonary rehabilitation (AT-PR) versus usual care (UC). METHODS: Adults with severe asthma were recruited and randomized 2:1 to AT-PR and UC. The primary outcomes were recruitment, retention, and serious adverse event rates. Secondary outcome measures included those for a future trial assessing the feasibility of collecting data. Assessments were performed at baseline, 12 weeks, and 9 months including measures of physical performance, health-related quality of life, and asthma control. A recruitment rate of 30% was estimated with 95% CI of ±7%, a retention rate of 75% ± 14% if we recruited 40 patients to AT-PR, and a serious adverse event rate of 2.5%. RESULTS: Sixty-one (26%) of 238 eligible patients were recruited (38 women; mean age, 54 ± 13 years; body mass index, 32 ± 7 kg/m2; FEV1, 1.9 ± 0.7 L; FEV1/forced vital capacity, 69% ± 11%). Fifty-one patients were randomized to AT-PR (n = 34) and UC (n = 17). The retention rate was 62% for the AT-PR group and 53% for the UC group, with a serious adverse event rate of 3.3% related to the study visits. Overall collection of the outcome measures was feasible. The results of the AT-PR group were suggestive of improvements in exercise performance, health-related quality of life, and asthma control, but the UC group results were either unchanged or worsened. CONCLUSIONS: Both recruitment and retention rates were within the a priori estimated 95% CI. Our results indicate that AT-PR may be efficacious for adults with severe asthma but any future intervention and trial design would need further modifications to improve acceptability and retention rate.

Severe exacerbations in moderate-to-severe asthmatics are associated with increased pro-inflammatory and type 1 mediators in sputum and serum.

BACKGROUND: Asthma is a heterogeneous disease and understanding this heterogeneity will enable the realisation of precision medicine. We sought to compare the sputum and serum inflammatory profiles in moderate-to-severe asthma during stable disease and exacerbation events. METHODS: We recruited 102 adults and 34 children with asthma. The adults were assessed at baseline, 3, 6, and 12-month follow-up visits. Thirty-seven subjects were assessed at onset of severe exacerbation. Forty sputum mediators and 43 serum mediators were measured. Receiver-operator characteristic (ROC) curves were constructed to identify mediators that distinguish between stable disease and exacerbation events. The strongest discriminating sputum mediators in the adults were validated in the children. RESULTS: The mediators that were significantly increased at exacerbations versus stable disease and by ≥1.5-fold were sputum IL-1ß, IL-6, IL-6R, IL-18, CXCL9, CXCL10, CCL5, TNFα, TNF-R1, TNF-R2, and CHTR and serum CXCL11. No mediators decreased ≥1.5-fold at exacerbation. The strongest discriminators of an exacerbation in adults (ROC area under the curve [AUC]) were sputum TNF-R2 0.69 (95% CI: 0.60 to 0.78) and IL-6R 0.68 (95% CI: 0.58 to 0.78). Sputum TNF-R2 and IL-6R were also discriminatory in children (ROC AUC 0.85 [95% CI: 0.71 to 0.99] and 0.80 [0.64 to 0.96] respectively). CONCLUSIONS: Severe asthma exacerbations are associated with increased pro-inflammatory and Type 1 (T1) immune mediators. In adults, sputum TNF-R2 and IL-6R were the strongest discriminators of an exacerbation, which were verified in children.

Lung Computational Models and the Role of the Small Airways in Asthma.

