title: High-throughput detection of clinically targetable alterations using next-generation sequencing.
abstract: Next-generation sequencing (NGS) has revolutionized the therapeutic care of patients by allowing high-throughput and parallel sequencing of large numbers of genes in a single run. However, most of available commercialized cancer panels target a large number of mutations that do not have direct therapeutic implications and that are not fully adapted to low quality formalin-fixed, paraffin-embedded (FFPE) samples. Here, we designed an amplicon-based NGS panel assay of 16 currently actionable genes according to the most recent recommendations of the French National Cancer Institute (NCI). We developed a panel of short amplicons (<150 bp) using dual-strand library preparation. The clinical validation of this panel was performed on well-characterized controls and 140 routine diagnostic samples, including highly degraded and cross-linked genomic DNA extracted from FFPE tumor samples. All mutations were detected with elevated inter-laboratory and inter-run reproducibility. Importantly, we could detect clinically actionable alterations in FFPE samples with variant allele frequencies as low as 1%. In addition, the overall molecular diagnosis rate was increased from 40.7% with conventional techniques to 59.2% with our NGS panel, including 41 novel actionable alterations normally not explored by conventional techniques. Taken together, we believe that this new actionable target panel represents a relevant, highly scalable and robust tool that is easy to implement and is fully adapted to daily clinical practice in hospital and academic laboratories.
authors.affiliation: CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Vendrell
authors.affiliation: Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France.
authors.forename: David
authors.initials: D
authors.lastname: Grand
authors.affiliation: Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rouquette
authors.affiliation: CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Costes
authors.affiliation: Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France.
authors.forename: Samira
authors.initials: S
authors.lastname: Icher
authors.affiliation: Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France.
authors.forename: Janick
authors.initials: J
authors.lastname: Selves
authors.affiliation: CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Larrieux
authors.affiliation: Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: Barbe
authors.affiliation: Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France.|Université de Montpellier, Montpellier, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Brousset
authors.affiliation: CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France.|INSERM, U1194, Montpellier, France.|Laboratoire d'excellence Labex TOUCAN, Toulouse, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Solassol
mesh_terms: D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D058990:Molecular Targeted Therapy / Q000379:methods*; D009154:Mutation / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm
keywords: NGS cancer panel; molecular diagnosis; routine practice; targeted therapies