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. 2022 May;16(10):1986-1999.
doi: 10.1002/1878-0261.13170. Epub 2022 Jan 10.

Copy number profiling of circulating free DNA predicts transarterial chemoembolization response in advanced hepatocellular carcinoma

Xiuqing Dong  1   2   3 , Geng Chen  2   3 , Xinghui Huang  3   4 , Zhenli Li  2   3 , Fang Peng  2   3 , Hengkai Chen  2   3   5 , Yang Zhou  2   3 , Lei He  2   3 , Liman Qiu  1   2   3 , Zhixiong Cai  2   3 , Jingfeng Liu  1   2   3   6 , Xiaolong Liu  1   2   3
Affiliations

Copy number profiling of circulating free DNA predicts transarterial chemoembolization response in advanced hepatocellular carcinoma

Xiuqing Dong et al. Mol Oncol. 2022 May.

Abstract

Transarterial chemoembolization (TACE) is the most commonly used treatment for advanced hepatocellular carcinoma (HCC), but still lacks accurate real-time biomarkers for monitoring its therapeutic efficacy. Here, we explored whether copy number profiling of circulating free DNA (cfDNA) could be utilized to predict responses and prognosis in HCC patients with TACE treatment. In total, 266 plasma cfDNA samples were collected from 64 HCC patients, 57 liver cirrhosis (LC) patients and 32 healthy volunteers. We performed low-depth whole-genome sequencing (LD-WGS) on cfDNA samples to conduct copy number variant (CNV) analysis and tumour fraction (TFx) quantification. Then, the correlation between TFx/CNVs and therapeutic efficacy, treatment outcomes and lipiodol deposition were explored. The change in TFx during TACE treatment was associated with patients' tumour burden, and could accurately and earlier predict treatment response and prognosis, providing an alternative strategy other than mRECIST. Meanwhile, the chromosomal 16q/NQO1 amplification indicated worse therapeutic response; in patients who underwent multiple TACE sessions, TFx change during their first TACE treatment reflected the long-term survival; additionally, the copy number amplification of chromosome 1q, 3p, 6p, 8q, 10p, 12q, 18p or 18q affected lipiodol deposition. Overall, we have provided a new liquid biopsy approach for future TACE management of HCC patients.

Keywords: circulating free DNA; copy number variants; hepatocellular carcinoma; transarterial chemoembolization; tumour fraction.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Fig. 1
Fig. 1
Overview of the study design, patient enrollment and clinical data collection. (A) The flow chart of study design. A total of 64 HCC patients receiving TACE treatment, 57 cirrhosis patients, and 32 healthy volunteers were enrolled. (B) Clinical features of enrolled 64 advanced HCC patients. The clinical and pathological data are coloured according to their categorization.
Fig. 2
Fig. 2
The correlation between TFx and patients' prognosis. (A) The distribution of TFx before TACE (pre‐TACE TFx) in each sample. (B) Pearson correlation and linear regression analysis to show the correlation between pre‐TACE TFx and matched tumour size. (C, D) Kaplan–Meier curves showing PFS(C) and OS(D) for enrolled patients stratified by pre‐TACE TFx, respectively. (E) The distribution of TFx after TACE (post‐TACE TFx) in each sample. (F) Pearson correlation and linear regression analysis to show the correlation between post‐TACE TFx and matched tumour size. (G, H) Kaplan–Meier curves showing PFS(G) and OS(H) for enrolled patients stratified by post‐TACE TFx, respectively. Statistical analysis for Kaplan–Meier plots used the log‐rank test. TFx, tumour fraction; TACE, transarterial chemoembolization; PFS, progression‐free survival; OS, overall survival.
Fig. 3
Fig. 3
Therapeutic efficacy assessment of TACE based on TFx in HCC patients. (A) Agreement between TFx variations and mRECIST criteria. The waterfall plot shows the change between pre‐TACE TFx and post‐TACE TFx. (B) TFx change in the PR/SD and PD group. The error bars indicate SEM. (C, D) Comparison of TFx with corresponding CT/MRI imaging during the TACE treatment. Red circle indicates the tumour location. Copy number amplification (red), copy number neutral (blue) and copy number deletion (green) are indicated. (E) Proportional representation of PR, SD, PD in TFx decline/stable and TFx increase groups. (F) Kaplan–Meier curves showing PFS in patients with TFx decline/stable versus TFx increase. (G) Copy number profile of the TFx decline/stable and TFx increase groups. Patterns above the x‐axes represent amplification, whereas opposite contours indicate deletion and horizontal black lines indicate loss of heterozygosity. (H, I) Kaplan–Meier curves showing PFS in patients with amplification versus nonamplification in the long arm of chromosome 16 and NQO1, respectively. (J) TFx change between pre‐TACE TFx and post‐TACE TFx in 4 patients with NQO1 amplification. Student t‐test was used to compare TFx change between 2 groups of PR/SD and PD. Chi‐squared test was used to compare proportional representation of PR, SD, PD between TFx decline/stable and TFx increase groups. Statistical analysis for Kaplan–Meier plots used the log‐rank test. TFx, tumour fraction; TACE, transarterial chemoembolization; PR, partial response; PD, progressive disease; SD, stable disease; CT, computed tomography; MRI, magnetic resonance imaging.
Fig. 4
Fig. 4
Comprehensive profiles of TFx in long‐term follow‐up patients. (A) The timeline presentation of follow‐up cfDNA samples in 24 HCC patients who received two or more TACE sessions. (B) The time‐course demonstration of TFx score (blue line), serum biomarker AFP level (orange line), tumour size (grey line) and corresponding CT/MRI imaging in patient 20. Red circle indicates the tumour location. (C, D) Kaplan–Meier curves showing PFS for long‐term follow‐up patients stratified by initial TFx change(C), secondary TFx change(D), respectively. (E) Kaplan–Meier curves showing PFS in patients with persistent TFx decline or stable versus initial and subsequent TFx increase. Statistical analysis for Kaplan–Meier plots used the log‐rank test. TFx: tumour fraction; AFP, alpha‐fetoprotein; CT, computed tomography; MRI, magnetic resonance imaging; PFS, progression‐free survival.
Fig. 5
Fig. 5
The correlation between lipiodol deposition and CNVs in cfDNA. (A) CT image showing lipiodol, left: lipiodol deposition <50%; right: lipiodol deposition ≥50%. (B) The size distribution of copy number amplification and deletion in each sample. (C) Copy number profile of lipiodol deposition <50% and lipiodol deposition ≥50% group. Patterns above the x‐axes represent amplification, whereas opposite contours indicate deletion and horizontal black lines indicate loss of heterozygosity. (D, E) The size of copy number amplification (D) and copy number deletions(E) was compared between lipiodol deposition < 50% and lipiodol deposition ≥ 50%, respectively. (F) Kaplan–Meier curves showing PFS for all enrolled patients stratified by chromosome 10p. Student's t‐test was used to compare the CNV alteration levels between the two lipiodol deposition groups. Statistical analysis for Kaplan–Meier plots used the log‐rank test. CNVs, copy number variants; CT, computed tomography.
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