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Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial
  1. Joshua R Kapp1,
  2. Tim Diss2,
  3. James Spicer3,
  4. Michael Gandy2,
  5. Iris Schrijver4,
  6. Lawrence J Jennings5,
  7. Marilyn M Li6,
  8. Gregory J Tsongalis7,
  9. David Gonzalez de Castro8,
  10. Julia A Bridge9,
  11. Andrew Wallace10,
  12. Joshua L Deignan11,
  13. Sandra Hing12,
  14. Rachel Butler13,
  15. Eldo Verghese14,
  16. Gary J Latham15,
  17. Rifat A Hamoudi1
  1. 1Division of Surgery and Interventional Sciences, University College London, London, UK
  2. 2University College London Advanced Diagnostics, University College London, London, UK
  3. 3Division of Research Oncology, Guy's and St. Thomas’ Hospital NHS Trust, London, UK
  4. 4Department of Pathology, Stanford University Medical Center, Stanford, USA
  5. 5Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, USA
  6. 6Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA
  7. 7Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, USA
  8. 8Institute of Cancer Research, The Royal Marsden Hospital NHS Trust, London, UK
  9. 9Department of Pathology, University of Nebraska Medical Center, Omaha, USA
  10. 10Regional Genetics Laboratory, Central Manchester University Hospital NHS Trust, Manchester, UK
  11. 11Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, USA
  12. 12Paediatric Malignancy Department, Great Ormond Street Hospital for Children NHS Trust, London, UK
  13. 13All Wales Genetics Laboratory, Cardiff and Vale NHS Trust, Cardiff, UK
  14. 14Pathology and Tumour biology, University of Leeds, Leeds, UK
  15. 15Asuragen, Austin, USA
  1. Correspondence to Dr Rifat A Hamoudi, Division of Surgery and Interventional Science. University College London, 67-73 Riding House Street, London, W1W 7EJ, UK; r.hamoudi{at}


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<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.

  • PCR
  • diagnostic screening

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