Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.

Keywords:
BRAF V600E mutation, Papillary thyroid carcinoma, Aspiration biopsy, Fine-needle aspiration, Thyroid cancer, Allele-specific PCR, Competitive probe blocking
1.
Demay R: The Art and Science of Cytopathology. Chicago, American Society of Clinical Pathology, 1996.
2.
Rowe LR, Bentz BG, Bentz JS: Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules. Cytojournal 2006;3:10.
3.
Rowe LR, Bentz BG, Bentz JS: Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis. J Clin Pathol 2007;60:1211–1215.
4.
Nikiforov YE: Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med 2011;135:569–577.
5.
Nikiforov YE: Molecular analysis of thyroid tumors. Mod Pathol 2011;24(suppl 2):S34–S43.
6.
Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, Zhu Z, Giannini R, Salvatore G, Fusco A, Santoro M, Fagin JA, Nikiforov YE: BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 2003;88:5399–5404.
7.
Wojciechowska K, Lewinski A: BRAF mutations in papillary thyroid carcinoma. Endocr Regul 2006;40:129–138.
8.
Xing M: Prognostic utility of BRAF mutation in papillary thyroid cancer. Mol Cell Endocrinol 2010;321:86–93.
9.
Ball DW: Selectively targeting mutant BRAF in thyroid cancer. J Clin Endocrinol Metab 2010;95:60–61.
10.
Ball DW, Jin N, Rosen DM, Dackiw A, Sidransky D, Xing M, Nelkin BD: Selective growth inhibition in BRAF mutant thyroid cancer by the mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244. J Clin Endocrinol Metab 2007;92:4712–4718.
11.
Espinosa AV, Porchia L, Ringel MD: Targeting BRAF in thyroid cancer. Br J Cancer 2007;96:16–20.
12.
Friday BB, Yu C, Dy GK, Smith PD, Wang L, Thibodeau SN, Adjei AA: BRAF V600E disrupts AZD6244-induced abrogation of negative feedback pathways between extracellular signal-regulated kinase and Raf proteins. Cancer Res 2008;68:6145–6153.
13.
Ellison G, Donald E, McWalter G, Knight L, Fletcher L, Sherwood J, Cantarini M, Orr M, Speake G: A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples. J Exp Clin Cancer Res 2010;29:132.
14.
Lee HJ, Choi J, Hwang TS, Shong YK, Hong SJ, Gong G: Detection of BRAF mutations in thyroid nodules by allele-specific PCR using a dual priming oligonucleotide system. Am J Clin Pathol 2010;133:802–808.
15.
Mancini I, Santucci C, Sestini R, Simi L, Pratesi N, Cianchi F, Valanzano R, Pinzani P, Orlando C: The use of COLD-PCR and high-resolution melting analysis improves the limit of detection of KRAS and BRAF mutations in colorectal cancer. J Mol Diagn 2010;12:705–711.
16.
Stadelmeyer E, Heitzer E, Wolf P, Dandachi N: COLD-HRM PCR versus conventional HRM PCR to detect the BRAF V600E mutation: a real improvement? J Mol Diagn 2011;13:243, author reply 244.
17.
Zhou L, Palais RA, Smith GD, Anderson D, Rowe LR, Wittwer CT: Enrichment and detection of rare alleles by means of snapback primers and rapid-cycle PCR. Clin Chem 2010;56:814–822.
18.
Zhou L, Wang Y, Wittwer CT: Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking, and melting analysis. Biotechniques 2011;50:311–318.
19.
Smith GD, Chadwick BE, Willmore-Payne C, Bentz JS: Detection of epidermal growth factor receptor gene mutations in cytology specimens from patients with non-small cell lung cancer utilising high-resolution melting amplicon analysis. J Clin Pathol 2008;61:487–493.
20.
Boulet GA, Horvath CA, Berghmans S, Moeneclaey LM, Duys IS, Arbyn M, Depuydt CE, Vereecken AJ, Sahebali S, Bogers JJ: Cervical cytology biobanking: quality of DNA from archival cervical pap-stained smears. J Clin Pathol 2008;61:637–641.
21.
Killian JK, Walker RL, Suuriniemi M, Jones L, Scurci S, Singh P, Cornelison R, Harmon S, Boisvert N, Zhu J, Wang Y, Bilke S, Davis S, Giaccone G, Smith WI Jr, Meltzer PS: Archival fine-needle aspiration cytopathology (FNAC) samples: untapped resource for clinical molecular profiling. J Mol Diagn 2010;12:739–745.
22.
Yip L, Nikiforova MN, Carty SE, Yim JH, Stang MT, Tublin MJ, Lebeau SO, Hodak SP, Ogilvie JB, Nikiforov YE: Optimizing surgical treatment of papillary thyroid carcinoma associated with BRAF mutation. Surgery 2009;146:1215–1223.
© 2011 S. Karger AG, Basel
2011
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.