**3. Eosinophils in nonallergic asthma**

The increase of the number of activated Th2 lymphocytes and eosinophils, as well as IL-5 levels, in both BAL fluid and bronchial biopsies from intrinsic asthmatics, has been extensively reported [45]. No difference between atopic and intrinsic asthmatics have been observed in studies examining the expression of high-affinity IgE receptor, IL-5 and IL-4 mRNA and protein expression in bronchial biopsies [16]. Actually, total serum IgE levels have been noted to be increased in the serum of patients with intrinsic asthma. This reflects the increases in Iå and Cå RNA+ cells in the bronchial mucosa and provides evidence for a local IgE synthesis even in the absence of a known antigen or allergen trigger.

Eosinophilic infiltration in nonallergic asthma can be even much more than in allergic asthma and this fact is reflected by the finding of a larger amount of RANTES in the bronchoalveolar lavage fluid of patients with nonallergic asthma compared with patients with allergic asthma [46].

Attempts to differentiate the inflammatory cascade between allergic and nonallergic asthma have proposed a different signal in the Th2 pathway of nonallergic asthma attributed to reduced signal transducer and activator of transcription 6 (STAT6) expression and consequently reduced IL-4R signaling in nonallergic asthma [47]. Another peculiar finding was the increased expression of GM-CSF receptor alpha expression in the macrophages detected in mucosa and BAL. Peripheral blood eosinophilia is present both in allergic and nonallergic asthma, in some studies being higher in the former compared to the latter group [48].

#### **4. The eosinophilic phenotype of asthma**

Different attempts have been found in order to identify an eosinophilic phenotype of asthma. Eosinophilic asthma is reported to account for approximately 50–60% of the total asthma population. The definition of eosinophilic asthma implies that eosinophils are the dominant cells responsible for the pathophysiological changes of the disease. The pathogenic role of eosinophils in these patients is

demonstrated by their increased number and status of activation in the airways, and consequently, they are detected in sputum, bronchoalveolar lavage, or bronchial mucosa or submucosa. These findings may be persistent and associated with severe or uncontrolled asthma [7].

Eosinophils may be demonstrated in the airways in the bronchial mucosa or submucosa or in the lumen (in the bronchial wash, BAL, or sputum). Bronchial biopsy is not routinely used as it is an invasive procedure and practicable only far from exacerbations to avoid dangerous complications and the quantification of eosinophils in BAL is not standardized and generally reflects samples of the peripheral airways.

Sputum examination is currently the most comprehensive and noninvasive method for measuring airway inflammation, processing and analysis being standardized and reliability, validity, and responsiveness proven [49].

The definition of "eosinophilic asthma" implies the existence of noneosinophilic asthma. A large cohort of patients with mild-to-moderate asthma in longitudinal studies resulted in approximately 50% of them with the absence of eosinophilic airway inflammation. The cellular pattern in noneosinophilic asthma may result in either predominant neutrophilic inflammation or normal sputum cell count. Within eosinophilic asthma, eosinophilia may result in persistent (22%) or on at least 1 occasion (intermittent eosinophilia, 31%) under multiple sputum examinations [50]. Sputum inflammatory granulocytes may identify phenotypic subgroups of differing pathology and clinical characteristics within asthmatics. Within the Severe Asthma Research Program (SARP), which included a population of severe and nonsevere patients with and without corticosteroid treatment, the stratification in four groups by granulocyte % in sputum showed significant clinical differences. Patients were divided combined for stratification by granulocytes in <2%Eos + <40%Neu, <2%Eos + ≥ 40%Neu, ≥2%Eos + <40% Neu, and ≥ 2%Eos + ≥40%Neu. In this study, eosinophilic asthma, indicated by ≥2%Eos, accounted for 31% of patients, those being with combined increased sputum eosinophils and neutrophils the most severe patients in terms of lowest lung function measures, worse asthma control, greatest symptoms and use of healthcare resources [51]. In another retrospective series of 508 asthmatics, the proportion of patients with raised sputum eosinophil counts ≥3% was 42% independent of the exclusion of steroid-treated patients. Eosinophilic phenotype exhibited higher atopy, levels of IgE, bronchial hyperresponsiveness to methacholine, FENO levels and lower asthma control, while the mixed granulocytic phenotype, with both eosinophilic and neutrophilic inflammation, had the lowest lung function and the highest degree of bronchial hyperresponsiveness to methacholine [52].

