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Pyrosequencing Analysis for Detection of a BRAFV600E Mutation in an FNAB Specimen of Thyroid Nodules

Kim, Suk Kyeong MD, PhD*; Kim, Dong-Lim MD, PhD*; Han, Hye Seung MD, PhD; Kim, Wan Seop MD, PhD; Kim, Seung Ja MD, PhD; Moon, Won Jin MD, PhD; Oh, Seo Young MT; Hwang, Tae Sook MD, PhD

Diagnostic Molecular Pathology: June 2008 - Volume 17 - Issue 2 - pp 118-125
doi: 10.1097/PDM.0b013e31815d059d
Original Articles

Background: Fine-needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant and of guiding therapeutic intervention in thyroid nodules. However, 10% to 30% of cases with indeterminate cytology in FNAB need other diagnostic tools to refine diagnosis.

Objective: We compared the pyrosequencing method with the conventional direct DNA sequencing analysis and investigated the usefulness of preoperative BRAFV600E mutation analysis as an adjunct diagnostic tool with routine FNAB.

Methods: A total of 103 surgically confirmed patients' FNA slides were recruited and DNA was extracted after atypical cells were scraped from the slides. BRAFV600E mutation was analyzed by pyrosequencing and direct DNA sequencing.

Results: Sixty-three (77.8%) of 81 histopathologically diagnosed malignant nodules revealed positive BRAFV600E mutation on pyrosequencing analysis. In detail, 63 (84.0%) of 75 papillary thyroid carcinoma (PTC) samples showed positive BRAFV600E mutation, whereas 3 follicular thyroid carcinomas, 1 anaplastic carcinoma, 1 medullary thyroid carcinoma, and 1 metastatic lung carcinoma did not show BRAFV600E mutation. None of 22 benign nodules had BRAFV600E mutation in both pyrosequencing and direct DNA sequencing. Out of 27 thyroid nodules classified as ‘indeterminate’ on cytologic examination preoperatively, 21 (77.8%) cases turned out to be malignant: 18 PTCs (including 2 follicular variant types) and 3 follicular thyroid carcinomas. Among these, 13 (61.9%) classic PTCs had BRAFV600E mutation. None of 6 benign nodules, including 3 follicular adenomas and 3 nodular hyperplasias, had BRAFV600E mutation. Among 63 PTCs with positive BRAFV600E mutation detected by pyrosequencing analysis, 3 cases did not show BRAFV600E mutation by direct DNA sequencing. Although it was not statistically significant, pyrosequencing was superior to direct DNA sequencing in detecting the BRAFV600E mutation of thyroid nodules (P=0.25).

Conclusion: Detecting BRAFV600E mutation by pyrosequencing is more sensitive, faster, and less expensive than direct DNA sequencing and is proposed as an adjunct diagnostic tool in evaluating thyroid nodules of indeterminate cytology.

Departments of *Internal Medicine

Pathology

Radiology, Konkuk University School of Medicine, Seoul, Korea

Supported by the Second-Phase BK (Brain Korea) 21 Project in 2006.

Reprints: Tae Sook Hwang, MD, PhD, Department of Pathology, Konkuk University Hospital, Konkuk University School of Medicine, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, Korea (e-mail: tshwang@kuh.ac.kr).

© 2008 Lippincott Williams & Wilkins, Inc.