Biomarkers Evaluation and Effect of Mepolizumab on Lower and Upper Airways Inflammation in Severe Refractory Eosinophilic Asthma
Conditions: Severe Eosinophilic Asthma
Sponsor: Istituti Clinici Scientifici Maugeri SpA

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STUDY TITLE:  
BIOMARKERS EVALUATION AND EFFECT OF MEPOLIZUMAB ON LOWER AND 
UPPER AIRWAYS INFLAMMATION IN SEVERE REFRACTORY EOSINOPHILIC ASTHMA 
AND NASAL POLYPOSIS 
 
NCT code: 
05063981 
Date last version: 
12/09/2020 
 

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Type of study: Prospective Observational study  
 
Partecipants:  
PI: Mauro Maniscalco  
 
ICS Maugeri IRCCS Telese 
Dina Visca and Antonio Spanevello  ICS Maugeri IRCCS Tradate 
Mauro Carone and Maria Aliani 
ICS Mageri IRCCS Bari 
Antonino di Stefano   
 
ICS Maugeri IRCCS Veruno 
Andrea Motta  
 
 
CNR Pozzuoli Napoli 
 
 
Scientific rationale 
Patients with severe or difficult-to-treat asthma represent a small amount of total asthmatic 
patients, but weight on the national health system for the costs of disease management. Chronic 
rhinosinusitis with nasal polyposis (CRSwNP) [1], which the Italian severe/uncontrolled asthma 
registry reported with a prevalence of 30% [2], represents a comorbidity that significantly impact 
lung function and asthma control in severe asthma [3]. Recent evidence indicates that there is a 
consistent heterogeneity regarding mucosal alterations present in subjects with nasal polyposis 
involving different pathways: inflammatory cells, remodeling, T cell activation, local IgE production, 
alteration induced by interactions between microorganisms and epithelial cells. 
 
Hypothesis 
Nasal, bronchial and systemic inflammation can differ among patients with severe refractory 
eosinophilic asthma with or without CRSwNP. Treatment with Mepolizumab could modify nasal, 
bronchial and systemic inflammation of patients with severe refractory eosinophilic asthma and 
nasal polyposis acting on disease control. 
 
Objectives (or Research questions) 
The aim of the study is to evaluate:  

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- 
differences in specific inflammatory pattern and metabolites between severe refractory 
asthma patients with or without nasal polyposis 
- 
nasal, bronchial and systemic inflammation and metabolomics profile in patients with severe 
refractory eosinophilic asthma before and after treatment with Mepolizumab.  
 
Study population 
We will enroll 10 patients with severe refractory eosinophilic asthma plus CRSwNP and 10 patients 
with severe refractory eosinophilic asthma and no CRSwNP, for whom treatment with mepolizumab 
is indicated, according to the AIFA recommendations.  
Inclusion criteria 
- age >18 years 
- diagnosis of severe refractory asthma according to the ERS/ATS criteria ATS  
- Blood eosinophils >150 cells/mcL at the screening visit and at least one value of 300 or more 
eosinophils/mcL during the previous year; 
- at least two exacerbations despite maximal therapy with long-acting bronchodilators and high 
dose inhaled corticosteroids in the previous year or the need for continued therapy with oral 
corticosteroids in addition to maximal inhaled therapy for at least 6 months in the previous year 
- written informed consent. 
- For CRSwNP diagnosis, the EPOS 2020 guidelines will be considered as reference. 
All the above-mentioned inclusion criteria must be met. 
Exclusion criteria 
- Pregnancy  
- Eosinophilic Granulomatosis with Polyangiitis (EGPA); 
- Serious life threatening cardiopulmonary disorders; 
- Systemic immunologic disorder in the last 12 months; 
- Positive history for malignant tumors ever in patient’s life; 
- Immunodeficiency. 

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Study design and methods 
Duration of the study.  
The study will last 2 years. The enrollment period will last 1 year. The follow-up period will last 1 
year. Visits and collection of biological fluids will be performed at baseline and at 6 and 12 months 
after initiation of treatment with Mepolizumab. 
The following parameters will be evaluated 
- Clinical history (age of asthma onset, duration of rhinosinusitis symptoms, ASA intolerance, 
number of asthma exacerbations in the previous year, ACT, ACQ, Morisky Medication Adherence 
Scale MMAS - 8, Lund-Mackay scores, treatment, etc..); 
- Snot 22; 
- Nasal polyps score; 
- Visual analogue scale (VAS) for main symptoms of CRSwNP 
- Lung function; 
- Nasal endoscopy evaluation. 
 
