Effect of Mepolizumab on Severe Eosinophilic Asthma (EMESEA)

Description

Hypotheses

Hº1. The levels of certain surface molecules on eosinophils or the presence or absence of certain proteins in the proteome of this leukocyte subset prior mepolizumab treatment can be used as predictive/prognostic markers of response to this biological.

Hº2. Mepolizumab alters the abundance of several surface or intracellular proteins in eosinophils as an outcome related to changes in their activation status, migratory ability, regulatory/effector function, or subset composition.

Hº3. Late-onset severe eosinophilic asthmatics have elevations in the serum concentration of different members of the IGF family (IGF-1, IGF-BP3, IGF-ALS) and mepolizumab treatment reduces these levels and behaves as a response-biomarker along with the number of eosinophils and clinical exacerbations.

Objectives or research questions

OB (Aspirational objective): Discovery of predictive/prognostic biomarkers of response to mepolizumab using flow cytometry, transcriptomic, and proteomic technologies.

• OB1.- To identify changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques. This objective is distributed in the following deliverables (DE):
• DE1.1: Selection of 15 healthy controls
• DE1.2: Diagnosis of late-onset severe eosinophilic asthma patients and selection of 15 patients who meet criteria, are scheduled to receive mepolizumab, and sign the informed consent.
• DE1.3: Generation of the initial database for healthy controls and asthma patients with demographic, clinical, haematologic, and biochemical information.
• DE1.4: Collection and processing of serum (1 SST tube) and whole blood samples (1-2 tubes) from healthy donors (T0) and mepolizumab-treated patients (T0, T4, T16, T32).
• DE1.5: Flow cytometry assays with multimarker panels 1 (regulatory), 2 (activation), and 3 (eosinophil subsets) (see below).
• DE1.6: Complete the database with clinical, hematological, biochemical and flow cytometry data generated at times T4, T16 and T32. Final uni- and multivariant statistical analysis.
• DE1.7: Publication of results.
• OB2.- Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab. This objective is distributed in the following deliverables (DE):
• DE2.1: Set up an eosinophil isolation protocol and check cell purity by flow cytometry.
• DE2.2: Purification of eosinophils from 15 healthy donors (T0) and 15 patients (T0, T4, T16, T32).
• DE2.3: Total RNA extraction from eosinophil lysates, assay of quality and quantity of RNA, and storage at -80ºC.
• DE2.4: Discovery-based/hypothesis-generating approach. Evaluation of the levels of 770 human protein-coding mRNAs linked to the recruitment, activation, and effector functions of myeloid cells by means of a direct multiplexed molecular measurement platform named nCounter® NanoString) in combination with a pre-made "nCounter® Human Myeloid Innate Immunity Panel (v2)" (www.nanostring.com/products/gene-expression-panels/gene-expression-panels-overview /ncounter-myeloid-innate-immunity-panel ). Due to the high economic cost, this part of the study will only be performed with eosinophil samples from healthy donors (T
  • 0) and two time points in patients (T = 0 and T = 16).
• DE2.5: Processing of the obtained data and initial statistical analysis.
• DE2.6: Validation of nCounter® data and hypothesis-driven approach with an established quantitative technique. Perform retrotranscription and qPCR analyses of those mRNAs in eosinophils displaying the greatest abundance changes in response to mepolizumab treatment according to the nCounter® study. In addition, some additional mRNAs not included in the "nanoString Myeloid Innate Immunity" panel, such as FOXP3 (regulatory function), CRLF2, ST2, or IL-7R (cytokine receptors; activation), will be analysed. HPRT1 gene will be used as a house-keeping gene in this set of RTqPCR experiments.
• DE2.7: Uni- and multivariant statistical analyses.
• DE2.8: Publication of transcriptomic results.
• OB3.- Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab. This objective is distributed in the following deliverables (DE):
• DE3.1: Lysis of eosinophils, insoluble material removal by centrifugation, protein quantification (BCA), and cell supernatants storage at -80°C.
• DE3.2: Develop a total proteome analysis protocol with a Data Dependent Acquisition (DDA) method using a liquid chromatography (LC)-MS / MS technology (Triple TOF 6600)
• DE3.3: Check the biological variability (biological replications) and technique (technical replicas) to check the reproducibility of the tests.
• DE3.4: Maintaining the standardized conditions of LC-MS / MS (TripleTOF), create a library for SWATH (sequential window acquisition of all theoretical mass spectra) with as many eosinophil proteins as possible.
• DE3.5: Perform SWATH-MS analysis in samples from 15 healthy donors and 15 patients (T0, T4, T16, T32) ("information-dependent acquisition" method or IDA; "Targeted label-free proteomics").
• DE3.6: Processing of the obtained data and initial statistical analysis.
• DE3.7: Checking the panel of biomarkers obtained with a different technology (e.g., Selected reaction monitoring / SRM, ELISA).
• DE3.8: Final uni- and multivariant statistical analysis. Identify proteins with significant differences between groups (P < 0.05) and at least a fold change ≥ 1.5.
• DE3.9: Publication of proteomic results
• OB4. Check whether late-onset severe eosinophilic asthmatics display elevated levels of IGF-1, IGF-BP3, IGF-ALS in serum samples, if the response of mepolizumab depends on the levels of this markers, and if treatment with this biological reduces the concentration in serum of these IGF-family members. This objective is distributed in the following deliverables (DE):
• DE4.1: Analysis of IGF-1, IGF-BP3 and IGF-ALS by ELISA
• DE4.2: Uni- and multivariant statistical analysis of experimental data
• DE4.3: Publication of results

