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, David Gonzalez De Castro, Julia A. Bridge, Andrew Wallace, Joshua L. Deignan, Sandra Hing, Rachel Butler, Eldo Verghese, Gary J. Latham, Rifat A. Hamoudi

Research output: Contribution to journalArticle

25 Citations (Scopus)

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<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.

Original languageEnglish (US)
Pages (from-to)111-118
Number of pages8
JournalJournal of Clinical Pathology
Volume68
Issue number2
DOIs
StatePublished - Feb 1 2015

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Paraffin
Formaldehyde
Polymerase Chain Reaction
Mutation
DNA
Spectrophotometry
Fluorometry
Molecular Pathology
Workflow
Mutation Rate
Analysis of Variance

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection : A diagnostic RING trial. / Kapp, Joshua R.; Diss, Tim; Spicer, James; Gandy, Michael; Schrijver, Iris; Jennings, Lawrence J.; Li, Marilyn M.; Tsongalis, Gregory J.; De Castro, David Gonzalez; Bridge, Julia A.; Wallace, Andrew; Deignan, Joshua L.; Hing, Sandra; Butler, Rachel; Verghese, Eldo; Latham, Gary J.; Hamoudi, Rifat A.

In: Journal of Clinical Pathology, Vol. 68, No. 2, 01.02.2015, p. 111-118.

Research output: Contribution to journalArticle

Kapp, JR, Diss, T, Spicer, J, Gandy, M, Schrijver, I, Jennings, LJ, Li, MM, Tsongalis, GJ, De Castro, DG, Bridge, JA, Wallace, A, Deignan, JL, Hing, S, Butler, R, Verghese, E, Latham, GJ & Hamoudi, RA 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, vol. 68, no. 2, pp. 111-118. https://doi.org/10.1136/jclinpath-2014-202644
Kapp, Joshua R. ; Diss, Tim ; Spicer, James ; Gandy, Michael ; Schrijver, Iris ; Jennings, Lawrence J. ; Li, Marilyn M. ; Tsongalis, Gregory J. ; De Castro, David Gonzalez ; Bridge, Julia A. ; Wallace, Andrew ; Deignan, Joshua L. ; Hing, Sandra ; Butler, Rachel ; Verghese, Eldo ; Latham, Gary J. ; Hamoudi, Rifat A. / Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection : A diagnostic RING trial. In: Journal of Clinical Pathology. 2015 ; Vol. 68, No. 2. pp. 111-118.
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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<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.",
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T1 - Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection

T2 - A diagnostic RING trial

AU - Kapp, Joshua R.

AU - Diss, Tim

AU - Spicer, James

AU - Gandy, Michael

AU - Schrijver, Iris

AU - Jennings, Lawrence J.

AU - Li, Marilyn M.

AU - Tsongalis, Gregory J.

AU - De Castro, David Gonzalez

AU - Bridge, Julia A.

AU - Wallace, Andrew

AU - Deignan, Joshua L.

AU - Hing, Sandra

AU - Butler, Rachel

AU - Verghese, Eldo

AU - Latham, Gary J.

AU - Hamoudi, Rifat A.

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N2 - 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.

AB - 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.

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