title: Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.
abstract: OBJECTIVE
  Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation.
  
  
  METHODS
  13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation.
  
  
  RESULTS
  Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation.
  
  
  CONCLUSIONS
  In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.
authors.affiliation: Division of Surgery and Interventional Sciences, University College London, London, UK.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Kapp
authors.affiliation: University College London Advanced Diagnostics, University College London, London, UK.
authors.forename: Tim
authors.initials: T
authors.lastname: Diss
authors.affiliation: Division of Research Oncology, Guy's and St. Thomas' Hospital NHS Trust, London, UK.
authors.forename: James
authors.initials: J
authors.lastname: Spicer
authors.affiliation: University College London Advanced Diagnostics, University College London, London, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Gandy
authors.affiliation: Department of Pathology, Stanford University Medical Center, Stanford, USA.
authors.forename: Iris
authors.initials: I
authors.lastname: Schrijver
authors.affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Jennings
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA.
authors.forename: Marilyn M
authors.initials: MM
authors.lastname: Li
authors.affiliation: Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, USA.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Tsongalis
authors.affiliation: Institute of Cancer Research, The Royal Marsden Hospital NHS Trust, London, UK.
authors.forename: David Gonzalez
authors.initials: DG
authors.lastname: de Castro
authors.affiliation: Department of Pathology, University of Nebraska Medical Center, Omaha, USA.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Bridge
authors.affiliation: Regional Genetics Laboratory, Central Manchester University Hospital NHS Trust, Manchester, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wallace
authors.affiliation: Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, USA.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Deignan
authors.affiliation: Paediatric Malignancy Department, Great Ormond Street Hospital for Children NHS Trust, London, UK.
authors.forename: Sandra
authors.initials: S
authors.lastname: Hing
authors.affiliation: All Wales Genetics Laboratory, Cardiff and Vale NHS Trust, Cardiff, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Butler
authors.affiliation: Pathology and Tumour biology, University of Leeds, Leeds, UK.
authors.forename: Eldo
authors.initials: E
authors.lastname: Verghese
authors.affiliation: Asuragen, Austin, USA.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Latham
authors.affiliation: Division of Surgery and Interventional Sciences, University College London, London, UK.
authors.forename: Rifat A
authors.initials: RA
authors.lastname: Hamoudi
mesh_terms: D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods / Q000592:standards*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D003951:Diagnostic Errors / Q000517:prevention & control; D066246:ErbB Receptors / Q000235:genetics*; D005404:Fixatives / Q000592:standards*; D005470:Fluorometry / Q000592:standards; D005557:Formaldehyde / Q000592:standards*; D006801:Humans; D059021:Laboratory Proficiency Testing; D009154:Mutation; D015588:Observer Variation; D016612:Paraffin Embedding / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000592:standards*; D011237:Predictive Value of Tests; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000592:standards; D016707:Tissue Fixation / Q000379:methods / Q000592:standards*; D014162:Transfection; D006113:United Kingdom; D014481:United States; D057188:Workflow
chemical_list: D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: LUNG CANCER; MELANOMA; MOLECULAR PATHOLOGY; PCR; diagnostic screening