Abstract 3259: Beyond tumor size: Biological and clinical predictors of ctDNA shedding in colon cancer

Abstract Introduction: Circulating tumor DNA (ctDNA) detection in liquid biopsies has multiple applications in oncology and is becoming part of routine diagnostics. However, the inter-patient variability regarding ctDNA presence and levels in blood hampers the interpretation of ctDNA testing results. A better understanding of the underlying biology of ctDNA shedding could facilitate ctDNA testing implementation, by identifying the patients for whom the test might be informative to guide clinical decisions. This study aims to explore the clinicopathological and molecular characteristics influencing ctDNA shedding in patients with colon cancer. Methods: Pre-surgery ctDNA levels were measured in 150 stage III colon cancer patients (PLCRC-PROVENC3 cohort) using Labcorp Plasma Detect, a highly sensitive tumor-informed, whole genome sequencing (WGS)-based ctDNA test. Chromosomal instability was assessed via WGS and SNVs using a 505-gene panel (PGDx elio tissue complete). RNA exome sequencing evaluated gene expression and immune cell composition. Features were analyzed independently and integrated using Lasso regression. Results: High ctDNA levels were associated with large tumor diameter (FDR<0.001), necrosis (FDR = 0.003), angioinvasion (FDR = 0.008), MSI status (FDR = 0.032) and more advanced substages (FDR = 0.004). In the MSS subgroup, genomic analyses showed correlation with more indels (FDR=0.001) and loss of chr10p/chr10q (FDR = 0.018). Transcriptomic analyses revealed association with epithelial-mesenchymal-transition (FDR < 0.001), TNF-alpha signaling (FDR < 0.001), angiogenesis (FDR < 0.001) and higher fraction of cancer-associated fibroblasts (FDR = 0.008) in high shedders, while low shedders showed enrichment in oxidative phosphorylation (FDR < 0.001). Importantly, factors influencing ctDNA shedding independent of tumor diameter included advanced lymph node involvement (FDR = 0.049), necrosis (FDR = 0.066), enrichment in the E2F-Targets (FDR = 0.006), G2M-checkpoint (FDR = 0.005), and Wnt-β- catenin (FDR = 0.022) pathways. Low ctDNA levels were associated with upregulation of KRAS signaling (FDR=0.049) and high expression of CPS1 (FDR < 0.001) and MUC4 (FDR=0.015). Lasso regression identified large tumor diameter and necrosis as the strongest overall predictors of ctDNA levels. Conclusion: Tumor diameter is the most important feature determining ctDNA levels in blood. High-shedding tumors are larger, show advanced lymph node involvement, necrosis, and activation of proliferation and invasion pathways driving ctDNA release. In contrast, low-shedding tumors are smaller, less aggressive, and metabolically distinct, relying on mitochondrial processes like oxidative phosphorylation. Our findings improve the understanding of the biology behind ctDNA shedding in non-metastatic colon cancer and have the potential to improve patient selection for ctDNA-based clinical decisions. Citation Format: Nerma Crnovsanin, Carmen Rubio Alarcon, Vincent Pappalardo, Ingrid Franken, Steven L. Ketelaars, Renske de Wit, Soufyan Lakbir, Marcos da Silva, Geraldine R. Vink, Gerrit A. Meijer, Mark Sausen, Renee Menezes, Jeanine M. Roodhart, Sanne Abeln, Remond J. Fijneman. Beyond tumor size: Biological and clinical predictors of ctDNA shedding in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3259.