Beyond Eosinophils: Proteomics to Identify Potential Biomarkers of Organ Damage and Response to MEPOLIZUMAB in EGPA
Phase: PHASE4
Conditions: EGPA - Eosinophilic Granulomatosis With Polyangiitis, Severe Asthma, Eosinophilic Asthma
Sponsor: Azienda Ospedaliero Universitaria di Cagliari

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
 
 
 
 
Beyond eosinophils: proteomics to identify potential biomarkers of organ damage 
and response to MEPOLIZUMAB in EGPA  
Protocol Number: 14918  
Compound Number or Name: BEOMICS study  
Brief Title: Beyond EOsinophils: proteoMICS to identify potential biomarker of organ damage and 
response to MEPOLIZUMAB in EGPA  
Principal investigator: Prof. Stefano Del Giacco, Cagliari, Italy 
Sub investigators: Giulia Costanzo, Davide Firinu, Andrea G. Ledda, Giada Sambugaro, Barbara  
Manconi, Alessandra Olianas, Tiziana Cabras. Index  
SCIENTIFIC RATIONALE.................................................................................................2  
OBJECTIVE/RESEARCH QUESTION...............................................................................2  
STUDY POPULATION......................................................................................................3  
• 
-  Inclusion criteria......................................................................................................3  
• 
-  Exclusion criteria....................................................................................................3  
STUDY DESIGN AND METHODS...................................................................................4  
• 
-  Demographics and medical history  
• 
-  Clinical Assessment and Questionnaires  
• 
-  Laboratory assessment and Samples preparation  
• 
-  Proteomic analysis  
• 
-  Safety  
STUDY ENDPOINTS................................................................................................................8 
STATISTICAL PLAN.................................................................................................................8 
LIMITATIONS..........................................................................................................................8  
 
