Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.
J Clin Pathol
; 68(2): 111-8, 2015 Feb.
Article
em En
| MEDLINE
| ID: mdl-25430497
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.Palavras-chave
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Análise Mutacional de DNA
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Reação em Cadeia da Polimerase
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Fixação de Tecidos
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Inclusão em Parafina
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Proteínas Proto-Oncogênicas B-raf
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Ensaio de Proficiência Laboratorial
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Fixadores
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Receptores ErbB
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Formaldeído
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Mutação
Tipo de estudo:
Clinical_trials
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Diagnostic_studies
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Guideline
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Prognostic_studies
Limite:
Humans
País como assunto:
America do norte
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Europa
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article