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The measurement of exhaled volatile organic compounds (VOCs) in exhaled breath (breathomics) represents an exciting biomarker matrix for airways disease, with early research indicating a sensitivity to airway inflammation. One of the key aspects to analytical validity for any clinical biomarker is an understanding of the short-term repeatability of measures. We collected exhaled breath samples on 5 consecutive days in 14 subjects with severe asthma who had undergone extensive clinical characterisation. Principal component analysis on VOC abundance across all breath samples revealed no variance due to the day of sampling. Samples from the same patients clustered together and there was some separation according to T2 inflammatory markers. The intra-subject and between-subject variability of each VOC was calculated across the 70 samples and identified 30.35% of VOCs to be erratic: variable between subjects but also variable in the same subject. Exclusion of these erratic VOCs from machine learning approaches revealed no apparent loss of structure to the underlying data or loss of relationship with salient clinical characteristics. Moreover, cluster evaluation by the silhouette coefficient indicates more distinct clustering. We are able to describe the short-term repeatability of breath samples in a severe asthma population and corroborate its sensitivity to airway inflammation. We also describe a novel variance-based feature selection tool that, when applied to larger clinical studies, could improve machine learning model predictions.

Background Sputum cytology is currently the gold standard to evaluate cellular inflammation in the airways and phenotyping patients with airways diseases. Sputum eosinophil proportions have been used to guide treatment for moderate to severe asthma. Furthermore, raised sputum neutrophils are associated with poor disease control and impaired lung function in both asthma and COPD and small airways disease in cystic fibrosis. However, induced-sputum analysis is subjective and resource heavy, requiring dedicated specialist processing and assessment; this limits its utility in most clinical settings. Indirect blood eosinophil measures have been adopted in clinical care. However, there are currently no good peripheral blood biomarkers of airway neutrophils. A resource-light sputum processing approach could thus help integrate induced sputum more readily into routine clinical care. New mechanical disruption (MD) methods can rapidly obtain viable single cell suspensions from sputum samples. Aims The aim of this study was to compare MD sputum processing to traditional methods for cell viability, granulocyte proportions and sputum cytokine analysis. Methods Sputum plugs were split and processed using traditional methods and the MD method, and samples were then compared. Results The MD method produced a homogeneous cell suspension in 62 s; 70 min faster than the standard method used. No significant difference was seen between the cell viability (p = 0.09), or the concentration of eosinophils (p = 0.83), neutrophils (p = 0.99) or interleukin-8 (p = 0.86) using MD. Conclusion This cost-effective method of sputum processing could provide a more pragmatic, sustainable means of directly monitoring the airway milieu. Therefore, we recommend this method be taken forward for further investigation.

Difficult asthma describes asthma in which comorbidities, inadequate treatment, suboptimal inhaler technique and/or poor adherence impede good asthma control. The association of anxiety and depression with difficult asthma outcomes (exacerbations, hospital admissions, asthma control, etc.) is unclear. This study assessed the clinical associations of anxiety and depression with difficult asthma outcomes in patients with a specialist diagnosis of difficult asthma. Using real-world data, we retrospectively phenotyped patients from the Wessex Asthma Cohort of Difficult Asthma (N = 441) using clinical diagnoses of anxiety and depression against those without anxiety or depression (controls). Additionally, we stratified patients by severity of psychological distress using the Hospital Anxiety and Depression Scale (HADS). We found that depression and/or anxiety were reported in 43.1% of subjects and were associated with worse disease-related questionnaire scores. Each psychological comorbidity group showed differential associations with difficult asthma outcomes. Anxiety alone (7.9%) was associated with dysfunctional breathing and more hospitalisations [anxiety, median (IQR): 0 (2) vs. controls: 0 (0)], while depression alone (11.6%) was associated with obesity and obstructive sleep apnoea. The dual anxiety and depression group (23.6%) displayed multimorbidity, worse asthma outcomes, female predominance and earlier asthma onset. Worse HADS-A scores in patients with anxiety were associated with worse subjective outcomes (questionnaire scores), while worse HADS-D scores in patients with depression were associated with worse objective (ICU admissions and maintenance oral corticosteroid requirements) and subjective outcomes. In conclusion, anxiety and depression are common in difficult asthma but exert differential detrimental effects. Difficult asthma patients with dual anxiety and depression experience worse asthma outcomes alongside worse measures of psychological distress. There is a severity-gradient association of HADS scores with worse difficult asthma outcomes. Collectively, our findings highlight the need for holistic, multidisciplinary approaches that promote early identification and management of anxiety and depression in difficult asthma patients.

Background: fungal sensitivity has been associated with severe asthma outcomes. However, the clinical implication of Aspergillus fumigatus sensitization in difficult-to-treat (or difficult) asthma is unclear.Objectives: to characterize the clinical implications of A fumigatus sensitization in a large difficult asthma cohort.Methods: participants who underwent both skin prick and specific IgE testing to A fumigatus (n = 318) from the longitudinal real-life Wessex AsThma CoHort of difficult asthma, United Kingdom, were characterized by A fumigatus sensitization (either positive skin prick test result or specific IgE) and allergic bronchopulmonary aspergillosis status using clinical/pathophysiological disease measures.Results: a fumigatus sensitization was found in 23.9% (76 of 318) of patients with difficult asthma. Compared with A fumigatus nonsensitized subjects, those with sensitization were significantly more often male (50% vs 31%), older (58 years) with longer asthma duration (33 years), higher maintenance oral corticosteroid (39.7%) and asthma biologic use (27.6%), raised current/maximum log10 total IgE+1 (2.43/2.72 IU/L), worse prebronchodilator airflow obstruction (FEV1 62.2% predicted, FEV1/forced vital capacity 61.2%, forced expiratory flow between 25% and 75% exhalation 30.9% predicted), and frequent radiological bronchiectasis (40%), but had less psychophysiologic comorbidities. Allergic bronchopulmonary aspergillosis diagnosis was associated with higher treatment needs and stronger eosinophilic signals. Factors independently associated with A fumigatus sensitization in difficult asthma included maintenance oral corticosteroid use (odds ratio [OR], 3.34) and maximum log10 total IgE+1 (OR, 4.30), whereas for allergic bronchopulmonary aspergillosis included maintenance oral corticosteroid use (OR, 6.98), maximum log10 total IgE+1 (OR, 4.65), and radiological bronchiectasis (OR, 4.08).Conclusions: a fumigatus sensitization in difficult asthma identifies a more severe form of airways disease associated with greater morbidity, treatment need, and airways dysfunction/damage, but fewer psychophysiologic comorbidities. Screening of A fumigatus status should be an early element in the comprehensive assessment of patients with difficult asthma.