1  
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
SCIENTIFIC RATIONALE  
Eosinophilic granulomatosis with polyangiitis (EGPA), former Churg Strauss Syndrome, is a systemic disease 
characterized by small vessel vasculitis, extravascular granulomas and peripheral blood eosinophilia, which 
typically occurs in the context of late-onset asthma and polypoid rhinosinusitis [1]. Although effective 
immunosuppressive treatment significantly improved the overall survival of EGPA patients, relapses are still 
common during follow-up [2], especially isolated asthma/rhinosinus exacerbations [3]. Recent evidence of 
pathogenetic mechanism shared among EGPA, severe asthma and other eosinophilic disorders, has 
supported the effective role of mepolizumab, an anti-IL-5 humanised monoclonal antibody, especially in 
the prevention of EGPA-related ENT and/or asthmatic relapses, with a significant GC- sparing effect [4–5]. 
There are still scant data on biomarkers that may predict of organ damage in EGPA patients and the 
heterogeneous spectrum of clinical symptoms makes more difficult the identification of a single biomarker.  
A better understanding of potential biomarkers of response to immunosoppressive drugs, as well as the 
impact of novel anti-IL-5 agents on these clinical domains is essential to guide treatment choice and to 
improve long-term management of the disease.  
In the OMICS era, an approach based on proteomics may highlight new insights of several molecular 
pathways all over different diseases. We previously applied this methods in diseases such as primary 
immunodeficiency, non-histaminergic angioedema and mastocytosis[6]. In EGPA there is by now no data 
about proteomics, and, similarly, in severe asthma there is still limited available data of proteomics 
profiling albeit asthma is the most common chronic respiratory disease and has been declared a global 
public health problem by the World Health Organization [7].  
RESEARCH QUESTION  
Only a few studies investigated potential biomarkers in EGPA patients and their relation and evolution 
during treatment.  
Aim of the study is to identify potential biomarkers, through a proteomic approach, which could be used to 
evaluate organ damage and predict the response to mepolizumab in a cohort of patients affected by EGPA. 
Proteomic analyses will be performed using a proteomic platform, based on a nano-HPLC- couplet to an 
high resolution ESI-MS device, on three types of biological matrices: blood, saliva and sputum samples in 
both EGPA and severe asthmatic patients (as controls) at baseline and at different time points after starting 
treatment with mepolizumab, an anti-IL-5 drug, in order to cluster patients and to analyze the effect of the 
therapy during treatment, assessing the disease progression on three key aspects: lung function and 
symptoms control, vasculitis and neuropathy. Plasma analysis will provide an overview of 
quantitative/qualitative proteomic variations at systemic level after drug administration; however, a less 
invasive procedure is often sufficient and would improve trial recruitment. On this regard, saliva is a 
biological fluid well suitable to be used in proteomic investigations for suggestion of potential disease 
biomarkers and includes various potential advantages compared with blood sample collection such as 
lower overall cost, lower infection risk, increased patient convenience, acceptability, compliance and 
uptake. Moreover, the protein composition of the human saliva includes both specific proteins of the oral 
cavity and proteins common to other tissues and bodily fluids, so saliva prognostic and diagnostic role is 
particularly interesting [8-9-10-]. Consequently, we plan to compare the proteomic results of the non-
invasive saliva testing to that of blood examination.  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
These data may be a further step to 
untangle the mechanisms of the disease and to characterize treatment’s response, in the contest of a 
phenotype/endotype asthma management.  
PRIMARY OBJECTIVES  
We plan to collect blood, salivary and sputum samples in both EGPA and severe asthmatic patients (as 
controls) at baseline and at different timepoints after starting treatment with mepolizumab, an anti-IL-5 
drug, in order to cluster patients and to analyse the effect of the therapy during treatment and assess the 
disease progression on three key aspects of the disease: lung function and symptoms control, vasculitis and 
neuropathy.  
SECONDARY OBJECTIVES  
to untangle the mechanisms of the disease and to characterize response to treatment, in the context of a 
phenotype/endotype asthma and EGPA management.  
STUDY POPULATION  
According to the patients’ groups evaluated in other publications with a robust statistical method and 
sample size calculation performed by the team, a sample of 90 patients can be considered sufficient to 
allow the realization of the study.  
We plan to enroll 90 patients followed in Allergy and Clinical Immunology Centers divided into three 
groups:  
• 
●  30 patients with EGPA with predominant ENT/asthmatic phenotype (longitudinal sampling 
before and after Mepolizumab treatment)  
• 
●  30 patients with EGPA with vasculitic phenotype before and after immunosuppressive drugs 
(CYC, AZA, MTX) and/or Mepolizumab.  
• 
●  30 patients with a diagnosis of severe eosinophilic asthma (longitudinal sampling before and 
after Mepolizumab treatment)  
Inclusion criteria  
A patient will be eligible for inclusion in this study if all the following criteria apply:  
1. Informed Consent: Prior to start any study related activities, participants must be able and 
willing to provide written informed consent;  
2. Participants must have a current clinical diagnosis of asthma or EGPA;  
3. Physician decision to initiate treatment with mepolizumab.  
4. Patient has to be in treatment with medium-high dose ICS plus an additional controller in the 
least 6 months before screening and, if on OCS therapy, a stable dosage of prednisone (or 
equivalent dose of other steroids) in the 4 weeks before screening will be allowed. The above-
indicated treatment has to be maintained by the patient all along the study.  
5. Adults aged 18 years or over.  
Exclusion Criteria  
A participant will not be eligible for inclusion in this study if any of the following criteria  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
apply: 
1. asthmatic patients receiving other biological treatment 
2. Participation in an interventional clinical trial in which the treatment regimen  
and/or monitoring is dictated by a protocol during the previous 12 months. 3. pregnant and breastfeeding 
woman  
STUDY DESIGN AND METHODS  
During months 1-2, the Allergy and Clinical Immunology Centers will prepare the documentations to obtain 
Ethics Committee approval; an electronic database will also be created from this group to collect the data 
obtained during the study. At the end of this time, we expect to have obtained approval of the Ethics 
Committee for samples and data collection.  
Primary END POINTS  
• 
Obtain a proteomic profile of EGPA patients and severe asthmatic patients from blood, saliva and 
sputum  
• 
Analyse the samples after starting anti IL – 5 treatment ( mepolizumab ) and evaluate the possible 
disease modifying effect of the mepoliumab on vasculitis and lung function.  
Secondary END POINTS 
• to untangle the mechanisms of the disease and to characterize response to  
treatment, in the context of a phenotype/endotype asthma and EGPA management.  
Patients will be screened at visit 1 (T0) and if eligible will be enrolled. After collecting the informed consent, 
we will collect the samples, respiratory lung function tests, and the clinical questionnaire. 
Mepolizumab administration will be done every 4 weeks (±7days), as per the drug administration plan (i.e. 
100mg SC Q4W). Other drugs will be administrated according to their administration plan and investigated 
in medical history. At each visit will be collected the clinical questionnaire and assessed the respiratory lung 
function. 
Patients will receive mepolizumab at visit 2 (after 4 weeks ±7days). At visit 3 (a time interval of 8 weeks 
±7days from visit 1), mepolizumab administration will happen, plus clinical assessment (questionnaire and 
respiratory lung function test). At visit 4 (also identified as T1, 12 weeks ±7days after visit 1), the same 
assessments and analysis as at T0 will be performed, as well as mepolizumab administration. Follow-up 
visit will be performed at visit 5 (after 24 weeks ±7days from visit 1) and patients will undergo 
somatosensory evoked potentials together with the collection of clinical questionnaire.  
Then, at visit 6, which corresponds to T2 (after 52 weeks _± 7 days from visit 1), the same assessments and 
analysis as at T0 and T1 will be performed, as well as mepolizumab administration. 
Study’s total duration is 52 weeks, with follow-up after 4 weeks.  
Safety profiles (AEs, SAEs and AEs of special interest) will be recorded throughout the study and at follow-
up visit. We plan to use our local template for adverse events. At the end of the study we will inform GSK 
about the reported adverse events. Also, for pregnancies, we would register them and strictly follow up the 
pregnancy during the delivery and the breastfeeding period. If an enrolled patient will be found on 
pregnancy, she will exit the study.  
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
The same timeline will be used for those patients with vasculitis phenotype and those affected by severe 
