title: Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.
abstract: OBJECTIVE
  Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling.
  
  
  METHODS
  TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75).
  
  
  RESULTS
  In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes.
  
  
  CONCLUSIONS
  TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.
authors.affiliation: Departments of Pathology, VU University Medical Center, PO Box 7057, 1007, MB, Amsterdam, The Netherlands.
authors.forename: Daoud
authors.initials: D
authors.lastname: Sie
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Snijders
authors.forename: Gerrit A
authors.initials: GA
authors.lastname: Meijer
authors.forename: Marije W
authors.initials: MW
authors.lastname: Doeleman
authors.forename: Marinda I H
authors.initials: MI
authors.lastname: van Moorsel
authors.forename: Hendrik F
authors.initials: HF
authors.lastname: van Essen
authors.forename: Paul P
authors.initials: PP
authors.lastname: Eijk
authors.forename: Katrien
authors.initials: K
authors.lastname: Grünberg
authors.forename: Nicole C T
authors.initials: NC
authors.lastname: van Grieken
authors.forename: Erik
authors.initials: E
authors.lastname: Thunnissen
authors.forename: Henk M
authors.initials: HM
authors.lastname: Verheul
authors.forename: Egbert F
authors.initials: EF
authors.lastname: Smit
authors.forename: Bauke
authors.initials: B
authors.lastname: Ylstra
authors.forename: Daniëlle A M
authors.initials: DA
authors.lastname: Heideman
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry; D045325:HCT116 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016612:Paraffin Embedding / Q000379:methods; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods; D018631:ras Proteins / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins