Methods
Trial design4
- Subjects with IPF and FVC ≥80% predicted were randomized 1:2 to receive nintedanib 150 mg bid or placebo for 12 weeks, followed by an open-label period during which all subjects received nintedanib for 40 weeks.
RNA sequencing
- Analyses were based on total RNA extracted from blood samples taken at baseline and week 12.
- RNA quantity and quality were measured using a NanoDrop spectrophotometer.
- Total RNA sequencing, with approximately 50 million reads per sample, was performed using the TruSeq Stranded Total RNA Kit with Ribo-Zero Globin and a HiSeq 4000 (Illumina).
Analyses
- We analyzed changes in gene expression from baseline at week 12 in the nintedanib and placebo groups:
- Data were log2 transformed prior to analysis.
- p-values were adjusted to control the false discovery rate (FDR) at 5%.
- Changes in gene expression over 12 weeks were considered significant if adjusted p≤0.05 and |log2fold change|≥0.5 (i.e., there was a ≥1.4-fold difference between baseline and week 12).
- Gene set variation analysis assessed the relative enrichment of differentially expressed genes. Enrichment scores were tested using a simple linear model and moderated t-statistics.
- Pathways analyses were performed using EnrichR. The network was generated using Ingenuity Pathway Analysis (QIAGEN, Inc).