Objectives

The ExpRess study is a multicentre prospective cohort study of adult patients with CRS recruited from at least five UK hospitals in parallel to the MACRO randomised controlled trial. Recruitment is currently ongoing at James Paget University Hospital, Norfolk and Norwich University Hospital, Guy’s and St Thomas’ NHS Foundation Trust, Imperial College Healthcare NHS Trust and Newcastle upon Tyne Hospitals NHS Foundation Trust. Study objectives include:

1. To quantify the cytokine expression pattern for MACRO trial participants,
2. To determine if cytokine expression varies between treatment responder and non-responder groups,
3. To identify if phenotypic characteristics collected from MACRO patients can be predicted by cytokine profiles,
4. To define the bacterial profiles of MACRO trial participants.

Participants

All participants are recruited from existing MACRO trial and are subject to the full eligibility criteria detailed in the MACRO protocol [6]. Selected eligibility criteria include:

• Adults aged 18 and over with a minimum of 12 weeks’ history of inflammation of the nose and paranasal sinuses characterised by two or more symptoms, one of which should be either (1) nasal blockage, obstruction or congestion or (2) nasal discharge (anterior or posterior nasal drip). Other symptoms may include facial pain or pressure and reduction or loss of smell.
• Moderate or severe symptoms within the last 3 months with a SNOT-22 score ≥ 20.
• Symptom control not achieved following previous alternative medical therapy, including intranasal corticosteroids, short courses of antibiotics, or saline rinses, and therefore considered eligible for ESS.

Participants will be excluded if they meet any of the following criteria or those detailed in the full MACRO trial protocol:

• Lund–Mackay non-contrast CT scan score < 4.
• Macrolide antibiotic treatment for >3 continuous weeks within the last 12 months.
• ESS in the previous 6 months or visible, open sinus cavities from previous surgery.
• Taking maintenance oral steroids or biologics
• Has received systemic corticosteroids in the last one month.
• Rare or complex sinus conditions, such as CRS secondary to systemic disease (e.g. ciliary dyskinesias or granulomatous diseases) or suspected malignancy.
• Allergic fungal rhinosinusitis confirmed or suspected on CT imaging necessitating immediate surgery.
• Severe asthma (taking high doses of inhaled steroids, i.e. >1.5 mg per day).

All participants will have received nasal endoscopy within 3 months prior to recruitment to confirm CRS phenotype, and a non-contrast CT scan within 18 months prior to recruitment to confirm suitability for ESS.

Recruitment

Participants will be recruited from January 2019 to September 2023 from participating UK sites. The recruitment target will be 120 patients in total across all three arms of the MACRO trial; equating to approximately 40 participants per arm. An a-priori sample size calculation was not possible due to lack of information around the baseline prevalence and variance of each cytokine included within the study groups and the likely difference between groups.

Patients will be approached for recruitment to ExpRess following randomisation to any of the three MACRO trial arms of surgery, macrolide antibiotics or placebo. Patients will be approached in clinic by the principal investigator or the research nurse and will be given a recruitment pack that will include a participant information sheet (S1 File) and consent forms. Following written informed consent, patients will have nasal tissue samples and endoscopically guided nasal swabs collected either at the point of randomisation or at their 6 month follow up visit. Participants randomised to the surgical arm of the MACRO trial are also eligible to have samples collected at the time of surgery instead, which is within 6 weeks of randomisation. This process is outlined in Fig 1.

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Fig 1. ExpRess study flowchart.

https://doi.org/10.1371/journal.pone.0289407.g001

Sample collection

Nasal tissue samples are collected by an otorhinolaryngologist either in clinic or in the operating theatre for participants randomised to ESS. Tissue is sampled during nasoendoscopy or ESS as a biopsy from the uncinate process of participants with CRS without nasal polyps (CRSsNP) or from nasal polyps for CRSwNP participants. Participants in clinic receive topical anaesthetic with 5% lidocaine and 0.5% phenylephrine up to five minutes prior to sample collection. Samples are stored in 2ml cryovials at -20°C at the collection site until transport to the Biorepository situated in Norwich Research Park at the University of East Anglia. Transport to the biorepository is assured by the local research teams and is performed in batches in a box filled with ice. After reception at the biorepository, samples are anonymised and stored at -80°C until analysis. Samples are pseudoanonymously linked to the MACRO trial database in Oxford.

Participants will also have endoscopically guided nasal swabs collected using DNA/RNA shield swabs (Cambridge Bioscience, Cambridge, UK). Nasal swabs for each participant will be taken from both the left and right middle meatus at a directional vector of 0–20 degrees in relation to the floor of the nose. Swabs will be stored at room temperature or colder and transported alongside nasal tissue samples to the Norwich Biorepository for storage at -80°C until analysis.

