title: Comparison Between Real-Time PCR and Pyrosequencing for Detection of BRAF V600E Mutation in Thyroid Fine-Needle Aspirates.
abstract: The BRAF V600E mutation test has proven diagnostic value in thyroid fine-needle aspiration. The real-time polymerase chain reaction (RT-PCR) has recently been introduced as a new method for BRAF mutation detection. We performed BRAF V600E detection in 126 cases of thyroid fine-needle aspiration, using RT-PCR and pyrosequencing. BRAF V600E mutation was detected in 78 (61.9%) of 126 cases by RT-PCR and in 74 (57.8%) by pyrosequencing. Of the 98 papillary thyroid carcinoma samples, the BRAF V600E mutation was identified in 72 by RT-PCR and in 70 by pyrosequencing (sensitivities of 71.6% and 71.4%, respectively). Among 28 benign nodules, 6 false-positive cases were detected by RT-PCR, whereas 4 false-positive cases were detected by pyrosequencing (specificities of 78.6% and 85.7%, respectively). When analyzing 104 cases after excluding equivocal samples, pyrosequencing had a marginally higher specificity than RT-PCR (100% vs. 78.3%, P=0.074). After modifying the cut-off criteria, the low RT-PCR specificity improved to a similar or a slightly lower specificity compared with that of pyrosequencing. In the titration assay mixing the mutant DNA with the wild-type DNA in varying proportions, RT-PCR was sensitive enough to detect the mutation in a mixture containing 0.001% mutant DNA, whereas the limit of detection was 10% for pyrosequencing. In conclusion, compared with pyrosequencing, RT-PCR was more sensitive, faster, and more convenient, but less specific, for detecting the BRAF V600E mutation. A readjustment or modification of the interpretation criteria may be necessary to reduce false-positive RT-PCR results and improve the specificity while maintaining the sensitivity.
authors.affiliation: *Department of Pathology, The Research Institute of Medical Science, Konkuk University School of Medicine †Department of Pathology, Konkuk University Medical Center, Seoul, Republic of Korea.
authors.forename: Wook Youn
authors.initials: WY
authors.lastname: Kim
authors.forename: Hyunkyung
authors.initials: H
authors.lastname: Kim
authors.forename: Tae Sook
authors.initials: TS
authors.lastname: Hwang
authors.forename: Seo Young
authors.initials: SY
authors.lastname: Oh
mesh_terms: D044963:Biopsy, Fine-Needle; D002291:Carcinoma, Papillary / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D017422:Sequence Analysis, DNA / Q000592:standards*; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics*
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf