Circulating DNA as prognostic biomarker in patients with advanced hepatocellular carcinoma: a translational exploratory study from the SORAMIC trial

Abstract

Background: Liquid biopsy based on cell-free DNA circulating in plasma has shown solid results as a non-invasive biomarker. In the present study we evaluated the utility of circulating free DNA (cfDNA) and the sub-type tumor DNA (ctDNA) in hepatocellular cancer (HCC) patients to assess therapy response and clinical outcome.

Methods: A cohort of 13 patients recruited in the context of the SORAMIC trial with unresectable, advanced HCC and different etiological and clinicopathological characteristics was included in this exploratory study. Plasma samples were collected between liver micro-intervention and beginning of sorafenib-based systemic therapy and then in correspondence of three additional follow-ups. DNA was isolated from plasma and next generation sequencing (NGS) was performed on a panel of 597 selected cancer-relevant genes.

Results: cfDNA levels showed a significant correlation with the presence of metastases and survival. In addition cfDNA kinetic over time revealed a trend with the clinical history of the patients, supporting its use as a biomarker to monitor therapy. NGS-based analysis on ctDNA identified 28 variants, detectable in different combinations at the different time points. Among the variants, HNF1A, BAX and CYP2B6 genes showed the highest mutation frequency and a significant association with the patients' clinicopathological characteristics, suggesting a possible role as driver genes in this specific clinical setting.

Conclusions: Taken together, the results support the prognostic value of cfDNA/ctDNA in advanced HCC patients with the potential to predict therapy response. These findings support the clinical utility of liquid biopsy in advanced HCC improving individualized therapy and possible earlier identification of treatment responders.

Keywords: Biomarkers; Circulating tumor DNA; Hepatocellular carcinoma; Liquid biopsy.

Fig. 1

SORAMIC trial design. Indicated in red are the time points chosen for discovery of the variants. SNV, single nucleotide variant; InDel, insertion and deletion; R, randomization; TX, therapy; RFA, radiofrequency ablation; ⁹⁰Y-RE, ⁹⁰Y-radioembolization

Fig. 2

Schematic description of the experimental workflow. Peripheral blood was collected and processed to separate plasma from buffy coat containing white blood cells (WBC). Whole circulating DNA was extracted and used for library preparation. Next generation sequencing was performed on Illumina platform using the 150 paired-end (PE) mode. To distinguish somatic from germline mutations, genomic DNA extracted from WBC was analyzed in parallel

Fig. 3

cfDNA concentrations at different time points and presence of metastases. The analysis shows that patients presenting metastases when recruited in the trial, had still a significant higher amount of plasmatic cfDNA in the timeframe between micro-interventional therapy and the beginning of sorafenib-based systemic therapy (T1, p = 0.012). Borderline significance (p = 0.073) was found after the beginning of systemic therapy (T2), while no significant difference was found at the two later time points. Comparison was performed using the Mann–Whitney U test (* ≤ 0.05)

Fig. 4

Survival plots for HCC patients grouped according to the amount of cfDNA at the different time points. Patients’ total population was grouped according to the corresponding cfDNA concentration with respect to the median value found at each time point. Higher cfDNA concentration at later time points (T3 and T4) was showing a trend with a shorter OS (p = 0.057 and p = 0.095) while no association (p > 0.1) was found and earlier time points (T1 and T2). High and low cfDNA levels were defined as being above or below the median values at the different time points. OS was analyzed using the Kaplan–Meier method and survival estimates in the different groups were compared using the log-rank test

Fig. 5

Comparison between cfDNA and AFP levels in three cases corresponding to patients showing different clinical characteristics. Panels a, b and c represent the described clinical cases of patients B, E and K, respectively

Fig. 6

Heatmaps showing the variants discovered in the two subgroups of patients (A–E: below 65, no liver cirrhosis, no past alcohol abuse and better response to therapies; F–M: over 65, with liver cirrhosis, past alcohol abuse and worse response to therapies) at the different time points. In (a) and in (b) SNV and InDel with relative mutation frequencies are reported, respectively

Fig. 7

Survival plots for HCC patients carrying the CP2B6 variant. Patients were grouped according to the time points (T1–T2, a; T3–T4, b). Patients carrying the variant at T1–T2, showed a worse survival with respect to those patients carrying the variant at T3–T4. OS was analyzed using the Kaplan–Meier method and survival estimates in the different groups were compared using the log-rank test