Rationale: Asthma is characterized by disease within the small airways. Several studies have suggested that forced oscillation technique-derived resistance at 5 Hz (R5) - resistance at 20 Hz (R20) is a measure of small airway disease; however, there has been limited validation of this measurement to date.Objectives: To validate the use of forced oscillation R5 - R20 as a measure of small airway narrowing in asthma, and to investigate the role that small airway narrowing plays in asthma.Methods: Patient-based complete conducting airway models were generated from computed tomography scans to simulate the impact of different degrees of airway narrowing at different levels of the airway tree on forced oscillation R5 - R20 (n = 31). The computational models were coupled with regression models in an asthmatic cohort (n = 177) to simulate the impact of small airway narrowing on asthma control and quality of life. The computational models were used to predict the impact on small airway narrowing of type-2 targeting biologics using pooled data from two similarly design randomized, placebo-controlled biologic trials (n = 137).Measurements and Main Results: Simulations demonstrated that narrowing of the small airways had a greater impact on R5 - R20 than narrowing of the larger airways and was associated (above a threshold of approximately 40% narrowing) with marked deterioration in both asthma control and asthma quality of life, above the minimal clinical important difference. The observed treatment effect on R5 - R20 in the pooled trials equated to a predicted small airway narrowing reversal of approximately 40%.Conclusions: We have demonstrated, using computational modeling, that forced oscillation R5 - R20 is a direct measure of anatomical narrowing in the small airways and that small airway narrowing has a marked impact on both asthma control and quality of life and may be modified by biologics.

Assessment of breath volatile organic compounds in acute cardiorespiratory breathlessness: a protocol describing a prospective real-world observational study.

INTRODUCTION: Patients presenting with acute undifferentiated breathlessness are commonly encountered in admissions units across the UK. Existing blood biomarkers have clinical utility in distinguishing patients with single organ pathologies but have poor discriminatory power in multifactorial presentations. Evaluation of volatile organic compounds (VOCs) in exhaled breath offers the potential to develop biomarkers of disease states that underpin acute cardiorespiratory breathlessness, owing to their proximity to the cardiorespiratory system. To date, there has been no systematic evaluation of VOC in acute cardiorespiratory breathlessness. The proposed study will seek to use both offline and online VOC technologies to evaluate the predictive value of VOC in identifying common conditions that present with acute cardiorespiratory breathlessness. METHODS AND ANALYSIS: A prospective real-world observational study carried out across three acute admissions units within Leicestershire. Participants with self-reported acute breathlessness, with a confirmed primary diagnosis of either acute heart failure, community-acquired pneumonia and acute exacerbation of asthma or chronic obstructive pulmonary disease will be recruited within 24 hours of admission. Additionally, school-age children admitted with severe asthma will be evaluated. All participants will undergo breath sampling on admission and on recovery following discharge. A range of online technologies including: proton transfer reaction mass spectrometry, gas chromatography ion mobility spectrometry, atmospheric pressure chemical ionisation-mass spectrometry and offline technologies including gas chromatography mass spectroscopy and comprehensive two-dimensional gas chromatography-mass spectrometry will be used for VOC discovery and replication. For offline technologies, a standardised CE-marked breath sampling device (ReCIVA) will be used. All recruited participants will be characterised using existing blood biomarkers including C reactive protein, brain-derived natriuretic peptide, troponin-I and blood eosinophil levels and further evaluated using a range of standardised questionnaires, lung function testing, sputum cell counts and other diagnostic tests pertinent to acute disease. ETHICS AND DISSEMINATION: The National Research Ethics Service Committee East Midlands has approved the study protocol (REC number: 16/LO/1747). Integrated Research Approval System (IRAS) 198921. Findings will be presented at academic conferences and published in peer-reviewed scientific journals. Dissemination will be facilitated via a partnership with the East Midlands Academic Health Sciences Network and via interaction with all UK-funded Medical Research Council and Engineering and Physical Sciences Research Council molecular pathology nodes. TRIAL REGISTRATION NUMBER: NCT03672994.

Airway pathological heterogeneity in asthma: Visualization of disease microclusters using topological data analysis.