In most mild-to-moderate asthma patients untreated with steroids, sputum eosinophilia >2% was significantly and inversely associated with PC20 methacholine identifying 69% of the asthma group [53]. Sputum eosinophils correlate, in addition, with symptom score and FEV% and, as previously reported, are increased by exposure to common allergens. The association between asthma exacerbations and sputum eosinophilia is suggested by different pieces of evidence. First, sputum eosinophil count is able to predict asthma deterioration after cessation of ICSs treatment in mild-to-moderate asthma, while it is decreased by treatment with corticosteroids [54]. Sputum eosinophilia may be a good additional predictor of FEV1, PC20 methacholine or quality of life of response to inhaled steroids [55].

Consequently, a clinical strategy, based on re-administration of ICSs when a change in sputum eosinophil percentage by using the 0.8% threshold was reached, could lower the rates of asthma deteriorations and the number of individuals treated with ICSs by 48%. In addition, an increase in sputum eosinophils is detected up to 3 months before the development of a clinical exacerbation [56]. The usefulness of sputum cell count to improve treatment has been shown by Green et al. that

**139**

*Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma*

increasing ICS dose independent of the level of symptom control.

uncontrolled, the diagnosis of severe asthma can be formulated [59].

needing more than three bursts of OCS in the previous year [64].

ally by a good response to glucocorticosteroids.

**5. Eosinophilic refractory severe asthma**

frequent asthma exacerbations [60].

showed the efficacy of reducing exacerbations when treatment was guided according to the sputum eosinophils (to achieve a sputum eosinophil count of less than 3%) [54]. A different study used sputum cell counts to guide corticosteroid therapy to keep eosinophils <2% in moderate-to-severe asthma resulting in the sputum strategy group lower number and milder exacerbations (overall risk of exacerbations by 49%, it reduced the number of severe exacerbations) that were prevalently

Management of asthma-inhaled corticosteroid treatment based on sputum eosinophil levels has been the object of a Cochrane review that concluded that actually the risk of exacerbations is significantly reduced compared to that based on clinical symptoms with or without lung function, as well as the rate and severity of exacerbations defined by requirement for use of oral corticosteroids and hospitalizations [58]. Sputum eosinophilia may, therefore, be considered a modifiable risk factor to reduce exacerbations. Small studies in selected populations have suggested

In this contest, the eosinophilic subtype of asthma may be defined as symptomatic asthma in the presence of airway eosinophilia and that is characterized gener-

When eosinophilic inflammation in asthma leads to uncontrolled disease, the patient is at risk of exacerbations. In a proportion of patients, asthma becomes difficult to be treated despite the adequate use of high-dose corticosteroid treatment. Once the management of modifiable factors such as incorrect inhaler technique, poor adherence, smoking or comorbidities is optimized but asthma remains still

In a subgroup of patients with severe asthma, eosinophilic inflammation is still active despite the high-dose ICS treatment or oral corticosteroid intake. The use of sputum cell counts was thus defined as a marker allowing the identification of a subgroup of subjects with severe eosinophilic asthma who were at risk of more

