Detection of BRAFV600E Mutation in Papillary Thyroid Carcinoma by Highly Sensitive Single Strand Conformation Polymorphism Analysis
C Flynn, C Theoharis, D Chhieng, M Prasad, P Hui. Yale New Haven Hospital, New Haven, CT
Background: Papillary thyroid carcinoma carries a high prevalence of BRAFV600E activating mutation, which may serve as a diagnostic molecular marker. When in doubt, particularly with fine needle aspiration (FNA) cytological specimens, detection of the mutation essentially confirms the presence of papillary thyroid carcinoma. However, FNA specimens frequently contain fewer tumor cells, requiring highly sensitive methods of BRAFV600E mutation detection.
Design: DNA samples were extracted from formalin-fixed paraffin embedded tissue samples of 39 classical papillary thyroid carcinomas. BRAF gene was amplified by PCR using primers flanking the exon 15. PCR products were then resolved by single strand conformation polymorphism (SSCP) gel electrophoresis and by direct sequencing of PCR products. Analytic sensitivity of SSCP detection of BRAFV600E was performed by serial dilution experiments using mutation positive samples admixed with wild-type tonsil DNA.
Results: 37 of 39 classical papillary thyroid carcinomas provided amplifiable DNA, leading to informative SSCP and sequencing results. BRAFV600E was detected in 28 of 37 cases (76%) by SSCP. Direct sequencing failed to identify four BRAFV600E positive cases detectable by SSCP. Dilution studies demonstrated that SSCP had an analytical sensitivity of 5% tumor cells for a definite identification of BRAFV600E.
Conclusions: BRAFV600E was seen in 76% of papillary thyroid carcinomas in our study cohort. SSCP is superior to direct sequencing of PCR product for the detection of the BRAFV600E with a high analytic sensitivity, making it an ideal method for molecular confirmation of papillary thyroid carcinoma, particularly when a specimen contains a few tumor cells.
Category: Head & Neck
Monday, March 22, 2010 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 168, Monday Morning