Two parts: A:Case-control study including 15 healthy adult donors and 15 severe adult
eosinophilic asthmatics selected for treatment with mepolizumab. B: A longitudinal cohort
study,where the same patients once on mepolizumab treatment are followed over time (0, 4, 16
and 32 weeks). SCOPE: response to mepolizumab in severe adult eosinophilic asthma.
INCLUSION CRITERIA: Male or female, 18-75 years-old, with severe eosinophilic asthma.
EXCLUSION CRITERIA: Smoking history, recent exacerbations, other pulmonary or systemic
disease with eosinophilia, malignancy, pregnancy, obesity (BMI >35). OBJECTIVES: General
objective: Discovery of predictive/prognostic biomarkers of response to mepolizumab using
flow cytometry, transcriptomic, and proteomic technologies. OTHER OBJECTIVES: 1.-To identify
changes in surface markers of eosinophils and eosinophil subpopulations in response to
treatment with mepolizumab using flow cytometry techniques. 2.-Transcriptomic analysis to
identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in
response to treatment with mepolizumab.3.-Proteomic profiling to identify proteins with
differential abundance within the eosinophils in response to treatment with
mepolizumab.4.-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.
MEASUREMENTS: Flow cytometry assays with multimarker panels 1 (regulatory), 2 (activation),
and 3 eosinophil subsets. Clinical, hematological, biochemical and flow cytometry data
generated at times T4, T16 and T32. Total RNA extraction from eosinophil lysates, assay of
quality and quantity of RNA, and storage at -80ºC. 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)". 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. 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").
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
- 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,
- 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
- 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
- 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)"
/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
- 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
- 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
- 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 .
- Diagnosis of severe uncontrolled asthma according to ERS/ATS criteria .
- 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.
- 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
- 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
- Participants who have received systemic corticosteroids within 30 days before Visit 1
- Pregnancy: Participants who are pregnant or breastfeeding.
- Obesity class 2 or higher (BMI≥ 35 kg/m2)
- 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
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)
- 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
- Total RNA purification from eosinophils and nCounter nanoString analysis
- 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;
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
). 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.
- 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 .
- 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].
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 , 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 .
- To achieve a reduction in the median annual dose of systemic corticosteroids of 50%
- 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 22.214.171.124
. 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=
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 .
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/ ) .
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.
- 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
- 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 . 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.