Objectives

INJUSTIS is a prospective multicentre observational cohort study that will be managed through the Nottingham Respiratory Research Unit and funded by the National Institute of Health Research (NIHR) through the Nottingham Biomedical Research Centre and an NIHR professorship (RGJ).

The primary objective is to:

• Identify biomarkers that determine progressive fibrotic lung disease irrespective of aetiology.

The secondary objectives are to:

• Identify biomarkers that predict all-cause mortality.

• Identify biomarkers that predict changes in QoL scores.

• Identify biomarkers that predict the development of disease complications (respiratory failure and acute exacerbations).

• Investigate genetic association and epigenetic modifications which affect fibrotic disease severity and progression.

• Prospectively evaluate longitudinal disease behaviour in patients with non-IPF fibrotic lung diseases with a view to developing composite clinical end points for subsequent use in intervention studies.

• Explore association of environmental exposures with disease progression and all-cause mortality.

• Investigate whether home handheld spirometry over 3 months predicts disease progression and survival.

The primary end point will be:

• Disease progression defined as relative forced vital capacity decline ≥10% or death within 12 months.

Secondary end points are:

• All-cause mortality at time of censoring.

• Number of acute exacerbations over 2 years.

• Change in Quality of Life (QoL) Questionnaire Scores from baseline to 12 weeks.

• Rate of change in biomarker activity from baseline to 12 weeks.

• Change in diffusing capacity of the lung for carbon monoxide (DLco) from baseline to 12 months.

• Change in 6 min walk distance from baseline to 12 months.

• Change in transcriptomic profiles from baseline to 12 weeks.

• Change in home handheld spirometry values from baseline to 12 weeks.

Selection of participants

Two hundred participants with recently diagnosed (within 18 months of study start date) fibrotic lung disease (50 each of rheumatoid-ILD, asbestosis, chronic HP and unclassifiable ILD) and 50 IPF participants as positive controls will be recruited from ILD clinics locally and across the UK. Only participants with MDT confirmed diagnosis of fibrotic ILD with radiological evidence of parenchymal lung fibrosis evidenced by reticulation and traction bronchiectasis, with or without honeycomb change will be recruited. Those with inflammatory radiological changes without evidence of fibrosis will not be deemed suitable regardless of clinical phenotype.

Recruitment will be reviewed on an ongoing basis and should rates fall below the expected levels, additional National Health Service (NHS) sites within the UK will be considered for participation. Eligible patients who meet the inclusion/exclusion criteria will be invited to consent. Most participants will be identified through outpatient clinics, but recruitment will not be restricted to this route. It will be explained to participants that entry into the study is entirely voluntary and that further treatment and care will not be affected by a decision to not partake. It will be clearly stated that participants are free to withdraw from the trial at any time. All participants will provide written informed consent, which will be countersigned by a member of the study team.

Inclusion criteria:

• Male or female aged ≥18 years.

• Able and willing to give written informed consent.

• Recently diagnosed (defined as diagnostic CT scan or surgical lung biopsy (if applicable) within 18 months of study start date).

• An MDT diagnosis of fibrotic ILD defined as the presence of traction change and reticulation with or without honeycombing within the lung parenchyma associated with either:

  • Rheumatoid arthritis (rheumatologist diagnosed with anti-cyclic citrullinated peptide antibodies and/or rheumatoid factor positive).

  • Asbestosis (appropriate occupational history and radiological evidence of asbestos exposure).

  • Chronic HP in accordance with consensus criteria11 (appropriate exposure history, radiological features ± avian and fungal precipitins).

  • Unclassifiable fibrotic lung disease (fibrotic lung disease otherwise unclassifiable despite extensive clinical and radiological examination).

  • IPF in accordance with consensus criteria (American Thoracic Society (ATS), European Respiratory Society (ERS), Japanese Respiratory Society (JRS, Latin American Thoracic Society (ALAT) guidelines).18 19

Exclusion criteria:

• Participating in an interventional clinic trial.

• Asymptomatic ILAs and normal lung function.

• Change in clinical phenotype from initial radiological diagnosis to screening.

• Any connective tissue disease other than rheumatoid arthritis.

• Acute HP.

• Participants who do not possess a smartphone cannot partake in the domiciliary spirometry.

Study regimen

Both cases and IPF controls will undertake the same investigations. Following informed consent, the following test results, previously carried out as part of the participant’s usual NHS care, will be used for the purposes of the study:

• HRCT findings.

• Blood results.

• Lung function tests.

• Bronchoscopy samples if already taken.