In patients with severe eosinophilic asthma, sputum eosinophils may be suppressed by using increasing doses of inhaled steroids reducing the number of subsequent exacerbations [54, 57]. Yet, the persistence of airway eosinophilia in these subjects reflects a failure of usually adequate doses of corticosteroid to suppress inflammation [61]. Corticosteroid insensitivity is therefore intrinsic in the definition of severe asthma resulting in persistent lack of control despite corticosteroid therapy or worsening of asthma control on reduction or discontinuation of corticosteroid therapy [62]. A majority of severe asthmatics are corticosteroid dependent, refractory or insensitive and require oral corticosteroids (OCS) in addition to ICS to maintain some degree of asthma control. Only a small portion of severe asthmatics can be considered completely "corticosteroid unresponsive" or resistant [63]. The proportion of asthmatics with corticosteroid insensitivity is confirmed from the fact that one-third of the current SARP cohort were on regular OCS, with over half

A dose-response relationship between the use of OCS in asthmatic patients and the risk of many adverse events has been documented. Long-term exposure to OCS leads to increased risk of osteoporosis, arterial hypertension, diabetes, metabolic syndrome, cataracts, gastrointestinal bleeding and neuropsychiatric diseases such as depression [65]. The negative effect of systemic corticosteroid is associated not only to its maintenance or use but also to cumulative prescriptions of OCS burst [66].

*DOI: http://dx.doi.org/10.5772/intechopen.92123*

noneosinophilic [57].

#### *Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma DOI: http://dx.doi.org/10.5772/intechopen.92123*

*Cells of the Immune System*

eral airways.

severe or uncontrolled asthma [7].

demonstrated by their increased number and status of activation in the airways, and consequently, they are detected in sputum, bronchoalveolar lavage, or bronchial mucosa or submucosa. These findings may be persistent and associated with

Eosinophils may be demonstrated in the airways in the bronchial mucosa or submucosa or in the lumen (in the bronchial wash, BAL, or sputum). Bronchial biopsy is not routinely used as it is an invasive procedure and practicable only far from exacerbations to avoid dangerous complications and the quantification of eosinophils in BAL is not standardized and generally reflects samples of the periph-

Sputum examination is currently the most comprehensive and noninvasive method for measuring airway inflammation, processing and analysis being stan-

The definition of "eosinophilic asthma" implies the existence of noneosinophilic asthma. A large cohort of patients with mild-to-moderate asthma in longitudinal studies resulted in approximately 50% of them with the absence of eosinophilic airway inflammation. The cellular pattern in noneosinophilic asthma may result in either predominant neutrophilic inflammation or normal sputum cell count. Within eosinophilic asthma, eosinophilia may result in persistent (22%) or on at least 1 occasion (intermittent eosinophilia, 31%) under multiple sputum examinations [50]. Sputum inflammatory granulocytes may identify phenotypic subgroups of differing pathology and clinical characteristics within asthmatics. Within the Severe Asthma Research Program (SARP), which included a population of severe and nonsevere patients with and without corticosteroid treatment, the stratification in four groups by granulocyte % in sputum showed significant clinical differences. Patients were divided combined for stratification by granulocytes in <2%Eos + <40%Neu, <2%Eos + ≥ 40%Neu, ≥2%Eos + <40% Neu, and ≥ 2%Eos + ≥40%Neu. In this study, eosinophilic asthma, indicated by ≥2%Eos, accounted for 31% of patients, those being with combined increased sputum eosinophils and neutrophils the most severe patients in terms of lowest lung function measures, worse asthma control, greatest symptoms and use of healthcare resources [51]. In another retrospective series of 508 asthmatics, the proportion of patients with raised sputum eosinophil counts ≥3% was 42% independent of the exclusion of steroid-treated patients. Eosinophilic phenotype exhibited higher atopy, levels of IgE, bronchial hyperresponsiveness to methacholine, FENO levels and lower asthma control, while the mixed granulocytic phenotype, with both eosinophilic and neutrophilic inflammation, had the lowest lung function and the highest degree of bronchial hyperresponsiveness to methacholine [52]. In most mild-to-moderate asthma patients untreated with steroids, sputum eosinophilia >2% was significantly and inversely associated with PC20 methacholine identifying 69% of the asthma group [53]. Sputum eosinophils correlate, in addition, with symptom score and FEV% and, as previously reported, are increased by exposure to common allergens. The association between asthma exacerbations and sputum eosinophilia is suggested by different pieces of evidence. First, sputum eosinophil count is able to predict asthma deterioration after cessation of ICSs treatment in mild-to-moderate asthma, while it is decreased by treatment with corticosteroids [54]. Sputum eosinophilia may be a good additional predictor of FEV1, PC20 methacholine or quality of life of response to inhaled steroids [55]. Consequently, a clinical strategy, based on re-administration of ICSs when a change in sputum eosinophil percentage by using the 0.8% threshold was reached, could lower the rates of asthma deteriorations and the number of individuals treated with ICSs by 48%. In addition, an increase in sputum eosinophils is detected up to 3 months before the development of a clinical exacerbation [56]. The usefulness of sputum cell count to improve treatment has been shown by Green et al. that