Background: Interleukin 5, an archetypal type 2 cytokine, is fundamental to eosinophilic airways disease, considered to signal the bone marrow to promote eosinophil progenitor maturation and increase levels of eosinophils in the circulation. Aim: To investigate the relationship between airway IL-5 concentrations and inflammatory phenotypes of severe asthma. Methods: Healthy volunteers and biologic naive severe asthma patients were clinically characterised and had blood and induced sputum samples collected. Sputum was analysed for differential cell count and supernatant (PBS processed) proteins, measured by single plex ELISA. Eosinophilic asthma was defined as sputum eosinophils >2%. Between group comparisons were assessed by Kruskal Wallis and relationships between variables by spearman rank correlations. Results: 17 healthy controls, 53 non-eosinophilic and 26 eosinophilic patients with severe asthma participated in the study. In severe asthma, there were modest correlations between sputum IL-5 and sputum eosinophils (r = 0.519, p Discussion: Despite high levels of steroid treatment, there is persistent IL-5 generation within the airways of severe asthma.

Background: blood eosinophil measurement is essential for the phenotypic characterization of patients with difficult asthma and in determining eligibility for anti-IL-5/IL-5Rα biological therapies. However, assessing such measures over limited time spans may not reveal the true underlying eosinophilic phenotype, as treatment, including daily oral corticosteroid therapy, suppresses eosinophilic inflammation and asthma is intrinsically variable.Methods: we interrogated the electronic healthcare records of patients in the Wessex AsThma CoHort of difficult asthma (WATCH) study (UK). In 501 patients being evaluated in this tertiary care centre for difficult to control asthma, all requested full blood count test results in a 10-year retrospective period from the index WATCH assessment were investigated (n = 11,176).Results: in 235 biological therapy-naïve participants who had 10 or more measures in this time period, 40.3% were eosinophilic (blood eosinophils ≥300 cells/µl) at WATCH enrolment whilst an additional 43.1%, though not eosinophilic at enrolment, demonstrated eosinophilia at least once in the preceding decade. Persistent eosinophilia was associated with worse post-bronchodilator airway obstruction and higher Fractional exhaled Nitric Oxide (FeNO). In contrast, the 16.6% of patients who never demonstrated eosinophilia at this blood eosinophil threshold showed preserved lung function and lower markers of Type 2 inflammation.Conclusions: this highlights the central role that type 2 inflammation, as indicated by blood eosinophilia, has in difficult asthma and suggests that longitudinal electronic healthcare record analysis can be an important tool in clinical asthma phenotyping, providing insight that may help understand disease progression and better guide more specific treatment approaches.

Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. To determine T cell subsets and effector molecules that drive pathogenesis of severe asthma, we performed single-cell transcriptome analysis of >50,000 airway CD4+ T cells isolated from bronchoalveolar lavage (BAL) samples from 30 patients with mild and severe asthma. We observed striking heterogeneity in the nature of CD4+ T cells present in asthmatics' airways with tissue-resident memory (TRM) cells making a dominant contribution. Notably, in severe asthmatics a subset of CD4+ TRM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4+ T cells in the airways of male patients with severe asthma when compared to mild asthma (13%). This subset was enriched for transcripts linked to T cell receptor (TCR) activation (HLA-DRB1, HLA-DPA1, CD40LG) and cytotoxicity (GZMB, GZMH), and following stimulation expressed high levels of transcripts encoding for pro-inflammatory non-TH2 cytokines (CCL3, CCL4, CCL5, TNF, LIGHT) that could fuel persistent airway inflammation and remodeling. Our findings indicate the need to look beyond the traditional T2 model of severe asthma to better understand the heterogeneity of this disease. This work was supported by (i) NIH research grant R01HL114093 (PV) and equipment grants (S10RR027366 - BD FACSAria Fusion, and S10OD025052 - Illumina Novaseq 6000); the William K. Bowes Jr. Foundation (P.V.). The WATCH study is supported by the Southampton NIHR Biomedical Research Centre and the Southampton NIHR Clinical Research Facility which are funded by the NIHR and are a partnership between the University of Southampton and University Hospital Southampton NHS Foundation Trust.

Introduction: Maintenance OCS use in severe asthma (SA) is associated with significant side-effects and has the potential to mask the systemic type-2 inflammatory biomarker signal required for initiation of biologic therapies. Method: WSAC is a large published cohort of 342 well characterised SA patients fulfilling the ATS/ERS 2014 definition of SA undertaken prior to the widespread use of biologic treatments. We compared the clinical, physiological & biological characteristics of patients requiring & not requiring maintenance OCS. Results: Discussion: Maintenance OCS use is associated with a significantly greater burden of comorbidities, poorer quality of life & disease control with more frequent exacerbations & ITU admissions. Despite this no significant differences were identified in lung function, atopic status, inflammatory cell counts or the other extensive biological measures assessed in the cohort. 44% of patients on maintenance OCS showed no biomarker signal of T2 inflammation. 77% of those on maintenance OCS with a sputum eos ≥2% had a blood eos count ≥0.3x109/L whilst only 60% had a FeNO ≥25ppb (43% FeNO ≥50ppb) suggesting blood eos remains a useful biomarker of T2 inflammation.

