Fulfilling the Promise of Breathomics: Considerations for the Discovery and Validation of Exhaled Volatile Biomarkers.

The exhaled breath represents an ideal matrix for non-invasive biomarker discovery, and exhaled metabolomics have the potential to be clinically useful in the era of precision medicine. In this concise translational review we will specifically address volatile organic compounds in the breath, with a view towards fulfilling the promise of these as actionable biomarkers, in particular for lung diseases. We review the literature paying attention to seminal work linked to key milestones in breath research; discuss potential applications for breath biomarkers across disease areas and healthcare systems, including the perspectives of industry; and outline critical aspects of study design that will need to be considered for any pivotal research going forward, if breath analysis is to provide robust validated biomarkers that meet the requirements for future clinical implementation.

Discovery and Validation of a Volatile Signature of Eosinophilic Airway Inflammation in Asthma.

RATIONALE: Volatile organic compounds (VOCs) in asthmatic breath may be associated with sputum eosinophilia. We developed a volatile biomarker-signature to predict sputum eosinophilia in asthma. METHODS: VOCs emitted into the space above sputum samples (headspace) from severe asthmatics (n=36) were collected onto sorbent tubes and analysed using thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Elastic net regression identified stable VOCs associated with sputum eosinophilia ≥3% and generated a volatile biomarker signature. This VOC signature was validated in breath samples from: (I) acute asthmatics according to blood eosinophilia ≥0.3x109cells/L or sputum eosinophilia of ≥ 3% in the UK EMBER consortium (n=65) and U-BIOPRED-IMI consortium (n=42). Breath samples were collected onto sorbent tubes (EMBER) or Tedlar bags (U-BIOPRED) and analysed by gas-chromatography-mass spectrometry (GC×GC-MS -EMBER or GC-MS -U-BIOPRED). MAIN RESULTS: The in vitro headspace identified 19 VOCs associated with sputum eosinophilia and the derived VOC signature yielded good diagnostic accuracy for sputum eosinophilia ≥ 3% in headspace (AUROC (95% CI) 0.90(0.80-0.99), p<0.0001), correlated inversely with sputum eosinophil % (rs= -0.71, p<0.0001) and outperformed FeNO (AUROC (95% CI) 0.61(0.35-0.86). Analysis of exhaled breath in replication cohorts yielded a VOC signature AUROC (95% CI) for acute asthma exacerbations of 0.89(0.76-1.0) (EMBER cohort) with sputum eosinophilia and 0.90(0.75-1.0) in U-BIOPRED - again outperforming FeNO in U-BIOPRED 0.62 (0.33-0.90). CONCLUSIONS: We have discovered and provided early-stage clinical validation of a volatile biomarker signature associated with eosinophilic airway inflammation. Further work is needed to translate our discovery using point of care clinical sensors.

Exhaled Volatile Organic Compounds for Asthma Control Classification in Children with Moderate to Severe Asthma: Results from the SysPharmPediA Study.

RATIONALE: Early identification of children with poorly controlled asthma is imperative for optimizing treatment strategies. The analysis of exhaled volatile organic compounds (VOCs) is an emerging approach to identify prognostic and diagnostic biomarkers in pediatric asthma. OBJECTIVES: To assess the accuracy of gas chromatography-mass spectrometry based exhaled metabolite analysis to differentiate between controlled and uncontrolled pediatric asthma. METHODS: This study encompassed a discovery (SysPharmPediA) and validation phase (U-BIOPRED, PANDA). Firstly, exhaled VOCs that discriminated asthma control levels were identified. Subsequently, outcomes were validated in two independent cohorts. Patients were classified as controlled or uncontrolled, based on asthma control test scores and number of severe attacks in the past year. Additionally, potential of VOCs in predicting two or more future severe asthma attacks in SysPharmPediA was evaluated. MEASUREMENTS AND MAIN RESULTS: Complete data were available for 196 children (SysPharmPediA=100, U-BIOPRED=49, PANDA=47). In SysPharmPediA, after randomly splitting the population into training (n=51) and test sets (n=49), three compounds (acetophenone, ethylbenzene, and styrene) distinguished between uncontrolled and controlled asthmatics. The area under the receiver operating characteristic curve (AUROCC) for training and test sets were respectively: 0.83 (95% CI: 0.65-1.00) and 0.77 (95% CI: 0.58-0.96). Combinations of these VOCs resulted in AUROCCs of 0.74 ±0.06 (UBIOPRED) and 0.68 ±0.05 (PANDA). Attacks prediction tests, resulted in AUROCCs of 0.71 (95% CI 0.51-0.91) and 0.71 (95% CI 0.52-0.90) for training and test sets. CONCLUSIONS: Exhaled metabolites analysis might enable asthma control classification in children. This should stimulate further development of exhaled metabolites-based point-of-care tests in asthma.

