title: Validation of an NGS mutation detection panel for melanoma.
abstract: Knowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.
  
  
  
  While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time. We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest.
  
  
  
  We have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status. There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR. However, these are often rare and of unknown significance for treatment.
  
  
  
  PCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup. The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK.|Centre for Research in Applied Biological and Exercise Sciences, Coventry University, Coventry, CV1 5FB, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Reiman
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK.|Centre for Research in Applied Biological and Exercise Sciences, Coventry University, Coventry, CV1 5FB, UK.
authors.forename: Hugh
authors.initials: H
authors.lastname: Kikuchi
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK.
authors.forename: Daniela
authors.initials: D
authors.lastname: Scocchia
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Smith
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK.
authors.forename: Yee Wah
authors.initials: YW
authors.lastname: Tsang
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK.
authors.forename: David
authors.initials: D
authors.lastname: Snead
authors.affiliation: Department of Pathology - Coventry and Warwickshire Pathology Services (CWPS), University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK. iancreephd@gmail.com.|Institute of Ophthalmology, University College London, Bath Street, London, EC1V 9EL, UK. iancreephd@gmail.com.|Centre for Technology Enabled Health Research (CTEHR), Faculty of Health & Life Sciences, Coventry University, Coventry, CV1 5FB, UK. iancreephd@gmail.com.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Cree
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D053263:Gene Regulatory Networks; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics*; D008875:Middle Aged; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D012878:Skin Neoplasms / Q000235:genetics*; D016707:Tissue Fixation; D014604:Uveal Neoplasms / Q000235:genetics*; D055815:Young Adult; D000098943:Uveal Melanoma
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; Melanoma; Mutation; NGS; NRAS; PCR