Laboratory analysis

Nasal tissue samples will be prepared on ice. Protein will be extracted using a small sterile stainless-steel bead within 2.0ml DNA LoBind® tubes (Eppendorf, Hamburg, Germany) which are pre-incubated for 15 minutes on ice. After addition of the nasal tissue sample, the tube will remain on ice for another 15 minutes. According to tissue sample volume, 0.1 to 1ml of buffer solution will be added, consisting of T-PER™ Tissue Protein Extraction Reagent (Thermofisher Scientific, Massachusetts, United States) and cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland), in a proportion of 1 tablet for 10 ml of T-PER buffer. Tissue samples will then be placed in a homogeniser at 50Hz for at least 7 minutes and then centrifuged for 10 minutes at 4000rpm at 4°C. The supernatant will then be extracted into clean LoBind® tubes and stored at -80°C prior to analysis. A Pierce™ bicinchoninic acid (BCA) protein assay (Thermofisher Scientific, Massachusetts, United States) will be run on an aliquot of each sample in duplicate to determine protein volumes.

Processed nasal tissue samples will be analysed in duplicate with multiplex detection via electrochemiluminescence, using the MESO QuickPlex SQ 120 (Meso Scale Diagnostics, Maryland, United States). R-PLEX assays for IgE and periostin will be used alongside a custom U-PLEX assay for ENA-78, eotaxin, eotaxin-2, G-CSF, IFN-β, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-22, IL-23, IL17E/IL25, IL-33, MCP-1, MCP-4, MIP-1α, MIP-3α, TNF-α, TSLP, VEGF-A and BAFF. 50–100μg of protein per sample will be used for each assay.

Microbiome analysis for nasal bacteriology swabs would be undertaken in conjunction with a bioinformatics team at the Quadram Institute Bioscience in Norwich. The LotuS2 pipeline developed in this group will be used to analyse these samples, having demonstrated that this pipeline produces more accurate microbiome reconstructions than contemporary pipelines [7]. Due to these samples likely being relatively low bacterial–high human biomass (compared to other environments), special care will be taken to avoid human contaminants using a technique developed in this group [8].

Outcome measures

The primary outcome measure will be mean expression for each cytokine listed within a core set of biomarkers for treatment responder and non-responder groups. Responders will be defined as participants with a minimum 10-point improvement from their baseline SNOT-22 score at 6 months from randomisation to the MACRO trial. The cytokine data and microbiome profiles will be linked with the MACRO trial data from the Oxford Surgical Intervention Trials Unit following completion of the MACRO trial. MACRO data includes participant demographic information, disease-specific health-related quality of life using the SNOT-22 at 6 months, endoscopic Lund–Kennedy score, Lildholdt polyp grading score, health-related quality of life and quality-adjusted life-years using the SF-12v2 and EQ-5D-5 L questionnaires, olfactory function measured using Sniffin’ Sticks, and health-care resource use recorded using patient questionnaires.

Statistical analysis

Data will be analysed using RStudio version 1.2.13 software (RStudio, Massachusetts, United States). For continuous variables, results will be expressed as mean and standard deviation. Data distribution will be tested for normality using a Shapiro-Wilk test. If the variables are not normally distributed, the following statistical tests will be used: for dichotomous variables, a chi-square test will be used to determine difference between groups; while, for continuous variables, a Kruskal-Wallis test will be used for between-group comparison. If expression levels of biomarkers are below the detection limit, they will be replaced by the value 0 for analysis purposes. A p-value of <0.05 is considered significant.

For objective 1 the expression of each cytokine will be expressed as mean and standard deviation for participants from each MACRO trial arm. For objective 2 each MACRO trial arm will be analysed separately. Logistic regression analysis will be performed to determine the impact of cytokine expression levels on binary responder status (defined as a minimum 10-point improvement in baselines SNOT-22 score at 6 months). For objective 3 logistic regression analysis will be performed to determine the impact of cytokine expression levels on phenotypic characteristics such as the presence of nasal polyps or raised total serum IgE (>100 IU/ml). For objective 4 microbiome profiles will be reported as mean distribution of bacterial strains. Bacterial profiles will be compared alongside outcomes from the MACRO trial database using univariate and multivariate statistics, associating microbiome profiles to cytokine profiles and treatment success on included patients.

Timescale

Sample recruitment began in January 2019 and will be completed by September 2023. Sample analysis is due to begin in March 2023. Final results will be available in March 2024 once results from the MACRO trial database are available and can be linked to the biomarker analysis.

Ethical approval

This study was approved by the NHS HRA East Midlands—Leicester Central Research Ethics Committee under the reference 16/EM/0468 (S2, S3 Files). All participants will provide written informed consent.