Biomarker Predictors of Clinical Efficacy of the Anti-IgE Biologic Omalizumab in Severe Asthma in Adults: Results of the SoMOSA Study.

Background: The anti-IgE monoclonal antibody omalizumab is widely used for severe asthma. This study aimed to identify biomarkers that predict clinical improvement during 1 year of omalizumab treatment. Methods: One-year open-label Study of Mechanisms of action of Omalizumab in Severe Asthma (SoMOSA) involving 216 patients with severe (Global Initiative for Asthma step 4/5) uncontrolled atopic asthma (at least two severe exacerbations in the previous year) taking high-dose inhaled corticosteroids and long-acting ß-agonists with or without maintenance oral corticosteroids. It had two phases: 0-16 weeks, to assess early clinical improvement by Global Evaluation of Therapeutic Effectiveness (GETE); and 16-52 weeks, to assess late responses based on ⩾50% reduction in exacerbations or mOCS dose. All participants provided samples (exhaled breath, blood, sputum, urine) before and after 16 weeks of omalizumab treatment. Measurements and Main Results: A total of 191 patients completed phase 1; 63% had early improvement. Of 173 who completed phase 2, 69% had reduced exacerbations by ⩾50% and 57% (37 of 65) taking mOCSs had reduced their dose by ⩾50%. The primary outcomes 2,3-dinor-11-ß-PGF2α, GETE score, and standard clinical biomarkers (blood and sputum eosinophils, exhaled nitric oxide, serum IgE) did not predict either clinical response. Five volatile organic compounds and five plasma lipid biomarkers strongly predicted the ⩾50% reduction in exacerbations (receiver operating characteristic areas under the curve of 0.780 and 0.922, respectively) and early responses (areas under the curve of 0.835 and 0.949, respectively). In an independent cohort, gas chromatography/mass spectrometry biomarkers differentiated between severe and mild asthma. Conclusions: This is the first discovery of omics biomarkers that predict improvement in asthma with biologic agent treatment. Prospective validation and development for clinical use is justified.

Breath Markers of Oxidative Stress in Children with Severe Viral Lower Respiratory Tract Infection.

Severe viral lower respiratory tract infection (LRTI), resulting in both acute and long-term pulmonary disease, constitutes a substantial burden among young children. Viral LRTI triggers local oxidative stress pathways by infection and inflammation, and supportive care in the pediatric intensive care unit may further aggravate oxidative injury. The main goal of this exploratory study was to identify and monitor breath markers linked to oxidative stress in children over the disease course of severe viral LRTI. Exhaled breath was sampled during invasive ventilation, and volatile organic compounds (VOCs) were analyzed using gas chromatography and mass spectrometry. VOCs were selected in an untargeted principal component analysis and assessed for change over time. In addition, identified VOCs were correlated with clinical parameters. Seventy breath samples from 21 patients were analyzed. A total of 15 VOCs were identified that contributed the most to the explained variance of breath markers. Of these 15 VOCs, 10 were previously linked to pathways of oxidative stress. Eight VOCs, including seven alkanes and methyl alkanes, significantly decreased from the initial phase of ventilation to the day of extubation. No correlation was observed with the administered oxygen dose, whereas six VOCs showed a poor to strong positive correlation with driving pressure. In this prospective study of children with severe viral LRTI, the majority of VOCs that were most important for the explained variance mirrored clinical improvement. These breath markers could potentially help monitor the pulmonary oxidative status in these patients, but further research with other objective measures of pulmonary injury is required.

