title: Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.
abstract: OBJECTIVE
  The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology.
  
  
  METHODS
  We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls.
  
  
  RESULTS
  Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA.
  
  
  CONCLUSIONS
  AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.
authors.affiliation: Departments of Oncology Biomarker Development, Biostatistics, and Pathology, Genentech Inc., South San Francisco, California, USA; Technology Development, Asuragen Inc., Austin, Texas, USA.
authors.forename: Liangxuan
authors.initials: L
authors.lastname: Zhang
authors.forename: Liangjing
authors.initials: L
authors.lastname: Chen
authors.forename: Sachin
authors.initials: S
authors.lastname: Sah
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Latham
authors.forename: Rajesh
authors.initials: R
authors.lastname: Patel
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Song
authors.forename: Hartmut
authors.initials: H
authors.lastname: Koeppen
authors.forename: Rachel
authors.initials: R
authors.lastname: Tam
authors.forename: Erica
authors.initials: E
authors.lastname: Schleifman
authors.forename: Haider
authors.initials: H
authors.lastname: Mashhedi
authors.forename: Sreedevi
authors.initials: S
authors.lastname: Chalasani
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.forename: Teiko
authors.initials: T
authors.lastname: Sumiyoshi
authors.forename: Rajiv
authors.initials: R
authors.lastname: Raja
authors.forename: William
authors.initials: W
authors.lastname: Forrest
authors.forename: Garret M
authors.initials: GM
authors.lastname: Hampton
authors.forename: Mark R
authors.initials: MR
authors.lastname: Lackner
authors.forename: Priti
authors.initials: P
authors.lastname: Hegde
authors.forename: Shidong
authors.initials: S
authors.lastname: Jia
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis*; D005557:Formaldehyde; D052138:Genes, Neoplasm / Q000235:genetics*; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation / Q000235:genetics; D010232:Paraffin; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D017422:Sequence Analysis, DNA; D016610:Tissue Embedding; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde; D010232:Paraffin; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human
keywords: AmpliSeq; Ion Torrent; Molecular diagnostics; Mutation; Next-generation sequencing; Precision oncology