Abstract
BackgroundTargeted next generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required. Establishing reliable FFPE QC materials for NGS can be challenging and/or expensive. Here, we established a reliable and cost-effective FFPE QC material for routine utilization in the Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHP2) assay.MethodsThe performance characteristics of the CHP2 assay were determined by sequencing various cell line mixtures and 55 different FFPE tumors on the Ion Torrent PGM platform. A FFPE QC material was prepared from a mixture of cell lines derived from different cancers, comprising single nucleotide variants and small deletions on actionable genes at different allelic frequencies.ResultsThe CHP2 assay performed with high precision and sensitivity when custom variant calling pipeline parameters where established. In addition, all expected somatic variants in the QC material were consistently called at variant frequencies ranging from 9.1 % (CV = 11.1 %) to 37.9 % (CV = 2.8 %).ConclusionsThe availability of a reliable and cost-effective QC material is instrumental in assessing the performance of this or any targeted NGS assay that detects somatic variants in fixed solid tumor specimens.
Highlights
Targeted generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required
The numerous cancer genome characterization efforts that have emerged in the past years [1,2,3] have promoted the development of targeted cancer therapeutics [4], including single or combined inhibitory agents [5], which reportedly are beneficial to individuals who have tumors harboring specific somatic mutations in genes encoding for proteins involved in cell growth, proliferation, and survival signaling pathways [6,7,8,9]
Performance of the Quality Control (QC) Material We have developed and implemented a high quality and cost-effective control material for routine utilization in the Cancer Hotspot Panel v2 (CHP2) assay on FFPE samples
Summary
Targeted generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required. We established a reliable and cost-effective FFPE QC material for routine utilization in the Ion AmpliSeqTM Cancer Hotspot Panel v2 (CHP2) assay. The recent technological advances in next-generation sequencing (NGS) and the applications in the field of oncology are revolutionizing clinical testing for personalized treatment decisions for oncology patients [10]; as well as the translational research field, where somatic variant findings may enhance the development of novel targeted cancer therapeutics, which could benefit individuals with tumors harboring such mutations. The Ion AmpliSeqTM Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA), which targets 207 exonic regions across 50 cancer-relevant genes, is producing robust results starting from 1 to 10 ng of DNA isolated from formalin-fixed, paraffin Amplifying discrete or targeted regions of the genome has allowed for the development of panels of “amplicon sequencing.” As an example, the Ion AmpliSeqTM Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA), which targets 207 exonic regions across 50 cancer-relevant genes, is producing robust results starting from 1 to 10 ng of DNA isolated from formalin-fixed, paraffin
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