Ignite Creation Date:
2024-05-06 @ 3:29 PM
Last Modification Date:
2024-10-26 @ 1:50 PM
Study NCT ID:
NCT04641741
Status:
RECRUITING
Last Update Posted:
2023-09-25
First Post:
2020-11-18
Brief Title:
Effect of Mepolizumab on Severe Eosinophilic Asthma
Sponsor:
Hospital Clinico Universitario de Santiago
Organization:
Hospital Clinico Universitario de Santiago
Study Overview
Official Title:
Effect of Mepolizumab on the PhenotypeProteomeTranscriptome of Eosinophils in Severe Eosinophilic Asthma
Status:
RECRUITING
Status Verified Date:
2023-09
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
EMESEA
Brief Summary:
Two parts ACase-control study including 15 healthy adult donors and 15 severe adult eosinophilic asthmatics selected for treatment with mepolizumab B A longitudinal cohort studywhere 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 predictiveprognostic 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 mepolizumab3-Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab4-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 5-Identify proteins with differential abundance within the deep serum proteome of patients with SEA in response to treatment with mepolizumab by means of non-targeted proteomic analysis
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 in eosinophil homogenates High abundant serum protein depletion using two protocols P1 affinity chromatography and P2 DTT precipitation and SWATH-MS analysis of medium-low abundant serum proteome in samples from 15 healthy donors and 15 patients T0 T4 T16 T32 information-dependent acquisition method or IDA Targeted label-free proteomics
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 predictiveprognostic 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 mepolizumab3-Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab4-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 5-Identify proteins with differential abundance within the deep serum proteome of patients with SEA in response to treatment with mepolizumab by means of non-targeted proteomic analysis
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 in eosinophil homogenates High abundant serum protein depletion using two protocols P1 affinity chromatography and P2 DTT precipitation and SWATH-MS analysis of medium-low abundant serum proteome in samples from 15 healthy donors and 15 patients T0 T4 T16 T32 information-dependent acquisition method or IDA Targeted label-free proteomics
Detailed 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 predictiveprognostic 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 regulatoryeffector 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
H4 Mepolizumab alters the abundance of several proteins in the medium-low abundance serum proteome of patients with SEA Therefore these proteins could be used as predictiveprognostic markers of response to this biological and could provide a better understanding of both eosinophilic and non-eosinophilic-related biological functions of IL-5 in SEA
Objectives or research questions
OB Aspirational objective Discovery of predictiveprognostic 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
DEDeliverable11 Selection of 15 healthy controls
DE12 15 patients with severe eosinophilic asthma patients are scheduled to receive mepolizumab and sign the informed consent
DE13 Generation of the initial database with demographic clinical haematologic and biochemical information
DE14 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
DE15 Flow cytometry assays see below
DE16 Complete the database with clinical hematological biochemical and flow cytometry data generated at times T4 T16 and T32 Final uni-multivariant statistical analysis
DE17 Publication of results
OB2- Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab
DE21 Set up an eosinophil isolation protocol
DE22 Purification of eosinophils from healthy donors T0 and patients T0 T4 T16 T32
DE23 RNA extraction from eosinophil lysates assay of quality and quantity of RNA and storage at -80ºC
DE24 Discovery-basedhypothesis-generating approach Evaluation of the levels of 770 mRNAs linked to the recruitment activation and effector functions of myeloid cells busing a pre-made nCounter Human Myeloid Innate Immunity Panel v2 wwwnanostringcomproductsgene-expression-panelsgene-expression-panels-overviewncounter-myeloid-innate-immunity-panel Eosinophil samples from healthy donors T 0 and patients T0 and T16
DE25 Processing of the obtained data and initial statistical analysis
DE26 Validation of nCounter data RTqPCR analyses of those mRNAs in eosinophils with the greatest abundance changes in response to mepolizumab treatment 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
DE27 Uni- and multivariant statistical analyses
DE28 Publication of results
OB3- Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab
DE31 Lysis of eosinophils protein quantification BCA and cell supernatants storage at -80C
DE32 Develop a total proteome analysis protocol with a Data Dependent Acquisition DDA method using LC-MSMS Triple TOF 6600
DE33 Check the biological biological replications and technical technical replicas variability
DE34 Create a library for SWATH with as many eosinophil proteins as possible