Background: In asthmatic patients, airway eosinophilia identifies a phenotype of patients that suffer from recurrent exacerbations and respond to biologic therapies. Direct measurement, through sputum induction, is impractical as part of routine clinical care so blood eosinophil counts are commonly used as a surrogate marker. We sought to assess the degree of concordance between these measures in our cohort of severe asthma patients. Methods: WATCH patients with severe asthma had blood tests, FeNO measurement and sputum induction performed on the same day. Spearman’s rank correlation coefficients and classification tables were calculated between sputum eosinophil, blood eosinophil, serum IgE and FeNO measures. Prediction of sputum eosinophilia (>3%) was assessed using receiver operating curves. Results: 203 patients, treated with high dose inhaled corticosteroids, were included in the analysis. Using commonly used cut-offs for blood eosinophilia (>300 cells/μL) and sputum eosinophilia (>3%), measurements were concordant in 138 patient (73.0%). 22 patients (11.6%) demonstrated blood eosinophilia alone and 29 patients (15.3%) sputum eosinophilia alone. Conclusions: We find that in a proportion of patients with severe asthma have sputum eosinophilia not recognised by blood. A better understanding of the nature of these airway eosinophils and the basis for the lack of apparent systemic IL5 signalling would help understand if these are a potentially biologically overlooked population or represent a different disease endotype.

Introduction: Depression is a well-recognised comorbidity in SA associated with poorer disease control & quality of life (QOL) but it remains unclear whether this is the consequence of altered pathobiology. Method: We compared the clinical, physiological & biological characteristics of patients with & without depression (defined by HADS depression score ≥8) within WSAC, a large published cohort of well characterised patients fulfilling the ATS/ERS 2014 definition of SA. Results: Discussion: SA patients with depression report poorer disease control & QOL associated with a greater burden of co-morbidities including obesity, rhinosinusitis & reflux. However, no statistically significant differences were identified in their other clinical or physiological characteristics. Inflammatory phenotypes did not differ between the groups but those with depression were found to have significantly higher serum hs-CRP & sputum IL-6 levels (not seen when subdivided for BMI). There is a well-recognised association between inflammation & depression with elevated serum IL-6 levels previously reported. Our findings raise the possibility that depression influences the pathobiology of asthma & thus reflects a targetable trait.

International audience; Background: Asthma is a diverse condition that differs with age and sex. However, it remains unclear how sex, age of asthma-onset, and/or their interaction, influence clinical expression of more problematic adult “difficult” asthma.Objectives: To better understand the clinical features of difficult asthma within a real-world clinical setting using novel phenotypic classification, stratifying subjects by sex and age of asthma-onset.Methods: Participants in a longitudinal difficult asthma clinical cohort study (Wessex AsThma CoHort of difficult asthma; WATCH), United Kingdom, (n=501) were stratified into 4 difficult asthma phenotypes based on sex and age of asthma-onset (early

BackgroundSevere asthma is associated with multiple comorbidities, including gastro-oesophageal reflux disease (GORD), which can contribute to exacerbation frequency and poor quality of life. Since epithelial dysfunction is an important feature in asthma, we hypothesised that in severe asthma the bronchial epithelium is more susceptible to the effects of acid reflux.MethodsWe developed an in vitro model of GORD using differentiated bronchial epithelial cells (BECs) from normal or severe asthmatic donors exposed to a combination of pepsin, acid pH and bile acids using a multiple challenge protocol (MCP-PAB). In addition, we analysed bronchial biopsies and undertook RNA sequencing of bronchial brushings from controls and severe asthmatics without or with GORD.ResultsExposure of BECs to the MCP-PAB caused structural disruption, increased permeability, interleukin (IL)-33 expression, inflammatory mediator release and changes in gene expression for multiple biological processes. Cultures from severe asthmatics were significantly more affected than those from healthy donors. Analysis of bronchial biopsies confirmed increased IL-33 expression in severe asthmatics with GORD. RNA sequencing of bronchial brushings from this group identified 15 of the top 37 dysregulated genes found in MCP-PAB treated BECs, including genes involved in oxidative stress responses.Conclusions and clinical implicationBy affecting epithelial permeability, GORD may increase exposure of the airway submucosa to allergens and pathogens, resulting in increased risk of inflammation and exacerbations. These results suggest the need for research into alternative therapeutic management of GORD in severe asthma.

Background: Fungal sensitisation is associated with asthma severity, but is not well described in difficult asthma. Aim: To assess the clinical characteristics of difficult asthma with Aspergillus fungal sensitisation in relation to asthma severity. Methods: Clinical characteristics of difficult asthma with Aspergillus fungal sensitisation, defined by positive Aspergillus skin test or serology, was assessed in the WATCH difficult asthma cohort. Results: 16.3% (80/490) had Aspergillus fungal sensitisation; 51.3% female, 48.8% male, with older mean ages (51y vs 61y). Median age of asthma diagnosis was 12.5 yrs, with disease duration of 34 yrs. Maintenance oral steroid use (48.8%) and mean number of acute courses in past year (3.0) were greater, whilst healthcare utilisation (20.3% ≥1 hospitalisation; 35.4% ≥1 ICU admission) and missed working days; p=0.25 were not significant. Poor asthma control was reflected by a mean ACQ-6 score 2.21, with significant difference in those with positive Aspergillus skin test; p=0.014. 28.2% had ABPA with 16.3% on itraconazole. Mean peripheral blood eosinophil count was 0.34; 44.1% being ≥0.3. Mean total IgE was raised; 684.2kU/L, with 26.3% on Omalizumab. There was greater airflow obstruction; FEV1 62.8%, FEV1/FVC 61.0%, FEF 25-75 35.9%, with mean FeNO 31.7 ppb. Conclusion: Aspergillus fungal sensitisation in difficult asthma was seen at an older age. Early-onset asthma with longer disease duration was prevalent. There was greater requirement for maintenance and acute oral steroids, but no significant impact on healthcare utilisation/missed working days. Poorer asthma control was observed, along with structural disease and greater airflow obstruction.

