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Circulating Cell-Free Tumor DNA Analysis of 50 Genes by Next Published OnlineFirst January 12, 2016; DOI: 10.1158/1078-0432.CCR-15-2470 Personalized Medicine and Imaging Clinical Cancer Research Circulating Cell-Free Tumor DNA Analysis of 50 Genes by Next-Generation Sequencing in the Prospective MOSCATO Trial Cecile Jovelet1, Ecaterina Ileana1,2, Marie-Cecile Le Deley3,4,5, Nelly Motte1, Silvia Rosellini3, Alfredo Romero1, Celine Lefebvre6, Marion Pedrero6,Noemie Pata-Merci7, Nathalie Droin7, Marc Deloger8, Christophe Massard2, Antoine Hollebecque2, Charles Ferte9, Amelie Boichard1, Sophie Postel-Vinay2,6, Maud Ngo-Camus2, Thierry De Baere10, Philippe Vielh11, Jean-Yves Scoazec1,5,11, Gilles Vassal12, Alexander Eggermont5,9, Fabrice Andre5,6,9, Jean-Charles Soria2,5,6, and Ludovic Lacroix1,6,11,13 Abstract Purpose: Liquid biopsies based on circulating cell-free DNA 48.4%–61.6%] at the patient level. Among the 220 patients with (cfDNA) analysis are described as surrogate samples for molecular mutations in tDNA, the sensitivity of cfDNA analysis was signif- analysis. We evaluated the concordance between tumor DNA icantly linked to the number of metastatic sites, albumin level, (tDNA) and cfDNA analysis on a large cohort of patients with tumor type, and number of lines of treatment. A sensitivity advanced or metastatic solid tumor, eligible for phase I trial and prediction score could be derived from clinical parameters. Sen- with good performance status, enrolled in MOSCATO 01 trial sitivity is 83% in patients with a high score (8). In addition, we (clinical trial NCT01566019). analyzed cfDNA for 51 patients without available tissue sample. Experimental Design: Blood samples were collected at inclu- Mutations were detected for 22 patients, including 19 oncogenic sion and cfDNA was extracted from plasma for 334 patients. variants and 8 actionable mutations. Hotspot mutations were screened using next-generation sequenc- Conclusions: Detection of somatic mutations in cfDNA is ing for 50 cancer genes. feasible for prescreening phase I candidates with a satisfactory Results: Among the 283 patients with tDNA–cfDNA pairs, specificity; overall sensitivity can be improved by a sensitivity 121 had mutation in both, 99 in tumor only, 5 in cfDNA only, score allowing to select patients for whom cfDNA constitutes a and for 58 patients no mutation was detected, leading to a reliable noninvasive surrogate to screen mutations. Clin Cancer Res; 55.0% estimated sensitivity [95% confidence interval (CI), 1–9. Ó2016 AACR. 1Laboratoire de Recherche Translationnelle et Centre de Ressources- Introduction Biologiques, AMMICA, INSERM US23/CNRS UMS3655, Gustave Roussy,Villejuif, France. 2Departement d'Innovation Therapeutique et Personalized medicine in oncology, which has seen a rapid d'Essais Precoces, Gustave Roussy,Villejuif, France. 3Departement de development in the last decade due to advances in cancer genomic Biostatistiques et Epidemiologie, Gustave Roussy, Villejuif, France. 4 5 research and technology, consists of proposing a treatment tai- INSERM U1018, Gustave Roussy, Villejuif, France. Facultede fi Medecine, Universite Paris Sud, Le Kremlin-Bicetre,^ Orsay, France. lored to the individual patient's molecular pro le (1). This 6INSERM U981, Gustave Roussy, Villejuif, France. 7Plateforme de assumes that the identified genomic mutations are driver altera- Genomique Fonctionnelle, AMMICA, INSERM US23/CNRS UMS3655, tions, and that matched targeted drugs are available (2). With the 8 Gustave Roussy, Villejuif, France. Plateforme de bioinformatique, use of high-throughput molecular technologies, the feasibility of AMMICA, INSERM US23/CNRS UMS3655, Gustave Roussy, Villejuif, France. 9Departement d'Oncologie Medicale,Gustave Roussy,Villejuif, such approach has been demonstrated in several pilot trials (3, 4) France. 10Departement de Radiologie, Gustave Roussy, Villejuif, as well as in large scale studies (5). 11 France. Departement de Biologie et Pathologie Medicales, Gustave To characterize the genomic alterations, tissue from primary Roussy,Villejuif, France. 12Direction de la Recherche Clinique, Gustave Roussy,Villejuif, France. 13Faculte de Pharmacie, Universite Paris Sud, tumor or metastasis is generally used for molecular analysis. In Chatenay-Malabry,^ France. the absence of suitable tissue samples from surgical or endo- Note: Supplementary data for this article are available at Clinical Cancer scopic resections, targeted biopsy is required. However, biopsy Research Online (http://clincancerres.aacrjournals.org/). techniques present ethical and logistic challenges, due to their invasiveness, morbidity, cost, variable accessibility of the tumor C. Jovelet and E. Ileana contributed equally and are the co-first authors of this article. sites, as well as potential sampling bias due to intratumor genetic heterogeneity (6,7). In contrast, tumor-derived nucleic J.