High-throughput detection of clinically targetable alterations using next-generation sequencing
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Julie A. Vendrell1,*, David Grand2,*, Isabelle Rouquette2, Valérie Costes1, Samira Icher2, Janick Selves2, Marion Larrieux1, Aurore Barbe2, Pierre Brousset2,5,** and Jérôme Solassol1,3,4,6,**
1CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France
2Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France
3IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France
4INSERM, Montpellier, France
5Laboratoire d’excellence Labex TOUCAN, Toulouse, France
6Université de Montpellier, Montpellier, France
*These authors have contributed equally to this work
**Equal senior contributors
Jérôme Solassol, email: firstname.lastname@example.org
Keywords: NGS cancer panel, molecular diagnosis, targeted therapies, routine practice
Received: July 20, 2016 Accepted: January 23, 2017 Published: March 03, 2017
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.
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