Peripheral blood mononuclear cell gene expression profiles predict poor outcome in idiopathic pulmonary fibrosis

Abstract

We aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) by performing microarray experiments of PBMCs in discovery and replication cohorts of IPF patients. Microarray analyses identified 52 genes associated with transplant-free survival (TFS) in the discovery cohort. Clustering the microarray samples of the replication cohort using the 52-gene outcome-predictive signature distinguished two patient groups with significant differences in TFS. We studied the pathways associated with TFS in each independent microarray cohort and identified decreased expression of "The costimulatory signal during T cell activation" Biocarta pathway and, in particular, the genes CD28, ICOS, LCK, and ITK, results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A proportional hazards model, including the qRT-PCR expression of CD28, ICOS, LCK, and ITK along with patient's age, gender, and percent predicted forced vital capacity (FVC%), demonstrated an area under the receiver operating characteristic curve of 78.5% at 2.4 months for death and lung transplant prediction in the replication cohort. To evaluate the potential cellular source of CD28, ICOS, LCK, and ITK expression, we analyzed and found significant correlation of these genes with the PBMC percentage of CD4(+)CD28(+) T cells in the replication cohort. Our results suggest that CD28, ICOS, LCK, and ITK are potential outcome biomarkers in IPF and should be further evaluated for patient prioritization for lung transplantation and stratification in drug studies.

Fig. 1. Study design and cohorts

The outline summarizes the studied cohorts, the experiments performed in each cohort, and the statistical analyses used. The horizontal arrows represent the confirmation of microarray and qRT-PCR experiments in both cohorts.

Fig. 2. Hierarchical clustering discriminates subgroups with outcome differences in the replication cohort

(A) Hierarchical clustering of IPF patients from the replication cohort (n = 75) based on the 52-gene signature found in the discovery cohort to be associated with TFS (FDR <5%, Cox score ≥2.5 and ≤−2.5). Two major clusters of IPF patients were identified. Every row represents a gene, and every column, a patient. Color scale is shown adjacent to heat map in log₂ scale; generally, yellow denotes increase over the geometric mean of samples, and purple, decrease. (B) TFS differs between clusters in the replication cohort; the median survival of each group is depicted in dotted vertical lines; n at risk is the number of IPF patients at risk of death or lung transplant at the beginning of each time point. P value was determined by the log-rank test.

Fig. 3. qRT-PCR confirms microarray findings in the discovery cohort

Correlation between log₂-transformed microarray gene expression values and corresponding SmartChip qRT-PCR expression levels for CD28, ICOS, LCK, and ITK in patients (n = 43) from the discovery cohort. P values were determined by Student’s t distribution for Pearson correlation.

Fig. 4. CD28, ICOS, LCK, and ITK are potential IPF outcome biomarkers

(A) TFS analysis in the replication cohort (n = 74) with available qRT-PCR data for CD28, ICOS, LCK, and ITK. In the Kaplan-Meier plots for each gene, the red lines are patients with expression levels above the ΔC_t median value (representing a decrease in gene expression); the black lines are patients with expression levels below the ΔC_t threshold (representing an increase in gene expression); the median survival of each group is depicted in dotted vertical lines. P values were determined by the log-rank test. (B) AUC of time-dependent ROC analysis for TFS based on clinical and/or genomic models in replication cohort subjects with all available variables (n = 72). Genomic model included continuous ΔC_t values of CD28, ICOS, LCK, and ITK. Clinical model included age, gender, and FVC%. P values were determined by the Wilcoxon signed rank test.

Fig. 5. Expression levels of CD28, ICOS, LCK, and ITK correlate with the number of circulating CD4⁺CD28⁺ T cells

Correlation between the percentage of CD4⁺CD28⁺ T cells in PBMCs and their corresponding 1 − ΔC_t SmartChip qRT-PCR expression levels of CD28, ICOS, LCK, and ITK in (n = 72) patients from the replication cohort. P values were determined by Student’s t distribution for Pearson correlation.