Biomarkers of Response to Mepolizumab Treatment in Patients With Nasal Polyps With or Without Bronchial Asthma

Hypothesis Identification of pathological and clinical characteristics of responders and non-responders to mepolizumab treatment in patients with chronic rhinosinusitis with nasal polyps with or without bronchial asthma regarding the airway remodeling, inflammatory cells and other biomarkers on a long-term basis.

Objectives/Endpoints

The primary objectives of this study are:

1. Evaluation of the airway remodeling in various anatomic locations of the airways (nose, bronchus) at baseline-before treatment initiation and after 156 weeks of mepolizumab treatment. 2. Identification of any characteristics in microbiome of various locations (nose, pharyngeal, bronchus) in responders and non-responders to mepolizumab treatment.

The key secondary objectives of this study are:

1. Combination of selected inflammatory biomarkers of various anatomic locations in the airways in tissue and secretion samples to identify non-responders and responders to mepolizumab after 152 weeks of mepolizumab treatment. 2. To demonstrate the effect of mepolizumab treatment in patients with nasal polyps with or without bronchial asthma through the reduction of patients requiring surgery after 52 weeks of treatment taking into consideration the airway remodeling, inflammatory cells and biomarkers of inflammation and clinical characteristics.

Other objectives/endpoints Any improvement in respiratory parameters in asthma control and in patients' quality of life including sleep quality during mepolizumab treatment in patients with nasal polyps with or without bronchial asthma.

Any possible biomarkers of response considering nasal polyps and bronchial asthma as a unified airway disease after 52 weeks of treatment and after 156 weeks of treatment.

Characterization of patients' compliance to mepolizumab doses for 156 weeks.

Study design and Methods A phase IV study, where patients will be collected from outpatient clinics of Bronchial Asthma and of Ear Nose Throat from each site which cover the population of Greece. Due to the unknown situation concerning Covid-19 pandemic, we assume that this clinical study does not interfere with the workload in our Clinics.

Overall, this is a prospective multicenter study including 7clinics. The study will include a screening period of up to 2 weeks to assess eligibility and obtain written informed consent and a mepolizumab treatment period of 156 weeks.

Sampling method, number of subjects and timelines Study population The study population will consist of approximately 57 patients with nasal polyposis with or without bronchial asthma receiving mepolizumab for three years aged 18 and above (no children). Clinical evaluation of patients with nasal polyposis with or without asthma will be made according to the European Position Paper on Rhinosinusitis and Nasal Polyps. Besides, standard of care for our patients apart from mepolizumab may include ICS, anti-histamine or LTRAs.

Target enrolment Our target sample size was calculated on the basis that 57 patients was the minimum sample size, computed for reticular basement membrane thickness from g-power analysis (as described in the paragraph of calculation of sample size). We add an additional 5% to ensure the validity of the results in case of potential patient withdrawals during our perspective study.

Study participation, assessment and follow up:

Vital signs, will be performed at site visits at screening visit, at baseline and every 3 months. Measurements will include systolic and diastolic blood pressure, pulse rate and body temperature.

Height in centimeters (cm) and body weight in kilograms (kg) will be measured at screening visits. Body Mass Index (BMI) will be calculated as the weight in kg divided by the height in meters squared

Laboratory evaluation (Hematology, Blood chemistry, Urinalysis) includes venous blood and urine will be collected at baseline. Collection of urine for female subjects will also evaluate pregnancy. IgE levels will be evaluated at screening visit using immunoCAP tests and will be reported as IU/mL.

Questionnaires The following will be administered: 1) the Asthma Control Questionnaire (ACQ-5) and Asthma Control Test (ACT) to assess current asthma control; 2) the Asthma Quality of Life Questionnaire (AQLQ+12) to assess quality of life and psychological morbidity. To further asses sleeping quality 1) Athens Insomnia Scale (AIS), 2) Epworth Sleepiness Scale (ESS), 3) St. George's Respiratory Questionnaire (SGRQ) and 4) WHO (Five) Well-Being Index (WHO-5), 5) Fatigue Severity Scale (FSS), 6) Visual Analogue Scale (VAS) score, 7) SinoNasal Outcome Test (SNOT-22) score, 8) University of Pennsylvania Smell Identification Test (UPSIT) score.

FeNO Exhaled nitric oxide (NO) will be measured at baseline and every 6 months according to the American Thoracic Society Guidelines and as specified by the manufacturer. FeNO levels will be reported as parts per billion. Measurements will be made before spirometry is performed.

Blood eosinophil levels Venous blood samples will be retrieved between 8 to 10 am, at baseline and every 3 months during the 156-week period with mepolizumab treatment. Absolute blood eosinophil count will be reported as cells/μl.

Spirometry Spirometry will be conducted, using the site's own equipment at the visits (baseline and every 3 months) as specified in the table 1. All clinic visits must occur in the morning. During the treatment period the spirometry will meet the current ATS/ERS guidelines and the spirometer must produce a printout of all data generated, which should be stored in the subject's notes. The spirometer will be calibrated in accordance with the manufacturer's instructions. Spirometry must be performed at the same time (±1 hour) of the Visit 0 spirometry. Subjects should try to withhold short-acting beta-2-agonists (SABAs) for ≥4 hours and LABAs for ≥15 hours prior to clinic visit, if possible (GINA 2019). Assessments to be recorded will include FEV1, FVC, FEV1/FVC%, PEF and FEF25-75. Post-bronchodilator measurements will be taken at every visit, using 200-400mcg albuterol or equivalent.

