Circulating Tumor DNA Methylation Markers for Differential Diagnosis of Hepatocellular Carcinoma

Background: An effective blood-based method for hepatocellular carcinoma (HCC) early detection has not yet been developed. Aberrant DNA methylation may offer opportunities in revolutionizing cancer screening and diagnosis. We sought to identified a non-invasive DNA methylation-based diagnostic approach using cell-free DNA for HCC early detection in high risk population. Methods: Differentially methylated DNA methylation blocks were determined by comparing methylation profiles of biopsy-proven HCC, cirrhotic and healthy liver tissues using targeted bisulfite sequencing. A multi-layer HCC screening model was subsequently constructed based on tissue-derived differentially methylated makers using plasma samples from 120 HCC patients, 92 cirrhotic patients and 290 healthy individuals. This model was independently validated in a cohort consisting of 67 HCC patients, 115 cirrhotic patients and 242 healthy individuals. The clinical parameters of misclassified samples were also investigated.  Findings: Based on methylation profiling of tissue samples, a total of 2,321 DNA methylation markers were derived, which were subsequently used to construct a cfDNA-based HCC screening model, achieving a sensitivity of 86% and specificity of 97% in the training cohort and a sensitivity of 82% and specificity of 96% in the independent validation cohort. The HCC screening model can effectively discriminate HCC patients from non-HCC patients, including patients with cirrhosis, HBV infection and healthy individuals, achieving an AUC of 0.956; whereas serum alpha-fetoprotein (AFP) achieved an AUC of 0.803. Interpretation: We have developed and validated an HCC screening model which can effectively distinguish early stage HCC patients from cirrhotic patients and symptomatic HBsAg-seropositive individuals. Funding: Key Research and Development Program of Hunan Province, Natural Science Foundation of Hunan Province, China. Declaration of Interest: None to declare. Ethical Approval: The study was approved by the ethic committee of The Second Xiangya Hospital of Central South University (KYLL2018072) and Chongqing University Cancer Hospital (2019167). All collection and usage of human sample and clinical data were in accordance with the principles of the Declaration of Helsinki. Written informed content was obtained from every participant for the use of their tissue or plasma samples.