DE35 Perform SWATH-MS analysis in samples from healthy donors and patients T0 T4 T16 T32 data-independent acquisition method or DIA Targeted label-free proteomics
DE36 Processing of the obtained data and initial statistical analysis
DE37 Validation of a panel of biomarkers with a different technology eg Selected reaction monitoringSRM ELISA
DE38 Final uni- and multivariant statistical analysis Identify proteins with significant differences between groups P 005 and a fold change 15
DE39 Publication of 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 these markers and if treatment with this biological reduces the concentration in serum of these IGF-family members
DE41 Analysis of IGF-1 IGF-BP3 and IGF-ALS by ELISA
DE42 Uni- and multivariant statistical analysis of experimental data
DE43 Publication of results
OB5- Identify proteins with differential abundance within the low abundant serum proteome of patients with severe eosinophilic asthma in response to treatment with mepolizumab
DE51 Optimization of protocols for serum pre-fractioning Protocol 1 depletion of high abundant proteins using affinity chromatography Protocol 2 depletion of high abundant proteins using DTT precipitation
DE52 Serum pre-fractioning protocol 1 To deplete the 14 highest abundant proteins in serum from healthy donors T0 and mepolizumab-treated severe eosinophilic asthma patients T0 T4 T32 using high abundant protein depletion columns
DE53 Serum pre-fractioning protocol 2 To deplete high abundant proteins in serum from healthy donors T0 and mepolizumab-treated severe eosinophilic asthma patients T0 T4 T32 using DTT precipitation
DE54-510 Same procedure after protocol 1 and protocol 2 in parallel
DE54 Development of a low abundant proteome analysis protocol with a DDA method using LC-MSMS Triple TOF 6600
DE55 Check the biological biological replications and technical technical replicas variability
DE56 Create a library for SWATH with as many low abundant serum proteins as possible
DE57 Perform SWATH-MS analysis in samples from healthy donors and patients T0 T4 T32 DIA
DE58 Processing of the obtained data and initial statistical analysis
DE59 Validation of a panel of biomarkers obtained using a different technology eg SRMMRM ELISA
DE510 Final uni- and multivariant statistical analysis Identify proteins with significant differences between groups P 005 and at least a fold change 15
DE511 Correlation analyses of proteins with changes Detected in protocol 1 andor protocol 2 with clinical haematological biochemical and flow cytometry data
DE512 Publication of results
Publication of results
We expect to present 2-3 communications to Spanish Respiratory Congress SEPAR and the European Respiratory Congress ERS resulting from the study of the clinical and experimental data In addition we expect to publish 3 publications in Q1 journals
Study population The study population will include healthy controls ie subjects without asthma allergy systemic diseases or scheduled for minor surgeries and 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
Inclusion criteria
Diagnosis of severe uncontrolled asthma according to ERSATS criteria
Persistent eosinophilia in blood 300 cellsμL on two occasions 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 andor an ED visit andor 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 day20 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 eg active lung infection 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 eg allergic bronchopulmonary aspergillosismycosis 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
Pregnancy Participants who are pregnant or breastfeeding
Obesity class 2 or higher BMI 35 kgm2 httpswwwwhointdietphysicalactivitychildhood_whaten
Sample size
Cohort of healthy controls n15 only for analysis at T 0
Cohort of n15 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 T0
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 Considering the recruitment and experimental parts we expect to complete the study in 120 weeks 30 months 25 years We also expect that at least 90 of subjects complete this study
Estimated study start date June 2021 Estimated study completion date 25 years 30 months
Study design and methods
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 the leader of the Translational Research in Airway Diseases TRIAD group Other members of the TRIAD group are Dr Francisco Javier Salgado Castro the Project Manager and Dr Juan José Nieto Fontarigo both experts in Immunology Biochemistry Proteomics and Respiratory Diseases Dra Marina Blanco Aparicio is responsible for the Asthma Unit at the University Hospital Complex of A Coruña CHUAC Dr Uxío Calvo Álvarez at the University Hospital Complex of Ferrol CHUF Dra Coral González Fernández at the University Hospital Complex of Ourense CHUO Dra Mar Mosteiro Añón at the University Hospital Complex of Vigo-Alvaro Cunqueiro CHUVI and Dolores Corbacho Abelaira at the POVISA Hospital Centre Vigo Proteomics experiments will be carried out by Dra Susana Belén Bravo López at FIDIS nCounter analysis will be carried out by GENVIP group at FIDIS