Background: The aim of this study was to evaluate the role of serum and sputum YKL-40 measurements as biomarkers in severe asthma phenotypes. Material and Methods: 243 adult subjects (48.9±14.2 years) from the Wessex Severe Asthma Cohort (WSAC), spirometry according to BTS/SIGN guidelines; sputum induction with inhaled hypertonic saline in 99 subjects. Serum and sputum levels of YKL-40 were determined by ELISA (Quidel corporation, San Diego, California, USA). Statistical analysis used SPSS (version 22) ; statistical significance at p Results: We observed correlation between markers of airway neutrophilia and sputum YKL-40 levels. Sputum YKL-40 was strongly correlated with both sputum MPO (Spearman’s rho 0.733; p≤0.001) and sputum absolute neutrophil count (Spearman’s rho 0.663; p≤0.001), but not serum levels of YKL-40. Sputum (p=0,025), but not serum(p=0,753), YKL-40 levels were also significantly different across the inflammatory cell subtypes in sputum. When comparing YKL-40 levels in serum and sputum between SARP clusters, serum ( p=0.002), but not sputum (p=0.081) YKL-40 levels were significantly different between phenotypes (namely differentiating clusters 1 and 2 from 3, 4 and 5). Conclusions: It is thus apparent that serum YKL-40 and sputum YKL-40 are reflecting different processes within the airways. Serum YKL-40 might reflect a more systemic inflammation opposed to sputum YKL-40 that might reflect more local: airway inflammation.

Rationale: Severe asthma is a heterogeneous disease with different inflammatory phenotypes based on blood or sputum measures. Increasingly blood eosinophils (Eos) are used to direct therapy choice but there is limited information how stable inflammatory measures in sputum and blood are over time and how they relate. Method: Sputum was successfully induced in 48 severe asthma patients at 2 timepoints within 2 years. At each timepoint a full blood count was completed. Patients were at least 4-weeks clear of exacerbation and considered stable. The percentage of granulocytes were reported and the stability at these two timepoints was assessed using Spearman’s rho. Results: In this cohort the sputum Eos and blood Eos significantly correlated between the two timepoints (sputum r=0.739,p= Discussion: These results demonstrate that airway neutrophilic inflammation is more dynamic than eosinophilic inflammation and poorly reflected in peripheral blood. A greater understanding of the underlying factors responsible for airway neutrophilic changes is needed if progress is to be made in addressing this severe asthma phenotype.

Background Asthma is now widely recognised to be a heterogeneous disease. The last two decades have seen the identification of a number of biological targets and development of various novel therapies. Despite this, asthma still represents a significant health and economic burden worldwide. Why some individuals should continue to suffer remains unclear. Methods The Wessex Asthma Cohort of Difficult Asthma (WATCH) is an ongoing ‘real-life’, prospective study of patients in the University Hospital Southampton Foundation Trust (UHSFT) Difficult Asthma service. Research data capture is aligned with the extensive clinical characterisation required of a commissioned National Health Service (NHS) Specialist Centre for Severe Asthma. Data acquisition includes detailed clinical, health and disease-related questionnaires, anthropometry, allergy and lung function testing, radiological imaging (in a small subset) and collection of biological samples (blood, urine and sputum). Prospective data are captured in parallel to clinical follow up appointments, with data entered into a bespoke database. Discussion The pragmatic ongoing nature of the WATCH study allows comprehensive assessment of the real world clinical spectrum seen in a Specialist Asthma Centre and allows a longitudinal perspective of deeply phenotyped patients. It is anticipated that the WATCH cohort would act as a vehicle for potential collaborative asthma studies and will build upon our understanding of mechanisms underlying difficult asthma. Electronic supplementary material The online version of this article (10.1186/s12890-019-0862-2) contains supplementary material, which is available to authorized users.

Previous publications have highlighted the disparity between research trial populations and those in clinical practice, but it has not been established how this relates to randomised controlled trials (RCTs) of phenotype-targeted biological therapies in severe asthma.Detailed characterisation data for 342 severe asthma patients within the Wessex Severe Asthma Cohort (WSAC) was compared against comprehensive trial eligibility criteria for published phase IIB and phase III RCTs evaluating biological therapies in severe asthma since 2000.37 RCTs evaluating 20 biological therapies were identified. Only a median of 9.8% (range 3.5–17.5%) of severe asthma patients were found to be eligible for enrolment in the phase III trials. Stipulations for airflow obstruction, bronchodilator reversibility and smoking history excluded significant numbers of patients. A median of 78.9% (range 73.2–86.6%) of patients with severe eosinophilic asthma would have been excluded from participation in the phase III licensing trials of interleukin (IL)-5/IL-5R targeted therapies.Despite including only well characterised and optimally treated severe asthmatics under specialist care within the WSAC study, the vast majority were excluded from trial participation by criteria designed to re-confirm diagnostic labels rather than by biomarker criteria that predict the characteristic addressed by the treatment.

The search for biomarkers that can guide precision medicine in asthma, particularly those that can be translated to the clinic, has seen recent interest in exhaled volatile organic compounds (VOCs). Given the number of studies reporting “breathomics” findings and its growing integration in clinical trials, we performed a systematic review of the literature to summarise current evidence and understanding of breathomics technology in asthma.A PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses)-oriented systematic search was performed (CRD42017084145) of MEDLINE, Embase and the Cochrane databases to search for any reports that assessed exhaled VOCs in adult asthma patients, using the following terms (asthma AND (volatile organic compounds AND exhaled) OR breathomics).Two authors independently determined the eligibility of 2957 unique records, of which 66 underwent full-text review. Data extraction and risk of bias assessment was performed on the 22 studies deemed to fulfil the search criteria. The studies are described in terms of methodology and the evidence narratively summarised under the following clinical headings: diagnostics, phenotyping, treatment stratification, treatment monitoring and exacerbation prediction/assessment.Our review found that most studies were designed to assess diagnostic potential rather than focus on underlying biology or treatable traits. Results are generally limited by a lack of methodological standardisation and external validation and by insufficiently powered studies, but there is consistency across the literature that exhaled VOCs are sensitive to underlying inflammation. Modern studies are applying robust breath analysis workflows to large multi-centre study designs, which should unlock the full potential of measurement of exhaled volatile organic compounds in airways diseases such as asthma.

Background The role of IL-17 immunity is well established in patients with inflammatory diseases, such as psoriasis and inflammatory bowel disease, but not in asthmatic patients, in whom further study is required. Objective We sought to undertake a deep phenotyping study of asthmatic patients with upregulated IL-17 immunity. Methods Whole-genome transcriptomic analysis was performed by using epithelial brushings, bronchial biopsy specimens (91 asthmatic patients and 46 healthy control subjects), and whole blood samples (n = 498) from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. Gene signatures induced in vitro by IL-17 and IL-13 in bronchial epithelial cells were used to identify patients with IL-17–high and IL-13–high asthma phenotypes. Results Twenty-two of 91 patients were identified with IL-17, and 9 patients were identified with IL-13 gene signatures. The patients with IL-17–high asthma were characterized by risk of frequent exacerbations, airway (sputum and mucosal) neutrophilia, decreased lung microbiota diversity, and urinary biomarker evidence of activation of the thromboxane B2 pathway. In pathway analysis the differentially expressed genes in patients with IL-17-high asthma were shared with those reported as altered in psoriasis lesions and included genes regulating epithelial barrier function and defense mechanisms, such as IL1B, IL6, IL8, and β-defensin. Conclusion The IL-17–high asthma phenotype, characterized by bronchial epithelial dysfunction and upregulated antimicrobial and inflammatory response, resembles the immunophenotype of psoriasis, including activation of the thromboxane B2 pathway, which should be considered a biomarker for this phenotype in further studies, including clinical trials targeting IL-17.

Background CD48 is a membrane receptor (mCD48) on eosinophils and mast cells and exists in a soluble form (sCD48). CD48 has a pivotal role in murine asthma and in the proinflammatory interactions of mast cells with eosinophils via its ligand CD244. Thus, CD48 might be important in human asthma. Methods Therefore, two separate cohorts (IL and UK) comprising mild, moderate, and severe asthma and healthy volunteers were evaluated for blood leukocyte mCD48 expression and sCD48 in serum. Asthmatic bronchial biopsies were immunostained for CD48. sCD48 effect on CD244-dependent eosinophil activation was evaluated. Results Eosinophil mCD48 expression was significantly elevated in moderate while downregulated in severe asthma. mCD48 expression on B, T, and NK cells and monocytes in severe asthma was significantly increased. sCD48 levels were significantly higher in mild while reduced in severe asthma. sCD48 optimal cutoff values for differentiating asthma from health were identified as >1482 pg/ml (IL) and >1619 pg/ml (UK). In asthmatic bronchial biopsies, mCD48 was expressed predominantly by eosinophils. sCD48 inhibited anti-CD244-induced eosinophil activation. Conclusions mCD48 and sCD48 are differentially expressed in the peripheral blood of asthma patients of varying severity. sCD48 inhibits CD244-mediated eosinophil activation. These findings suggest that CD48 may play an important role in human asthma.

Background: Phenotypic differences between early-onset (EODA) and adult-onset (AODA) difficult asthma in adulthood are not well described. Aim: To characterise EODA and AODA in difficult asthma. Methods: The Wessex AsThma CoHort of difficult asthma (WATCH) is a longitudinal study at University Hospital Southampton (UHS) UK set up in 2015. To date, 380 patients are enrolled from the UHS tertiary difficult asthma clinic. Clinical features of EODA (age ≤18 yrs) and AODA (age >18 yrs) are presented here. Results: Of 368 patients with available data, 51.6% had EODA and 48.4% AODA (median age of onset 4.0 yrs v 40.5 yrs respectively (p EODA had a greater proportion with eczema ever (30.6% v 10.3% p Functional comorbidities were more prevalent in EODA; dysfunctional breathing (61.9% v 45.9% p=0.003), depression (42.9% v 32.1% p=0.045) and anxiety (40.0% v 24.4% p=0.003). Rhinitis, GORD and obesity did not differ, but COPD (14.3% v 3.7% p EODA had more ICU admissions in the past year (33.0% v 21.1% p=0.011) and intubation ever for asthma (17.1% v 9.7% p=0.040). Conclusion: EODA is commoner in females and associated with more atopy, functional comorbidity, biologic therapy and healthcare need. AODA is commoner in males associated with smoking and comorbid lung disease.

Background: Asthma prognosis is thought to be influenced in some part by age of onset and female hormonal status but the mechanisms remain unclear. Aim: To explore differences in clinical presentation between men and women in a population of adult onset difficult asthmatics. Methods: 380 patients aged 17-85 years were recruited into the Wessex AsThma CoHort of difficult asthma (WATCH). Asthma history, questionnaires and biological measurements were taken at enrolment. Those with onset ≥19 were split by sex to explore differences in disease. Results: Age of asthma diagnosis was available in 335 of 380 patients with 48% reporting diagnosis at 19 years or older. Men with adult onset difficult asthma had significantly later age of diagnosis and higher FeNO than women. Their perception of disease severity was less and BMI significantly lower than females. Men had higher IgE, lower propensity to salicylate sensitivity and greater level of airway obstruction though this was not specific to age of onset. Additional sex differences in lung function reported within the total WATCH cohort were not seen in this subset of difficult asthmatics. Significance a= Conclusion: Several features of difficult adult onset asthma differ by sex, suggesting potential sex-specific disease influences. This supports the need for further sex-specific studies that might result in new sex-stratified asthma treatments.

Background: The Wessex AsThma CoHort of difficult asthma (WATCH) is a large prospective observational cohort study established at University Hospital Southampton (UHS) UK in 2015. Patients are enrolled from the UHS tertiary difficult asthma clinic. Aim: WATCH aims to improve understanding of the nature of difficult asthma in clinical practice. Here we report enrolment clinical characteristics. Methods: Patients undergo extensive clinical characterisation and phenotyping at enrolment using collated historical data plus objective clinical measurements. 380 patients have been enrolled to date with annual follow-up thereafter. Results: (as below) Conclusion: The cohort predominantly consists of female, middle-aged, atopic and obese patients whose disease often began before adulthood. The cohort showed high prevalence for maintenance oral steroids and omalizumab. WATCH provides a well-characterised cohort of patients to facilitate real-life difficult asthma research.