BACKGROUND: Asthma is a complex chronic disease underpinned by pathological changes within the airway wall. How variations in structural airway pathology and cellular inflammation contribute to the expression and severity of asthma are poorly understood. OBJECTIVES: Therefore we evaluated pathological heterogeneity using topological data analysis (TDA) with the aim of visualizing disease clusters and microclusters. METHODS: A discovery population of 202 adult patients (142 asthmatic patients and 60 healthy subjects) and an external replication population (59 patients with severe asthma) were evaluated. Pathology and gene expression were examined in bronchial biopsy samples. TDA was applied by using pathological variables alone to create pathology-driven visual networks. RESULTS: In the discovery cohort TDA identified 4 groups/networks with multiple microclusters/regions of interest that were masked by group-level statistics. Specifically, TDA group 1 consisted of a high proportion of healthy subjects, with a microcluster representing a topological continuum connecting healthy subjects to patients with mild-to-moderate asthma. Three additional TDA groups with moderate-to-severe asthma (Airway Smooth MuscleHigh, Reticular Basement MembraneHigh, and RemodelingLow groups) were identified and contained numerous microclusters with varying pathological and clinical features. Mutually exclusive TH2 and TH17 tissue gene expression signatures were identified in all pathological groups. Discovery and external replication applied to the severe asthma subgroup identified only highly similar "pathological data shapes" through analyses of persistent homology. CONCLUSIONS: We have identified and replicated novel pathological phenotypes of asthma using TDA. Our methodology is applicable to other complex chronic diseases.

A CEACAM6-High Airway Neutrophil Phenotype and CEACAM6-High Epithelial Cells Are Features of Severe Asthma.

Severe asthma represents a major unmet clinical need; understanding the pathophysiology is essential for the development of new therapies. Using microarray analysis, we previously found three immunological clusters in asthma: Th2-high, Th17-high, and Th2/17-low. Although new therapies are emerging for Th2-high disease, identifying molecular pathways in Th2-low disease remains an important goal. Further interrogation of our previously described microarray dataset revealed upregulation of gene expression for carcinoembryonic Ag cell adhesion molecule (CEACAM) family members in the bronchi of patients with severe asthma. Our aim was therefore to explore the distribution and cellular localization of CEACAM6 using immunohistochemistry on bronchial biopsy tissue obtained from patients with mild-to-severe asthma and healthy control subjects. Human bronchial epithelial cells were used to investigate cytokine and corticosteroid in vitro regulation of CEACAM6 gene expression. CEACAM6 protein expression in bronchial biopsies was increased in airway epithelial cells and lamina propria inflammatory cells in severe asthma compared with healthy control subjects. CEACAM6 in the lamina propria was localized to neutrophils predominantly. Neutrophil density in the bronchial mucosa was similar across health and the spectrum of asthma severity, but the percentage of neutrophils expressing CEACAM6 was significantly increased in severe asthma, suggesting the presence of an altered neutrophil phenotype. CEACAM6 gene expression in cultured epithelial cells was upregulated by wounding and neutrophil elastase. In summary, CEACAM6 expression is increased in severe asthma and primarily associated with airway epithelial cells and tissue neutrophils. CEACAM6 may contribute to the pathology of treatment-resistant asthma via neutrophil and airway epithelial cell-dependent pathways.

Emphysema- and airway-dominant COPD phenotypes defined by standardised quantitative computed tomography.

EvA (Emphysema versus Airway disease) is a multicentre project to study mechanisms and identify biomarkers of emphysema and airway disease in chronic obstructive pulmonary disease (COPD). The objective of this study was to delineate objectively imaging-based emphysema-dominant and airway disease-dominant phenotypes using quantitative computed tomography (QCT) indices, standardised with a novel phantom-based approach.441 subjects with COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 1-3) were assessed in terms of clinical and physiological measurements, laboratory testing and standardised QCT indices of emphysema and airway wall geometry.QCT indices were influenced by scanner non-conformity, but standardisation significantly reduced variability (p<0.001) and led to more robust phenotypes. Four imaging-derived phenotypes were identified, reflecting "emphysema-dominant", "airway disease-dominant", "mixed" disease and "mild" disease. The emphysema-dominant group had significantly higher lung volumes, lower gas transfer coefficient, lower oxygen (PO2 ) and carbon dioxide (PCO2 ) tensions, higher haemoglobin and higher blood leukocyte numbers than the airway disease-dominant group.The utility of QCT for phenotyping in the setting of an international multicentre study is improved by standardisation. QCT indices of emphysema and airway disease can delineate within a population of patients with COPD, phenotypic groups that have typical clinical features known to be associated with emphysema-dominant and airway-dominant disease.

