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

Abstract

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 patients with severe asthma (n = 36) were collected onto sorbent tubes and analyzed using thermal desorption gas chromatography-mass spectrometry (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: 1) patients with acute asthma according to blood eosinophilia ⩾0.3 × 10⁹cells/L or sputum eosinophilia of ⩾3% in the UK EMBER (East Midlands Breathomics Pathology Node) consortium (n = 65) and 2) U-BIOPRED-IMI (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes Innovative Medicines Initiative) consortium (n = 42). Breath samples were collected onto sorbent tubes (EMBER) or Tedlar bags (U-BIOPRED) and analyzed by GC-MS (GC × GC-MS for EMBER or GC-MS for U-BIOPRED). Measurements and 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 (area under the receiver operating characteristic curve [AUROC] 0.90; 95% confidence interval [CI], 0.80-0.99; P < 0.0001), correlated inversely with sputum eosinophil percentage (r_s = -0.71; P < 0.0001), and outperformed fractional exhaled nitric oxide (AUROC 0.61; 95% CI, 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 fractional exhaled nitric oxide 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.

Keywords: eosinophilic airway inflammation; severe asthma; volatile organic compound biomarkers.

Figure 1.

(A) Graphical summary of the discovery cohort and identification of canonical eosinophil-associated volatile organic compounds (VOCs) using elastic net (eNET) regression and generation of volatile biomarker score using the regression coefficient and VOC concentrations in tissue headspace. (B) Summary of EMBER (East Midlands Breathomics Pathology Node) and U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) replication cohorts for exhaled breath validation of eosinophil-associated VOCs identified in the discovery cohort. GC-MS = gas chromatography–mass spectrometry; TD = thermal desorption.

Figure 2.

(A) An illustration of discovered eosinophilic breath volatile biomarkers, their chemical structures, and the metabolic pathways that could potentially be related to each chemical group. (B) Previously identified breath biomarkers in severe asthma studies characterizing patients according to sputum eosinophilia and arranged to show the possible chemical relationship and similarities with the reported discovery biomarkers in this study.

Figure 3.

(A) Histogram of the normalized peak area values of the 19 discovered eosinophilic volatile organic compounds (VOCs) summarized as median and interquartile range and compared by Mann-Whitney test (*P < 0.05 and **P < 0.01). (B) Box plot of VOC biomarker score derived from elastic net regression in the eosinophil-enriched and non–eosinophil-enriched sputum samples (median, Q1, Q3, min and max) (Mann-Whitney test: **P < 0.0001). (C) Receiver operating characteristic (ROC) curve to evaluate the discriminatory performance of VOC scores in differentiating between eosinophil-enriched and non–eosinophil-enriched sputa. (D) Spearman’s correlation coefficient between VOC biomarker scores and the percentage of sputum eosinophils (r_s = −0.71; two-tailed t test: P < 0.0001). AUC = area under the ROC curve; CI = confidence interval; Fe_NO = fractional exhaled nitric oxide; GINA = Global Initiative for Asthma; ICS = inhaled corticosteroids; LABA =  long-acting β antagonist; mOCS = maintenance oral corticosteroids.

Figure 4.

Visual summary of the diagnostic accuracy for both sputum and blood eosinophilia of eosinophil-associated volatile organic compounds (VOCs) in exhaled breath in the EMBER (East Midlands Breathomics Pathology Node) and U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) cohorts. AUC = area under the receiver operating characteristic curve; CI = confidence interval.

Figure 5.

(A) Ln (x + 1) transformed exhaled volatile organic compound (VOC) biomarker concentrations (y-axis), exhaled VOC (eVOC), in patients with eosinophilia (red) and without eosinophilia (blue) U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) characterized by sputum eosinophilia. *P < 0.05 (unadjusted) when comparing patients with or without eosinophilia. (B) Correlation (Pearson’s r) heatmap for the exhaled VOCs that individually constitute the eVOC score. *P < 0.05 for the correlation with the eVOC biomarker score. (C) Elastic net (eNET) biomarker score values for patients with or without eosinophilia according to sputum eosinophilia in U-BIOPRED. **P < 0.001. (D) Receiver operating characteristic (ROC) curves for classification of severe eosinophilic asthma, according to sputum eosinophilia, with fractional exhaled nitric oxide (Fe_NO) (orange), eVOC score (green), and combination of Fe_NO and eVOC score (blue). Area under ROC curve (AUROC) for eVOC score is presented in green. SpEos = sputum eosinophil count (%).