Development of a Novel Biomarker for the Progression of Idiopathic Pulmonary Fibrosis

Abstract

The progression of idiopathic pulmonary fibrosis (IPF) is diverse and unpredictable. We identified and validated a new biomarker for IPF progression. To identify a candidate gene to predict progression, we assessed differentially expressed genes in patients with advanced IPF compared with early IPF and controls in three lung sample cohorts. Candidate gene expression was confirmed using immunohistochemistry and Western blotting of lung tissue samples from an independent IPF clinical cohort. Biomarker potential was assessed using an enzyme-linked immunosorbent assay of serum samples from the retrospective validation cohort. We verified that the final candidate gene reflected the progression of IPF in a prospective validation cohort. In the RNA-seq comparative analysis of lung tissues, CD276, COL7A1, CTSB, GLI2, PIK3R2, PRAF2, IGF2BP3, and NUPR1 were up-regulated, and ADAMTS8 was down-regulated in the samples of advanced IPF. Only CTSB showed significant differences in expression based on Western blotting (n = 12; p < 0.001) and immunohistochemistry between the three groups of the independent IPF cohort. In the retrospective validation cohort (n = 78), serum CTSB levels were higher in the progressive group (n = 25) than in the control (n = 29, mean 7.37 ng/mL vs. 2.70 ng/mL, p < 0.001) and nonprogressive groups (n = 24, mean 7.37 ng/mL vs. 2.56 ng/mL, p < 0.001). In the prospective validation cohort (n = 129), serum CTSB levels were higher in the progressive group than in the nonprogressive group (mean 8.30 ng/mL vs. 3.00 ng/mL, p < 0.001). After adjusting for baseline FVC, we found that CTSB was independently associated with IPF progression (adjusted OR = 2.61, p < 0.001). Serum CTSB levels significantly predicted IPF progression (AUC = 0.944, p < 0.001). Serum CTSB level significantly distinguished the progression of IPF from the non-progression of IPF or healthy control.

Keywords: IPF; cathepsin; prediction; prognosis; progression.

Figure 1

Workflow of this study. We first independently compared the different gene expression levels among a total of 78 lung tissue samples from control, early IPF, and advanced IPF patients in the GSE10667, GSE24206, and PNU datasets. We found common DEGs that were significantly 13 up-regulated or three down-regulated in the control, advanced IPF, and early IPF datasets. Among the 16 genes common to the three datasets, gene validation was conducted for only nine genes, including eight up-regulated genes (CD276, COL7A1, CTSB, GLI2, PIK3R2, PRAF2, IGF2BP3, and NUPR1) and one down-regulated gene (ADAMTS8). Immunohistochemical staining was performed on 15 lung tissue samples (control three vs. early IPF 6 vs. advanced IPF 6). Western blot analysis was also performed on 12 lung tissue samples (control three vs. early IPF 5 vs. advanced IPF 4). To test the possibility of using CTSB as a biomarker, serum CTSB was retrospectively measured in the test cohort (n = 78) using ELISA. Finally, to validate the possibility of using CTSB as a biomarker, serum CTSB levels were prospectively measured in the independent IPF clinical cohort (validation cohort; n = 129). E: early IPF; A: advanced IPF; DEGs: differentially expressed genes; IHC: immunohistochemistry; ELISA: enzyme-linked immunosorbent assay; NP: nonprogressive IPF; P: progressive IPF; IPF: idiopathic pulmonary fibrosis.

Figure 2

Identification of DEGs. (A) Venn diagrams show the overlapping DEGs among the three datasets: up-regulated genes (left) and down-regulated genes (right). We used the ANOVA or Kruskal–Wallis rank sum test to compare different expression levels among the three groups (control, early IPF, and advanced IPF). Each circle represents the number of independently up or down-regulated genes in each dataset. Heatmap of scaled gene expression of 16 common DEGs in the three datasets: (B) GSE10667, (C) GSE24206, and (D) PNU. Red indicates high relative expression, and blue indicates low relative expression. The rows represent 13 up-regulated genes and three down-regulated genes in order, and the gray, yellow, and orange bars at the top of the heatmap represent control, early, and advanced IPF samples, respectively. DEGs: differentially expressed genes; E: early IPF; A: advanced IPF; DEGs: differentially expressed genes; IPF: idiopathic pulmonary fibrosis; ANOVA: analysis of variance.

Figure 3

Representative image of immunohistochemistry CTSB and Western blot. (A) Control; (B) early IPF; (C) advanced IPF; (D) control; (E) early IPF; (F) advanced IPF; IHC staining and Western blot were performed on tissue from the right lower lobe area of control, early IPF, and advanced IPF. The staining of CTSB was increased in advanced IPF compared to that in the control and in early IPF, marking macrophages in the honeycomb region. Arrows indicate macrophages stained for CTSB. A negative control was added, as shown in Figure S2. (G) Western blot. In the Western blot, each group was marked separately using a dividing line. (H) CTSB expression levels in control vs. early IPF vs. advanced IPF. CTSB was increased in advanced IPF compared to the control and early IPF. Each short bar below and above represents the first and third quartiles of expression, and each central long bar represents the median value. E: early IPF; A: advanced IPF; IPF: idiopathic pulmonary fibrosis; n.s.: no significance.

Figure 4

CTSB in progressive IPF. Data represent normalized values relative to the control, which is considered to have a value of 1. (A) Bar charts with error bars are presented as the median and 95% confidence intervals around the median. (B) ROC curve of CTSB. CTSB significantly predicted progressive IPF (AUC = 0.944, p < 0.001). (C) There was a significant correlation between CTSB and decreased FVC per year (r = 0.723, p < 0.001). NP: nonprogressive IPF; P: progressive IPF; AUC: area under the curve; FVC: forced vital capacity; ROC: receiver operating characteristic.

Figure 5

Logistic regression analysis. (A) Forest plot for univariate logistic regression analysis. As a result, age (OR = 0.94, 95% CI 0.90–0.98, p = 0.006), BMI (OR = 0.89, 95% CI 0.81–0.99, p = 0.024), baseline FVC (OR = 0.96, 95% CI 0.94–0.98, p < 0.001), and CTSB level (OR = 2.57, 95% CI 1.95–3.38, p < 0.001) were significantly associated with progressive IPF. (B) Forest plot for adjusted OR analysis. After we adjusted for baseline FVC, CTSB was significantly associated with progressive IPF (adjusted OR = 2.61, 95% CI 1.94–3.51, p < 0.001). OR: odds ratio; CI: confidence interval; FVC: forced vital capacity; GAP: gender-age-physiology.