Background: Difficult asthmatics with a history of smoking, experience increased lung reactivity. Associations between related comorbidities need further investigation. Aim: To evaluate the symptoms of difficult asthmatics in relation to smoking status, in the Wessex AsThma Cohort of difficult asthma (WATCH) Southampton, UK. Methods: The smoking status of 377 adult asthmatics in WATCH was assessed for asthma control, symptoms of hyperventilation and depression. A retrospective analysis compared smoking status of difficult asthmatics using Chi square (categorical data) and Kruskall-Wallis (continuous data). Results: 180 (48%) had a history of smoking, of whom 13 continued to smoke. Differences in smoking status were not associated with BMI, but did differ by sex, age and age at diagnosis. Asthma control and symptoms of hyperventilation in ex-smokers was comparable to that of never smokers. Current smoking was associated with increased symptoms of depression and decreased attentional control than ever smokers. Conclusion: Prevalence of smoking in difficult asthma is low, but is associated with lack of asthma and attentional control, hyperventilation and depression. These findings highlight the benefits of smoking cessation.

Background: Difficult asthma is associated with increased prevalence of gastro-oesophageal reflux disease (GORD). However, it is unclear how controlling comorbid GORD impacts difficult asthma. Aim: To evaluate the impact of effectively controlling GORD symptoms in difficult asthmatics in the Wessex AsThma Cohort of difficult asthma (WATCH) Southampton UK. Methods: A retrospective analysis was undertaken to compare the reported symptoms of GORD in 311 adult asthmatics on treatment for GORD. We compared patients with ongoing GORD symptoms against those with no symptoms using Chi square (categorical data) and Mann-Whitney U tests (continuous data). Results: 208 (67%) difficult asthmatics had GORD, of whom 175 were receiving treatment for GORD. Prevalence of symptomatic GORD in those having treatment did not differ by sex, but was associated with higher BMI, worse asthma control and symptoms of depression, rhinitis and cough. Conclusion: Inadequately controlled GORD in difficult asthma is associated with increased symptoms of asthma, and related comorbidities. This highlights multiple potential benefits of optimising GORD treatment in difficult asthmatics.

Background: Whether the nature of early-onset difficult adult asthma differs by sex remains unclear. Aim: To assess clinical characteristics of early-onset difficult asthma in adulthood stratified by sex. Method: The Wessex AsThma CoHort of difficult asthma (WATCH) at University Hospital Southampton (UHS) UK has enrolled 380 patients from the UHS tertiary difficult asthma clinics. We compared clinical characteristics between sexes in patients with early-onset difficult asthma (age of diagnosis ≤18 years). Results: 190 patients had early-onset difficult asthma; 72.1% female (F) and 27.9% male (M). Median age of diagnosis (F vs M) was 5.0 yrs v 3.0 yrs (p=0.033) with a disease duration of 30.0 yrs v 41.5 yrs (p=0.047). Significant differences in comorbidities (F vs M) existed for salicylate sensitivity (29.9% v 8.0% p=0.002), depression (49.6% v 23.8% p=0.004) and bronchiectasis (7.4% v 21.6% p=0.006). No significant differences were seen for atopy, rhinitis, GORD, obesity, other functional comorbidities (dysfunctional breathing, vocal cord dysfunction and anxiety) or healthcare utilisation. F had a higher prevalence for maintenance oral steroids use (41.6% v 24.5% p=0.029). M showed significantly greater airflow obstruction (M vs F); FEV1 67.9% pred. v 81.7%, FEV1/FVC 62.1% pred. v 71.8% and FEF25-75 35.9% pred. v 56.8% (p Conclusion: Early-onset difficult asthma in adulthood was predominantly female, but showed different features by sex. Females had higher prevalence of depression, salicylate sensitivity and steroid dependency, while males were diagnosed earlier, had higher smoking prevalence, worse lung function and associated bronchiectasis.

Background: Asthma is widely considered an eosinophilic disease however severe asthma is frequently non-eosinophilic. Treatment options can depend on airway cell counts but proximal and distal airways may not be concordant in terms of cell differential. The Wessex Severe Asthma Cohort (WSAC) recruited 342 participants with severe asthma for detailed phenotyping with some participants undergoing sputum induction and/or bronchoscopy with bronchoalveolar lavage (BAL). We aimed to use data from the WSAC to assess the agreement between sputum and BAL cell differentials. Methods: WSAC participants with severe asthma were included if they underwent both BAL and successful sputum induction. Correlation coefficients and classification tables were used to calculate the agreement between the proximal (sputum) and distal (BAL) compartments. Results: 38 participants were included in the analysis. Correlation coefficients were 0.429 for neutrophil count (p=0.007) and 0.569 for eosinophil count (p Conclusions: Sputum and BAL cell counts showed moderate correlation, but significant classification discrepancies were found when using recognised thresholds. Sampling of multiple compartments may be required for optimal assessment of individuals with severe asthma.

Background: In severe asthma, the presence of airway eosinophilia is a key “treatable trait”. Airway eosinophilia may be proximal or distal, however work on predictors of airway eosinophilia have focussed on the proximal compartment which is less invasive to sample. The Wessex Severe Asthma Cohort (WSAC) included 342 severe asthmatics for detailed phenotyping, including bronchoalveolar lavage (BAL) and sputum induction in some participants. We aimed to use data from WSAC to determine the most accurate non-invasive predictor of eosinophilia in participants with severe asthma. Method: Severe asthmatics in WSAC were included if they underwent BAL or successful sputum induction. The value of Fraction of Exhaled Nitric Oxide (FeNO), blood eosinophil count and periostin in predicting sputum or BAL eosinophilia (3% or 1% respectively) were assessed using Receiver Operating Characteristic (ROC) curves. Results: All three measures were more accurate at predicting BAL than sputum eosinophilia. FeNO demonstrated a higher predictive value than the other measures in both compartments. Optimal thresholds were similar across compartments for FeNO but more divergent for periostin and blood. Conclusion: Within this cohort, FeNO was the strongest predictor of sputum or BAL eosinophilia. All factors assessed were stronger at predicting BAL eosinophilia than sputum.

