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Circulating DNA As Prognostic Biomarker in Patients With Alunni‑Fabbroni et al. J Transl Med (2019) 17:328 https://doi.org/10.1186/s12967‑019‑2079‑9 Journal of Translational Medicine RESEARCH Open Access Circulating DNA as prognostic biomarker in patients with advanced hepatocellular carcinoma: a translational exploratory study from the SORAMIC trial Marianna Alunni‑Fabbroni1* , Kerstin Rönsch2, Thomas Huber1,3, Clemens C. Cyran1, Max Seidensticker1, Julia Mayerle4, Maciej Pech5, Bristi Basu6, Chris Verslype7, Julia Benckert8, Peter Malfertheiner4 and Jens Ricke1 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 diferent 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 signifcant 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 identifed 28 variants, detectable in diferent combinations at the diferent time points. Among the variants, HNF1A, BAX and CYP2B6 genes showed the highest mutation frequency and a signifcant association with the patients’ clinicopathological characteristics, suggesting a possible role as driver genes in this specifc 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 fndings support the clinical utility of liquid biopsy in advanced HCC improving individualized therapy and possible earlier identifcation of treatment responders. Keywords: Liquid biopsy, Circulating tumor DNA, Hepatocellular carcinoma, Biomarkers Background stress [4], is recognized as the most frequent risk factor, Hepatocellular carcinoma (HCC) is the sixth most com- patients are often diagnosed with HCC in an already mon cancer and the second leading cause of cancer- advanced phase, when treatment is limited and resec- related death globally, with a median survival of only tion or transplantation are not any longer an option. Tis 7 months, if untreated [1–3]. Although cirrhosis, gener- is in part due to the limited sensitivity and specifcity of ally resulting from chronic infammation and oxidative the standard diagnostic modalities of imaging and bio- markers, such as serum alpha-fetoprotein (AFP) [5], in the detection of small tumors during surveillance of high *Correspondence: [email protected]‑muenchen.de risk populations. Advanced HCC often shows a poor out- 1 Department of Radiology, University Hospital, LMU Munich, Marchioninistrasse 15, Munich, Germany come, with very limited beneft from cytotoxic agents. Full list of author information is available at the end of the article Targeted therapy with the multi-tyrosine kinase inhibitor © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Alunni‑Fabbroni et al. J Transl Med (2019) 17:328 Page 2 of 15 sorafenib has been the mainstay of systemic treatment, the systemic therapy with sorafenib (T1), approximately nevertheless resulting in modest improvements in over- 8 weeks after beginning of systemic therapy (T2, median all survival over placebo and often only in subgroups of numbers of days between T1 and T2: 112), at frst follow patients [6]. Identifcation of early predictors of therapy up (FU) (T3, median numbers of days between T1 and response would be highly desirable in order to move T3: 224) and at second FU (T4, median numbers of days quickly patients to more efective treatments. between T1 and T4: 297). Te SORAMIC trial design is Circulating cell-free DNA (cfDNA) is detectable in the given in Fig. 1, where the time points relative to blood plasma and serum of healthy individuals as consequence collection are indicated. Te study was approved by all of cellular necrosis or apoptosis. Cancer patients show a the involved ethical boards and conducted in accordance higher amount of cfDNA since tumor cells divide faster with the Declaration of Helsinki [22]. Before entering the than normal cells, and cfDNA is released in higher pro- study, all patients gave their written informed consent. portion [7–9]. Te fraction of cfDNA derived from the tumor is indicated as circulating tumor DNA (ctDNA) DNA isolation, library preparation and next‑generation [10, 11]. In the last years, analysis of both cfDNA and sequencing (NGS) ctDNA has gained considerable attention as novel blood Peripheral blood (5 ml) was drawn in EDTA tubes (Bec- biomarkers, since quantifcation and kinetic analysis of ton–Dickinson, Heidelberg, Germany) and processed cfDNA [12, 13] and molecular profling of ctDNA [14] immediately (centrifugation 3000 rpm, 5 min, 4 °C) to have demonstrated both a predictive and prognostic collect plasma and bufy coats, which were aliquoted value. Venipuncture, with capacity for serial sampling and stored at − 80 °C until further use. Plasma was used over time, may ofer advantages over standard biopsy, for extraction of cell-free DNA (cfDNA) and bufy coats avoiding complications from incisions and refecting the were used for extraction of genomic DNA (gDNA). DNA genetic heterogeneity of the whole tumor. In addition, extraction, quality control and NGS were performed at due to the short half-life of circulating DNA [15], genetic Eurofns Genomics GmbH (Konstanz, Germany). Te analysis may ofer “real time” insights into the kinetic quality and quantity of the extracted DNA was assessed via mutations arising during therapy. Te prognostic value of electropherograms and fuorometer concentration deter- cfDNA/ctDNA in early HCC has been previously shown minations (for details, see Additional fle 1: Figure S1). Te [16, 17]. Te aim of the present study was to evaluate if extracted DNA was used for library preparation and sub- cfDNA quantifcation over time may be a valid monitor- sequent sequencing. A summary of the depth of coverage ing strategy to predict clinical outcome in advanced HCC is shown in Additional fle 2: Table S1. patients in the context of the SORAMIC trial [18, 19], Te genes were selected according to their importance and whether molecular profling of ctDNA may track the and in accordance with clinical guidelines such as the genetic evolution of the tumor during the combined sys- European Society for Medical Oncology (ESMO), the temic and liver-directed micro-interventional treatment. National Comprehensive Cancer Network (NCCN), and the College of American Pathologists (CAP). Methods NGS was performed on Illumina platform using the Patients and therapy 150 paired-end mode. Te sequencing reads were qual- A total of 13 male patients (median age (years ± SD): ity checked and mapped to the human genome using 68 ± 8.91, range 46–82) from the prospective rand- BWA with default parameters [23]. After target process- omized multicenter phase II SORAMIC trial (EudraCT ing and alignment refnement, variants (single nucleotide 2009-012576-27, NCT01126645) were included in this variants, SNV; Insertions and deletions, InDel; and gene exploratory study [18, 19]. Participants were recruited fusions according to Chimer DB 2.0 [24]) were discovered between 2011 and 2016 at diferent European study cent- and annotated. To distinguish somatic from germline ers. Advanced, unresectable HCC patients received radi- mutations, genomic DNA extracted from bufy coats oembolization with Yttrium-90 (90Y-RE) in combination was analyzed in parallel (Additional fle 3: Table S2). Te with systemic treatment with sorafenib (n = 10) or radiof- whole experimental workfow is schematically described requency ablation in combination with sorafenib (n = 2) in Fig. 2. or placebo (n = 1). Treatment efcacy was evaluated based on imaging and alfa-fetoprotein (AFP) [20, 21]. Statistical analysis Te median overall survival was 23.8 months (range 11.9 All statistical analyses were performed using IBM – 41.4) and only two patients showed recurrence with a SPSS Statistics 21.0.0 (IBM Corporation, New York, progression free survival of 22.3 and 26.8 months. Blood N.Y.). Results for numerical data are given as median samples were collected at four diferent time points: together with minimum and maximum of the sam- after micro-interventional therapy and before starting ple (i.e. range). Te correlation between tumor size and Alunni‑Fabbroni et al. J Transl Med (2019) 17:328
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