dardized and reliability, validity, and responsiveness proven [49].

**138**

showed the efficacy of reducing exacerbations when treatment was guided according to the sputum eosinophils (to achieve a sputum eosinophil count of less than 3%) [54]. A different study used sputum cell counts to guide corticosteroid therapy to keep eosinophils <2% in moderate-to-severe asthma resulting in the sputum strategy group lower number and milder exacerbations (overall risk of exacerbations by 49%, it reduced the number of severe exacerbations) that were prevalently noneosinophilic [57].

Management of asthma-inhaled corticosteroid treatment based on sputum eosinophil levels has been the object of a Cochrane review that concluded that actually the risk of exacerbations is significantly reduced compared to that based on clinical symptoms with or without lung function, as well as the rate and severity of exacerbations defined by requirement for use of oral corticosteroids and hospitalizations [58]. Sputum eosinophilia may, therefore, be considered a modifiable risk factor to reduce exacerbations. Small studies in selected populations have suggested increasing ICS dose independent of the level of symptom control.

In this contest, the eosinophilic subtype of asthma may be defined as symptomatic asthma in the presence of airway eosinophilia and that is characterized generally by a good response to glucocorticosteroids.

## **5. Eosinophilic refractory severe asthma**

When eosinophilic inflammation in asthma leads to uncontrolled disease, the patient is at risk of exacerbations. In a proportion of patients, asthma becomes difficult to be treated despite the adequate use of high-dose corticosteroid treatment. Once the management of modifiable factors such as incorrect inhaler technique, poor adherence, smoking or comorbidities is optimized but asthma remains still uncontrolled, the diagnosis of severe asthma can be formulated [59].

In a subgroup of patients with severe asthma, eosinophilic inflammation is still active despite the high-dose ICS treatment or oral corticosteroid intake. The use of sputum cell counts was thus defined as a marker allowing the identification of a subgroup of subjects with severe eosinophilic asthma who were at risk of more frequent asthma exacerbations [60].

In patients with severe eosinophilic asthma, sputum eosinophils may be suppressed by using increasing doses of inhaled steroids reducing the number of subsequent exacerbations [54, 57]. Yet, the persistence of airway eosinophilia in these subjects reflects a failure of usually adequate doses of corticosteroid to suppress inflammation [61]. Corticosteroid insensitivity is therefore intrinsic in the definition of severe asthma resulting in persistent lack of control despite corticosteroid therapy or worsening of asthma control on reduction or discontinuation of corticosteroid therapy [62]. A majority of severe asthmatics are corticosteroid dependent, refractory or insensitive and require oral corticosteroids (OCS) in addition to ICS to maintain some degree of asthma control. Only a small portion of severe asthmatics can be considered completely "corticosteroid unresponsive" or resistant [63]. The proportion of asthmatics with corticosteroid insensitivity is confirmed from the fact that one-third of the current SARP cohort were on regular OCS, with over half needing more than three bursts of OCS in the previous year [64].