Impulse Oscillometry Impulse Oscillometry (IOS) will be conducted at baseline, after 52 weeks of treatment and after 156 weeks of treatment.

Lund Mackay Score Lund-Mackay computed tomography (CT) score will be evaluated by CT sinus at baseline, after 52 and 156 weeks of treatment as specified in table 1. Preoperative and follow up CT sinus are used in clinical practice in CRS patients as standard of care in sponsor's country (radiation dose about 50mGy). Lund-Mackay CT staging will be evaluated blindly by 2 different clinicians.

Peak nasal inspiratory flow (PNIF) PNIF will be applied at baseline and every 6 months until 156-week of treatment. PNIF will be evaluated using Olympus ENF type P4 fibroscope and a Clement Clark International Limited model IN-CHECK ORAL ATM device.

Anterior Rhinomanometry Anterior Rhinomanometry will be conducted at baseline and every 6 months until 156-week of treatment by rhinomanometry system PDD-301/sr.

Therapy with biologics Upon completion of the screening visits the participant may initiate treatment with biologic therapy (mepolizumab) within a month. During therapy subjects will be under the care of the pulmonologist who had referred them to the research group for taking part into the study. These pulmonologists will be termed as 'associate pulmonologists. Treatment will be provided as per product manufacturer instructions.

Associate pulmonologists and otorhinologists providing biologic treatment during the study will require to sign an agreement to conduct the study in compliance with the protocol, to acknowledge that he/she is responsible for medical conduct and for study conduct, and to ensure that the associate (and their colleagues and employees) assisting in the conduct of the study are informed about their obligations.

Mechanisms will be in place to ensure that associate staff receives the appropriate information and support by the research group throughout the study.

Follow up during biologic therapy: At initiation and at every visit for biologic therapy administration collection of information (see table 1) will be performed by the associate pulmonologist, and with support by the research group if requested by the associate pulmonologist e.g., researchers via telephone or face-to-face contact will interview the patient. Furthermore, compliance will be recorded form the physician based on patient's oral confirmation. Date form of drug administration will be recorded at every visit, as well as immediate and delayed adverse effects.

Safety monitoring Recorded adverse events (AEs) will be monitored and collated as required in study reports.

Adverse events An adverse event is the appearance or worsening of any undesirable sign, symptom, or medical condition occurring after starting the study entry. Medical conditions/diseases present before starting study drug are only considered adverse events if they worsen after starting study drug. Abnormal laboratory values or test results constitute adverse events only if they induce clinical signs or symptoms, are considered clinically significant, or require therapy. Also, adverse events related to Covid-19 are included. If a partner pulmonologist or research staff becomes aware of an AE associated with a product, it can be reported to the manufacturer according to their local standard practice for spontaneous AE reporting. A copy of the report should also be sent to the study coordinator. Serious adverse events (SAE) will be reported in the eCRF pages and within 24 hours all the investigators will have the responsibility to inform the Central Committee in Thessaloniki. Then the Central Committee will inform the scientific board and the Greek Medical Agency. Also, medical and scientific judgment will be exercised by the principal investigator of each center with the Central Committee, in deciding whether the subject should stop the treatment or not.

Independent Data Monitoring Committee/Data and Safety Monitoring Board (IDMC/ DSMB) is the central research personnel in Thessaloniki (Central Committee). For a successful monitoring of protocol assessments, IDMC/DSMB will schedule a phone-call every month to all the sites included.

Konstantinos Porpodis -Principal Investigator (Associate Professor, Pulmonary Department, Aristotle University of Thessaloniki, George Papanikolaou Hospital, Thessaloniki), Kalliopi Domvri - Clinical Research Associate-Study Manager-Sub Investigator, Laboratory of Histology-Embryology, Medical School, Aristotle University of Thessaloniki.

Pregnancy report Initially, female patients at screening will have to do a pregnancy test. During treatment, if a female subject reports a pregnancy to us, she will be asked to withdraw from the study (to prevent bias) Then, she will be asked if she would like to keep getting the treatment outside of this protocol and be monitored at each center's outpatient clinic.

Pre-screening and Screening Failures Subjects will be assigned a study number at the time of signing the consent. Subjects who signed but do not progress to the screening visits assessment will be deemed a pre-screen failure. No data will be captured in the database for these subjects.

Subjects that complete at least one of the two screening visits, but do not initiate biologic therapy will be designated as screening failures.

Information to be collected for pre-screening and screening failures will be detailed in the database completion.

Withdrawal Visit If a participant decides to withdraw from the study during follow-up (for any reason other than loss to follow up), they may be asked to attend a withdrawal visit (if deemed necessary by investigator and subject to consent). During the visit, the participant may be asked questions about their current condition

• Change of cytokine and protein levels in serum [Time frame: through study completion, 156 weeks]
• Change in exacerbation rate [Time frame: through study completion, 156 weeks]
• Change in requiring surgery [Time frame: through study completion, 156 weeks]
• Change of cytokine and protein levels in bronchial washing [Time frame: through study completion, 156 weeks]