Publication of results:

We will endeavour to present a communication during the first year of the study in a Spanish Respiratory Congress (SEPAR) as well as in the European Respiratory Congress (ERS) resulting from the study of the clinical data of patients before and after the administration of mepolizumab. In addition, we expect to publish 3 publications in Q1 journals as well as other 2 or 3 congress communications resulting from experimental studies.

Study population The study population will include healthy controls (i.e., subjects without asthma, allergy, systemic diseases or scheduled for minor surgeries) and late-onset severe eosinophilic asthma patients, who will be recruited from different areas of Galicia (Santiago de Compostela, A Coruña, Lugo, Vigo, and Ourense), Spain. Diagnosis of severe eosinophilic asthma patients at screening will be based on several inclusion criteria and exclusion criteria that we describe below [12].

Inclusion criteria:

• Diagnosis of severe uncontrolled asthma according to ERS/ATS criteria [52].
• Persistent eosinophilia in blood (>300 cells/μL) on ≥ two occasions (more than 4 weeks between each measurement).
• Frequent exacerbations (≥ two per year), defined as a period for ≥ 3 days of lack of asthma control requiring treatment with systemic corticosteroids and/or an emergency department (ED) visit and/or hospitalization.
• Signature of informed consent and agree to comply with all the visits of the study and all the procedures that this entails.

Exclusion criteria:

• Smoking history: Current smokers or former smokers with a smoking history of ≥10 pack-years (number of pack years = (number of cigarettes per day/20) x number of years smoked). A former smoker is defined as a participant who quit smoking at least 6 months prior to Visit 1.
• Clinically important pulmonary disease other than asthma (e.g. active lung infection, Chronic Obstructive Pulmonary Disease (COPD), bronchiectasis, pulmonary fibrosis, cystic fibrosis, hypoventilation syndrome associated with obesity, lung cancer, alpha 1 anti-trypsin deficiency, and primary ciliary dyskinesia) or ever been diagnosed with pulmonary or systemic disease, other than asthma, that are associated with elevated peripheral eosinophil counts (e.g. allergic bronchopulmonary aspergillosis/mycosis, Churg- Strauss syndrome, hypereosinophilic syndrome).
• Any disorder, including, but not limited to, cardiovascular, gastrointestinal, hepatic, renal, neurological, musculoskeletal, infectious, endocrine, metabolic, haematological, psychiatric, or major physical impairment that is not stable in the opinion of the Investigator.
• Malignancy: A current malignancy or previous history of cancer in remission.
• Acute upper or lower respiratory infections requiring antibiotics or antiviral medication within 30 days prior to the Visit 1.
• Xolair: Participants who have received omalizumab (Xolair) or another monoclonal antibody previously.
• Participants who have received systemic corticosteroids within 30 days before Visit 1 [53].
• Pregnancy: Participants who are pregnant or breastfeeding.
• Obesity class 2 or higher (BMI≥ 35 kg/m2) (https://www.who.int/dietphysicalactivity/childhood_what/en/).