Background: Severe asthma is a complex amalgam of phenotypes and comorbidities. Most novel therapies target type 2 disease so determining the optimal treatment for each patient will likely require detailed phenotyping. The WSAC study recruited 491 participants split between severe asthma (n=342), mild asthma (n=69) and healthy controls (n=80) for detailed phenotyping; inclusion criteria were asthma diagnosed as per guidelines and at least 1 exacerbation in 12 months. Smokers and those with comorbidity were included in order to reflect clinical practice. We aimed to assess the differences in demographic and clinical characteristics between participants with eosinophilic and non-eosinophilic severe asthma in the WSAC. Methods: WSAC participants with severe asthma (BTS Step 4-5) were included for analysis if they underwent successful sputum induction. Demographics, medication use, comorbidities, FEV1, FeNO, Asthma Control Questionnaire 6 (ACQ6) score and exacerbation frequency were compared between eosinophilic and non-eosinophilic groups. Results: 210 participants provided sputum, of which 86 (41%) had sputum eosinophils over 3%. These participants were more likely to be male (52% vs 29%) and never smokers(59% vs 45%), had lower median FEV1(68% vs 77% predicted) and BMI(28.2 vs 31.8) but higher FeNO(32 vs 16ppb) and were less likely to use antidepressants(13% vs 27%) or reflux treatment(39% vs 55%) (all P Conclusion: Severe asthmatics with sputum eosinophilia despite high-dose ICS have poorer lung function but less obesity, depression and reflux.

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalised management approach can be provided.OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinico-physiological parameters and performed an -omics analysis of sputum.METHODS: Partition-around-medoid clustering was applied to a training set of 266 asthma participants from the European U-BIOPRED adult cohort using 8 pre-specified clinic-physiological variables. This was repeated in a separate validation set of 152 asthmatics. The clusters were compared based on sputum proteomic and transcriptomic data.RESULTS: Four reproducible and stable clusters of asthmatics were identified. The training set cluster T1 consists of well-controlled moderate-to-severe asthmatics, while cluster T2 is a group of late-onset severe asthmatics with history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of non-smokers. Cluster T4 is predominantly composed of obese female uncontrolled severe asthmatics with increased exacerbations, but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters, T2, T3 and T4, had higher sputum eosinophilia than T1 with no differences in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels.CONCLUSION: Clustering based on clinico-physiological parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.CLINICAL IMPLICATIONS: The definition of four distinct clusters of asthma linked to different pathobiological pathways provides a better template for the phenotyping and personalised treatment of severe asthma, where high unmet needs remain.CAPSULE SUMMARY: Unsupervised clustering of asthma on clinical features alone has led to the definition of four phenotypes. Sputum 'omics' analysis has revealed different biological pathways pointing towards potential new treatments.

WATCH is a longitudinal observational study of patients under the Difficult Asthma Clinic at University Hospital Southampton (UHS), UK. Enhanced asthma phenotyping in the study helps improve patient care, as well as identifying patients suitable for research. These patients may otherwise be missed due to divide between clinical and research goals. One objective was to measure the impact of large outpatient cohort recruitment on patient enrolment rates in asthma research. Method: Adult Severe asthmatics (British Thoracic Society (BTS) step 4 or 5) were recruited from the Difficult Asthma clinic at UHS. Monthly recruitment numbers are shown for adult severe asthmatics PreWATCH (Jun 2014-Dec 2014) and PostWATCH (Jun 2015-Dec 2015). Figure1Download figure | Open in new tab | Download powerpoint Results: Total number of patients recruited PreWATCH was 21. Total number of patients recruited in PostWATCH group was 201. This includes patients recruited to WATCH itself (n=161) but also those recruited to non-WATCH adult severe asthma studies (n=40). Percentage improvement in recruitment PostWATCH was 47.5%. Overall improvement in recruitment (including WATCH) was 111.67%. Conclusion: Preliminary analysis suggests improvement to overall recruitment following commencement of WATCH. Further planned analysis will include qualitative questionnaires, and will hopefully show both positive impact on recruitment rates, as well as improvements to patient care and clinic/research experience.

The WATCH Study is a longitudinal study of patients under the Difficult Asthma Clinic at University Hospital Southampton, UK. To create a better real life understanding of difficult asthma (British Thoracic Society (BTS) step 4/5), a process of clinical dataset compilation, meeting clinical and research needs, was adopted. One objective was to trial feasibility of longitudinal data collection in a large outpatient clinic cohort. Other aims included; a) Merging research and clinical objectives; meeting needs of the BTS Difficult Asthma registry plus data collation needed for research purposes b) Ensuring all eligible patients have the opportunity to participate in research, current and future. c) Gathering all relevant clinical data to support clinical phenotyping d) Ensuring data was collected in a time efficient manner Number of patients recruited to date (175). Recruitment rate (18.2 per month). Total recruitment failures; due to lack of asthma severity (10). Number declining participation (8). Number withdrawn (1) (no reason given). Using a method combining clinical and research services to collect longitudinal data from this large cohort of patients attending the Difficult Asthma clinic was feasible and successful. The majority of patients approached were consented, participated and stayed in the study.

