Plasma cell-free RNA transcriptome analysis reveals transcriptional dysregulation and immune remodeling in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer due to poor early detection and frequent treatment resistance. Current tumor markers, including carbohydrate antigen 19-9 (CA19-9), show limited diagnostic and prognostic accuracy, underscoring the need for efficient biomarkers. Cell-free RNA (cfRNA), which circulates in body fluids and can be obtained through a minimally invasive approach, reflects tumor- and tissue-specific gene expression, offering strong potential as a biomarker across various cancer types. In brief, whole blood samples were collected from PDAC patients (n=63) and healthy controls (n=8), followed by plasma isolation via centrifugation prior to cfRNA extraction. Cell-free nucleic acids were isolated using a standardized circulating nucleic acid protocol, and cfRNA purity was enhanced through additional DNase treatment and a cleanup step. Complementary DNA (cDNA) libraries were generated using a low-input, strand-specific RNA sequencing method and were paired-end sequenced on a high-throughput next-generation sequencing platform.cfRNA transcriptomic analysis identified 541 differentially expressed genes (adjusted p < 0.05, |log₂FC| > 1), of which 496 were upregulated and 45 were downregulated in PDAC compared with healthy controls. Although most genes were not correlated with tumor stage, a subset displayed progressive, stage-dependent expression changes. Such gradual increases or decreases across tumor stages may reflect tumor burden and disease advancement, suggesting their potential as molecular indicators of PDAC progression. Functional enrichment analysis revealed pathways related to extracellular matrix remodeling, immune regulation, and cell proliferation. Additionally, cfRNA deconvolution analysis inferred shifts in immune cell composition. Notably, the proportion of neutrophils increased from approximately 7-9% in healthy controls to 10-15% across PDAC stages, whereas naïve CD4 T cells showed a marked decline from about 26% to 10-15%, and naïve B cells decreased from roughly 13-14% to 5-10%. In contrast, regulatory T cells exhibited a relative increase across PDAC stages. Together, these alterations reflect an immune landscape progressively skewed toward immunosuppressive features in PDAC. These findings collectively suggest cfRNA transcriptomic profiling captures tumor-derived transcriptional dysregulation and systemic immune remodeling, highlighting its promise as a biomarker source and a longitudinal monitoring tool for PDAC. Funding: This work was supported in part by the National Cancer Center, Korea (No. 2510590).