A dose-response relationship between the use of OCS in asthmatic patients and the risk of many adverse events has been documented. Long-term exposure to OCS leads to increased risk of osteoporosis, arterial hypertension, diabetes, metabolic syndrome, cataracts, gastrointestinal bleeding and neuropsychiatric diseases such as depression [65]. The negative effect of systemic corticosteroid is associated not only to its maintenance or use but also to cumulative prescriptions of OCS burst [66].

In the global strategy for asthma management and prevention (GINA) 2019 update, sputum eosinophilia ≥2% is presented as a criteria to identify patients with severe asthma with refractory type 2 inflammation despite high-dose ICS or daily OCS treatment [1, 59].

Type 2 high asthma was initially used to identify the eosinophilic phenotype of asthma. The current concept of type 2 asthma includes a phenotype, characterized by the release of signature cytokines like interleukin IL-4, IL-5 and IL-13 from cells of both the innate and the adaptive immune systems. Th2 cells and type 2 innate lymphoid cells (ILC2s) are primarily responsible for the production of high levels of T2 cytokines in the airways. The demonstration of this cytokine pathway from cellular to molecular and transcriptomic levels represents the signature for type 2 (T2) asthma [67]. The importance of identifying different phenotypes of asthma has been addressed by hypothesis-based and unbiased analyses that showed different characteristics of asthma phenotype in terms of severity, functional and clinical features, comorbidities, prognosis and response to treatment [68].

#### **5.1 Severe eosinophilic asthma in cluster analysis**

Asthma phenotyping has involved biased and unbiased approaches with the aim of grouping clinical, physiological and genetic characteristics.

In the TENOR study (the epidemiological and natural history of asthma: outcome and treatment regiments), a severe allergic asthma phenotype emerged as a high-risk group of patients for severe exacerbations with early-onset, IgE and allergen sensitization [69]. The existence of this population was confirmed in the cluster analysis by Haldar P and coworkers who found an early-onset atopic asthma cluster in which a concordance between symptom expression and eosinophilic airway inflammation is present and a symptom-based approach to therapy titration may be sufficient. On the other side, a marked discordant cluster with late-onset active predominant eosinophilic inflammation emerged as a refractory phenotype of severe asthma [70].

The predominance of sputum eosinophilia in the inflammatory patterns of severe asthmatic subphenotypes is confirmed in the unsupervised hierarchical cluster analysis of the Severe Asthma Research Program cohort where cluster 4 of severe asthmatics was associated to atopic disease and reversible severe reductions in pulmonary function, while cluster 5 was characterized mainly by later-onset disease and airflow limitations that remain with a FEV1 < 80% predicted [71].

The expansion of this analysis using a supervised learning approach that included blood, bronchoscopic, exhaled nitric oxide and clinical data gave a further focus on severe asthma phenotypes. Therefore, while cluster 4 resembled that previously described with early-onset allergic asthma with low lung function and eosinophilic inflammation, the eosinophilic refractory asthma could be further split into cluster 5, characterized by late-onset severe asthma with nasal polyps and eosinophilia and cluster 6 with persistent inflammation in blood and bronchoalveolar lavage fluid, increased FENO levels and exacerbations despite high systemic corticosteroid use and side effects. Consequently, cluster 5 was characterized as more prone to respond to corticosteroid treatment, even if rapidly deteriorated after discontinuation (corticosteroid dependent), while cluster 6 was characterized to be corticosteroid complete insensitivity [72].

