Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study
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Jessica Garcia1,2,3,4,**, Eric Dusserre1,3,4,**, Valérie Cheynet3,5, Pierre Paul Bringuier6, Karen Brengle-Pesce3,5, Anne-Sophie Wozny1,7, Claire Rodriguez-Lafrasse1,3,4,7, Gilles Freyer4,8,9, Marie Brevet4,6,9, Léa Payen1,2,3,4,7,* and Sébastien Couraud4,9,10,*
1Laboratoire de Biochimie et Biologie Moléculaire, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, 69310, Pierre Bénite, France
2Centre de Recherche en Cancérologie de Lyon, INSERM 1052, CNRS 5286, Université Claude Bernard Lyon 1, Lyon, 69003, France
3Laboratoire Commun de Recherche Hospices Civils de Lyon – BioMérieux, Centre Hospitalier Lyon Sud, 69310, Pierre Bénite, France
4Institut de Cancérologie des Hospices Civils de Lyon, CIRculating CANcer Program (CIRCAN), 69002 Lyon, France
5Medical Diagnostic Discovery Department, BioMérieux, 69290 Craponne, France
6Service d’Anatomie et de Cytologie Pathologiques, Groupement Hospitalier Est, Hospices Civils de Lyon, 69500, Bron, France
7Faculté de Pharmacie de Lyon (IPSB), Université de Lyon1, Lyon, 69008, France
8Service d’Oncologie Médicale, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, 69003, France
9EMR 3738 Ciblage Thérapeutique en Oncologie, Faculté de Médecine Lyon Sud, Université Lyon 1, 69600, Oullins, France
10Service de Pneumologie Aigue Spécialisée et Cancérologie Thoracique, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, 69003, France
*Authors share co-senior authorship
**Authors share co-first authorshop
Léa Payen, email: [email protected]
Keywords: lung cancer, EGFR mutation, circulating-free DNA, liquid biopsy, digital PCR
Received: December 22, 2016 Accepted: June 26, 2017 Published: September 21, 2017
Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20.
Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.
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