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High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens.
Cheng, Lauren Y; Haydu, Lauren E; Song, Ping; Nie, Jianyi; Tetzlaff, Michael T; Kwong, Lawrence N; Gershenwald, Jeffrey E; Davies, Michael A; Zhang, David Yu.
Afiliação
  • Cheng LY; Department of Bioengineering, Rice University, 65000 Main St, Houston, TX, 77030, USA.
  • Haydu LE; Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
  • Song P; Department of Bioengineering, Rice University, 65000 Main St, Houston, TX, 77030, USA.
  • Nie J; Department of Bioengineering, Rice University, 65000 Main St, Houston, TX, 77030, USA.
  • Tetzlaff MT; Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
  • Kwong LN; Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
  • Gershenwald JE; Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
  • Davies MA; Department of Melanoma Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
  • Zhang DY; Department of Bioengineering, Rice University, 65000 Main St, Houston, TX, 77030, USA. dyz1@rice.edu.
Sci Rep ; 11(1): 9043, 2021 04 27.
Article em En | MEDLINE | ID: mdl-33907234
Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.
Assuntos

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Neoplasias Cutâneas / Análise Mutacional de DNA / Inclusão em Parafina / Análise de Sequência de DNA / Proteínas Proto-Oncogênicas B-raf / Melanoma / Mutação Tipo de estudo: Diagnostic_studies / Observational_studies Limite: Humans Idioma: En Revista: Sci Rep Ano de publicação: 2021 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Neoplasias Cutâneas / Análise Mutacional de DNA / Inclusão em Parafina / Análise de Sequência de DNA / Proteínas Proto-Oncogênicas B-raf / Melanoma / Mutação Tipo de estudo: Diagnostic_studies / Observational_studies Limite: Humans Idioma: En Revista: Sci Rep Ano de publicação: 2021 Tipo de documento: Article País de afiliação: Estados Unidos