Abstract A018: Epigenetic silencing of <i>TMEM240</i> drives metastatic progression in advanced pancreatic cancer: A promising cfDNA biomarker for monitoring intra-abdominal metastatic spread and clinical progression

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies, with a five-year survival rate below 3% in stage IV disease. Liver and peritoneal metastases are the most common sites of distant spread, occurring in approximately 74% and 48% of cases, respectively. Given the high burden of intra-abdominal metastasis, there is an urgent need for non-invasive biomarkers to detect and monitor metastatic progression in PDAC. This study investigated the role of TMEM240, a gene previously implicated in breast and colon cancer metastasis, and evaluated its potential as a circulating DNA methylation biomarker for liver and/or peritoneal metastases in PDAC. Methods and Results Genome-wide methylation analysis identified TMEM240 as frequently silenced by promoter hypermethylation in PDAC. Methylation-specific PCR confirmed promoter hypermethylation in 76.9% (10/13) of tumor tissues, consistent with TCGA data (70%, 7/10). Circulating cell-free DNA (cfDNA) from plasma showed elevated TMEM240 methylation in all stage IV patients with liver and/or peritoneal metastases, but not in those with lung-only metastasis, indicating strong association with intra-abdominal spread. Longitudinal blood sampling every 3 months further demonstrated that cfDNA methylation levels correlated with disease progression. Transcriptomic data (GEPIA and Human Protein Atlas) revealed significant TMEM240 downregulation in PDAC and association with poor overall survival (P = 0.0099 and 0.0012, respectively). Functional restoration of TMEM240 in PDAC cell lines (AsPC-1, PANC-1, MIA PaCa-2) reduced cell viability by 14.7–31% (p &amp;lt; 0.01), and significantly suppressed migration and invasion. Immunofluorescence analysis showed decreased F-actin polymerization and cytoskeletal remodeling. TMEM240 localized to both the plasma membrane and cytoplasm. RNA sequencing revealed that TMEM240 restoration led to the upregulation of genes related to extracellular matrix organization, adhesion, and ion transport, while repressing genes involved in EMT and metastasis, including pathways associated with NF-κB, TGF-β/SMAD, and integrin–FAK signaling—indicating reversal of the metastatic phenotype. Conclusion TMEM240 is epigenetically silenced in PDAC and plays a functional role in limiting metastatic traits such as cell motility, viability, and invasion. Its promoter methylation in cfDNA is strongly associated with liver and/or peritoneal metastases, supporting its potential as a clinically relevant, non-invasive biomarker for monitoring intra-abdominal metastatic progression in advanced pancreatic cancer. Citation Format: Ruo-Kai Lin, Yao-Yu Hsieh. Epigenetic silencing of TMEM240 drives metastatic progression in advanced pancreatic cancer: A promising cfDNA biomarker for monitoring intra-abdominal metastatic spread and clinical progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A018.