All participants will have baseline investigations at the first visit having provided informed consent. At the first visit, 40 mL of blood will be obtained, full lung function tests and a 6 min walk test will be performed. Participants will also be asked to complete QoL Questionnaires (Medical Research Council Dyspnoea Scale,20 Leicester Cough Questionnaire,21 IPF-abridged Profile for Assessment and Referral to Care,22 King’s Brief ILD Questionnaire23 and EQ-5D-5L).24 If consent is given for optional bronchoscopy, this will be subsequently performed and bronchoalveolar lavage carried out. Participants with a smartphone will be given the option of a home handheld spirometer and asked to provide daily FVC readings for the first 3 months of the study period.

Further visits at 3 months, 12 months and 24 months will include further 40 mL blood sampling, QoL and full lung function analysis. At 12 months and 24 months a 6 min walk test will be repeated (figure 1).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Legend – participant flow through the study.

The majority of initial sample processing will be performed at the participant’s local NHS hospital trust via the Clinical Research Network. Participation in the study will be for 2 years. Other than initial assessment and follow-up visits at 3 months, 12 months and 24 months, there will be no further assessments. Only participants recruited at Nottingham University Hospitals NHS Trust will be offered a study bronchoscopy. It is not anticipated that any data will be obtained before completion of study that would lead to discontinuation of the study. For individual participants, discontinuation will be decided on an individual basis. The end of study will be defined as the last patient to complete the 2 years of follow-up.

Specimen processing and analysis

All human tissue (blood, lavage, biopsy, etc.) samples will be processed and stored in accordance with the Human Tissue Act (2004). All biological samples will be processed within 2 hours of being obtained according to local standard operating procedures and stored in 500 µl aliquots at −80°C until analysis. Sample analysis will take place either at the University of Nottingham or in the laboratories of third party national, or international, academic partners or contract research organisations following appropriate tissue transfer agreements.

The primary analysis will include measurement of epithelial biomarkers [matrix metalloproteinase-7 (MMP-7), cancer antigen 125 (CA-125), carbohydrate antigen 19-9 (CA19-9), surfactant protein D (SP-D)]) and markers of matrix turnover [C-reactive protein degraded by MMP-1/8 (CRPM), collagen 3 degraded by MMP-9 (C3M) and collagen 6 degraded by MMP-2/9 (C6M)) as well as genotyping for mucin 5B (MUC5B), desmoplakin (DSP) and A-kinase anchoring protein 13 (AKAP13). Exploratory analysis will include whole genome sequencing, RNA sequencing, proteomic and metabolomic analysis to identify novel biomarkers that predict fibrotic disease behaviour. Biopsy material will be used to culture cellular components, frozen for extraction of protein, RNA and DNA or formalin-fixed paraffin embedded.

Any additional samples will be archived for future genetic and biomarker studies in the University of Nottingham premises at the Nottingham City Hospital. The Human Tissues Authority license number is 12 265. Where participants do not agree to the future use of the samples they will be destroyed in accordance with the Human Tissue Act, 2004.

Statistics

The primary end point of disease progression (10% relative FVC decline or death within 12 months) will be used dichotomously across all subgroups collectively analysed together, with the exception of IPF controls. Association of baseline biomarker levels with dichotomous disease progression and overall survival will be analysed using binomial family of generalised linear models, while Gaussian family or otherwise appropriate models will be used to assess associations with continuous secondary end points. The Benjamini-Hochberg procedure will be applied to account for multiplicity as appropriate. Repeated measures mixed models will be used to assess associations over time, which includes time points within one model and circumvents correction for multiple testing. Fixed factors will include baseline demographic information. Where putative biomarkers are identified, end point data will be used to compare biomarker levels by time-to-event through proportional hazard models; comparable ability to predict end points will be assessed through sensitivity and specificity analyses (receiver operator characteristic curves).

To identify transcriptomic biomarkers, RNA-seq libraries will be prepared and entered into a workflow for read count normalisation enabling quantification of transcript expression; normalisation ensures length and abundance of cDNA reads are corrected according to other genes (reads per kilobase per million) and further library scaling can occur.25 26 Libraries, aligned to an appropriate reference genome, will enable detection of differential gene expression and SNPs/variants according to primary end point and secondary measures of disease severity. Transcript-discovery artefacts, transcripts that remain below detectable levels of change across compared samples, as well as any with zero mapped reads will be excluded. R statistical packages will be used for these bioinformatics workflows.