The research project will be minimally invasive eg 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 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 FEV1FVC 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 ie absence of exacerbations for at least 4 weeks before sample collection Exacerbations will be managed in accordance with standard clinical guidelines Patients n15 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 T0 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 at least 10-40 x 106 cells from 30 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-IIGF-1 DuoSet ELISA RD Systems catalog DY291 and IGF-BP3 Human IGFBP-3 DuoSet ELISA RD Systems catalog DY675 by means of ELISA
Total RNA purification from eosinophils and nCounter nanoString analysis Discovery-basedhypothesis-generating approach
Purified eosinophils from healthy controls T0 and patients T0 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 httpswwwnanostringcomscientific-contenttechnology-overviewncounter-technology 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 eg eosinophils with no need for other requirements such as mRNA-cDNA conversion RT or DNA-amplification qPCR leading to less data variability httpswwwnanostringcomscientific-contenttechnology-overviewchallenges-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-7RαCD127 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 n15 T0 and mepolizumab-treated patients n15 T0 T4 T16 T32
Label 100 μLtube 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 FSCSSC 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-110
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 CD62LL-selectin and IL-5Rα
Analysis of eosinophil proteome Discovery-basedhypothesis-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 -80C
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
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 P005 and a fold change 15
Analysis of medium-low abundant serum proteome Discovery-basedhypothesis-generating approach
Serum from healthy controls T0 and mepolizumab-treated patients T0 4 and 32 weeks will be isolated from peripheral venous blood Once the blood has been collected in a BD Vacutainer SST Serum Separation Tube blood coagulation will be allowed to proceed for a minimum of 45 min in a vertical position at room temperature RT Afterward the tube will be centrifugated at 1100-1300 xg for 10 min RT in swinging bucket rotor units After centrifugation the serum will be located above the polymer barrier The serum will be harvested and aliquoted into Eppendorf tubes
After that two different protocols for high abundant protein removal will be performed
Pre-fractioning protocol 1 High abundant serum protein removal using affinity chromatography 100 μL of serum will be applied to High Select Top14 Abundant Protein Depletion Midi Spin Columns from ThermoFisher Scientific according to manufacturers instructions
Pre-fractioning protocol 2 High abundant serum protein removal using DTT precipitation Use 30 μL of serum according to the protocol we have published
Low abundant serum protein concentration from protocol 1 and 2 will be quantified by RCDC Protein Assay Kit BIORAD as per the manufacturers protocol After depletion of the highest abundant proteins 99 of total protein 60-80 mgmL we expect to have around 60-80 μg of low abundant proteins
Proteome characterization will be made following the same protocol as the one described for eosinophil homogenates
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 FEV1FVC biochemical and haematological parameters
Peripheral blood and serum samples will be collected and eosinophils will be magnetically purified at T0 T4 T16 and T32 Flow cytometry RTqPCR and proteomic analyses as well as immunoassays will be performed All the experimental variables eg the abundance of eosinophil proteins in proteomic assays the abundance of serum proteins eosinophil activation markers will be correlated with clinical parameters eg 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 nanoStringmRNA expression data FOXP3 CRLF2 ST2 IL-7R
Proteomic data
Flow cytometry data Expression of CD16 galectins-110 CD48 CD44 CD11b Siglec-8 CD62L and IL-5Rα
Study secondary endpoints
Lung function parameters FEV1 FEV1FVC
Haematological parameters eg eosinophils number
Other clinical and biochemical variables eg 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 220 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 GPower 3194 64 During these analyses we calculate N necessary get statistical significance in a F test ANOVA Repeated measures within factors given α 005 power 1-β 095 the number of measurements T0 T4 T16 and T32 and the effect size f 04 large effect size which gives as a more clinically relevant results The output N was 15 with a critical F 282705