eosinophilic asthma, collecting biological samples before and after immunosuppressive and mepolizumab 
treatment. 
Samples obtained from the patients at T0, T1 and T2, treated as reported in “Laboratory assessment and 
Samples preparation” section, will be sent to the proteomics unit for analysis.  
Demographics and medical history  
At study visits, patients’ demographics and medical history (asthma disease and therapy history, 
immunosuppressive drugs) will be documented. Comorbidities in association with severe eosinophilic 
asthma, in particular chronic rhinosinusitis (CRS), with or without polyps (NP), will be recorded as analysis 
will be performed taking into account those three subgroups (i.e. according to the presence of CRS and/or 
NP).  
Clinical Assessment and Questionnaires  
Patients will be clinically evaluated. Clinical assessment will include physical examination, spirometry, 
patient’s reports outcome [Asthma control test (ACT), Asthma Control Questionnaire 5 (ACQ-5), Sino- nasal 
outcome test 22 (if patient with CRSwNP), BIVAS, and VDI [10] [11]. Spirometry measurements will be 
obtained using spirometry equipment that meets or exceeds the minimal performance recommendations 
of the ATS [Miller, 2005]. All subjects will have spirometry performed at Screening and scheduled clinic 
visits during the treatment period as indicated in the Time and event table. 
For FEV1 and FVC determinations, at least three acceptable spirometry efforts (with no more than 8) 
should be obtained. Acceptable spirometry efforts should have a satisfactory start of test and end of test 
(i.e. a plateau in the volume-time curve) and be free from artifacts due to cough, early termination, poor 
effort, obstructed mouthpiece, equipment malfunction, or other reasons [Miller, 2005]. The largest FEV1 
and FVC from the three acceptable efforts should be recorded, even if they do not come from the same 
effort.  
Spirometry must be performed as follows: After withholding salbutamol for 4 hours and ICS/LABA for 12 h. 
FeNO assessment will be performed after spirometry and even after four puffs of salbutamol.  
In order to assess the adequacy of asthma control and the modification of rhinosinusitis, symptoms ACT, 
ACQ-5 and SNOT-22, respectively, will be submitted to patients at every visit. ACT and ACQ scores at T1 and 
T2 will be compared to baseline. The current dose of OCS and/or the withdrawal of OCS will be recorded at 
T0, T1, and T2.  
The number of asthma exacerbations will be recorded and evaluated during the12 months. 
We assessed asthma exacerbations, defined as any of the following events: asthma attack needing an 
increase in oral prednisone dose, asthma related emergency department admission, and/or use of acute 
oral glucocorticoids, antibiotics, or short-acting beta agonists. 
For vasculitic patients, similarly to the previous published MIRRA trial a complete response to treatment 
was defined as no disease activity (BVAS = 0) and a prednisolone or prednisone dose (or equivalent) of ≤4.0 
mg/day. 
Blood samples, salivary samples and sputum samples are collected in fasting condition before respiratory 
measurements.  
LABORATORY ASSESSMENT AND SAMPLES PREPARATION  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
Plasma, saliva and sputum proteome will be analyzed on a proteomic platform based on a nano-HPLC-high 
resolution ESI-MS device (LTQ Orbitrap Elite, Thermo Fisher Scientific) available at the “proteomic 
laboratory” of the University Service Center for Research of Cagliari (CeSAR) and by a Surveyor HPLC system 
connected to a low resolution LTQ XL mass spectrometer (Thermo Fisher Scientific San Jose, CA, USA), 
available at the proteomic laboratory of the Dept. of life and Environmental Sciences.  
Peripheral blood samples of each patient will be collected into BD Vacutainer EDTA. To obtain cell-free 
plasma samples, the blood samples will be centrifuged at 1600 g for 10 minutes at +4°C. The plasma 
fraction of each patient will be stabilized by protease inhibitor, divided into aliquots, transferred to clean 
tubes and stored at –80°C for subsequent studies.  
Unstimulated whole saliva will be collected during the morning and in fasting conditions with a plastic 
Pasteur pipette and will be added with an equal amount of PBS containing protease inhibitor and stored at 
-80°C (whole saliva for bottom-up proteomics).  
Sputum samples will be collected after 15–30 min inhalation of hypertonic saline in concentrations of 3%, 
treated as salivary samples and stored at -80°C. the procedure for analyzing the proteomic profile in the 
sputum is the same as saliva samples. The traditional use of sputum induction is not the goal of our 
research, indeed the eosinophils count in the sputum is not one of our purposes.  
 