Sample size

• Cohort of healthy controls (n=15) only for analysis at T = 0.
• Cohort of n=15 subjects with severe eosinophilic asthma that start with mepolizumab therapy with no modification to their currently prescribed medications. Follow-up study visits at 4 (T4), 16 (T16) and 32 (T32) weeks after the original study visit (T=0).
• The rationale for sample size is explained in the statistical section.

Anticipated rate of enrolment Since this will be a multicentre study, we expect to reach a rate of enrolment of at least 2 severe eosinophilic asthmatics beginning with mepolizumab therapy per month (4 weeks) in each hospital (Total = 8 per month). This means that the 15 subjects should be scheduled to receive mepolizumab along the first 36 weeks of this study, having enough time to complete the study in 72 weeks (1.5 years). We also expect that at least 90% subjects complete this study.

Estimated study start date: December 2020 Estimated study completion date: 1.5 years (72 weeks)

Study design and methods

Figure 2. Study design. This figure represents in a schematic way both the clinical and the experimental parts of the study.

This is an observational, longitudinal, prospective, and multicentre study to evaluate both the early response (4 weeks) and late-response (16 and 32 weeks) to mepolizumab therapy in severe eosinophilic asthmatics. The study will be headed by Dr. Francisco Javier González Barcala (Pneumology Service at CHUS). Dr. Barcala was national coordinator in one clinical trial as well as principal investigator and sub-investigator in 41 and 19 clinical trials, respectively. In addition, Dr. Barcala has been principal investigator of 7 research projects, collaborator in other 7 projects, and published more than 120 relevant publications (peer-reviewed and JCR-indexed) in the field of respiratory diseases, mainly asthma.

The study also involves other members of the Multidisciplinary Asthma Unit (Dr. Francisco Javier Salgado Castro, Dr. Juan José Nieto Fontarigo). In particular, the Project Manager Dr. Salgado has been involved in 11 research projects, has 28 research papers in high impact journals belonging to fields of Immunology, Biochemistry, Proteomics and Respiratory Diseases. In addition, the other participants in this project have a broad experience in their respective fields, both basic and translational research. They are Dra. Marina Blanco Aparicio, responsible for the Asthma Unit at the University Hospital Complex of A Coruña (CHUAC), Dr. Uxío Calvo, at the University Hospital Complex of Ferrol (CHUF), and Coral González at the University Hospital Complex of Ourense (CHUO), Mar Mosteiro at Hospital Alvaro Cunqueiro of Vigo; Dolores Corbacho at Hospital Povisa-Vigo . It will be necessary to hire a researcher in the predoctoral phase for 12 months to carry out sample preparation in transcriptomic and proteomic studies as well as the RTqPCR, flow cytometry and ELISA assays. This researcher will be under the supervision of Dr. Francisco Javier Salgado Castro and Dr. Juan José Nieto-Fontarigo (Multidisciplinary Asthma Unit, CHUS). Proteomics experiments will be carried out by Dra. Susana Belén Bravo López and María García Vence, who work at the Proteomic Platform at Sanitary Research Foundation of Santiago de Compostela (FIDIS). The nCounter® analysis will be carried out through a service offered by the GENVIP group (Group of Genetics, Vaccines and Infections in Pediatrics; https://nanostringenvip.com/), FIDIS.

