Genome-wide copy number alteration and VEGFA amplification of circulating cell-free DNA as a biomarker in advanced hepatocellular carcinoma patients treated with Sorafenib

Abstract

Background: Although sorafenib is the global standard first-line systemic treatment for unresectable hepatocellular carcinoma (HCC), it does not have reliable predictive or prognostic biomarkers. Circulating cell-free DNA (cfDNA) has shown promise as a biomarker for various cancers. We investigated the use of cfDNA to predict clinical outcomes in HCC patients treated with sorafenib.

Methods: This prospective biomarker study analyzed plasma cfDNA from 151 HCC patients who received first-line sorafenib and 14 healthy controls. The concentration and VEGFA-to-EIF2C1 ratios (the VEGFA ratio) of cfDNA were measured. Low depth whole-genome sequencing of cfDNA was used to identify genome-wide copy number alteration (CNA), and the I-score was developed to express genomic instability. The I-score was defined as the sum of absolute Z-scores of sequenced reads on each chromosome. The primary aim of this study was to develop cfDNA biomarkers predicting treatment outcomes of sorafenib, and the primary study outcome was the association between biomarkers with treatment efficacy including disease control rate (DCR), time to progression (TTP) and overall survival (OS) in these patients.

Results: The cfDNA concentrations were significantly higher in HCC patients than in healthy controls (0.71 vs. 0.34 ng/μL; P < 0.0001). Patients who did not achieve disease control with sorafenib had significantly higher cfDNA levels (0.82 vs. 0.63 ng/μL; P = 0.006) and I-scores (3405 vs. 1024; P = 0.0017) than those achieving disease control. The cfDNA-high group had significantly worse TTP (2.2 vs. 4.1 months; HR = 1.71; P = 0.002) and OS (4.1 vs. 14.8 months; HR = 3.50; P < 0.0001) than the cfDNA-low group. The I-score-high group had poorer TTP (2.2 vs. 4.1 months; HR = 2.09; P < 0.0001) and OS (4.6 vs. 14.8 months; HR = 3.35; P < 0.0001). In the multivariable analyses, the cfDNA remained an independent prognostic factor for OS (P < 0.0001), and the I-score for both TTP (P = 0.011) and OS (P = 0.010). The VEGFA ratio was not significantly associated with treatment outcomes.

Conclusion: Pretreatment cfDNA concentration and genome-wide CNA in cfDNA are potential biomarkers predicting outcomes in advanced HCC patients receiving first-line sorafenib.

Keywords: Biomarker; Circulating cell-free DNA; Genome-wide copy number alteration; Hepatocellular carcinoma; Sorafenib; Vascular endothelial growth factor-a.

Conflict of interest statement

Ethics approval and consent to participate

This prospective biomarker study was conducted under approval from the Institutional Review Board (IRB) at Asan Medical Center, Korea. All patients provided written informed consent before study enrollment.

Consent for publication

Not applicable.

Competing interests

EHC, JHJ, and JL are employees of Genome Research Center, Green Cross Genome. All remaining authors declare no actual or potential conflicts of interest.

Not applicable.

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Figures

Fig. 1

Patients flow chart for the study

Fig. 2

(a) Total cfDNA concentration and (b) VEGFA ratio in healthy controls and HCC patients. A two-tailed Mann-Whitney U test was performed to compare the median values. The horizontal line in the middle of each box indicates the median, and the top and bottom borders of the box mark the 75th and 25th percentiles, respectively. The whiskers above and below the box mark the ranges. Abbreviations: cfDNA, cell-free DNA; VEGFA, vascular endothelial growth factor-A; HCC, hepatocellular carcinoma

Fig. 3

CNA profiles for hepatocellular carcinoma cfDNA. (a) Circos plot of the distribution of CNA in the chromosomes of 151 patients. The chromosome map is located on the external periphery with the centromere in blue. The relative chromosomal deviations of individual cfDNA samples from the means of reference samples, expressed as Z-scores (red represents gain; blue represents loss) are illustrated as inner wheels. (b–c) Representative I-score profiles of three patients. Each point represents the normalized read count ratio of a 1 Mb-sized bin. Separate chromosomes from 1 to 22 are shown, and a Z-score of zero corresponds to a copy number of 2. Abbreviations: CNA, copy number alteration; cfDNA, cell-free DNA

Fig. 4

Treatment outcomes according to the cfDNA level and I-score. Comparison of (a) the cfDNA level and (b) the I-score between patients who achieved disease control and patients who did not. (c–h) Kaplan–Meier for (c) TTP and (d) OS according to high vs. low cfDNA level; and (e) TTP and (f) OS to high vs. low I-score; and (g) TTP and (h) OS according to I-score quartile. Abbreviations: cfDNA, cell-free DNA; TTP, time to progression; OS, overall survival; PD, progressive disease