PROTEOMIC ANALYSIS  
Proteomic analysis based on mass spectrometry (MS) analysis will be performed to evaluate qualitative and 
quantitative alterations in the saliva, sputum and plasma proteomes associated with:  
• EGPA with predominant ENT/asthmatic phenotype (before and after Mepolizumab treatment)  
• EGPA with vasculitic phenotype before and after immunosuppressive drugs (CYC, AZA, MTX) and/or 
Mepolizumab.  
• Severe eosinophilic asthma (already with established Mepolizumab treatment)  
1.  saliva and sputum: the comparison of the protein profiles among the three groups of samples will 
allow us to highlight quali/quantitative changes associated with EGPA before and after 
Mepolizumab treatment, immunosuppressive drugs and eosinophilic asthma. The approach will be 
suitable to identify peptides/proteins that are potentially useful as biomarkers for EGPA diagnosis. 
Salivary and sputum samples (n=30 from each patient group at T0 T1 and T2 of medical treatment, 
total 300 samples) will be submitted to protein technique processing in bottom-up proteomics by 
filter aided sample preparation (FASP), including tryptic digestion, after reduction/alkylation of Cys 
residues, recovery of tryptic fragments and their analysis by HPLC-ESI-MS for quantification and 
identification.  
2.  Plasma: plasma is a very complex protein matrix where the MS signals of high- abundance proteins 
hide those from other proteins, so plasma samples (n=30 from each patient group at T0 T1 and T2 
of medical treatment, total 180 samples) will undergo to depletion of albumin and the 20 most 
abundant plasma proteins with a commercial kit to improve the detectability of low abundance 
proteins. Plasma samples will be used to specifically analyze the peptides and/or proteins 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
individuated as potential 
biomarkers in EGPA samples. Thus, to detect them, depending on their molecular weight (MW), 
suitability for top-down proteomics, and abundance, we will apply one of these different 
approaches:  Bottom-up proteomics based on FASP processing of plasma proteins and HPLC-  
ESI-MS analysis to detect proteins not suitable for a top-down proteomic analysis or with medium/high 
abundance;   ultrafiltration with 30 KDa cut-off membranes to obtain two fractions with a simpler 
composition that facilitates the detection of low-abundance proteins. A protein fraction <30 kDa is useful 
for low-MW protein and peptide characterization by a top- down approach, and a protein fraction >30 kDa 
is useful for proteins with high MW, which will be analyzed by HPLC-ESI-MS analysis with a bottom-up 
approach.  Before any MS analysis, the total protein concentration will be measured in each sample to 
normalize quantitative data. Proteomic analysis will be performed by both high-resolution (HR), 120,000 at 
m/z 200 in full MS mode, and low-resolution (LR) mass spectrometry, 60,000 at m/z 200 in full MS mode. 
For HR-MS we will use the nano- HPLC-ESI-LTQ Orbitrap Elite (Thermo Fisher Scientific San Jose, CA, USA) 
available by payment at the University Service Center for Research of Cagliari (CeSAR); for the LR-MS, we 
will use the HPLC- ESI-LTQ XL-MS instrument (Thermo Scientific) available in the Proteomic Lab of the 
Department of Life and Environmental Sciences, Biomedical section.  
-The HR-MS platform will be used for the identification of peptides/proteins, either in top-down or in 
bottom-up proteomics modalities, through amino acid sequencing based on data-dependent MS/MS 
experiments. In addition, post-translational modifications will be characterized. All MS/MS data will be 
elaborated by the ProteomeDiscoverer software v.2.4 (Thermo Scientific). Spectra will be recorded in the 
300–2000 m/z scanning range in the positive ion mode and ions fragmented with higher energy collisional 
dissociation (HCD) or collision induced dissociation (CID). Protein identification will be performed using the 
reviewed Swiss-Prot human database with a precision tolerance of 10 ppm for peptide masses and 0.02 Da 
for fragment ion masses. Proteins will be identified with at least two unique peptides and a significance 
threshold FDR = 0.01. In plasma samples, beyond full protein profiling, targeted peptides/proteins will be 
selectively searched and identified by consecutive reaction monitoring.  
- The LR-MS platform will be used for explorative experiments and to quantify target proteins/peptides, 
due to the high sensitivity of the instruments, which are able to detect up to femtomolar concentrations.  
Moreover, target components detectable either as intact proteins or peptides in top- down mode or 
through their unique tryptic fragments in bottom-up mode, can also be quantified by the LR-MS platform 
by measuring the AUC (area under the curve) of either multiple components with the extracted ion current 
technique, or few components with the selected ion monitoring technique.  
- Quantification of peptides and proteins can be performed in label-free way (Levin, Y.; Schwarz, E.; Wang, 
L.; Leweke, F. M.; Bahn, S. Label-free LC-MS/MS quantitative proteomics for large-scale biomarker 
discovery in complex samples. J. Sep. Sci. 2007, 30 (14), 2198–203.) or via selected reaction monitoring 
(SRM) by spiking unlabeled samples with known concentrations of isotopically-labeled synthetic peptides 
[Allergy. 2020 Dec;75(12):3171-3183.doi: 10.1111/all.14406.].  
d) Quantitative MS data will be compared by statistical analyses with GraphPad Prism 5.0 and Perseus 
software, to highlight significant differences between the protein profiles of:  
• EGPA with predominant ENT/asthmatic phenotype (before and after Mepolizumab treatment)  
•EGPA with vasculitic phenotype before and after immunosuppressive drugs (CYC, AZA, MTX) and/or 
Mepolizumab.  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
•severe eosinophilic asthma 
(already with established Mepolizumab treatment).  
-MS results will undergo technical verification by immune-detection methods (western-blot or dot-blot) in 
the same samples for target proteins and peptides individuated as candidate biomarkers.  
 
