From the 7220 COPD patients in TARDIS, 178120 stable and 15826 exacerbation associated BNCs were obtained (Figure 1); their clinical characteristics at index are shown in Table 1. The median age of patients at index was 75 years (interquartile range (IQR) 65-81), 48% were male, 4525 (63%) were ICS users and 3494 (49%) were current smokers whilst median FEV1 was 1.6L (IQR 1.1-2.0). Comparing the normal and elevated groups, the elevated group were older, more likely to be smokers, receiving ICS, and had more exacerbations and a lower index FEV1 (Table 1). 2% of stable BNCs were classed as low; 50% as normal, 43% as elevated and 4% as extreme. Less than 1% of the exacerbation BNCs were classed as low; 38% as normal, 56% as elevated and 6% extreme.
Blood eosinophil analysis
The association between index blood eosinophil counts and mortality was weak and only evident in patients with blood eosinophil counts <100cells/µL (Figure 2A). No significant difference in all-cause mortality, mortality due to COPD or change in FEV1 was observed between the different blood eosinophil count groups. The total exacerbation rates were also indistinguishable, though the rate of severe exacerbations was lower among those with elevated index blood eosinophils than in all other groups (e-Figure 1 and e-Table 2).
Elevated BNCs and increased mortality.
BNC were significantly higher during exacerbations (P<0.001; Figure 2B), though this difference was small compared to the within-individual variability (median coefficient of variation 0.36; IQR 0.26-0.48; e-Figure 2). Despite this variability, a pattern of mortality risk being higher for those with BNC outside the normal range, was evident. Unsurprisingly, the association of mortality with index BNC (Figure 2C) was less strong than that for the final BNC (Figure 2D). These patterns were similar for all-cause mortality and that recorded as due to COPD. The patterns in the raw data (Figures 2C & 2D) remained as statistically significant differences (P<0.001) in survival between the BNC groups even after adjustment for age, sex and smoking history, with 10 year survival for those with an elevated BNC being about 2/3 of that for those with normal BNC at index (Figures 3A and 3B). Very similar results were obtained for unadjusted models, and those considering all respiratory (ICD code J) deaths (e-Figure 3). The adjusted model with BNC as a linear term estimated the hazard ratio for all-cause mortality at 1.067 (95%CI: 1.060-1.073 per 1,000cells/µL), and that for COPD mortality at 1.091 (95%CI: 1.082-1.100). These estimates, while still significant, are lower than the between group differences (e-Table 3) because there is high mortality among the group with low index BNC. Additional models did not identify any important confounding effects (e-Figures 4 and 5).
The elevated BNC group had more exacerbations than the normal BNC group in both the year before index (mean 2.3 vs 1.3 exacerbations/year, P<0.001) and the year following index (1.9 vs 1.5, P<0.001). The incident rate ratios for all exacerbations or severe exacerbations alone in the 12 months following index date were determined, with the normal BNC group as reference; two models are shown (Figure 3C), one unadjusted and the second adjusted for age, sex and smoking history. The adjusted models indicated that, for patients with an elevated BNC, there was a 40% increase in total exacerbations, and a 170% increase in severe exacerbations in the year from index relative to the rates for those with normal BNC (P<0.001 in both cases). The adjusted model with BNC as a linear term, estimated that each increase of 1000cells/µL in BNC was associated with the total exacerbation rate increasing by a factor of 1.05 (95%CI: 1.04-1.06). For severe exacerbations, the figure was 1.17 (95%CI: 1.14-1.20). In the year from index date the much smaller group of patients with an extreme BNC had an increase in all and severe exacerbations of 96% and 450% (P<0.001 in both cases; e-Figure 6).
Additional GLMs did not identify any important confounding effects (e-Figures 7 and 8). When looking at all exacerbations, the neutrophil effect remained significant in every case, with only the total number of exacerbations in the year up to index date attenuating the magnitude of the effect. (e-Figure 7). Restricting the analysis to the younger half of the cohort did not change the results (e-Figure 9). Changing the upper limit of the normal range from 6000 to 8000cells/µL halved the size of the elevated BNC group, and widened confidence intervals, without changing the overall results or conclusions (e-Figure 10).
The overall rate of change in FEV1, across the whole cohort was -0.038 (95%CI: -0.034, -0.041) L/yr or 38mL/yr. The rate within the elevated BNC group was indistinguishable from the normal BNC group (Figure 3D). While estimated rate of decline for the elevated BNC group was slightly faster (difference -0.004; 95%CI: -0.010, 0.003), the difference was not statistically significant, and was reversed when consideration was restricted to only the first 3 years after the index date (difference between groups 0.002; 95%CI: -0.012, 0.015). However, these confidence intervals are too wide to entirely rule out the existence of a clinically meaningful difference.
273 patients provided at least one sputum sample, when clinically stable, of which 246 samples passed sequencing quality control to be included in the analysis. Utilizing the same cut offs as the main TARDIS cohort, 1 patient had a low BNC, 159 were normal, 79 elevated and 7 extreme. For the purpose of the microbiome analysis, low and normal patients were grouped as <6000cells/µL, elevated and extreme as >6000cells/µL. At the phylum level, the microbiome was dominated by Proteobacteria and Firmicutes, corresponding to dominant genera of Haemophilus (n=59 patients) and Streptococcus (n=78) at the genus level (e-Figure 11). Additional dominant genera observed included: Veillonella (n=55), Neisseria (n=12), Pseudomonas (n=11), Moraxella (n=8) and Prevotella (n=7). The BNC count of the samples was significantly associated with increased % Proteobacteria (P=0.027) and reduced Shannon Wiener Diversity Index (P=0.0017) (Figure 4 A and B). Beta diversity was assessed using the Bray Curtis Dissimilarity Index and Weighted UNIFRAC (Figure 4C). Between patients, the microbiome composition was shown to be significantly different by PERMANOVA when comparing patients with BNCs <6000cells/µL to patients with BNCs >6000cells/µL (P=0.014), which remained statistically significant (P=0.021) when adjusted for age, sex and smoking status (Figure 4D).
ECLIPSE validation cohort
The observation that BNC was associated with mortality in COPD was confirmed in the ECLIPSE cohort. From 2164 COPD patients enrolled in ECLIPSE, 1934 participants were alive at the end of the original 3 year study period. 1555 participants consented to an assessment of survival at 8 years; the 379 patients who did not participate in the 8 year survival assessment were censored. In the Cox proportional hazards model adjusted for age, prior hospitalization and BODE index, BNC was independently associated with decreased survival as a linear variable (HR 1.20 95%CI 1.11-1.29, P<0.0001).