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

doi:10.1136/jclinpath-2014-202644

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

School of Medicine, Dentistry and Biomedical Sciences

Research output: Contribution to journal › Article

26 Citations (Scopus)

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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 language	English
Pages (from-to)	111-118
Number of pages	8
Journal	Journal of Clinical Pathology
Volume	68
Issue number	2
Early online date	27 Nov 2014
DOIs	https://doi.org/10.1136/jclinpath-2014-202644
Publication status	Published - 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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Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial
Copyright the authors 2015. This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial.
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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

Kapp, Joshua R. ; Diss, Tim ; Spicer, James ; Gandy, Michael ; Schrijver, Iris ; Jennings, Lawrence J. ; Li, Marilyn M. ; Tsongalis, Gregory J. ; de Castro, D.G. ; 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.

@article{f60decd77cea4c9ca710cbfef0b0ef9b,

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

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",

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",

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

year = "2015",

month = feb,

doi = "10.1136/jclinpath-2014-202644",

language = "English",

volume = "68",

pages = "111--118",

journal = "Journal of Clinical Pathology",

issn = "0021-9746",

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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

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, D.G.; 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, 02.2015, p. 111-118.

Research output: Contribution to journal › Article

TY - JOUR

T1 - Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: 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, D.G.

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.

PY - 2015/2

Y1 - 2015/2

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

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

KW - LUNG CANCER

KW - MELANOMA

KW - MOLECULAR PATHOLOGY

KW - PCR

KW - diagnostic screening

KW - Cell Line, Tumor

KW - DNA Mutational Analysis

KW - DNA, Neoplasm

KW - Diagnostic Errors

KW - Fixatives

KW - Fluorometry

KW - Formaldehyde

KW - Great Britain

KW - Humans

KW - Laboratory Proficiency Testing

KW - Mutation

KW - Observer Variation

KW - Paraffin Embedding

KW - Polymerase Chain Reaction

KW - Predictive Value of Tests

KW - Proto-Oncogene Proteins B-raf

KW - Receptor, Epidermal Growth Factor

KW - Reproducibility of Results

KW - Spectrophotometry

KW - Tissue Fixation

KW - Transfection

KW - United States

KW - Workflow

U2 - 10.1136/jclinpath-2014-202644

DO - 10.1136/jclinpath-2014-202644

M3 - Article

VL - 68

SP - 111

EP - 118

JO - Journal of Clinical Pathology

JF - Journal of Clinical Pathology

SN - 0021-9746

IS - 2

ER -