 
 
SAFETY MONITORING AND REPORTING 
Definition of adverse events and reporting requirements Adverse events (AEs) 
 
An AE is any untoward medical occurrence (e.g. any unfavorable and unintended sign [including abnormal 
laboratory findings], symptom or disease) in a subject or clinical investigation subject after providing 
written informed consent for participation in the  
study. Therefore, an AE may or may not be temporally or causally associated with the use of a medicinal 
product. The investigator has the responsibility for managing the safety of individual subject and identifying 
AEs. The occurrence of AEs must be sought by non-directive questioning of the subject at each visit or, for 
self-administering subjects at each scheduled safety phone call, during the study. AEs also may be detected 
when they are volunteered by the subject during or between visits or telephone calls or through physical 
examination findings, laboratory test findings, or other assessments. AEs must be recorded under the signs, 
symptoms or diagnosis associated with them, accompanied by the following information (as far as possible) 
(if the event is serious refer to Section 1.2): the Common Toxicity Criteria (CTC) AE grade (version 5 or 
higher);  
- its relationship to the investigational study treatment and other study treatment.  
-its duration (start and end dates), or if the event is ongoing, an outcome of not recovered/not resolved, 
must be reported;  
- whether it constitutes a SAE (see Section 1.2 for definition of SAE) and which seriousness criteria have 
been met.  
- action taken regarding with study treatment 
- its outcome i.e., its recovery status or whether it was fatal. 
All AEs must be treated appropriately. Treatment may include 1 or more of the following: Dose not 
changed - Dose Reduced/increased - Drug interrupted/permanently discontinued;  
Relevant conditions that were already present at the time of informed consent should be recorded in medical 
history of the subject. AEs (including lab abnormalities that constitute AEs) should be described using a 
diagnosis whenever possible, rather than individual underlying signs and symptoms. Once an AE is detected, 
it must be followed until its resolution or until it is judged to be permanent (e.g., continuing at the end of the 
study), and assessment must be made at each visit (or more frequently, if necessary) of any changes in 
severity, the suspected relationship to the interventions required to treat it, and the outcome.  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
Serious adverse events (SAEs)  
An SAE is defined as any AE, i.e. appearance of (or worsening of any pre-existing) undesirable sign(s), 
symptom(s), or medical conditions(s) which meets any one of the following criteria: fatal OR life-
threatening.  
Life-threatening in the context of a SAE refers to a reaction in which the subject was at  risk of death at the 
time of the reaction; it does not refer to a reaction that hypothetically might have caused death if it were 
more severe (please refer to the ICH- E2D Guidelines) results in persistent or significant disability/incapacity 
constitutes a congenital anomaly/birth defect requires inpatient hospitalization or prolongation of existing 
hospitalization, unless hospitalization is for: routine treatment or monitoring of the studied indication, not 
associated with any deterioration in condition elective or pre- planned treatment for a pre-existing condition 
that is unrelated to the indication under study and has not worsened since signing the informed consent 
social reasons and respite care in the absence of any deterioration in the subject’s general condition 
treatment on an emergency outpatient basis for an event not fulfilling any of the definitions of a SAE given 
above and not resulting in hospital admission is medically significant, e.g. defined as an event that jeopardizes 
the subject or may require medical or surgical intervention to prevent one of the outcomes listed above. 
Medical and scientific judgment should be exercised in deciding whether other situations should be 
considered serious reactions, such as important medical events that might not be immediately life 
threatening or result in death or hospitalization but might jeopardize the subject or might require 
intervention to prevent one of the other outcomes listed above. Such events should be considered as 
“medically significant.” Examples of such events are intensive treatment in an emergency room or at home 
for allergic bronchospasm, blood dyscrasias, or convulsions that do not result in hospitalization or 
development of dependency or abuse (please refer to the ICH-E2D Guidelines).  
All malignant neoplasms will be assessed as serious under “medically significant” if other seriousness 
criteria are not met. Any suspected transmission via a medicinal product of an infectious agent is also 
considered a serious adverse reaction.  
All reports of intentional misuse and abuse of the product are also considered SAE irrespective of whether a 
clinical event has occurred or not.  
STATISTICAL PLAN  
Data will be analyzed by SPSS for MS Windows, version 18.0 (SPSS, Inc, Chicago, IL, USA).  
The primary analysis is to determine the difference of proteomic profile at T1 and T2 versus T0 (baseline) 
and to evaluate this percentage in relation to subject condition (CRS, NP). This will be addressed using 
parametric and non-parametric tests and clustering techniques on flow cytometry data (cytograms and 
sample composition). Results will be expressed as mean values SEM. Mann-Whitney U test will be used for 
two-group comparisons. Wilcoxon signed-rank test will be used for paired data. When more than two 
groups will need to be analyzed, ANOVA and Dunnett’s multiple comparison test will be used. Correlation 
will be evaluated by the Spearman rank test.  
Differences will be considered significant if P values were <.05.  
LIMITATIONS  
The study could be limited from the relatively small size group of vasculitis patients.  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
Another limit could be due to the 
collection of anamnestic data regarding the disease onset from patients already in mepolizumab treatment 
or under immunosuppressive drug(s).  
Funding  
The study is supported by GSK.  
Ethics  
The study will be performed after collecting a valid informed consent in accordance with the Good Clinical 
Practice and with the Declaration of Helsinki.  
Data policy and informed consent  
At visit 1 the study is presented to potential eligible patients. They will be suggested to read with attention 
the information sheet and the informed consent. Only those patients who read and sign the informed 
consent can be enrolled in the study.  
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Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum. 2013;65:1–11. 
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with eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) enrolled in two prospective 
trials. J Autoimmun. 2013;43:60–9.  
3. Puéchal X, Pagnoux C, Baron G, Lifermann F, Geffray L, Quémeneur T, et al. Non-severe eosinophilic 
granulomatosis with polyangiitis: long-term outcomes after remission- induction trial. Rheumatol Oxf Engl. 
2019;58:2107–16. 
4. Kahn J-E, Grandpeix-Guyodo C, Marroun I, Catherinot E, Mellot F, Roufosse F, et al. Sustained response to 
mepolizumab in refractory Churg-Strauss syndrome. J Allergy Clin Immunol. 2010;125:267–70.  
5. Wechsler ME, Akuthota P, Jayne D, Khoury P, Klion A, Langford CA, et al. Mepolizumab or Placebo for 
Eosinophilic Granulomatosis with Polyangiitis. N Engl J Med. 2017;376:1921–32.  
6. Serrao S, Firinu D, Olianas A, Deidda M, Contini C, Iavarone F, Sanna MT, Boroumand M, Amado F, 
Castagnola M, Messana I, Del Giacco S, Manconi B, Cabras T. Top-Down Proteomics of Human Saliva 
Discloses Significant Variations of the Protein Profile in Patients with Mastocytosis. J Proteome Res. 2020 
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PMC8008451.  
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Long & Tao Peng (2021) New advances in quantitative proteomics research and current applications in 
asthma, Expert Review of Proteomics, 18:12, 1045- 1057, DOI: 10.1080/14789450.2021.2017777  
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9. Cabras, T.; Iavarone, F.; Manconi, B.; Olianas, A.; Sanna, M. T.; Castagnola, M.; Messana,I. Top-down 
analytical platforms for the characterization of the human salivary proteome.Bioanalysis 2014, 6, 563, 581.  

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
10. Bandhakavi, S.; Stone, M. D.; 
Onsongo, G.; Van Riper, S. K.; Griffin, T.J. A dynamic range compression and three-dimensional peptide 
fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva. J. 
Proteome Res. 2009, 8, 5590-5600.  
11. Steinfeld, J., Bradford, E. S., Brown, J., Mallett, S., Yancey, S. W., Akuthota, P., Cid, M. C., Gleich, G. J., 
Jayne, D., Khoury, P., Langford, C. A., Merkel, P. A., Moosig, F., Specks, U., Weller, P. F., & Wechsler, M. E. 
(2019). Evaluation of clinical benefit from treatment with mepolizumab for patients with eosinophilic 
granulomatosis with polyangiitis. The Journal of allergy and clinical immunology, 143(6), 2170–2177. 
https://doi.org/10.1016/j.jaci.2018.11.041  
12. Doubelt, I., Cuthbertson, D., Carette, S., Chung, S. A., Forbess, L. J., Khalidi, N. A., Koening, C. L., 
Langford, C., McAlear, C. A., Moreland, L. W., Monach, P. A., Seo, P., Specks, U., Spiera, R. F., Springer, J. M., 
Sreih, A. G., Warrington, K. J., Merkel, P. A., Pagnoux, C., & Vasculitis Clinical Research Consortium (2021). 
Clinical Manifestations and Long-Term Outcomes of Eosinophilic Granulomatosis With Polyangiitis in North 
America. ACR open rheumatology, 3(6), 404–412. https://doi.org/10.1002/acr2.11263  
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
X1: all the therapy taken by the patient, like asthma therapy or immunosoppressive drug, and their specific 
dose. 
X2: not applicable in patients without a diagnosis of EGPA 
X3: if the patient has EGPA with vasculitic phenotype and takes immunosuppressive drugs (CYC, AZA, MTX) 
without taking Mepolizumab, don’t administrate it.  
 
 
Monserrato (CA), 12.0 2.202 3  
Prof. Stefano Del Giacco  
.  
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108 
 
 

                                  
 
 
 
 
PRGM- C Allergologia e Immunologia Clinica 
Direttore Prof. Stefano Del Giacco  
07051096108