Nasal inflammation. Nasal cytology: cytospin preparations for differential cell count of eosinophils, 
neutrophils, macrophages, T-cells. Nasal secretions by rhinowash: ELISA tests for identification and 
quantitation of IL-4, IL-5, IL-3, IL-13, IFγ, eotaxin (CCL11), TSLP GMCSF. 
 
Bronchial inflammation. In induced sputum cytospin preparations: differential cell count of 
eosinophils, neutrophils, macrophages, T-cells. 
In bronchial sputum supernatants by ELISA tests: IL-4, IL-5, IL-3, IL-13, IFγ, eotaxin (CCL11), TSLP 
GMCSF. 
In cytospin preparations some selected receptors expressed by eosinophils, neutrophils or 
macrophages will be analyzed after evaluation of the prevalent secreted cytokines in the 
supernatants. 
 
Systemic inflammation. Blood eosinophils and neutrophil count; serum specific IgE evaluation with 
microarray technology; ELISA tests for IL-4, IL-5, IL-3, IL-13, IFγ, eotaxin (CCL11), TSLP GMCSF. 
 

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Metabolomic analysis in exhaled breath condensate (EBC) 
Pulmonary function tests. Flow rates will be determined using automated equipment (V Max 22 
System Sensor Medics, Milan, Italy) according to ATS/ERS recommendations. Static lung volumes 
will be determined by the helium dilution method. 
Nasal cytology. Nasal cytology will be assessed with nasal brushing. Nasal blown secretions will be 
collected by rhino-wash and processed as previously reported and supernatants stored at -80°C for 
soluble mediator evaluations [4]. 
Induced sputum. Sputum induction will be performed using a modification of the method suggested 
by prof. Howarth. The supernatant will be stored at -80°C for soluble markers determination by high 
sensitive tests ELISA  
Exhaled NO. Exhaled will be measured by an electrochemical analyzer (Hypair FeNO medisoft 
Exp’air, 2010) according to ATS-ERS recommendations for online measurement of FeNO in adults 
[5] and as previously shown [6]. 
Inflammatory markers. Blood will be withdrawn, percentage of blood eosinophils will be determined 
and serum and plasma stored at -20°C for soluble biomarkers determinations. IL-4, IL-5, IL-3, IL-13, 
IFγ, eotaxin (CCL11), TSLP GMCSF, will be evaluated in serum or plasma, induced sputum and nasal 
secretion supernatants with commercially available ELISA [7] . 
Furhermore the Humanitas Laboratory under direction of prof. Heffler will assess the following 
biological markers: Innate Lymphoid Cells 2 (ILC2) in sputum, Migration Stimulating Factor (MSF) 
Pentatraxin 3 (PTX3) Galectin 3 (GAL3) in plasma and sputum. Interleukina-5 (IL-5) in EBC.  
 
EBC sampling. EBC will be collected with a TURBO-DECCS condenser set at -5.0 ± 1.0 °C (Medivac, 
Pilastrello, Parma, Italy, Medivac.it). Briefly, all subjects will be asked to breathe at tidal volume 
through the mouthpiece for 15 min while sitting comfortably and wearing a nose-clip. We will 
obtain, on average, 2.0 ± 0.3 ml (mean ± SD) of EBC from each subject. EBC samples will be 
immediately sealed in polypropylene tubes and stored first in dry ice and then at -80°C until NMR 
acquisition [8]. 
The salivary contamination of the samples will be tested by measuring their α-amylase activity. 
NMR-based metabolomics. Metabolomic analysis will be performed by NMR spectroscopy. 1D and 
2D NMR spectra will be recorded at 27°C in a randomized sequential order on a 600-MHz Bruker 
Avance-III spectrometer equipped with a CryoProbe. Metabolites will be identified by resorting to 
2D experiments and comparison with metabolite data banks. 

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Study Endpoints 
The primary outcomes will be: 
Evaluation of the differences in specific inflammatory pattern and metabolites between severe 
refractory asthma patients with or without nasal polyposis and correlation with clinical data. 
Variation of specific inflammatory pattern and metabolites between severe asthmatic patients with 
or without nasal polyposis metabolites after treatment with Mepolizumab. 
 
