title: A more sensitive platform for the detection of low-abundance BRAF(V⁶⁰⁰E) mutations.
abstract: Identifying low-abundance mutations is important for the therapy and diagnose of cancer. Since the potential for tumor heterogeneity, the efficient detection of cancer-relevant mutations largely depends on the sensitivity of the methods employed. To confirm whether the mutation detection platforms affect the perceived prevalence of the BRAF(V600E) and its correlation with clinicopathologic features in papillary thyroid carcinomas (PTC), we compared Sanger Sequencing (SS), Pyrosequencing (PS), and a newly built allele-specific real-time PCR (AS-qPCR) apparatus for the detection of BRAF(V600E) in a Chinese cohort of conventional variant PTC. Accurate plasmid standards were built to assess the limit of detection of the three platforms. In this research, AS-qPCR has been found both the most sensitive and reliable at detecting mutation. The mutations detected by AS-qPCR which were not detected by SS or PS due to low abundance were confirmed by mutation enrichment platform COLD-PCR followed by SS. When analyzed by AS-qPCR, BRAF(V600E) was associated with a more aggressive phenotype. Our results indicate that the reported prevalence of the BRAF(V600E) mutations in PTC has been underestimated and more sensitive methods such as AS-qPCR should be applied in clinical settings.
authors.affiliation: Cancer Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
authors.forename: Weiqin
authors.initials: W
authors.lastname: Jiang
authors.forename: Weibin
authors.initials: W
authors.lastname: Wang
authors.forename: Fangfang
authors.initials: F
authors.lastname: Fu
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Teng
authors.forename: Haohao
authors.initials: H
authors.lastname: Wang
authors.forename: Haiyong
authors.initials: H
authors.lastname: Wang
authors.forename: Lisong
authors.initials: L
authors.lastname: Teng
mesh_terms: D000328:Adult; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002291:Carcinoma, Papillary; D004252:DNA Mutational Analysis / Q000592:standards; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D010957:Plasmids / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012372:ROC Curve; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf