title: Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.
abstract: BACKGROUND
  Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.
  
  
  METHODS
  We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.
  
  
  RESULTS
  Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.
  
  
  CONCLUSIONS
  Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.
authors.affiliation: Department of Pathology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Wendy W J
authors.initials: WW
authors.lastname: de Leng
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Oncology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Christa G
authors.initials: CG
authors.lastname: Gadellaa-van Hooijdonk
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Genetics, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Françoise A S
authors.initials: FA
authors.lastname: Barendregt-Smouter
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Genetics, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Marco J
authors.initials: MJ
authors.lastname: Koudijs
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Genetics, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Ies
authors.initials: I
authors.lastname: Nijman
authors.affiliation: Department of Pathology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: John W J
authors.initials: JW
authors.lastname: Hinrichs
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Genetics, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Genetics, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Stef
authors.initials: S
authors.lastname: van Lieshout
authors.affiliation: Medical Sciences, Amgen Inc., Thousand Oaks, CA, 91320-1799, United States of America.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Loberg
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, 3075 EA Rotterdam, The Netherlands.
authors.forename: Maja
authors.initials: M
authors.lastname: de Jonge
authors.affiliation: Department of Medical Oncology, Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.
authors.forename: Emile E
authors.initials: EE
authors.lastname: Voest
authors.affiliation: Department of Pathology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Roel A
authors.initials: RA
authors.lastname: de Weger
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Oncology, Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.
authors.forename: Neeltje
authors.initials: N
authors.lastname: Steeghs
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Oncology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
authors.forename: Marlies H G
authors.initials: MH
authors.lastname: Langenberg
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, 3075 EA Rotterdam, The Netherlands.
authors.forename: Stefan
authors.initials: S
authors.lastname: Sleijfer
authors.affiliation: Department of Pathology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.|Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Willems
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Utrecht, The Netherlands.|Department of Medical Oncology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.|Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, 3075 EA Rotterdam, The Netherlands.
authors.forename: Martijn P
authors.initials: MP
authors.lastname: Lolkema
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D015203:Reproducibility of Results; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde