A five-gene qPCR signature can classify type 2 asthma comparably to microscopy of induced sputum from severe asthma patients

Figures & data

Figure 1. Plots showing the median target/β-actin-ratio visualised on a logarithmic conversion on the Y-axis in each group on the X-axis. Mann-Whitney U test performed for each target under the null hypothesis that the observations in each group come from the same population. CPA3: carboxypeptidase A3. EA: eosinophilic asthma. GATA2: GATA binding protein 2. HDC: histidine decarboxylase. MS4A2: membranes spanning 4A2. NEA: Non-eosinophilic asthma. TPSAB1/TPSB2: tryptase α/β-1/Tryptase β2.

Table 1. Demographics from the EA and NEA patients. Values shown as mean ± Standard deviation. Sex and smoking are shown as proportions. BMI: Body mass index. EA: Eosinophilic asthma. NEA: Non-eosinophilic asthma. FEV1: Forced expiratory volume in 1 sec. FVC: Functional vital capacity. ICS: inhaled corticosteroid. Ppb: parts per billion. FENO: Fraction of Exhaled Nitric Oxide. *P<0.05. ^§ICS dose in beclomethasone (standard particle) equivalent. ^ΔData only available for 10 EA patients and 13 NEA patients. ^◊Data only available for 11 EA patients and 11 NEA patients.

	EA	NEA	p-value
Number (N=30)	14	16
Age (years)	62.7 ± 13.8	54.4 ± 14.6	0.10
Sex (male:female)	9:5	6:16	0.16
FEV1 predicted (%)	76.8 ± 28.9	79.3 ± 20.0	0.78
FEV1 volume (L)	2.30 ± 1.14	2.38 ± 0.78	0.83
FVC predicted (%)	98.4 ± 27.0	98.7 ± 14.0	0.97
FVC volume (L)	3.60 ± 1.35	3.56 ± 0.90	0.93
BMI (kg/m^2)	26.2 ± 5.0	30.0 ± 8.8	0.13
Smoking (ex:never)	6:8	8:8	0.70
ICS dose (µg)^§	1140 ± 677	1196 ± 727	0.60
Sputum eosinophils (%)	36.8 ± 30.2	0.6 ± 0.8	<0.001*
Sputum neutrophils (%)	41.3 ± 27.8	53.9 ± 32.6	0.25
Blood eosinophils (10^9/L)^Δ	0.71 ± 0.92	0.29 ± 0.27	0.16
FeNO (ppb)^◊	57.5 ± 43.0	16.3 ± 9.2	0.002*

Figure 2. Spearman correlation heat map with correlation coefficients. FENO: fraction of Exhaled Nitric Oxide.

Figure 3. Scatterplot. X-axis: FeNO values in parts per billion(ppb), segmented axis. Y-axis: relative target mRNA on a logarithmic conversion. Spearman’s rank correlation coefficient (r) with 95% confidence intervals and p values. Only data from 22 patients. *p < 0.05. CPA3: carboxypeptidase A3. GATA2: GATA binding protein 2. HDC: histidine decarboxylase. MS4A2: membrane spanning 4A2. TPSAB1/TPSB2: tryptase α/β-1/Tryptase β2. FENO: fraction of Exhaled Nitric Oxide. mRNA: messenger ribonucleic acid.

Figure 4. Scatterplot. X-axis: blood eosinophil values in 10⁹/L, segmented axis. Y-axis: relative target mRNA on a logarithmic conversion. Spearman’s rank correlation coefficient (r) with 95% confidence intervals and p values. Only data from 23 patients. *p < 0.05. CPA3: carboxypeptidase A3. GATA2: GATA binding protein 2. HDC: histidine decarboxylase. MS4A2: membrane spanning 4A2. TPSAB1/TPSB2: tryptase α/β-1/Tryptase β2. mRNA: messenger ribonucleic acid.

Figure 5. ROC curves for each target along with AUC displaying diagnostic ability. Y-axis: sensitivity X-axis: 1 – specificity. CPA3: carboxypeptidase A3. GATA2: GATA binding protein 2. HDC: histidine decarboxylase. MS4A2: membranes spanning 4A2. TPSAB1/TPSB2: tryptase α/β-1/Tryptase β2. mRNA: messenger ribonucleic acid.

Table 2. Best cut-off value represented for each target. CPA3: Carboxypeptidase A3. GATA2: GATA binding protein 2. HDC: Histidine decarboxylase. MS4A2: Membranes spanning 4A2. TPSAB1/TPSB2: Tryptase α/β-1/Tryptase β2. mRNA: messenger ribonucleic acid.

Target	Best Cut-off value
CPA3	6.6
GATA2	2
MS4A2	1
HDC	8.5
TPSAB1/TPSB2	2.15

Figure 6. Green curve: ROC curve using CPA3, GATA2, MS4A2 and HDC. Red curve: ROC curve using CPA3, GATA2, MS4A2, HDC and TPSAB1/TPSB2. Y-axis: sensitivity. X-axis: 1 – specificity.

Table 3. qPCR’s ability to classify patients with EA and NEA by using ≥2 out of 5 targets Sensitivity, specificity, PPV and NPV were calculated. EA: Eosinophilic asthma. NEA: Non-eosinophilic asthma. NPV: Negative predictive value. qPCR: Quantitative polymerase chain reaction.

≥2 targets	Microscopy
qPCR		EA	NEA	Total
	EA	12	1	13
	NEA	2	15	17
	Total	14	16	30
Sensitivity	87%
Specificity	94%
PPV	92%
NPV	88%

Figure 7. Scatter plot. X-axis: sputum eosinophils in percentage. Y-axis: targets above cut-off. Targets included: GATA2, HDC, MS4A2 and CPA3.

Figure 8. Scatter plot. X-axis: fraction of Exhaled Nitric Oxide in parts per billion, segmented axis. Y-axis: targets above cut-off. Targets included: GATA2, CPA3, MS4A2 and TPSAB1/TPSB2.

Figure 9. Figure 8: scatter plot. X-axis: blood eosinophils in 10⁹/L, segmented axis. Y-axis: targets above cut-off. Targets included: GATA2, CPA3, HDC MS4A2 and TPSAB1/TPSB2.