Accelerated variant of idiopathic pulmonary fibrosis: clinical behavior and gene expression pattern

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by the insidious onset of dyspnea or cough. However, a subset of patients has a short duration of symptoms with rapid progression to end-stage disease. In this study, we evaluated clinical and molecular features of "rapid" and "slow" progressors with IPF.

Methods and findings: 26 patients with <6 months of symptoms before first presentation [rapid progressors] and 88 patients with >24 months of symptoms [slow progressors] were studied. Survival was analyzed by the Kaplan-Meyer method and proportional hazard's model. Lung microarrays and tissue proteins were measured in a subset of patients. No differences were found in age, physiologic impairment and bronchoalveolar lavage (BAL) cellular profile. There were more males (OR = 6.5; CI:1.4-29.5; p = 0.006) and smokers (OR = 3.04; CI:1.1-8.3; p = 0.04) in the rapid progressors group. Survival from the beginning of symptoms was significantly reduced in rapid progressors (HR = 9.0; CI:4.48-18.3; p<0.0001) and there was a tendency for decreased survival from the time of diagnosis (HR = 1.5; CI:0.81-2.87; p = 0.18). We identified 437 differentially expressed genes. Lungs of rapid progressors overexpressed genes involved in morphogenesis, oxidative stress, migration/proliferation, and genes from fibroblasts/smooth muscle cells. Upregulation of two of these genes, adenosine-2B receptor and prominin-1/CD133, was validated by immunohistochemistry and were expressed by alveolar epithelial cells. BAL from rapid progressors showed a >2-fold increase of active matrix metalloproteinase-9, and induced a higher fibroblast migration compared with slow progressors and controls [238+/-98% versus 123+/-29% (p<0.05) and 30+/-17% (p<0.01)].

Conclusions/significance: A subgroup of IPF patients, predominantly smoking males, display an accelerated clinical course and have a gene expression pattern that is different from those with slower progression and longer survival. These findings highlight the variability in the progression of IPF, and may explain, in part, the difficulty in obtaining significant and reproducible results in studies of therapeutic interventions in patients with IPF.

Figure 1. Survival rate.

Kaplan-Meier plot of cumulative survival of the whole cohort (n = 167) divided into three groups by the time of the onset of symptoms (≤6 months; 7 to 23 months; and ≥24 months).

Figure 2. Distributions of oxygen saturation at rest at baseline and after 6 months follow-up.

Box-and-whisker plots show the distributions for “rapid” progressors (red) and “slow” progressors (blue). Boxes indicating the middle 50% of data points extend from the first (25%) quartile to the third (75%) quartile. The height of each box is the interquartile range (IQR). The second (50%) quartile, or median, is indicated by a line within each box. Whiskers extend out to the smallest and largest data points within 1.5 IQRs of the first and third quartiles, respectively. Observations beyond the whiskers are potential outliers, indicated here by only two dashed lines, among slow progressors at baseline.

Figure 3. Gene expression patterns that distinguish “rapid” progressors from “slow” progressors.

(A) Infogram of 437 differentially expressed genes in individual rapid and slow progressor patients. The expression levels for each gene were normalized to the geometric mean of all the samples for each gene. Increased genes are shown in progressively brighter shades of yellow, and decreased genes are shown in progressively darker shades of blue. Genes shown in gray are not different between the groups. The genes were ranked according to their significance level. (B) A log scale scatter plot of the average of intensity of all the genes on the arrays in rapid progressors (X-axis) and slow progressors (Y-axis). Colored points-437 genes that were significantly changed (p-value <0.05 in TNoM and t-test and fold ratio >2). Points are colored by their fold ratios; progressive shades of blue indicate increase and progressive shades of red indicate decrease. Points colored in gray did not reach significance. Adenosine A_2B receptor (ADORA2B) and prominin-1/CD133 (PROM1) were among the most upregulated genes in the “rapid” progressor group.

Figure 4. Localization of adenosine A_2B receptor in IPF lungs.

Immunoreactive protein was revealed with 3-amino-9-ethyl-carbazole and samples were counterstained with hematoxylin. Panels A and B show two different IPF lungs from rapid progressors exhibiting strong epithelial staining of A_2B AR (original magnification, 10 and 40×). Stained fibroblasts are also seen in panel B. Panel C: A_2B AR staining in an IPF lung from slow progressor. Panel D: Lung specimen from hypersensitivity pneumonitis displaying positive macrophage staining for A_2B AR (10×, inset 40×). Panel E: Control lung (10×). Panel F: Negative control section from IPF lung in which the primary antibody was replaced with non-immune serum (40×).

Figure 5. Immunolocalization of prominin-1/CD133 in IPF lungs.

Panels A, B, and C: Three different IPF lungs from “rapid” progressor patients showing staining in an area of bronchiolar metaplasia (A) and in reactive alveolar epithelial cells (B and C). Panels D and E: “Slow” progressor IPF lung (D) and normal control (E) showing no staining.

Figure 6. Immunoblotting of adenosine-2B receptor.

Top: Western blot analysis of BAL fluid proteins (35 µg/line) using an anti-A_2B receptor antibody. Samples were from normal individual (lane 1), “rapid” progressors (lanes 2–5) and “slow” progressors (lanes 6–9). Bottom: Quantitative densitometry of the bands shown in top panel.

Figure 7. Fibroblast migration.

Fibroblasts were placed in the upper compartment of a Boyden-type chamber, and F-12 medium containing 5% BSA alone or with 50% BAL fluid was added to the lower compartment. After 8 h of incubation, the migrating cells were stained and the absorbance of the stained solution was measured by ELISA. *p<0.01 versus controls and p<0.05 versus slow progressors.

Figure 8. Identification of gelatinolytic activities in bronchoalveolar lavage from controls, and rapid and slow progressors IPF patients.

Supernatants were resolved by SDS-PAGE gels (8.5%) containing gelatin (1 mg/ml) and a final concentration of 0.3 mg/ml heparin. Std = MMP-9 and MMP-2 zymography standards. The relative variations of gelatinase levels were analyzed by densitometry. Densitometric analysis of pro-MMP-9 and active MMP-9 is shown in the bottom. *P<0.05.