#### **5.2 Blood eosinophilia as a biomarker of severe eosinophilic asthma**

The question of whether blood eosinophilia may be considered, in this contest, a surrogate marker of airway eosinophilia, is debated. The measurement of

**141**

*Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma*

both children and adults, independently of atopy [73].

eosinophil counts in blood is inexpensive and widely available. However, blood eosinophilia is nonspecific for asthma and often asthmatic patients have normal levels of eosinophils. In asthmatics with increased blood eosinophilia, there exists a direct correlation with symptom scores and an inverse correlation with FEV1 in

Blood eosinophilic counts have been reported to exhibit a moderate-to-good correlation with sputum eosinophils in asthma in large cohorts of asthmatics. A

eosinophilia ≥3% as revealed by an AUC of ROC curves of 79% that yielded 77% sensitivity and 70% specificity and an independent factors associated with the presence of sputum eosinophilic inflammation in multiple logistic regression models [52]. Other studies confirmed that blood eosinophils are an accurate biomarker of eosinophilic airway inflammation comparing two independent cohorts, mild-tomoderate asthma versus moderate-to-severe asthma. The authors used a cut-off

inflammation of ≥3% with a sensitivity of 78% and specificity of 91% [74].

In a multiple clinical variable analysis within the SARP cohort, the sensitivity and specificity of blood eosinophil counts of greater than 300/mL to detect an "eosinophil phenotype" based on sputum eosinophil counts of greater than 2% were 59% and 65%, respectively. This means that a blood eosinophil count of less than 300/L yields a 41% false-negative that has yet a sputum eosinophil percentage of greater than 2%, and likewise, many subjects with sputum eosinophil count of less than 2% would also be misclassified with a false-positive rate of 35%. Therefore, although statistically significant, the AUC of the ROC curve for predicting sputum eosinophil percentages of less than 2% or 2% and greater shows fair-to-poor accuracy and positive predictive values. These results are not improved when the cut-off of sputum eosinophil counts is more than 3% or whether the analysis is restricted to subjects with severe asthma only [51]. The stratification of SARP subjects based on blood eosinophil counts of less than 300 or 300/mL and greater showed significant differences only in methacholine bronchial hyperresponsiveness (log PC20), FEV1 percent predicted and FEV1/FVC ratio, neither in any variable related to overall asthma health care use or frequency and severity of exacerbations. This notenthusiastic result has been confirmed both in patients with mild-to-moderate or in those with severe asthma who entered a clinical trial for mepolizumab for severe

In a study that evaluated 75 uncontrolled asthmatic patients, a significant positive relationship between the percentage of sputum eosinophils and the percentage of blood eosinophils (r = 0.3647; p = 0.0013) was demonstrated. An important limits of this study were the cut-off point of blood eosinophils of 2% of WBC and

/L) yielded a significant higher sensitivity and specificity and AUC

A systematic review and meta-analysis estimated the diagnostic accuracy of markers for airway eosinophilia in patients with asthma. Looking at the 14 studies that investigated blood eosinophils as a predictor marker, an overall modest ability to distinguish between patients with or without airway eosinophilia was reported

Increasing the peripheral blood eosinophil cut-off percentage (2.7% or

as a diagnostic biomarker of sputum eosinophilia (≥3%) in a population of uncontrolled asthmatics [76] suggesting that blood eosinophils can be used in the clinic for detecting airway eosinophilia in uncontrolled asthma. These data are confirmed when looking at the population selected for treatment with reslizumab, another anti-IL-5 mAb, in which blood eosinophil counts of greater than 400/mL might be able to improve the prediction of sputum eosinophil counts of greater

resulted in good predictors of sputum

/L blood eosinophils that were able to differentiate eosinophilic

*DOI: http://dx.doi.org/10.5772/intechopen.92123*

high blood eosinophil count >220/mm3

point of ≥0.27 × 109

eosinophilic asthma [6].

0.26 × 109

than 3% [77].

again the low accuracy of ROC curves [75].

#### *Eosinophilic Phenotype: The Lesson from Research Models to Severe Asthma DOI: http://dx.doi.org/10.5772/intechopen.92123*

*Cells of the Immune System*

OCS treatment [1, 59].

of severe asthma [70].