The research project will be minimally invasive (e.g., no bronchoscopic examinations) but the protocol needs to be reviewed and approved by the Ethics Committee of Clinical Research of Galicia, Spain. Only fifteen patients who meet the late-onset severe asthma diagnosis criteria, are scheduled to receive mepolizumab, and sign the informed consent will be enrolled in this study. The same protocol will be followed by the different clinical teams. Demographic, as well as clinical, haematological, and biochemical variables will be included in a database. Skin prick test to common allergens and the presence of allergen-specific IgE (ImmunoCAP, Thermo Fisher) will be used to check for allergic sensitization. Lung function parameters (forced expiratory volume in the 1st second (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio) also will be analysed. Spirometry will be performed before and after use of a bronchodilator. The Asthma Control Test (ACT) and the Asthma Quality of Life Questionnaire (AQLQ) questionnaire will be performed. Asthmatics must be in a stable phase of the disease (i.e. absence of exacerbations for at least 4 weeks before sample collection). Exacerbations will be managed in accordance with standard clinical guidelines. Patients (n=15) will receive 100 mg subcutaneous injection of mepolizumab at 4 weeks intervals, and blood and serum samples (2-3 EDTA tubes; 1 SST tube) will be withdrawn at T=0, 4, 16 and 32 weeks, in order to evaluate both early-response (4 weeks) and late-response (16 and 32 weeks) to treatment.

