Abstract Rationale While technology has improved our ability to identify subtypes of interstitial lung disease (ILD), predicting how patients will respond to treatments like steroids and antifibrotic drugs, and ultimately determining their long-term prognosis, remains a significant challenge. Analyzing RNA from affected lung tissue is a powerful tool for ILD subtyping and holds promise for predicting patient outcomes. To make this type of analysis widely available, researchers often need to use formalin-fixed, paraffin-embedded (FFPE) specimens. However, the RNA in these samples is typically degraded, making it difficult to obtain both sufficient quality and quantity for standard RNA sequencing. Our study specifically investigated whether MACE-Seq, a sequencing method optimized for degraded RNA, could effectively be used to predict ILD prognosis using these challenging samples. Methodsand Results We analyzed FFPE tissue sections from 29 ILD patients (collected between 2018-2019 at Haruhi Respiratory Hospital) who had comprehensive clinical follow-up—14 with poor prognosis and 15 with good prognosis—after excluding one case with lung cancer. Using MACE-Seq, we successfully extracted and sequenced RNA from these archived, years-old FFPE samples. The sequencing yielded a high number of reads (average of 9.8 million) and detected an average of 15,423 genes per sample. Differential expression analysis identified 30 genes whose expression levels significantly differed between the good and poor prognosis groups. Notably, 27 genes were upregulated in patients with poor prognosis. Further analysis revealed that the genes upregulated in the poor prognosis group were enriched for functions related to extracellular matrix components. Thirteen of the identified genes have been previously linked to ILD in scientific literature, while the other 17 represent novel potential biomarkers without a prior reported association with ILD prognosis. Conclusion Our findings successfully demonstrate that MACE-Seq can be utilized to obtain valuable RNA sequencing data from FFPE ILD tissue samples. This technique allowed us to identify a distinct set of 30 differentially expressed genes associated with poor prognosis in ILD patients, opening avenues for future research into reliable prognostic biomarkers. This abstract is funded by: None
Fujita et al. (Fri,) studied this question.