In the global strategy for asthma management and prevention (GINA) 2019 update, sputum eosinophilia ≥2% is presented as a criteria to identify patients with severe asthma with refractory type 2 inflammation despite high-dose ICS or daily

Type 2 high asthma was initially used to identify the eosinophilic phenotype of asthma. The current concept of type 2 asthma includes a phenotype, characterized by the release of signature cytokines like interleukin IL-4, IL-5 and IL-13 from cells of both the innate and the adaptive immune systems. Th2 cells and type 2 innate lymphoid cells (ILC2s) are primarily responsible for the production of high levels of T2 cytokines in the airways. The demonstration of this cytokine pathway from cellular to molecular and transcriptomic levels represents the signature for type 2 (T2) asthma [67]. The importance of identifying different phenotypes of asthma has been addressed by hypothesis-based and unbiased analyses that showed different characteristics of asthma phenotype in terms of severity, functional and clinical

Asthma phenotyping has involved biased and unbiased approaches with the aim

In the TENOR study (the epidemiological and natural history of asthma: outcome and treatment regiments), a severe allergic asthma phenotype emerged as a high-risk group of patients for severe exacerbations with early-onset, IgE and allergen sensitization [69]. The existence of this population was confirmed in the cluster analysis by Haldar P and coworkers who found an early-onset atopic asthma cluster in which a concordance between symptom expression and eosinophilic airway inflammation is present and a symptom-based approach to therapy titration may be sufficient. On the other side, a marked discordant cluster with late-onset active predominant eosinophilic inflammation emerged as a refractory phenotype

The predominance of sputum eosinophilia in the inflammatory patterns of severe asthmatic subphenotypes is confirmed in the unsupervised hierarchical cluster analysis of the Severe Asthma Research Program cohort where cluster 4 of severe asthmatics was associated to atopic disease and reversible severe reductions in pulmonary function, while cluster 5 was characterized mainly by later-onset disease and airflow limitations that remain with a FEV1 < 80% predicted [71]. The expansion of this analysis using a supervised learning approach that included blood, bronchoscopic, exhaled nitric oxide and clinical data gave a further focus on severe asthma phenotypes. Therefore, while cluster 4 resembled that previously described with early-onset allergic asthma with low lung function and eosinophilic inflammation, the eosinophilic refractory asthma could be further split into cluster 5, characterized by late-onset severe asthma with nasal polyps and eosinophilia and cluster 6 with persistent inflammation in blood and bronchoalveolar lavage fluid, increased FENO levels and exacerbations despite high systemic corticosteroid use and side effects. Consequently, cluster 5 was characterized as more prone to respond to corticosteroid treatment, even if rapidly deteriorated after discontinuation (corticosteroid dependent), while cluster 6 was characterized

features, comorbidities, prognosis and response to treatment [68].

of grouping clinical, physiological and genetic characteristics.

**5.1 Severe eosinophilic asthma in cluster analysis**

to be corticosteroid complete insensitivity [72].

**5.2 Blood eosinophilia as a biomarker of severe eosinophilic asthma**

The question of whether blood eosinophilia may be considered, in this contest, a surrogate marker of airway eosinophilia, is debated. The measurement of

**140**

eosinophil counts in blood is inexpensive and widely available. However, blood eosinophilia is nonspecific for asthma and often asthmatic patients have normal levels of eosinophils. In asthmatics with increased blood eosinophilia, there exists a direct correlation with symptom scores and an inverse correlation with FEV1 in both children and adults, independently of atopy [73].

Blood eosinophilic counts have been reported to exhibit a moderate-to-good correlation with sputum eosinophils in asthma in large cohorts of asthmatics. A high blood eosinophil count >220/mm3 resulted in good predictors of sputum eosinophilia ≥3% as revealed by an AUC of ROC curves of 79% that yielded 77% sensitivity and 70% specificity and an independent factors associated with the presence of sputum eosinophilic inflammation in multiple logistic regression models [52]. Other studies confirmed that blood eosinophils are an accurate biomarker of eosinophilic airway inflammation comparing two independent cohorts, mild-tomoderate asthma versus moderate-to-severe asthma. The authors used a cut-off point of ≥0.27 × 109 /L blood eosinophils that were able to differentiate eosinophilic inflammation of ≥3% with a sensitivity of 78% and specificity of 91% [74].

