High-throughput detection of clinically targetable alterations using next-generation sequencing

// Julie A. Vendrell 1, * , David Grand 2, * , Isabelle Rouquette 2 , Valerie Costes 1 , Samira Icher 2 , Janick Selves 2 , Marion Larrieux 1 , Aurore Barbe2 , Pierre Brousset 2, 5, ** and Jerome Solassol 1, 3, 4, 6, ** 1 CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France 2 Department of Pathology, Institut Universitaire du Cancer Toulouse Oncopole, CHU de Toulouse, Toulouse, France 3 IRCM, Institut de Recherche en Cancerologie de Montpellier, Montpellier, France 4 INSERM, Montpellier, France 5 Laboratoire d’excellence Labex TOUCAN, Toulouse, France 6 Universite de Montpellier, Montpellier, France * These authors have contributed equally to this work ** Equal senior contributors Correspondence to: Jerome Solassol, email: j-solassol@chu-montpellier.fr Keywords: NGS cancer panel, molecular diagnosis, targeted therapies, routine practice Received: July 20, 2016     Accepted: January 23, 2017     Published: March 03, 2017 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.