Methods

• Tubes: EDTA (complete blood) and SST (serum)
• Eosinophils purification
• Eosinophils can be isolated from whole blood (heparin tubes) using the Miltenyi Human Eosinophil Isolation Kit (Catalog #130-104-466) or the EasySep™ Human Eosinophil Isolation Kit (Catalog #17956), both negative selection procedures that yield untouched subsets of these leukocytes. We expect around at least 1.0-4.0 x 106 cells from ∼20 ml blood, but also high viability and purity (>95%).
• ELISA studies.
• Serum sample collection: Measurement of IGF-ALS (GENOIT4078 Immunotag Human IGFALS 96 well), IGF-1 (Human IGF-I/IGF-1 DuoSet ELISA, R&D Systems, catalog #DY291), and IGF-BP3 (Human IGFBP-3 DuoSet ELISA, R&D Systems, catalog #DY675) by means of ELISA.
• Total RNA purification from eosinophils and nCounter nanoString analysis (Discovery-based/hypothesis-generating approach):
• Purified eosinophils from healthy controls (T=0) and patients (T=0, 4, 16 and 32 weeks) will be stored at -80ºC in RNAlater solution (Ambion, Paisley, UK). Total RNA will be isolated by means of a RNeasy Mini kit (Qiagen) and stored at -80ºC after checking RNA quality and concentration (Nanodrop).
• The nCounter® platform (nanoString; https://www.nanostring.com/scientific-content/technology-overview/ncounter-technolo gy) is a multiplex methodology that allows the quantification of up to 800 RNA, DNA, or protein targets. Regarding mRNA molecules, this technology is based on the in-solution hybridization of every mRNA to two complementary oligonucleotides: a biotinylated mRNA-specific probe and a mRNA-specific oligonucleotide containing a sequential combination of six fluorochromes (four different colours) that create a fluorescent barcode that identifies the specific mRNA being detected. Once the excess of both probes is removed, the hybridised complexes are captured through a biotin-streptavidin interaction and aligned on cartridge in order to the nCounter instrument can read those "barcodes". To carry out these steps, the nCounter platform consists of two instruments the Prep Station, which performs the purification of the hybridized complexes and their immobilization onto the surface of a cartridge, and the Digital Analyzer (DA), a scanner that identifies and counts the barcodes captured for each sample. This quantitative analysis Therefore, each miRNA can be quantified individually (absolute quantification; counts) from difficult samples (e.g., eosinophils) with no need for other requirements such as mRNA-cDNA conversion (RT) or DNA-amplification (qPCR), leading to less data variability (https://www.nanostring.com/scientific-content/technology-overview/challenges-of-rt ). In addition, the amount of input material is low (25 ng-300 ng mRNA) and can be derived from FFPE-derived RNA, total RNA, fragmented RNA, cell lysates, and sorted cells. Afterwards, nCounter data will be normalized, background noise subtracted, and further correction performed to account for the efficiency of the extraction (calculated based on the expression of spike-in miRNAs that will be added to the sample in a defined amount before the miRNA extraction). Normalizations will be done using the R NanoStringNorm package. After normalization, a log2 transformation of the data will be made and subsequently analysed by means of the LIMMA Bioconductor package to identify those mRNAs displaying a differential abundance upon mepolizumab treatment. This analysis will take no longer than 24 hours.
• RTqPCR studies (Hypothesis-driven approach):
• To analyse the levels of mRNAs encoding proteins related with alarmin-mediated activation of eosinophils (CRLF2, ST2, IL-7Rα/CD127) and with the regulatory function of eosinophils (FOXP3) from patients treated with mepolizumab, total RNA will be transcribed into cDNA (QuantiTect Rev. Transcription Kit; Qiagen) and stored at -80ºC. qPCR (QuantiTect SYBR Green PCR Kit; Qiagen) will be performed in a LightCycler® 96 Instrument (Roche Life Science) and used to analyse the expression of FOXP3, CRLF2, ST2, IL-7R and the HPRT1 gene (endogenous control).
• Flow cytometry studies (Hypothesis-driven approach):
• EDTA-treated peripheral blood samples from healthy controls (n=15; T0) and mepolizumab-treated patients (n=15; T0, T4, T16, T32).
• Label 100 μL/tube of whole peripheral blood (EDTA) with both specific and isotype-matched control antibodies (BD). Red cells lysis with FACSlyse (BD). Analysis with a FACSCalibur flow cytometer (BD). Use FSC/SSC to select granulocytes; then SSC vs CCR3 (FITC) to separate eosinophils from neutrophils. Gate eosinophils:
• Multimarker panel 1 (Regulatory proteins in eosinophils): Measurement of CD16 and galectins-1/10 [41-44].
• Multimarker panel 2 (Activation receptors in eosinophils): Measurement of CD48 (reduced in total eosinophils with moderated-severe asthma compared to healthy controls (HC) [our studies, 54]), CD44, and CD11b.
• Multimarker panel 3 (Eosinophils subsets): Analysis of subsets based on the expression of Siglec-8, CD62L(L-selectin), and IL-5Rα [40].
• Analysis of eosinophil proteome (Discovery-based/hypothesis-generating approach):
• As much as 50 x 103 cells will be necessary to perform proteomic assays. We expect around 50-400 x 103 cells from ∼1 mL of blood.
• Isolated eosinophils (50 x 103 cells) will be collected by centrifugation, washed, and resuspended in lysis buffer with proteinase inhibitors. After that, insoluble material will be removed by centrifugation and cell supernatants stored at -80°C.
• For eosinophils total proteome characterization will be made after trypsin digestion using a DDA method in a LC-MSMS system. For this approach we will use samples from 15 healthy donors and 15 patients (T0, T4, T16, T32). The proteins selected will be only those that reported a 1% Global false discovery rate (FDR) or better [55, 56].
• Protein "pools" from the 5 groups of study (healthy donors and patients at time T0, T4, T16, and T32 after treatment) will be used, dividing them (1-DE) in 5-6 bands, extracting the proteins from each band, generating the corresponding peptides and analysing them by MS / MS to produce a library for SWATH with a high number of proteins, on which then the quantification will be carried out. Once the library was made and maintaining the standardized conditions of LC-MS / MS (TripleTOF), we will perform a SWATH-MS analysis ("information-dependent acquisition" method or IDA; "Targeted label-free proteomics") in samples from 15 healthy donors and 15 patients (T0, T4, T16, T32). This assay will let us identify proteins with significant differences between the groups of study. The proteins selected will be only those with a P<0.05 and a fold change ≥1.5 [56-59].

Study endpoints:

Demographic data for all individuals enrolled in the study will be obtained at basal. In addition, several data will be collected, including asthma history, lung function parameters, skin prick test, allergen-specific IgE, AQLQ score, ACT score, the number of exacerbations, and consumption of prednisone. During the following visits to the Pneumology service at T0, 4, 16, and 32, patients treated with mepolizumab will be followed up. This includes measurements of lung function (FEV1, FEV1/FVC), biochemical and haematological parameters.

