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

Oncotarget. 2017 Jun 20;8(25):40345-40358. doi: 10.18632/oncotarget.15875.

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.

Keywords: NGS cancer panel; molecular diagnosis; routine practice; targeted therapies.

MeSH terms

  • DNA, Neoplasm / genetics
  • Female
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Lung Neoplasms / drug therapy
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / pathology
  • Male
  • Melanoma / drug therapy
  • Melanoma / genetics*
  • Melanoma / pathology
  • Molecular Diagnostic Techniques / methods*
  • Molecular Targeted Therapy / methods*
  • Mutation / genetics

Substances

  • DNA, Neoplasm