Longitudinal changes in sputum and blood inflammatory mediators during FeNO suppression testing

To explore whether fractional exhaled nitric oxide (FeNO) non-suppression identifies corticosteroid resistance, we analysed inflammatory mediator changes during a FeNO suppression test with monitored high-intensity corticosteroid therapy. In linear mixed-effects models analysed over time, the 15 clinically distinct ‘suppressors’ (ie, ≥42% FeNO suppression) normalised Asthma Control Questionnaire scores (mean±SD, start to end of test: 2.8±1.4 to 1.4±0.9, p<0.0001) and sputum eosinophil counts (median (IQR), start to end of test: 29% (6%–41%) to 1% (1%–5%), p=0.0003) while significantly decreasing sputum prostaglandin D2 (254 (89–894) to 93 (49–209) pg/mL, p=0.004) and numerically decreasing other type-2 cytokine, chemokine and alarmin levels. In comparison, the 19 non-suppressors had persistent sputum eosinophilia (10% (1%–67%) despite high-intensity therapy) with raised end-test inflammatory mediator levels (1.9 (0.9–2.8)-fold greater than suppressors). FeNO non-suppression during monitored treatment implies biological corticosteroid resistance.


Content
Page Appendix 1 Flowchart of patient enrolment process E3 Appendix 2 Detailed outputs from linear mixed effects modelling E4 Appendix 3 Results of pooled linear mixed effect models exploring the continuous relationships between FeNO and key analytes E10 Appendix 4 Sensitivity analyses for different test durations and optimisation methods E11 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Methods
Analytes found to suppress significantly in the primary longitudinal subgroup analyses on FeNO suppressors and non-suppressors (ACQ-5, sputum eosinophils, sputum PGD2, sputum IL-4) were submitted to further analyses exploring their continuous relationship between log-FeNO suppression and analyte-suppression. The 34 patients were analysed in linear mixed effect models with a random intercept for patient to be able to consider each available timepoint, with the independent variable the natural logarithm of FeNO. An unstructured covariance structure was assumed based on AIC and modeling assumptions were all verified visually. Coefficients and their 95% confidence interval are presented to assess the association between significant markers and FeNO suppression.

Summary of different optimisation methods and test durations
An overview of the FeNO suppression test Oxford protocol is provided in Figure E2. Briefly, patients with asthma underwent 7 to 35 days of additional inhaled and/or systemic corticosteroids (+1000μg inhaled fluticasone propionate per day and, if FeNO did not suppress on day 7 according to the equation provided in study methods, +80mg intramuscular (IM) triamcinolone with followup 28 days later). Some FeNO suppression tests stopped after 7 days due to patient availability, physician decision, or transition to research bronchoscopy protocols.

E12
Assessing impact of the optimisation method Sensitivity analyses of positive findings were conducted to assess whether the final optimisation method for FeNO suppression (i.e.: +1000μg inhaled fluticasone propionate or +80mg triamcinolone intramuscularly) resulted in significantly different before/after changes. All study patients were grouped based on the optimisation method. Before vs after changes in log10transformed FeNO and differences in ACQ-5 scores were compared within each group using paired t-tests. Fold-changes in FeNO and differences in ACQ (afterbefore) were compared between the two groups with a Mann-Whitney test and an unpaired t-test, respectively. Furthermore, areas under the curve (AUC) were computed for the log-transformed FeNO values (dependent variable) vs time course of FeNO suppression testing (independent variable; segmented in days 0 to 7 and days 7 to 35), with their 95% CI analysed for differences.

Assessing the impact of the test duration
To assess if study duration method (i.e. 7-or 35-day test) impacted degree of FeNO suppression and ACQ-5 improvements, all included patients were grouped based on study duration. Before vs after changes in log10-transformed FeNO and differences in ACQ-5 scores were compared within each group using paired t-tests. Fold-changes in FeNO and differences in ACQ (afterbefore) were compared between the two groups with a Mann-Whitney test and an unpaired t-test, respectively.
Statistics were analysed with a two-sided α of 0.05. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Sensitivity analyses on the optimisation method
Patients who did and did not suppress FeNO received triamcinolone 80mg intramuscularly on day 7 in similar proportions (3/15 vs 6/19; see main manuscript text, Table 1). The results of the sensitivity analyses are shown in Figure E3 and suggest that, although both optimisation methods were used in different circumstances to ensure optimal FeNO suppression, the magnitude of  (Table E4).