Integrative Analysis of 5-Hydroxymethylcytosine and Transcriptional Profiling Identified 5hmC-Modified lncRNA Panel as Non-Invasive Biomarkers for Diagnosis and Prognosis of Pancreatic Cancer

Pancreatic adenocarcinoma (PAAD) is the fourth leading cause of cancer-related deaths worldwide. 5-Hydroxymethylcytosine (5hmC)-mediated epigenetic regulation has been reported to be involved in cancer pathobiology and has emerged to be promising biomarkers for cancer diagnosis and prognosis. However, 5hmC alterations at long non-coding RNA (lncRNA) genes and their clinical significance remained unknown. In this study, we performed the genome-wide investigation of lncRNA-associated plasma cfDNA 5hmC changes in PAAD by plotting 5hmC reads against lncRNA genes, and identified six PAAD-specific lncRNAs with abnormal 5hmC modifications compared with healthy individuals. Then we applied machine-learning and Cox regression approaches to develop predictive diagnostic (5hLRS) and prognostic (5hLPS) models using the 5hmC-modified lncRNAs. The 5hLRS demonstrated excellent performance in discriminating PAAD from healthy controls with an area under the curve (AUC) of 0.833 in the training cohort and 0.719 in the independent testing cohort. The 5hLPS also effectively divides PAAD patients into high-risk and low-risk groups with significantly different clinical outcomes in the training cohort (log-rank test p = 0.04) and independent testing cohort (log-rank test p = 0.0035). Functional analysis based on competitive endogenous RNA (ceRNA) and enrichment analysis suggested that these differentially regulated 5hmC modified lncRNAs were associated with angiogenesis, circulatory system process, leukocyte differentiation and metal ion homeostasis that are known important events in the development and progression of PAAD. In conclusion, our study indicated the potential clinical utility of 5hmC profiles at lncRNA loci as valuable biomarkers for non-invasive diagnosis and prognostication of cancers.