Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

Joshua R. Kapp, Tim Diss, James Spicer, Michael Gandy, Iris Schrijver, Lawrence J. Jennings, Marilyn M. Li, Gregory J. Tsongalis, D.G. de Castro, Julia A. Bridge, Andrew Wallace, Joshua L. Deignan, Sandra Hing, Rachel Butler, Eldo Verghese, Gary J Latham, Rifat A. Hamoudi

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Abstract

AIMS: 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
Original languageEnglish
Pages (from-to)111-118
Number of pages8
JournalJournal of Clinical Pathology
Volume68
Issue number2
Early online date27 Nov 2014
DOIs
Publication statusPublished - Feb 2015

Keywords

  • LUNG CANCER
  • MELANOMA
  • MOLECULAR PATHOLOGY
  • PCR
  • diagnostic screening
  • Cell Line, Tumor
  • DNA Mutational Analysis
  • DNA, Neoplasm
  • Diagnostic Errors
  • Fixatives
  • Fluorometry
  • Formaldehyde
  • Great Britain
  • Humans
  • Laboratory Proficiency Testing
  • Mutation
  • Observer Variation
  • Paraffin Embedding
  • Polymerase Chain Reaction
  • Predictive Value of Tests
  • Proto-Oncogene Proteins B-raf
  • Receptor, Epidermal Growth Factor
  • Reproducibility of Results
  • Spectrophotometry
  • Tissue Fixation
  • Transfection
  • United States
  • Workflow

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  • Cite this

    Kapp, J. R., Diss, T., Spicer, J., Gandy, M., Schrijver, I., Jennings, L. J., Li, M. M., Tsongalis, G. J., de Castro, D. G., Bridge, J. A., Wallace, A., Deignan, J. L., Hing, S., Butler, R., Verghese, E., Latham, G. J., & Hamoudi, R. A. (2015). Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial. Journal of Clinical Pathology, 68(2), 111-118. https://doi.org/10.1136/jclinpath-2014-202644