Further statistical analyses will be performed to determine associations between identified biomarkers and exploratory secondary objectives. Statistical approaches include, but are not limited to, correlation and analyses of variance between biomarker levels and patient-reported QoL outcomes or disease exacerbations; machine learning strategies on home spirometry to detect decline in lung function, and subsequent comparison of sensitivity with traditional spirometry measures at predicting end points. Factor analysis approaches will be used to reduce dimensions of data sets and assess clustering; generalised linear models will be used to test associations.

Confounding factors are particularly important in studies that hope to offer causal inference of biomarkers on clinical outcomes,27 and corrections to adjust for these will be applied in the analysis as proposed by the directed acyclic graph (figure 2).28 Fixed factors in mixed models will include baseline demographic information: age, gender, body mass index and smoking status. Exposure history in patients with chronic HP will be collected as will time from diagnosis to enrolment across all subgroups and adjusted for as a potential confounder. Some participants may be on background therapies such as immunosuppression and corticosteroids. While these therapies are likely to be confounders, it is possible they may decrease or increase progression of fibrosis based on the PANTHER (Prednisone, Azathioprine, and N-Acetylcysteine: A Study That Evaluates Response in Idiopathic Pulmonary Fibrosis) Study.29 Therefore, models will include treatment strategy as part of the minimal set of adjustment for estimating direct effect on end point. The objectives of the study do not extend to assessing treatment effect. Further unknown confounders will be identified from the spirometry smartphone application that will collect various data such as air quality index to inform limitations and future study.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Directed acyclic graph (DAG) illustrates proposed model of causal inference and minimal sufficient adjustment sets (confounders; red) for estimating the direct effect of biomarker expression profiles (exposure; green) on disease progression (outcome; blue). Image generated using DAGitty.net environment. QoL, quality of life. BMI, body mass index; ILD, interstitial lung disease.

Sample size justification

A power calculation has been conducted using prior data obtained at 3 months in the PROFILE (Prospective Observation of Fibrosis in the Lung Clinical Endpoints) study of patients with IPF.8 The study demonstrated that 90 stable and 90 progressive individuals were sufficient to detect a twofold change in MMP7 with >90% power and 5% type I error rate. MMP7 was selected for power calculation as the most conservative of the biomarkers identified in the PROFILE Study, with the lowest threshold for biomarker change and least statistical precision, thus powering on MMP7 ensures that other biomarkers will be analysed with adequate power. The calculation demonstrates that a sample of 100 participants with progressive fibrosis and 100 with stable fibrosis will be large enough to detect dynamic differences in biomarkers over 3 months with 80% power and a type I error rate of 5% (α=0.05). Therefore 200 participants with ILD will be recruited assuming 50% disease progression. Fifty positive IPF controls will be recruited to take part in the study to benchmark progressive fibrotic lung disease but will not be included in the final analysis.

The study is also appropriately powered for genetic risk scores. An individual SNP with 25% minor allele frequency would have >70% power to detect an OR of 1.8 for stable versus progressive disease. The power for a risk score comprising multiple variants is expected to be greater. This assumes an additive genetic model, p<0.05, in 100 stable versus 100 progressive patients whereby with each additional marker added to the model the power is actually increased rather than reduced. The markers used will all be defined a priori based on data obtained from the PROFILE Study7 8 but are likely to include MUC5B, DSP, AKAP13, SP-D, CA-125, CA19-9, MMP-7, CRPM and C6M.

Following the recruitment of 100 participants who complete 1 year in the study, an interim sample size re-estimation by an independent data monitoring committee will be conducted to determine the progression status between blinded subgroups. If the blinded progression status is approximately 50%, recruitment will continue as described. If however, any subgroups show a relatively stable phenotype they will be excluded from subsequent recruitment, after being unblinded to the data monitoring committee. To attain an adequately powered sample size with 50% progressive fibrosis, further recruitment will be enriched with participants from the progressive phenotypical subgroups relative to rate of progression in interim analysis. Those recruited from subsequently excluded subgroups will be removed from the primary analysis, as they will be sources of inconsistency. However, if all subgroups progress at a substantially lower rate than expected and conditional power calculations at interim analysis suggest futility then all subgroups will be included in an analysis that demonstrates the null hypothesis (fibrotic lung disease progress at a rate specific to aetiology) could not be rejected. The study is not statistically powered to detect differences between ILD subgroups, although exploratory analyses will be carried out to inform future studies and support replication studies.

Patient and public involvement

The Action for Pulmonary Fibrosis (APF)30 charity have been consulted during the design of the study and will sit on the steering committee as patient representatives, which will inform study conduct and recruitment. All patient information material has been reviewed by patient representatives. Study findings will be communicated to participants, and the APF will also support the dissemination of the study’s finding to patients with pulmonary fibrosis and their families.