Increased monocyte count as a cellular biomarker for poor outcomes in fibrotic diseases: a retrospective, multicentre cohort study

Abstract

Background: There is an urgent need for biomarkers to better stratify patients with idiopathic pulmonary fibrosis by risk for lung transplantation allocation who have the same clinical presentation. We aimed to investigate whether a specific immune cell type from patients with idiopathic pulmonary fibrosis could identify those at higher risk of poor outcomes. We then sought to validate our findings using cytometry and electronic health records.

Methods: We first did a discovery analysis with transcriptome data from the Gene Expression Omnibus at the National Center for Biotechnology Information for 120 peripheral blood mononuclear cell (PBMC) samples of patients with idiopathic pulmonary fibrosis. We estimated percentages of 13 immune cell types using statistical deconvolution, and investigated the association of these cell types with transplant-free survival. We validated these results using PBMC samples from patients with idiopathic pulmonary fibrosis in two independent cohorts (COMET and Yale). COMET profiled monocyte counts in 45 patients with idiopathic pulmonary fibrosis from March 12, 2010, to March 10, 2011, using flow cytometry; we tested if increased monocyte count was associated with the primary outcome of disease progression. In the Yale cohort, 15 patients with idiopathic pulmonary fibrosis (with five healthy controls) were classed as high risk or low risk from April 28, 2014, to Aug 20, 2015, using a 52-gene signature, and we assessed whether monocyte percentage (measured by cytometry by time of flight) was higher in high-risk patients. We then examined complete blood count values in the electronic health records (EHR) of 45 068 patients with idiopathic pulmonary fibrosis, systemic sclerosis, hypertrophic cardiomyopathy, or myelofibrosis from Stanford (Jan 01, 2008, to Dec 31, 2015), Northwestern (Feb 15, 2001 to July 31, 2017), Vanderbilt (Jan 01, 2008, to Dec 31, 2016), and Optum Clinformatics DataMart (Jan 01, 2004, to Dec 31, 2016) cohorts, and examined whether absolute monocyte counts of 0·95 K/μL or greater were associated with all-cause mortality in these patients.

Findings: In the discovery analysis, estimated CD14+ classical monocyte percentages above the mean were associated with shorter transplant-free survival times (hazard ratio [HR] 1·82, 95% CI 1·05-3·14), whereas higher percentages of T cells and B cells were not (0·97, 0·59-1·66; and 0·78, 0·45-1·34 respectively). In two validation cohorts (COMET trial and the Yale cohort), patients with higher monocyte counts were at higher risk for poor outcomes (COMET Wilcoxon p=0·025; Yale Wilcoxon p=0·049). Monocyte counts of 0·95 K/μL or greater were associated with mortality after adjusting for forced vital capacity (HR 2·47, 95% CI 1·48-4·15; p=0·0063), and the gender, age, and physiology index (HR 2·06, 95% CI 1·22-3·47; p=0·0068) across the COMET, Stanford, and Northwestern datasets). Analysis of medical records of 7459 patients with idiopathic pulmonary fibrosis showed that patients with monocyte counts of 0·95 K/μL or greater were at increased risk of mortality with lung transplantation as a censoring event, after adjusting for age at diagnosis and sex (Stanford HR=2·30, 95% CI 0·94-5·63; Vanderbilt 1·52, 1·21-1·89; Optum 1·74, 1·33-2·27). Likewise, higher absolute monocyte count was associated with shortened survival in patients with hypertrophic cardiomyopathy across all three cohorts, and in patients with systemic sclerosis or myelofibrosis in two of the three cohorts.

Interpretation: Monocyte count could be incorporated into the clinical assessment of patients with idiopathic pulmonary fibrosis and other fibrotic disorders. Further investigation into the mechanistic role of monocytes in fibrosis might lead to insights that assist the development of new therapies.

Funding: Bill & Melinda Gates Foundation, US National Institute of Allergy and Infectious Diseases, and US National Library of Medicine.

Figure 1

Analysis overview Overview of discovery and validation analyses (A) and detailed information for each validation cohort including the number of samples, diagnostic criteria for idiopathic pulmonary fibrosis (IPF), and data modality used for measuring monocytes (B). EHR=electronic health records. PBMC=peripheral blood mononuclear cell. FACs=fluorescence-activated cell sorting. CyTOF=cytometry by time-of-flight. ATS and ERS=American Thoracic Society and European Respiratory Society. HRCT=high-resolution CT. ACR=American College of Rheumatology. SSc-ILD=scleroderma-associated interstitial lung disease. HCM=hypertrophic cardiomyopathy. ICD=International Classification of Diseases. GAP=gender, age, physiology. *Of the 153 patients in the Stanford cohort initially identified with ICD codes as having idiopathic pulmonary fibrosis, 151 had charts available, of whom 130 had confirmed idiopathic pulmonary fibrosis. We removed data for the remaining 21 patients from all analyses.

Figure 2

Classic monocyte (CD14+ CD16–) count association with poor outcomes in patients with idiopathic pulmonary fibrosis (A) In the discovery analysis, patients with an estimated proportion of classical monocytes greater than the mean had reduced transplant-free survival. (B) Patients with progressive idiopathic pulmonary fibrosis in the COMET trial had higher CD14+ monocyte counts than patients with non-progressive disease. (C) Patients with idiopathic pulmonary fibrosis in the Yale cohort had higher proportions of CD14+ monocytes (of total peripheral blood mononuclear cells) compared with healthy controls. Each circle represents an individual patient. Horizontal lines within boxes indicate the median. The bottom and top edges of each box indicate the first and third quartiles of the data, respectively. Vertical lines indicate the datapoints within 1·5 times of the IQR of the data.

Figure 3

Survival of patients with idiopathic pulmonary fibrosis patients up to 5 years after diagnosis Data are stratified by absolute monocyte count ≥0·95 K/μL or <0·95 K/μL. With lung transplantation as a censoring event and after adjusting for age and sex, monocyte count ≥0·95 K/μL was significantly associated with increased risk of mortality in the Stanford (A), Vanderbilt (B), and Optum cohorts (C). Hazard ratios (HRs) for monocyte counts and outcomes across the COMET, Stanford, and Northwestern systemic sclerosis with interstitial lung disease (SSc-ILD) cohorts, adjusted for forced vital capacity (FVC) and gender, age, and physiology (GAP) index (D). Bold text shows the combined HRs for poor outcomes across the three cohorts. There was no heterogeneity in FVC-adjusted and GAP-adjusted HRs across the three cohorts.

Figure 4

Association between high monocyte count in complete blood count at diagnosis with mortality in hypertrophic cardiomyopathy, systemic sclerosis, and myelofibrosis Data are from electronic health records in the Stanford, Vanderbilt, and Optum cohorts. Absolute monocyte count ≥0·95 K/uL was significantly associated with reduced survival for patients with hypertrophic cardiomyopathy in Stanford (A), Vanderbilt (B), and Optum (C); systemic sclerosis in Stanford (D), Vanderbilt (E), and Optum (F); and myelofibrosis in Stanford (G), Vanderbilt (H), and Optum (I).