Breath metabolomics for diagnosis of acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) poses challenges in early identification. Exhaled breath contains metabolites reflective of pulmonary inflammation. AIM: To evaluate the diagnostic accuracy of breath metabolites for ARDS in invasively ventilated intensive care unit (ICU) patients. METHODS: This two-center observational study included critically ill patients receiving invasive ventilation. Gas chromatography and mass spectrometry (GC-MS) was used to quantify the exhaled metabolites. The Berlin definition of ARDS was assessed by three experts to categorize all patients into "certain ARDS", "certain no ARDS" and "uncertain ARDS" groups. The patients with "certain" labels from one hospital formed the derivation cohort used to train a classifier built based on the five most significant breath metabolites. The diagnostic accuracy of the classifier was assessed in all patients from the second hospital and combined with the lung injury prediction score (LIPS). RESULTS: A total of 499 patients were included in this study. Three hundred fifty-seven patients were included in the derivation cohort (60 with certain ARDS; 17%), and 142 patients in the validation cohort (47 with certain ARDS; 33%). The metabolites 1-methylpyrrole, 1,3,5-trifluorobenzene, methoxyacetic acid, 2-methylfuran and 2-methyl-1-propanol were included in the classifier. The classifier had an area under the receiver operating characteristics curve (AUROCC) of 0.71 (CI 0.63-0.78) in the derivation cohort and 0.63 (CI 0.52-0.74) in the validation cohort. Combining the breath test with the LIPS does not significantly enhance the diagnostic performance. CONCLUSION: An exhaled breath metabolomics-based classifier has moderate diagnostic accuracy for ARDS but was not sufficiently accurate for clinical use, even after combination with a clinical prediction score.

The Non-Invasive Detection of Pulmonary Exacerbations in Disorders of Mucociliary Clearance with Breath Analysis: A Systematic Review.

Background: Disorders of mucociliary clearance, such as cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and bronchiectasis of unknown origin, are characterised by periods with increased respiratory symptoms, referred to as pulmonary exacerbations. These exacerbations are hard to predict and associated with lung function decline and the loss of quality of life. To optimise treatment and preserve lung function, there is a need for non-invasive and reliable methods of detection. Breath analysis might be such a method. Methods: We systematically reviewed the existing literature on breath analysis to detect pulmonary exacerbations in mucociliary clearance disorders. Extracted data included the study design, technique of measurement, definition of an exacerbation, identified compounds and diagnostic accuracy. Results: Out of 244 identified articles, 18 were included in the review. All studies included patients with CF and two also with PCD. Age and the definition of exacerbation differed between the studies. There were five that measured volatile organic compounds (VOCs) in exhaled breath using gas chromatography with mass spectrometry, two using an electronic nose and eleven measured organic compounds in exhaled breath condensate. Most studies showed a significant correlation between pulmonary exacerbations and one or multiple compounds, mainly hydrocarbons and cytokines, but the validation of these results in other studies was lacking. Conclusions: The detection of pulmonary exacerbations by the analysis of compounds in exhaled breath seems possible but is not near clinical application due to major differences in results, study design and the definition of an exacerbation. There is a need for larger studies, with a longitudinal design, international accepted definition of an exacerbation and validation of the results in independent cohorts.

Exhaled breath analyses for bronchial thermoplasty in severe asthma patients.

