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.
Highlights
The concept of personalized and targeted therapies has become a reality of practice for medical oncologists with the administration of specific molecules according to somatic genetic alterations [1, 2]
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
To circumvent the problem of analyzing highly degraded FFPE samples, we developed a panel with an amplicon median size of 130 bp
Summary
The concept of personalized and targeted therapies has become a reality of practice for medical oncologists with the administration of specific molecules according to somatic genetic alterations [1, 2]. Guidelines recommend that exon 3 and exon 4 KRAS and NRAS mutation status should be determined [6] This change illustrates that the number (or the extent) of biomarkers that will need to be assessed in clinical daily practice in molecular pathology is rapidly increasing. This increase requires the implementation of methods probing the detection of multiple genes and mutations, including rarely encountered or unexpected genetic variations. This increase in the number of genes to be tested is associated with a decrease in the number of analyzable samples and a need to optimize technical procedures. As the number of clinically relevant genetic variants has increased, routine laboratory tests have evolved, moving from single mutations to multiplex hotspot evaluations in multiple cancer genes
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