RESUMO
OBJECTIVES: To compare next-generation sequencing (NGS) platforms with mutation-specific analysis platforms in a clinical setting, in terms of sensitivity, mutation specificity, costs, capacity, and ease of use. METHODS: We analyzed 25 formalin-fixed, paraffin-embedded lung cancer samples of different size and tumor percentage for known KRAS and EGFR hotspot mutations with two dedicated genotyping platforms (cobas [Roche Diagnostics, Almere, The Netherlands] and Rotor-Gene [QIAGEN, Venlo, The Netherlands]) and two NGS platforms (454 Genome Sequencer [GS] junior [Roche Diagnostics] and Ion Torrent Personal Genome Machine [Life Technologies, Bleiswijk, The Netherlands]). RESULTS: All platforms, except the 454 GS junior, detected the mutations originally detected by Sanger sequencing and high-resolution melting prescreening and detected an additional KRAS mutation. The dedicated genotyping platforms outperformed the NGS platforms in speed and ease of use. The large sequencing capacity of the NGS platforms enabled them to deliver all mutation information for all samples at once. CONCLUSIONS: Sensitivity for detecting mutations was highly comparable among all platforms. The choice for either a dedicated genotyping platform or an NGS platform is basically a trade-off between speed and genetic information.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/métodos , Receptores ErbB/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Custos e Análise de Custo , Análise Mutacional de DNA/economia , DNA de Neoplasias/química , DNA de Neoplasias/isolamento & purificação , Educação Médica Continuada , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Neoplasias Pulmonares/diagnóstico , Mutação , Inclusão em Parafina , Proteínas Proto-Oncogênicas p21(ras) , Sensibilidade e Especificidade , Análise de Sequência de DNA , Fatores de TempoRESUMO
AIMS: In surgical pathology, formalin-fixed paraffin-embedded tissues are increasingly being used as a source of DNA and RNA for molecular assays in addition to histopathological evaluation. However, the commonly used formalin fixative is carcinogenic, and its crosslinking impairs DNA and RNA quality. METHODS: The suitability of three new presumably less toxic, crosslinking (F-Solv) and non-crosslinking (FineFIX, RCL2) alcohol-based fixatives was tested for routine molecular pathology in comparison with neutral buffered formalin (NBF) as gold standard. Size ladder PCR, epidermal growth factor receptor sequence analysis, microsatellite instability (MSI), chromogenic (CISH), fluorescence in situ hybridisation (FISH) and qPCR were performed. RESULTS: The alcohol-based non-crosslinking fixatives (FineFIX and RCL2) resulted in a higher DNA yield and quality compared with crosslinking fixatives (NBF and F-Solv). Size ladder PCR resulted in a shorter amplicon size (300 bp) for both crosslinking fixatives compared with the non-crosslinking fixatives (400 bp). All four fixatives were directly applicable for MSI and epidermal growth factor receptor sequence analysis. All fixatives except F-Solv showed clear signals in CISH and FISH. RNA yield and quality were superior after non-crosslinking fixation. qPCR resulted in lower Ct values for RCL2 and FineFIX. CONCLUSION: The alcohol-based non-crosslinking fixatives performed better than crosslinking fixatives with regard to DNA and RNA yield, quality and applicability in molecular diagnostics. Given the higher yield, less starting material may be necessary, thereby increasing the applicability of biopsies for molecular studies.