In a multiple clinical variable analysis within the SARP cohort, the sensitivity and specificity of blood eosinophil counts of greater than 300/mL to detect an "eosinophil phenotype" based on sputum eosinophil counts of greater than 2% were 59% and 65%, respectively. This means that a blood eosinophil count of less than 300/L yields a 41% false-negative that has yet a sputum eosinophil percentage of greater than 2%, and likewise, many subjects with sputum eosinophil count of less than 2% would also be misclassified with a false-positive rate of 35%. Therefore, although statistically significant, the AUC of the ROC curve for predicting sputum eosinophil percentages of less than 2% or 2% and greater shows fair-to-poor accuracy and positive predictive values. These results are not improved when the cut-off of sputum eosinophil counts is more than 3% or whether the analysis is restricted to subjects with severe asthma only [51]. The stratification of SARP subjects based on blood eosinophil counts of less than 300 or 300/mL and greater showed significant differences only in methacholine bronchial hyperresponsiveness (log PC20), FEV1 percent predicted and FEV1/FVC ratio, neither in any variable related to overall asthma health care use or frequency and severity of exacerbations. This notenthusiastic result has been confirmed both in patients with mild-to-moderate or in those with severe asthma who entered a clinical trial for mepolizumab for severe eosinophilic asthma [6].

In a study that evaluated 75 uncontrolled asthmatic patients, a significant positive relationship between the percentage of sputum eosinophils and the percentage of blood eosinophils (r = 0.3647; p = 0.0013) was demonstrated. An important limits of this study were the cut-off point of blood eosinophils of 2% of WBC and again the low accuracy of ROC curves [75].

Increasing the peripheral blood eosinophil cut-off percentage (2.7% or 0.26 × 109 /L) yielded a significant higher sensitivity and specificity and AUC as a diagnostic biomarker of sputum eosinophilia (≥3%) in a population of uncontrolled asthmatics [76] suggesting that blood eosinophils can be used in the clinic for detecting airway eosinophilia in uncontrolled asthma. These data are confirmed when looking at the population selected for treatment with reslizumab, another anti-IL-5 mAb, in which blood eosinophil counts of greater than 400/mL might be able to improve the prediction of sputum eosinophil counts of greater than 3% [77].

A systematic review and meta-analysis estimated the diagnostic accuracy of markers for airway eosinophilia in patients with asthma. Looking at the 14 studies that investigated blood eosinophils as a predictor marker, an overall modest ability to distinguish between patients with or without airway eosinophilia was reported

with a summary estimate of AUC of 0.78 [78]. To be noticed that among the different studies, five different definitions of airway eosinophilia had been used, but either eosinophils ≥2% or 3% was used and this did not affect the accuracy of the test. Moreover, a subanalysis of the study showed the forest plots for blood eosinophilia in detecting sputum eosinophilia in subgroup populations of asthmatics. Smoking habit, steroid-treated or untreated and asthma severity revealed a considerable variability of positive thresholds of the marker. In severe asthma, only groups with the cut-point between 275 and 315 μL gave the highest sensibility and specificity [79]. As the most robust clinical value of sputum eosinophilia is tailoring inhaled corticosteroids and reducing the frequency of asthma exacerbations, it is expected that blood eosinophilia to replace induced sputum in this context should yield a sensitivity and specificity of at least 90%, so that only a small portion of patients will be misclassified. One of the most evident limits in the role of blood eosinophilia as a biomarker comes for the cross-sectional nature of the study populations. Significant variability of blood eosinophil count in the same patient over time and according to treatment status must be taken into account.