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 eg 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 501 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 httpfunrichorgindexhtml For statistics FunRich uses hypergeometric test BH and Bonferroni 66 67 We will use DAVID httpsdavidncifcrfgovtoolsjsp or GO httpgeneontologyorgpagego-enrichment-analysis for gene ontology enrichment and for protein-protein interaction network construction and clustering we will use String httpsstring-dborg or Cytoscape 37 httpscytoscapeorg 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 Students 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 005 with a 15 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 Targetedhypothesis-driven or broaderuntargeted -omics technologies approaches We are aware that it might be challenging to find predictive markers in this small and prospectiveproof of concept study 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 and reduce technical variance eg nCounter nanoString in order to shorten sample sizes On the other hand hypothesis-driven approaches eg flow cytometry RT-qPCR ELISA with the advantages of greater credence less risk of type I 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 005 differences obtained with untargeted transcriptomicproteomic approaches Finally we purpose the use of two independent but complementary protocols for serum pre-fractioning This will increase the probability of reaching a higher number of proteins with changes in serum and this will therefore increase the probability of discovering a higher number of biomarkers
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 predictiveprognostic 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 regulatoryeffector 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
H4 Mepolizumab alters the abundance of several proteins in the medium-low abundance serum proteome of patients with SEA Therefore these proteins could be used as predictiveprognostic markers of response to this biological and could provide a better understanding of both eosinophilic and non-eosinophilic-related biological functions of IL-5 in SEA
Objectives or research questions
OB Aspirational objective Discovery of predictiveprognostic 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
DEDeliverable11 Selection of 15 healthy controls
DE12 15 patients with severe eosinophilic asthma patients are scheduled to receive mepolizumab and sign the informed consent
DE13 Generation of the initial database with demographic clinical haematologic and biochemical information
DE14 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
DE15 Flow cytometry assays see below
DE16 Complete the database with clinical hematological biochemical and flow cytometry data generated at times T4 T16 and T32 Final uni-multivariant statistical analysis
DE17 Publication of results
OB2- Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab
DE21 Set up an eosinophil isolation protocol
DE22 Purification of eosinophils from healthy donors T0 and patients T0 T4 T16 T32
DE23 RNA extraction from eosinophil lysates assay of quality and quantity of RNA and storage at -80ºC
DE24 Discovery-basedhypothesis-generating approach Evaluation of the levels of 770 mRNAs linked to the recruitment activation and effector functions of myeloid cells busing a pre-made nCounter Human Myeloid Innate Immunity Panel v2 wwwnanostringcomproductsgene-expression-panelsgene-expression-panels-overviewncounter-myeloid-innate-immunity-panel Eosinophil samples from healthy donors T 0 and patients T0 and T16
DE25 Processing of the obtained data and initial statistical analysis
DE26 Validation of nCounter data RTqPCR analyses of those mRNAs in eosinophils with the greatest abundance changes in response to mepolizumab treatment 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
DE27 Uni- and multivariant statistical analyses
DE28 Publication of results
OB3- Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab
DE31 Lysis of eosinophils protein quantification BCA and cell supernatants storage at -80C
DE32 Develop a total proteome analysis protocol with a Data Dependent Acquisition DDA method using LC-MSMS Triple TOF 6600
DE33 Check the biological biological replications and technical technical replicas variability
DE34 Create a library for SWATH with as many eosinophil proteins as possible
DE35 Perform SWATH-MS analysis in samples from healthy donors and patients T0 T4 T16 T32 data-independent acquisition method or DIA Targeted label-free proteomics
DE36 Processing of the obtained data and initial statistical analysis
DE37 Validation of a panel of biomarkers with a different technology eg Selected reaction monitoringSRM ELISA
DE38 Final uni- and multivariant statistical analysis Identify proteins with significant differences between groups P 005 and a fold change 15
DE39 Publication of 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 these markers and if treatment with this biological reduces the concentration in serum of these IGF-family members
DE41 Analysis of IGF-1 IGF-BP3 and IGF-ALS by ELISA
DE42 Uni- and multivariant statistical analysis of experimental data
DE43 Publication of results
OB5- Identify proteins with differential abundance within the low abundant serum proteome of patients with severe eosinophilic asthma in response to treatment with mepolizumab
DE51 Optimization of protocols for serum pre-fractioning Protocol 1 depletion of high abundant proteins using affinity chromatography Protocol 2 depletion of high abundant proteins using DTT precipitation