Statistical Plan 
We will use multivariate analysis to discriminate signals/metabolites to identify hidden phenomena 
and trends in ensembles of NMR spectra. Subregions (buckets) of 0.02-ppm width of the spectra will 
be integrated using the AMIX 3.9.15 software (Bruker Biospin GmbH, Rheinstetten, Germany). Each 
bucket will be normalized to the total spectrum area, and the data matrix analyzed with multivariate 
statistical regressions. Principal Component Analysis (PCA) and Orthogonal Projection to Latent 
Structures Discriminant Analysis (OPLS-DA) will be carried out with the SIMCA P+14 package 
(Umetrics, Umeå, Sweden). PCA will first be applied to detect EBC metabolites trends and clusterings 
in an unsupervised (i.e., no prior group knowledge is used in the calculation) manner, while OPLS-
DA will be used to correlate the metabolic variations to pathophysiological changes. 
We will also apply pathway topology and biomarker analysis on selected and more representative 
metabolites in class separation by resorting to MetaboAnalyst 4.0 software. Discriminating 
metabolites will be selected by evaluating both Variable Importance in Projection values greater 
than 1 in class discrimination and correlation values |ρ| greater than 0.6. 
Data will be reported as mean ± standard deviation or median with interquartile range. Between-
group comparisons will be performed by using Analysis of Variance (cross-sectional evaluation) and 
Analysis of Covariance (longitudinal study) with baseline values as covariates. Depending on the 
distribution of variable, the use of transformations or non parametric tests will be evaluated. 
Categorical variables will be reported as percentage and their associations will be analysed by chi-
squared or Fisher exact test as appropriate. Because of the exploratory nature of the study, sample 
size is not formally computed but decided a priori. A p value of <0.05 will be considered statistically 
significant. The statistical analyses will be carried out using professional statistical software. 
 
Pharmacovigilance 

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All adverse reactions encountered during the study for both the biological drug and the concomitant 
drugs will be reported to local pharmacovigilance officers, as required. 
 
Limitations 
Patients will be asked to return to the clinic with a frequency not much different to that of routine 
disease control to reduce possible drop out. 
A possible problem could be the unsuitable sputum sample or patient with too compromised lung 
functions to undergo sputum induction function (FEV1 < 1.5L). We will collect spontaneous sputum 
when available, while if the patient cannot expectorate or the induced sputum collected is not 
suitable (sample mainly constituted by saliva), FeNO measurement will be the surrogate evaluation 
for eosinophilic airway inflammation. 
At the co-investigator unit, a specific asthma school is present in order to properly train severe 
asthmatic patients to manage the disease and to reduce non adherence. This approach will be useful 
to avoid drop-out and loss of adherence to the protocol. 
 
References  
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Wu W, Bleecker E, Moore W, Busse WW, Castro M, Chung KF, Calhoun WJ, Erzurum S, 
Gaston B, Israel E et al: Unsupervised phenotyping of Severe Asthma Research Program 
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Maio S, Baldacci S, Bresciani M, Simoni M, Latorre M, Murgia N, Spinozzi F, Braschi M, 
Antonicelli L, Brunetto B et al: RItA: The Italian severe/uncontrolled asthma registry. 
Allergy 2018, 73(3):683-695. 
3. 
Novelli F, Bacci E, Latorre M, Seccia V, Bartoli ML, Cianchetti S, Dente FL, Franco AD, Celi A, 
Paggiaro P: Comorbidities are associated with different features of severe asthma. Clin 
Mol Allergy 2018, 16:25. 
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Maniscalco M, Sofia M, Faraone S, Carratu L: The effect of platelet-activating factor (PAF) 
on nasal airway resistance in healthy subjects is not mediated by nitric oxide. Allergy 
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5. 
ATS/ERS recommendations for standardized procedures for the online and offline 
measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J 
Respir Crit Care Med 2005, 171(8):912-930. 
6. 
Maniscalco M, Faraone S, Sofia M, Molino A, Vatrella A, Zedda A: Extended analysis of 
exhaled and nasal nitric oxide for the evaluation of chronic cough. Respir Med 2015, 
109(8):970-974. 
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Di Stefano A, Coccini T, Roda E, Signorini C, Balbi B, Brunetti G, Ceriana P: Blood MCP-1 
levels are increased in chronic obstructive pulmonary disease patients with prevalent 
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8. 
de Laurentiis G, Paris D, Melck D, Maniscalco M, Marsico S, Corso G, Motta A, Sofia M: 
Metabonomic analysis of exhaled breath condensate in adults by nuclear magnetic 
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