Circulating-tumor DNA and cancer-induced gene expression as novel liquid biomarkers of liver cancers

Liver cancer is one of the most cancer-related mortality with high incidence worldwide. Hepatocellular carcinoma (HCC) is the most common liver cancer type. Currently, Liquid biopsy is known as a non-invasive biomarker tool that specifically represents tumor appearance, leading to improved patient outcomes. Therefore, the novel biomarkers of liquid biopsy in Thai HCC patients are still unexplored and needed for precision medicine. The main aims of this study were First, to explore genetic profiles from liquid biopsies of circulating cell-free DNA (cfDNA) and peripheral blood mononuclear cells (PBMCs) using next-generation sequencing (NGS) and identify novel biomarkers for HCC from these liquid biopsies. The comprehensive analysis of cfDNA from a total of 60 HCC patients of different stages and 17 chronic hepatitis (CH) patients was performed by whole-exome sequencing (WES). We found that the level of cfDNA in HCC was higher than CH and associated with tumor size and stage of HCC. The 8 highest mutated genes (ZNF814, HRNR, ZNF492, ADAMTS12, FLG, OBSCN, TP53, and TTN) were found in HCC patients (62%) of the TCGA database. Interestingly, patients with co-occurrence of HRNR and TTN mutations in cfDNA associated with short-time overall survival. Second, to identify biomarkers from transcription profiling of PBMCs in eight HCC patients and a co-cultured model were performed using RNA-sequencing (RNA-seq). The transcription profiles were cross compared with the published microarray to identify differentially expressed genes (DEGs). Eighteen upregulated and six downregulated DEGs in HCC were proposed as cancer-induced genes in PBMCs. Five up-regulated candidate genes including BHLHE40, AREG, SOCS1, CCL5, and DDIT4 were selected and further validated in PBMCs. BHLHE40 and DDIT4 displayed superior diagnostic performance than alpha-fetoprotein (AFP). BHLHE40 also emerged as an independent prognostic factor of the overall survival of HCC. Based on our findings, liquid biopsies of cfDNA and cancer-induced genes in PBMCs could serve as promising novel biomarkers for diagnosis and prognosis in HCC patients and had the potential to reflect the tumor genetic of HCC when tumor tissue unavailable.