Peripheral blood and serum samples will be collected, and eosinophils will be magnetically purified, at T0, T4, T16, and T32, and flow cytometry, RTqPCR, and proteomic analyses, as well as immunoassays, will be performed. All the experimental variables (e.g., the abundance of eosinophil proteins in proteomic assays, eosinophil activation markers, …) will be correlated with clinical parameters (e.g., lung function, asthma control, number of exacerbations) in order to assess the association of these variables with the response to treatment. We will consider a favourable response to mepolizumab if one of the following criteria is met:

• To obtain adequate asthma control ACT ≥20 [60], or/ a change of ≥3 points representing a minimally important difference.
• To achieve a reduction in the annual rate of exacerbations of 48%. Exacerbation is defined as the increase in symptoms requiring treatment with systemic corticosteroids for ≥3, or an unscheduled medical consultation, similar to that reflected in clinical trials with mepolizumab [20, 61].
• Get a 50% reduction in the annual rate of hospital admissions due to asthma exacerbation, similar to that reflected in clinical trials [62].
• To achieve a reduction in the median annual dose of systemic corticosteroids of 50% [63].
• Study primary endpoints:
• IGF-1, IGF-BP3 and IGF-ALS levels in serum
• Transcriptomic (nanoString)/mRNA expression data: FOXP3, CRLF2, ST2, IL-7R
• Proteomic data
• Flow cytometry data: Expression of CD16, galectins-1/10, CD48, CD44, CD11b, Siglec-8, CD62L, and IL-5Rα
• Study secondary endpoints:
• Lung function parameters (FEV1, FEV1/FVC)
• Haematological parameters (e.g., eosinophils number).
• Other clinical and biochemical variables (e.g., IgE or other immunoglobulins).
• Number of exacerbations, prednisone consumption, ACT score, AQLQ score.

Statistical plan or data analysis:

Graph Pad Prism will be used to create graphics. IBM SPSS, Statistics 22.0, or R. will be used for the statistical study. During the analyses, we will be assisted by the USC Statistics and Operational Research area (Dr. Rosa María Crujeiras Casais).

Sample size The calculation of sample size (N) has been carried out by using G*Power 3.1.9.4 [64]. During these analyses we calculate N necessary get statistical significance in a F test (ANOVA: Repeated measures, within factors), given α (0.05), power (1-β, 0.95), the number of measurements (T0, T4, T16 and T32), and the effect size (f = 0.4; large effect size, which gives as a more clinically relevant results). The output N was 15, with a critical F= 2.82705.

For clinical, flow cytometry and transcriptomic data. Cross-sectional comparisons between HC and patients in T0 (before treatment) following a normal distribution and having homogeneity of variances will be made by using t-test. For non-normal distributed variables, we will use Mann-Whitney U test. Changes in the different study variables in response to treatment with mepolizumab (longitudinal study; T0, T4, T16, and T32) will be tested using RM-ANOVA. Multivariate analysis (e.g., PCA, unsupervised clustering) as well as functional enrichment analysis will be performed with flow cytometry, and above all, transcriptomic data.

For total proteome characterization and quantitative SWATH analysis We will use ProteinPilotTM 5.0.1 software from ABSciex which have the algorithm ParagonTM for database search and ProgroupTM for data grouping. Data will be searched using a Human specific Uniprot database. False discovery rate will be performed using a non-lineal fitting method displaying only those results that reported a 1% Global false discovery rate or better [65].

Functional analysis will be performed by different open-access software. FunRich (Functional Enrichment analysis tool) for functional enrichment and interaction network analysis (http://funrich.org/index.html). For statistics, FunRich uses hypergeometric test, BH and Bonferroni [66, 67]. We will use DAVID (https://david.ncifcrf.gov/tools.jsp) or GO (http://geneontology.org/page/go-enrichment-analysis) for gene ontology enrichment and for protein-protein interaction, network construction and clustering, we will use String (https://string-db.org/) or Cytoscape 3.7 (https://cytoscape.org/ ) [68].