DE52 Serum pre-fractioning protocol 1 To deplete the 14 highest abundant proteins in serum from healthy donors T0 and mepolizumab-treated severe eosinophilic asthma patients T0 T4 T32 using high abundant protein depletion columns
DE53 Serum pre-fractioning protocol 2 To deplete high abundant proteins in serum from healthy donors T0 and mepolizumab-treated severe eosinophilic asthma patients T0 T4 T32 using DTT precipitation
DE54-510 Same procedure after protocol 1 and protocol 2 in parallel
DE54 Development of a low abundant proteome analysis protocol with a DDA method using LC-MSMS Triple TOF 6600
DE55 Check the biological biological replications and technical technical replicas variability
DE56 Create a library for SWATH with as many low abundant serum proteins as possible
DE57 Perform SWATH-MS analysis in samples from healthy donors and patients T0 T4 T32 DIA
DE58 Processing of the obtained data and initial statistical analysis
DE59 Validation of a panel of biomarkers obtained using a different technology eg SRMMRM ELISA
DE510 Final uni- and multivariant statistical analysis Identify proteins with significant differences between groups P 005 and at least a fold change 15
DE511 Correlation analyses of proteins with changes Detected in protocol 1 andor protocol 2 with clinical haematological biochemical and flow cytometry data
DE512 Publication of results
Publication of results
We expect to present 2-3 communications to Spanish Respiratory Congress SEPAR and the European Respiratory Congress ERS resulting from the study of the clinical and experimental data In addition we expect to publish 3 publications in Q1 journals
Study population The study population will include healthy controls ie subjects without asthma allergy systemic diseases or scheduled for minor surgeries and 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
Inclusion criteria
Diagnosis of severe uncontrolled asthma according to ERSATS criteria
Persistent eosinophilia in blood 300 cellsμL on two occasions 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 andor an ED visit andor 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 day20 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 eg active lung infection 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 eg allergic bronchopulmonary aspergillosismycosis 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
Pregnancy Participants who are pregnant or breastfeeding
Obesity class 2 or higher BMI 35 kgm2 httpswwwwhointdietphysicalactivitychildhood_whaten
Sample size
Cohort of healthy controls n15 only for analysis at T 0
Cohort of n15 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 T0
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 Considering the recruitment and experimental parts we expect to complete the study in 120 weeks 30 months 25 years We also expect that at least 90 of subjects complete this study
Estimated study start date June 2021 Estimated study completion date 25 years 30 months
Study design and methods
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 the leader of the Translational Research in Airway Diseases TRIAD group Other members of the TRIAD group are Dr Francisco Javier Salgado Castro the Project Manager and Dr Juan José Nieto Fontarigo both experts in Immunology Biochemistry Proteomics and Respiratory Diseases Dra Marina Blanco Aparicio is responsible for the Asthma Unit at the University Hospital Complex of A Coruña CHUAC Dr Uxío Calvo Álvarez at the University Hospital Complex of Ferrol CHUF Dra Coral González Fernández at the University Hospital Complex of Ourense CHUO Dra Mar Mosteiro Añón at the University Hospital Complex of Vigo-Alvaro Cunqueiro CHUVI and Dolores Corbacho Abelaira at the POVISA Hospital Centre Vigo Proteomics experiments will be carried out by Dra Susana Belén Bravo López at FIDIS nCounter analysis will be carried out by GENVIP group at FIDIS
The research project will be minimally invasive eg 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 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 FEV1FVC 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 ie absence of exacerbations for at least 4 weeks before sample collection Exacerbations will be managed in accordance with standard clinical guidelines Patients n15 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 T0 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 at least 10-40 x 106 cells from 30 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-IIGF-1 DuoSet ELISA RD Systems catalog DY291 and IGF-BP3 Human IGFBP-3 DuoSet ELISA RD Systems catalog DY675 by means of ELISA
Total RNA purification from eosinophils and nCounter nanoString analysis Discovery-basedhypothesis-generating approach
Purified eosinophils from healthy controls T0 and patients T0 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 httpswwwnanostringcomscientific-contenttechnology-overviewncounter-technology 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 eg eosinophils with no need for other requirements such as mRNA-cDNA conversion RT or DNA-amplification qPCR leading to less data variability httpswwwnanostringcomscientific-contenttechnology-overviewchallenges-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-7RαCD127 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 n15 T0 and mepolizumab-treated patients n15 T0 T4 T16 T32
