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An epithelial biomarker signature for idiopathic pulmonary fibrosis: an analysis from the multicentre PROFILE cohort study

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Summary

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disorder with a variable disease trajectory. The aim of this study was to assess potential biomarkers to predict outcomes for people with IPF.

Methods

PROFILE is a large prospective longitudinal cohort of treatment-naive patients with IPF. We adopted a two-stage discovery and validation design using patients from the PROFILE cohort. For the discovery analysis, we examined 106 patients and 50 age and sex matched healthy controls from Nottingham University Hospitals NHS Trust and the Royal Brompton Hospital. We did an unbiased, multiplex immunoassay assessment of 123 biomarkers. We further investigated promising novel markers by immunohistochemical assessment of IPF lung tissue. In the validation analysis, we examined samples from 206 people with IPF from among the remaining 212 patients recruited to PROFILE Central England. We used the samples to attempt to replicate the biomarkers identified from the discovery analysis by use of independent immunoassays for each biomarker. We investigated the predictive power of the selected biomarkers to identify individuals with IPF who were at risk of progression or death. The PROFILE studies are registered on ClinicalTrials.gov, numbers NCT01134822 (PROFILE Central England) and NCT01110694 (PROFILE Royal Brompton Hospital).

Findings

In the discovery analysis, we identified four serum biomarkers (surfactant protein D, matrix metalloproteinase 7, CA19-9, and CA-125) that were suitable for replication. Histological assessment of CA19-9 and CA-125 suggested that these proteins were markers of epithelial damage. Replication analysis showed that baseline values of surfactant protein D (46·6 ng/mL vs 34·6 ng/mL, p=0·0018) and CA19-9 (53·7 U/mL vs 22·2 U/mL; p<0·0001) were significantly higher in patients with progressive disease than in patients with stable disease, and rising concentrations of CA-125 over 3 months were associated with increased risk of mortality (HR 2·542, 95% CI 1·493–4·328, p=0·00059).

Interpretation

We have identified serum proteins secreted from metaplastic epithelium that can be used to predict disease progression and death in IPF.

Funding

GlaxoSmithKline R&D and the UK Medical Research Council.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal condition with a variable disease trajectory. The advent of effective therapy for IPF has generated an urgent need to identify biomarkers for various aspects of disease behaviour, particularly those that could be used to stratify therapy, to enable the delivery of personalised medicine, and to provide robust and reliable measures of response to antifibrotic therapy.

The accepted understanding of the pathogenesis of IPF suggests that it occurs in genetically susceptible individuals following repeated or persistent epithelial injury. This combination of susceptibility and injury in turn activates fibroblasts with an invasive phenotype that secrete abundant extra-cellular matrix proteins, resulting in the progressive parenchymal destruction characteristic of IPF.1

Several serum biomarkers have been consistently described in case-control and point of diagnosis studies, including markers reflective of epithelial dysfunction: matrix metalloproteinase 7 (MMP7), surfactant protein D (SP-D), and Krebs von den Lungen 6 (KL-6; the antibody that recognises the human protein synthesised by the MUC1). Putative inflammatory molecules such osteopontin and CCL18 have also been identified as potential prognostic biomarkers.2 More recently we described biomarkers of matrix turnover which, when measured longitudinally, identify individuals with increased risk of disease progression and death.3

In this study, by use of a two-stage discovery and validation analysis from the PROFILE study cohort, we sought to identify potential biomarkers of prognosis and disease progression in IPF. This is, to our knowledge, the first study to adopt such an approach to biomarker discovery in a prospectively enrolled, longitudinally sampled cohort of patients with IPF.

Research in context

Evidence before this study

We searched PubMed without language restrictions for reports published up to July 20, 2017, using the search terms “idiopathic pulmonary fibrosis”, “biomarkers”, “surfactant protein D”, “matrix metalloproteinase”, “CA-125”, and “CA19-9”. At the time of the search, we found no biomarker studies that had used a two-stage hypothesis-free discovery and targeted validation design. Only one study (our previous report from the PROFILE study) had assessed longitudinal change of biomarkers in a treatment-naive cohort of patients with idiopathic pulmonary fibrosis (IPF). Although tumour markers have been assessed in patients with IPF, no studies had identified CA-125 as a dynamic biomarker or CA19-9 as a biomarker of disease progression for IPF.

Added value of this study

To our knowledge, this is the largest study to prospectively and systematically assess the role of known and potentially novel serum biomarkers in patients with IPF. Furthermore, the two-stage discovery and validation design; the use of two distinct assays (one multiplex and four individual ELISAs); prospective, systematic longitudinal sample measurement; assessment of more than 300 patients with long-term follow-up data; appropriately powered sample size for an a-priori-defined outcome; and the thorough assessment of four epithelial-derived biomarkers represent a step-change over the currently available literature in terms of both the methodological approach and the findings. Thus, our results help to clarify the roles of known biomarkers and highlight the importance of two previously unvalidated biomarkers for IPF.

These data represent the largest prospective analysis of serum biomarkers in IPF. The two-stage design permitted us to adopt an unbiased approach to identifying the most promising biomarkers on the basis of their ability to distinguish individuals with disease from matched healthy controls, identify individuals with IPF who have progressive disease, and identify changes in biomarkers over time to predict death as an outcome (potentially acting as theranostic biomarkers). The most promising biomarkers in the discovery set were independently validated using individual ELISAs, including those available in routine clinical practice.

Implications of all the available evidence

These results identified epithelium-derived proteins as promising biomarkers in all three of our categories. Validation analysis supported the finding that high concentrations of baseline surfactant protein D and matrix metalloproteinase 7 can be used to distinguish individuals with disease from controls and predict outcome. By contrast, high concentrations of CA19-9 were able to predict 12-month disease progression, whereas rising concentrations of CA-125 after 3 months were predictive of 12 month disease progression and overall mortality. Changes in these markers were independent of baseline physiological parameters. These data identify biomarkers of IPF that could potentially be used to streamline clinical trial designs, identify individuals at high risk of progression, and even assess clinical response to therapy.

Section snippets

Study design and participants

This two-stage study consisted of a discovery analysis using samples from patients with IPF (diagnoses confirmed by a multidisciplinary team) and age and sex matched healthy controls, and a validation analysis with samples taken from other patients with confirmed IPF (appendix). The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consent.

PROFILE is a prospective, multicentre, observational cohort study of incident cases of

Results

The first patient was enrolled at Royal Brompton Hospital was on Sept 1, 2010, and at the Nottingham University Hospitals on Sept 27, 2010. The last patient included in these analyses was enrolled at Nottingham University Hospitals on Oct 15, 2014. The healthy controls were enrolled from Oct 8, 2012, to Sept 3, 2014. We included 106 patients with a diagnosis of IPF confirmed by multidisciplinary team in the discovery analyses. Of the 252 eligible patients recruited to PROFILE Central England

Discussion

We have identified a novel epithelial biomarker signature that might be predictive of prognosis, disease progression, and risk of death in patients with IPF. In this analysis from the PROFILE study, we systematically assessed 123 serum proteins using a two-stage experimental approach in patients with incident IPF, starting with a discovery analysis with a multiplex platform followed by specific replication with individual ELISAs for the most promising candidate biomarkers. With this approach,

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