For SWATH data, MarkerView software will give us a multivariate statistical analysis using principal component analysis (PCA) to compare the data across the samples. The average MS peak area of each protein will be derived from the replicates of the SWATH-MS of each sample followed by Student's t-test analysis using the MarkerView software for comparison among the samples based on the averaged area sums of all the transitions derived for each protein. The t-test will indicate how well each variable distinguishes the two groups, reported as a P-value. For the library, its set of differentially abundant proteins (p-value <0.05) with a 1.5 up-regulated or down-regulated proteins will be selected.

Limitations

• As previously commented, 15 subjects will be scheduled to receive mepolizumab during the first half of the study (36 weeks). We expect that at least 90% subjects complete this study. However, patient dropouts and non-adherence (or non-compliance) are common events in clinical studies. In such a case, sample size will be proportionally inflated.
• The present project has been proposed as a study of the discovery of molecular biomarkers in response to mepolizumab. This kind of studies can be boarded through Targeted/hypothesis-driven or broader/untargeted ("-omics" technologies) approaches. We are aware that it might be challenging to find predictive markers in this small and prospective/proof of concept study. In the present project we propose a double approach to minimize this risk. On the one hand, modern and untargeted methodologies to work and highly sensitive to detect low-abundant proteins (SWATH MS) or simplified protocols to work with difficult samples (generation of good quality RNA from eosinophils is always challenging due to the presence of abundant proteins like EDN, a member of the RNase family) and reduce technical variance (e.g., nCounter nanoString) in order to shorten sample sizes. On the other hand, hypothesis-driven approaches (e.g., flow cytometry, RT-qPCR, ELISA), with the advantages of a greater credence, less risk of type I (i.e., false discovery) and II errors, and easy to future replication of results. These targeted-methodologies will be also used to confirm only clinically relevant (high effect-size) and significant (p-value < 0.05) differences obtained with untargeted transcriptomic/proteomic approaches.
• Another potential limitation is the number of eosinophils, which could be potentially low in some patient after treatment with mepolizumab (50 x 103 or lower). Therefore, it could be necessary to purify more than 1 or 2 mL of blood in those patients in order to achieve enough cells to perform the proteomic and transcriptomic experiments. We have considered this issue in the study protocol and in the budget of the project.
• Finally, the TripleTOF is highly sensitive mass spectrometer designed to dig deeper into complex samples like the eosinophil proteome. The high sensitivity of current mass spectrometers combined with bidimensional schemes of nanoLC allows the detection higher numbers of proteins (>1000) and analytes at concentrations in the attomolar range (10-18), enough to detect low abundant intracellular proteins like chemokines or cytokines. Thus, TGFα, TGFβ1, CCL5, CCL23, CSF1, CCL18, CCL24, CXCL12, and IL-18 have been detected by nanoLC-MS/MS (Q-TOF), but not others like IL-5 or IL-13 [69]. This expected limitation is due to the presence of peptides from highly abundant proteins (e.g., eosinophil granule proteins) that suppress the ionization of peptides from lo w abundant proteins in LC-MS/MS applications. This leads to the overrepresentation in the list of detected proteins of abundant species such as the Charcot-Leyden crystal protein (CLC, galectin-10), which is still interesting for the present project since identifies eosinophils with regulatory capacities. However, in order to reduce sample complexity and improve the detection of low-abundance proteins, there are different depletion (e.g., ACN-depletion, ultrafiltration, 1-DE) and enrichment methods (e.g., CPLLs) that we could use to gain detection sensitivity. Optionally, targeted approaches such as high-multiplex immunoassays (e.g., the Olink Immune Response, Olink Inflammation, or Olink Immuno-Oncology panels; https://www.olink.com/) have the advantage of a high detection sensitivity with low volume of biological samples (e.g., cell lysates), even though only allows the measurement of 92 protein biomarkers at once.

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