Label 100 μLtube 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 FSCSSC 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-110
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 CD62LL-selectin and IL-5Rα
Analysis of eosinophil proteome Discovery-basedhypothesis-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 -80C
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
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 P005 and a fold change 15
Analysis of medium-low abundant serum proteome Discovery-basedhypothesis-generating approach
Serum from healthy controls T0 and mepolizumab-treated patients T0 4 and 32 weeks will be isolated from peripheral venous blood Once the blood has been collected in a BD Vacutainer SST Serum Separation Tube blood coagulation will be allowed to proceed for a minimum of 45 min in a vertical position at room temperature RT Afterward the tube will be centrifugated at 1100-1300 xg for 10 min RT in swinging bucket rotor units After centrifugation the serum will be located above the polymer barrier The serum will be harvested and aliquoted into Eppendorf tubes
After that two different protocols for high abundant protein removal will be performed
Pre-fractioning protocol 1 High abundant serum protein removal using affinity chromatography 100 μL of serum will be applied to High Select Top14 Abundant Protein Depletion Midi Spin Columns from ThermoFisher Scientific according to manufacturers instructions
Pre-fractioning protocol 2 High abundant serum protein removal using DTT precipitation Use 30 μL of serum according to the protocol we have published
Low abundant serum protein concentration from protocol 1 and 2 will be quantified by RCDC Protein Assay Kit BIORAD as per the manufacturers protocol After depletion of the highest abundant proteins 99 of total protein 60-80 mgmL we expect to have around 60-80 μg of low abundant proteins
Proteome characterization will be made following the same protocol as the one described for eosinophil homogenates
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 FEV1FVC biochemical and haematological parameters
Peripheral blood and serum samples will be collected and eosinophils will be magnetically purified at T0 T4 T16 and T32 Flow cytometry RTqPCR and proteomic analyses as well as immunoassays will be performed All the experimental variables eg the abundance of eosinophil proteins in proteomic assays the abundance of serum proteins eosinophil activation markers will be correlated with clinical parameters eg 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 nanoStringmRNA expression data FOXP3 CRLF2 ST2 IL-7R
Proteomic data
Flow cytometry data Expression of CD16 galectins-110 CD48 CD44 CD11b Siglec-8 CD62L and IL-5Rα
Study secondary endpoints
Lung function parameters FEV1 FEV1FVC
Haematological parameters eg eosinophils number
Other clinical and biochemical variables eg 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 220 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 GPower 3194 64 During these analyses we calculate N necessary get statistical significance in a F test ANOVA Repeated measures within factors given α 005 power 1-β 095 the number of measurements T0 T4 T16 and T32 and the effect size f 04 large effect size which gives as a more clinically relevant results The output N was 15 with a critical F 282705
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 eg 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 501 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 httpfunrichorgindexhtml For statistics FunRich uses hypergeometric test BH and Bonferroni 66 67 We will use DAVID httpsdavidncifcrfgovtoolsjsp or GO httpgeneontologyorgpagego-enrichment-analysis for gene ontology enrichment and for protein-protein interaction network construction and clustering we will use String httpsstring-dborg or Cytoscape 37 httpscytoscapeorg 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 Students 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 005 with a 15 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 Targetedhypothesis-driven or broaderuntargeted -omics technologies approaches We are aware that it might be challenging to find predictive markers in this small and prospectiveproof of concept study 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 and reduce technical variance eg nCounter nanoString in order to shorten sample sizes On the other hand hypothesis-driven approaches eg flow cytometry RT-qPCR ELISA with the advantages of greater credence less risk of type I 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 005 differences obtained with untargeted transcriptomicproteomic approaches Finally we purpose the use of two independent but complementary protocols for serum pre-fractioning This will increase the probability of reaching a higher number of proteins with changes in serum and this will therefore increase the probability of discovering a higher number of biomarkers
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
True
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
False
Is an FDA AA801 Violation?:
None