BACKGROUND: Bronchial thermoplasty (BT) is a bronchoscopic treatment for severe asthma. Although multiple trials have demonstrated clinical improvement after BT, optimal patient selection remains a challenge and the mechanism of action is incompletely understood. The aim of this study was to examine whether exhaled breath analysis can contribute to discriminate between BT-responders and non-responders at baseline and to explore pathophysiological insights of BT. METHODS: Exhaled breath was collected from patients at baseline and six months post-BT. Patients were defined as responders or non-responders based on a half point increase in asthma quality of life questionnaire scores. Gas chromatography-mass spectrometry was used for volatile organic compounds (VOCs) detection and analyses. Analytical workflow consisted of: 1) detection of VOCs that differentiate between responders and non-responders and those that differ between baseline and six months post-BT, 2) identification of VOCs of interest and 3) explore correlations between clinical biomarkers and VOCs. RESULTS: Data was available from 14 patients. Nonanal, 2-ethylhexanol and 3-thujol showed a significant difference in intensity between responders and non-responders at baseline (p = 0.04, p = 0.01 and p = 0.03, respectively). After BT, no difference was found in the compound intensity of these VOCs. A negative correlation was observed between nonanal and IgE and BALF eosinophils (r = -0.68, p < 0.01 and r = -0.61, p = 0.02 respectively) and 3-thujol with BALF neutrophils (r = -0.54, p = 0.04). CONCLUSIONS: This explorative study identified discriminative VOCs in exhaled breath between BT responders and non-responders at baseline. Additionally, correlations were found between VOC's and inflammatory BALF cells. Once validated, these findings encourage research in breath analysis as a non-invasive easy to apply technique for identifying airway inflammatory profiles and eligibility for BT or immunotherapies in severe asthma.

Volatile Organic Compounds in Cellular Headspace after Hyperbaric Oxygen Exposure: An In Vitro Pilot Study.

Volatile organic compounds (VOCs) might be associated with pulmonary oxygen toxicity (POT). This pilot study aims to identify VOCs linked to oxidative stress employing an in vitro model of alveolar basal epithelial cells exposed to hyperbaric and hyperoxic conditions. In addition, the feasibility of this in vitro model for POT biomarker research was evaluated. The hyperbaric exposure protocol, similar to the U.S. Navy Treatment Table 6, was conducted on human alveolar basal epithelial cells, and the headspace VOCs were analyzed using gas chromatography-mass spectrometry. Three compounds (nonane [p = 0.005], octanal [p = 0.009], and decane [p = 0.018]), of which nonane and decane were also identified in a previous in vivo study with similar hyperbaric exposure, varied significantly between the intervention group which was exposed to 100% oxygen and the control group which was exposed to compressed air. VOC signal intensities were lower in the intervention group, but cellular stress markers (IL8 and LDH) confirmed increased stress and injury in the intervention group. Despite the observed reductions in compound expression, the model holds promise for POT biomarker exploration, emphasizing the need for further investigation into the complex relationship between VOCs and oxidative stress.

HS-GC-MS analysis of volatile organic compounds after hyperoxia-induced oxidative stress: a validation study.

BACKGROUND: Exhaled volatile organic compounds (VOCs), particularly hydrocarbons from oxidative stress-induced lipid peroxidation, are associated with hyperoxia exposure. However, important heterogeneity amongst identified VOCs and concerns about their precise pathophysiological origins warrant translational studies assessing their validity as a marker of hyperoxia-induced oxidative stress. Therefore, this study sought to examine changes in VOCs previously associated with the oxidative stress response in hyperoxia-exposed lung epithelial cells. METHODS: A549 alveolar epithelial cells were exposed to hyperoxia for 24 h, or to room air as normoxia controls, or hydrogen peroxide as oxidative-stress positive controls. VOCs were sampled from the headspace, analysed by gas chromatography coupled with mass spectrometry and compared by targeted and untargeted analyses. A secondary analysis of breath samples from a large cohort of critically ill adult patients assessed the association of identified VOCs with clinical oxygen exposure. RESULTS: Following cellular hyperoxia exposure, none of the targeted VOCs, previously proposed as breath markers of oxidative stress, were increased, and decane was significantly decreased. Untargeted analysis did not reveal novel identifiable hyperoxia-associated VOCs. Within the clinical cohort, three previously proposed breath markers of oxidative stress, hexane, octane, and decane had no real diagnostic value in discriminating patients exposed to hyperoxia. CONCLUSIONS: Hyperoxia exposure of alveolar epithelial cells did not result in an increase in identifiable VOCs, whilst VOCs previously linked to oxidative stress were not associated with oxygen exposure in a cohort of critically ill patients. These findings suggest that the pathophysiological origin of previously proposed breath markers of oxidative stress is more complex than just oxidative stress from hyperoxia at the lung epithelial cellular level.