Peripheral blood proteomic profiling of idiopathic pulmonary fibrosis biomarkers in the multicentre IPF-PRO Registry

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease for which diagnosis and management remain challenging. Defining the circulating proteome in IPF may identify targets for biomarker development. We sought to quantify the circulating proteome in IPF, determine differential protein expression between subjects with IPF and controls, and examine relationships between protein expression and markers of disease severity.

Methods: This study involved 300 patients with IPF from the IPF-PRO Registry and 100 participants without known lung disease. Plasma collected at enrolment was analysed using aptamer-based proteomics (1305 proteins). Linear regression was used to determine differential protein expression between participants with IPF and controls and associations between protein expression and disease severity measures (percent predicted values for forced vital capacity [FVC] and diffusion capacity of the lung for carbon monoxide [DLco]; composite physiologic index [CPI]). Multivariable models were fit to select proteins that best distinguished IPF from controls.

Results: Five hundred fifty one proteins had significantly different levels between IPF and controls, of which 47 showed a |log2(fold-change)| > 0.585 (i.e. > 1.5-fold difference). Among the proteins with the greatest difference in levels in patients with IPF versus controls were the glycoproteins thrombospondin 1 and von Willebrand factor and immune-related proteins C-C motif chemokine ligand 17 and bactericidal permeability-increasing protein. Multivariable classification modelling identified nine proteins that, when considered together, distinguished IPF versus control status with high accuracy (area under receiver operating curve = 0.99). Among participants with IPF, 14 proteins were significantly associated with FVC % predicted, 23 with DLco % predicted, 14 with CPI. Four proteins (roundabout homolog-2, spondin-1, polymeric immunoglobulin receptor, intercellular adhesion molecule 5) demonstrated the expected relationship across all three disease severity measures. When considered in pathways analyses, proteins associated with the presence or severity of IPF were enriched in pathways involved in platelet and haemostatic responses, vascular or platelet derived growth factor signalling, immune activation, and extracellular matrix organisation.

Conclusions: Patients with IPF have a distinct circulating proteome and can be distinguished using a nine-protein profile. Several proteins strongly associate with disease severity. The proteins identified may represent biomarker candidates and implicate pathways for further investigation.

Trial registration: ClinicalTrials.gov (NCT01915511).

Keywords: Interstitial lung diseases; Observational study; Proteome; Registries.

Conflict of interest statement

JLT, MLN, RO, LKN and SMP are employees of the Duke Clinical Research Institute, which receives funding support from Boehringer Ingelheim Pharmaceuticals, Inc. to coordinate the IPF-PRO Registry. MG reports personal fees, non-financial support, and other support from the France Foundation; grants, non-financial support and other support from Boehringer Ingelheim and the Pulmonary Fibrosis Foundation; and personal fees from Genentech. HH is on a speaker panel for Boehringer Ingelheim. JRoman reports grants and personal fees from Boehringer Ingelheim; and grants from Genentech, the Department of Veterans Affairs, and the National Institutes of Health. Until the end of 2017, JRoman served on the board of the American Lung Association - Midland States, and chaired the American Thoracic Society Committee on Health Equality and Inclusion. KRF reports grants and personal fees from Boehringer Ingelheim and Roche/Genentech; and personal fees from FibroGen, Sanofi Genzyme, and Veracyte. JRoy is an employee of Staburo GmbH, which was contracted by Boehringer Ingelheim for this work. IN reports personal fees from Boehringer Ingelheim, Genentech and ImmuneWorks. JAB has no disclosures. KD, RV, YL, RS, BS, CH and TBL are employees of Boehringer Ingelheim.

Figures

Fig. 1

Operating characteristics of linear and non-linear models to differentiate patients with IPF from controls in training set (a) and receiver operating curve for the test set (b)

Fig. 2

Heat map indicating expression of most frequently observed proteins of importance across the linear and non-linear models in patients with IPF versus controls

Fig. 3

Proteins significantly associated with measures of disease severity in patients with IPF. All proteins presented had an FDR-corrected p Value < 0.05 and a > 5-unit difference in the respective disease severity measure per unit change in log2RFU (i.e., doubling of protein concentration)

Fig. 4

Top 12 pathways/gene sets related to proteins observed at higher (black) or lower (hatched) levels in patients with IPF versus controls (Benjamini-Hochberg corrected p Value for enrichment in respective pathway using Fisher’s exact test < 4.40E-5) (a) or observed at higher levels in more severe disease (black) or less severe disease (hatched) in patients with IPF (corrected p Value for enrichment < 0.029) (b) as identified by EnrichR, sorted according to the combined score15