Ann Lab Med.  2021 Mar;41(2):198-206. 10.3343/alm.2021.41.2.198.

Targeted Next-Generation Sequencing of Plasma CellFree DNA in Korean Patients with Hepatocellular Carcinoma

• Chae H
• Sung PS
• Choi H
• Kwon A
• Kang D
• Kim Y
• Kim M
• Yoon SK

• KMID: 2512666
• DOI: http://doi.org/10.3343/alm.2021.41.2.198

Abstract

Background
Hepatocellular carcinoma (HCC) is the second-most-common cause of cancer-related deaths worldwide, and an accurate and non-invasive biomarker for the early detection and monitoring of HCC is required. We assessed pathogenic variants of HCC driver genes in cell-free DNA (cfDNA) from HCC patients who had not undergone systemic therapy.
Methods
Plasma cfDNA was collected from 20 HCC patients, and deep sequencing was performed using a customized cfDNA next-generation sequencing panel, targeting the major HCC driver genes (TP53, CTNNB1, TERT) that incorporates molecular barcoding.
Results
In 13/20 (65%) patients, we identified at least one pathogenic variant of two major HCC driver genes (TP53 and CTNNB1), including 16 variants of TP53 and nine variants of CTNNB1. The TP53 and CTNNB1 variants showed low allele frequencies, with median values of 0.17% (range: 0.06%–6.99%) and 0.07% (range: 0.05%–0.96%), respectively. However, the molecular coverage of variants was sufficient, with median values of 5,543 (range: 2,317–9,088) and 7,568 (range: 2,400–9,633) for TP53 and CTNNB1 variants, respectively.
Conclusions
Our targeted DNA sequencing successfully identified low-frequency pathogenic variants in the cfDNA from HCC patients by achieving high coverage of unique molecular families. Our results support the utility of cfDNA analysis to identify somatic gene variants in HCC patients.

Keyword

Hepatocellular carcinoma; Cell-free DNA; Next-generation sequencing; Molecular barcoding; Pathogenic variants; TP53; CTNNB1; TERT

Figure

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  Fig. 1 Mean observed molecular count (VAF) compared with the designed VAF of the reference material for the six variants. Abbreviation: VAF, variant allele frequency.

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  Fig. 2 Passing–Bablok regression analysis plots for comparison of the observed and designed VAFs for each of the six variants are shown. The slope of the linear regression line for each variant ranged from 0.57 to 1.03, and, for TP53 p.R248Q and TP53 p.C242fs*5 variants, the 95% confidence interval (CI) of the slope contained 1.0. Abbreviation: VAF, variant allele frequency.

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