Rationale: While sputum cell counts and proportions have been used to characterise asthma the utility of BAL in phenotyping asthmatic inflammation is less well understood. Method: BAL differential cell counts were obtained from 61 healthy volunteers(H) and 70 mild atopic asthmatics(MA),BTS step 1, 28 moderate asthmatics(MO), BTS step 3, and 81 severe asthmatics(SA), BTS steps 4-5. Median [IQR] percentage differential counts were calculated and group differences were analysed using Mann Whitney U test. Results H median% [IQR] MA median% [IQR] MO median% [IQR] SA median% [IQR] Macrophages 90.3 [83.3-94.1] 86.5 [80.7-91.0]b 81.8 [76.0-89.9]b 78.0 [56.0-90.4]a,c Neutrophils 1.5 [0.8-4.3] 2.0 [1.25-3.0] 2.4 [1.3-7.8] 4.5 [2.0-12.5]a,c,d Epithelial cells 3.6 [0.8-10.0] 7.0 [4.3-10.8] b 7.65 [5.1-17.6]b 9.5 [2.5-20.0]b Eosinophils 0.1 [0.0-0.5] 1.0 [0.3-2.7]a 0.60 [0.0-1.5]b 0.5 [0.1-1.5]a Lymphocytes 1.2 [0.3-3.1] 1.0 [0.3-1.5] 0.625 [0.0-1.5]b 0.5 [0.0-1.3]b,c •a p= Conclusion: Asthma, independent of severity, was associated with eosinophilic inflammation and increased epithelial shedding in BAL. Neutrophilic airway inflammation was, by contrast, only significantly different from health in severe asthma. In this respect, SA was also different from MA and MO. When the cut off for neutrophilic, eosinophilic and mixed granulocytic BAL was defined as median+2CI for the H patient population, 24% MA, 50% MO and 43% SA samples have results for one of these parameters that was outside of this normal range. These findings indicate the need for at a greater understanding of the relevance of distal airway inflammation to disease persistence in asthma.

Background: Surfactant protein D (SP-D) is an essential component of the innate immune defence against pathogens within the airways. In addition SP-D regulates allergic inflammation and promotes the removal of apoptotic cells. SP-D dysregulation is evident in several pulmonary diseases. Our aim was to investigate whether airway and serum levels of SP-D are altered in treatment-resistant severe asthma.Methods: SP-D concentrations were measured in matched serum and bronchoalveolar lavage (BAL) samples collected from 10 healthy controls (HC) and 50 asthmatics (22 mild [MA] and 28 severe [SA]). These samples were also evaluated by Western blots to investigate variations in SP-D size.Results: SP-D levels in BAL were significantly lower in SA compared to HC and MA (P < .001) and inversely correlated with BAL eosinophil cationic protein (ECP) concentrations in severe asthma (P < .01). Serum SP-D was significantly increased in SA when compared to HC and MA (P < .001), BAL/serum ratios were significantly lower in SA compared to HC and MA (P < .001). Reduced SP-D levels in in BAL with concomitant rises in serum in SA were associated with degraded fragments of SP-D in the serum and increased BAL neutrophils and lipopolysaccharide levels.Conclusions: These findings suggest defective innate immunity within the airways in severe asthma, as reflected by low BAL SP-D concentrations and altered bacterial presence with airway neutrophilia. Furthermore, BAL SP-D leakage into the serum in severe asthmatics may provide a peripheral blood biomarker reflecting increased epithelial damage and/or epithelial permeability within the peripheral airways.

Introduction: Sputum inflammometry has been used to describe asthma phenotypes and severe asthma is associated with neutrophilia based on the percentage of cells present. Sputum density is not accounted for in clinical practice. Methods: Induced sputum was obtained from 110 severe asthmatics (BTS step 4/5) of the Wessex Severe Asthma Cohort. Inflammatory biological markers were measured in sputum supernatant with Multiplex ELISA. Biomarker concentrations were compared to sputum neutrophil density using Spearmans correlation. Results: The median [IQR] cell number (x 106/g sputum) was significantly lower in P phenotype (n=41 0.64 \[0.96]) than N (n=29 2.1 [2.6\] (p

Rationale: Asthma is a disease of the conducting airways. Small airway events are poorly reflected by standard lung function.Methods: IOS, spirometry and FeNO measures were made in healthy controls (HC, n=76), non-steroid treated mild asthmatics (MA, n=30), steroid treated step 2/3 asthmatics (ModA, n=35) and treatment-resistant step4/5 severe asthmatics (SA n=71), to assess disease related small airways dysfunction. Group comparisons and correlations were undertaken using both parametric and non-parametric statistical analyses.Results: Maximum airway nitric oxide flux (JawNO) , reflecting central airways inflammation, was increased in MA (median 2740, p 10% in absolute values being present in 53.3% of MA, 74.3% of ModA and 84.5% of SA.. A significant negative correlation was noted between R5-R20 and MEF25-75(r=-0.537), p

Introduction: Gastro-oesophageal reflux disease (GORD) is a common co-morbidity in severe asthmatics. Its role in the description of the disease process and its mechanisms remains to be confirmed. Methods: We analysed the MRC Wessex severe asthma cohort data focussing on severe asthmatics. 324 patients were classified on the basis of treatment with anti-acid treatment (Proton pump inhibitors, H2 receptor blockers) as those with and without GORD. Data were analysed with appropriate statistical tests using SPSS and Graphpad Prism Results: Gender distribution showed a prevalence of females compared to males (GORD-76.6% vs 23.4%; p Conclusions: This study data highlights the importance of GORD as potentially describing a phenotype of asthma. Better definition of this phenotype has the potential to identify disease pathways associated with GORD that could help define new strategies for the management of severe asthma.

Data from a multicentre survey based on three London teaching hospitals on the relative safety and clinical utility of intravenous carotid digital subtraction angiography (DSA) over intra-arterial DSA and conventional carotid angiography are presented. The incidence of stroke during intra-arterial DSA was 0.7% (n = 538) and during conventional angiography was 0.8% (n = 780). The incidence of stroke during intravenous DSA was zero (n = 3710). When it constituted the initial investigation, intravenous DSA achieved a 93.8% replacement value over intra-arterial studies as a whole (n = 474) and 89% replacement value for patients having carotid endarterectomy (n = 99). It was also noted that the installation of DSA equipment at one unit coincided with a sixfold increase in the number of carotid angiographic examinations and an almost threefold increase in carotid endarterectomies.