Tracing the Tissue of Origin in Cancers of Unknown Primary Using Cell-Free DNA (cfDNA)

Abstract: Cancers of unknown primary (CUP) are metastatic cancers in which the origin cannot be identified despite thorough diagnostic evaluations. This lack of knowledge delays the institution of specific treatment protocols and is linked with unfavorable clinical outcomes. One of the solutions to this problem lies in the application of liquid biopsy, which is a minimally invasive process that analyzes circulating tumor DNA fragments present in the blood (Hum & Lee, 2025). The principal purpose of liquid biopsy is to identify the origin of cancer by recognizing specific DNA methylation signatures. The systematic review provides a summary of 56 research articles that have used DNA methylation profiling of cell-free DNA (cfDNA) for identifying the tissue of origin of cancers classified as tumors of unknown primary. Across these studies, researchers employed targeted methylation panels, whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and Illumina EPIC arrays. The researchers applied logistic regression, support vector machines (SVM), random forests, and deep learning, trained on cancers with known primaries and tested on CUP samples (Jia et al., 2021). Most of the classifiers performed well, with areas under the curves (AUC) measures exceeding 0.90 (Li et al., 2025). The CUPiD study reported an AUC of 0.99 and predicted the probable tumor origin in almost 80% of patients with CUP (Stanley et al., 2024). Collectively, the results indicate that cfDNA methylation profiling is a potentially useful and less invasive strategy to enhance the precision of diagnosis and treatment planning for patients with CUP (Liu, 2022).