-C. Soria and L. Lacroix share senior authorship acids in plasma (also known as circulating cell-free tumor DNA Corresponding Author: Cecile Jovelet, Laboratoire de Recherche Translation- - cfDNA), only requires a blood sample, can be collected easily nelle, 114 rue Edouard Vaillant, Villejuif 94800, France. Phone: 331-4211-4364; and repeatedly, and may be a noninvasive alternative to tissue Fax: 331-4211-4364; E-mail: [email protected] biopsy (8). doi: 10.1158/1078-0432.CCR-15-2470 cfDNA is composed of small circulating DNA fragments shed Ó2016 American Association for Cancer Research. into the plasma following apoptosis or necrosis of tumor cells, or www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 12, 2016; DOI: 10.1158/1078-0432.CCR-15-2470 Jovelet et al. (ECOG ¼ 0 or 1) and a primary tumor or metastasis accessible to Translational Relevance biopsy. The study was approved by the local Institutional review Molecular screening using next-generation sequencing board and all patients provided written informed consent for (NGS) of tumor-derived nucleic acids in plasma or circulating genetic analysis of their tumor and plasma samples prior to cell-free DNA (cfDNA) only requires a blood sample, and participation in this study. CT scan or ultrasound-guided biopsies appears as an attractive noninvasive alternative to DNA anal- were performed in metastatic or primary tumor sites to carry out a ysis extracted from tissue sample (tDNA). cfDNA-based anal- comprehensive molecular characterization. The percentage of ysis might also overcome the issue of sampling bias. This tumor cells out of all nucleated cells was assessed on a 3-mm article confirms the feasibility of NGS mutations screening section after a hematoxylin and eosin staining by a senior pathol- based on cfDNA samples on a large cohort of phase I clinical ogist. Samples with at least 10% of tumor cells were considered for trial. Although cfDNA specificity was very high, sensitivity further tDNA analysis through NGS; at least 30% of tumor cells appeared limiting for using this surrogate analysis in some within the sample was required for CGH analysis. Patients with patients. We consequently developed a sensitivity prediction less than 10% of tumor cells on the tissue sample underwent only score, which allowed appropriately selecting patients for cfDNA analysis. A weekly molecular tumor board reviewed all the cfDNA analysis. The analysis of cfDNA using NGS represents NGS and CGH results and determined the most relevant targeted an attractive noninvasive option for patients in whom a tumor therapy available through early clinical trials according to the molecular profile is required. molecular profile. Treatment efficacy was evaluated by Response Evaluation Criteria in solid tumors (RECIST) version 1.1 (18). Peripheral blood samples were collected just before the tissue biopsy. through spontaneous release from cancer tissue, and possibly fi circulating tumor cells (CTC; refs. 9, 10). The detection and Extraction and quanti cation of cfDNA – clinical use of cfDNA have long remained challenging due to the Blood samples (3 10 mL) were collected in EDTA tubes (BD mixture between tumor circulating free DNA and normal circu- Vacutainer, Beckton Dickinson and Company) and centrifuged  lating free DNA, the low levels of cfDNA, and the difficulty to for 10 minutes at 1,000 g within 4 hours of the blood draw. The accurately quantify the number of DNA fragments. However, supernatant containing the plasma was further centrifuged at  recent technological advances now enable the detection, quanti- 14,000 g for 10 minutes at room temperature and stored at À fication, and analysis of cfDNA (11), thereby opening up possi- 80 C until analysis. Initially, the plasma were selected on the bilities for clinical use. Added to the minimal invasiveness of basis of their availability, and then, consecutively. m blood sampling, the attractiveness of liquid biopsy also consists in DNA was extracted from 500 L of plasma using the QIAamp avoiding cost and delay linked to the tissue biopsy. DSP virus kit (Qiagen), according to the manufacturer's instruc- m The potential applications of cfDNA are currently under exten- tions, and resuspended in 40 L of elution buffer. A real-time sive investigations, and include diagnosis, patient selection for quantitative PCR TaqManassay targeting GAPDH was used to targeted therapies, prognostic assessment, monitoring of treat- measure plasma DNA concentration. ment response and resistance, as well as screening for acquired tDNA was extracted from the fresh frozen
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