TH2 and TH17 inflammatory pathways are reciprocally regulated in asthma.

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.

COPD exacerbation severity and frequency is associated with impaired macrophage efferocytosis of eosinophils.

BACKGROUND: Eosinophilic airway inflammation is observed in 10-30% of COPD subjects. Whether increased eosinophils or impairment in their clearance by macrophages is associated with the severity and frequency of exacerbations is unknown. METHODS: We categorised 103 COPD subjects into 4 groups determined by the upper limit of normal for their cytoplasmic macrophage red hue (<6%), an indirect measure of macrophage efferocytosis of eosinophils, and area under the curve sputum eosinophil count (≥ 3%/year). Eosinophil efferocytosis by monocyte-derived macrophages was studied in 17 COPD subjects and 8 normal controls. RESULTS: There were no differences in baseline lung function, health status or exacerbation frequency between the groups: A-low red hue, high sputum eosinophils (n=10), B-high red hue, high sputum eosinophils (n=16), C-low red hue, low sputum eosinophils (n=19) and D- high red hue, low sputum eosinophils (n=58). Positive bacterial culture was lower in groups A (10%) and B (6%) compared to C (44%) and D (21%) (p=0.01). The fall in FEV1 from stable to exacerbation was greatest in group A (ΔFEV1 [95 % CI] -0.41 L [-0.65 to -0.17]) versus group B (-0.16 L [-0.32 to -0.011]), C (-0.11 L [-0.23 to -0.002]) and D (-0.16 L [-0.22 to -0.10]; p=0.02). Macrophage efferocytosis of eosinophils was impaired in COPD versus controls (86 [75 to 92]% versus 93 [88 to 96]%; p=0.028); was most marked in group A (71 [70 to 84]%; p=0.0295) and was inversely correlated with exacerbation frequency (r=-0.63; p=0.006). CONCLUSIONS: Macrophage efferocytosis of eosinophils is impaired in COPD and is related to the severity and frequency of COPD exacerbations.

Lung function decline and variable airway inflammatory pattern: longitudinal analysis of severe asthma.

BACKGROUND: Eosinophilic airway inflammation measured by using induced sputum is an important treatment stratification tool in patients with severe asthma. In addition, sputum eosinophilia has been shown to be associated with severe exacerbations and airflow limitation. OBJECTIVES: We sought to identify whether eosinophilic inflammation in sputum is associated with FEV1 decrease in patients with severe asthma and whether we could identify subgroups of decrease behavior based on the variation of eosinophilic airway inflammation over time. METHODS: Ninety-seven patients with severe asthma from the Glenfield Asthma Cohort were followed up with scheduled 3-month visits; the median duration of follow-up and number of visits was 6 years (interquartile range, 5.6-7.6 years) and 2.7 visits per year. Induced sputum was analyzed for eosinophilic inflammation at scheduled visits. Linear mixed-effects models were used to identify variables associated with lung function and overall decrease. In addition, using individual patients' mean and SD sputum eosinophil percentages over time, a 2-step cluster analysis was performed to identify patient clusters with different rates of decrease. RESULTS: FEV1 decrease was -25.7 mL/y in the overall population. Postbronchodilator FEV1 was also dependent on exacerbations, age of onset, height, age, sex, and log10 sputum eosinophil percentages (P < .001). Three decrease patient clusters were identified: (1) noneosinophilic with low variation (mean decrease, -14.0 mL/y), (2) eosinophilic with high variation (mean decrease, -40.9 mL/y), and (3) hypereosinophilic with low variation (mean decrease in lung function, -19.2 mL/y). CONCLUSION: The amplitude of sputum eosinophilia was associated with postbronchodilator FEV1 in asthmatic patients. In contrast, high variability rather than the amplitude at baseline or over time of sputum eosinophils was associated with accelerated FEV1 decrease.