Cell-Free DNA Analysis by Whole-Exome Sequencing for Hepatocellular Carcinoma: A Pilot Study in Thailand

SIMPLE SUMMARY: Liquid biopsy for cell-free DNA (cfDNA) is a non-invasive technique to characterize the genetic profile of a tumor. Despite being a valuable tool, there is no mutational profile of cfDNA from hepatocellular carcinoma (HCC) in patients from Thailand, where HCC is prevalent. The present study aimed to demonstrate the utility of using whole-exome sequencing of cfDNA to define the somatic mutation profiles of HCC in Thai patients who underwent nonoperative therapies. The level of cfDNA was higher in HCC patients than in chronic hepatitis patients. Single nucleotide variations were present in somatic genes in cfDNA, including in ZNF814, HRNR, ZNF492, ADAMTS12, FLG, OBSCN, TP53, and TTN. The co-occurrence of HRNR and TTN mutations in cfDNA was associated with shorter overall survival. These findings indicate that the mutational profiles of cfDNA reflected those of HCC tissue, and cfDNA could serve as a useful biomarker for diagnosis and prognosis in HCC patients. ABSTRACT: Cell-free DNA (cfDNA) has been used as a non-invasive biomarker for detecting cancer-specific mutations. However, the mutational profile of cfDNA in Thai patients with hepatocellular carcinoma (HCC) has not been investigated. Here, we demonstrated the utility of using whole-exome sequencing (WES) of cfDNA to define the somatic mutation profiles of HCC in Thai patients. The comprehensive profile of cfDNA was determined with WES to identify variants in matched cfDNA and germline DNA from 30 HCC patients in Thailand who underwent nonoperative therapies. The level of cfDNA was higher in HCC patients compared with chronic hepatitis patients (p-value < 0.001). Single nucleotide variants were present in somatic genes in cfDNA, including in ZNF814 (27%), HRNR (20%), ZNF492 (20%), ADAMTS12 (17%), FLG (17%), OBSCN (17%), TP53 (17%), and TTN (17%). These same mutations were matched to HCC mutation data from The Cancer Genome Atlas (TCGA) and a previous Thai HCC study. The co-occurrence of HRNR and TTN mutations in cfDNA was associated with shorter overall survival in HCC patients (hazard ratio = 1.60, p-value = 0.0196). These findings indicate that the mutational profile of cfDNA accurately reflected that of HCC tissue and suggest that cfDNA could serve as a useful biomarker for diagnosis and prognosis in Thai HCC patients. In addition, we demonstrated the use of the pocket-sized sequencer of Oxford Nanopore Technology to detect copy-number variants in HCC tissues that could be applied for onsite clinical detection/monitoring of HCC.