Analysis of BRAF V600E mutation in thyroid aspirates for establishing a pre-operative diagnosis of thyroid tumours

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Analysis of BRAF V600E mutation in thyroid aspirates for establishing a pre-operative diagnosis of thyroid tumours

 

Author: Law, Ying
Title: Analysis of BRAF V600E mutation in thyroid aspirates for establishing a pre-operative diagnosis of thyroid tumours
Degree: M.Sc.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
Thyroid gland -- Cancer.
Thyroid gland -- Diseases -- Diagnosis.
Cyclin-dependent kinases.
Protein kinases.
Cancer cells -- Motility.
Department: Dept. of Health Technology and Informatics
Pages: xiii, 71 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2305602
URI: http://theses.lib.polyu.edu.hk/handle/200/4610
Abstract: Numerous molecular markers have been studied to enhance the accuracy of fine needle aspiration in the diagnostic and prognostic evaluation of thyroid cancers. BRAF V600E mutation is the most common mutation found in papillary thyroid carcinoma (PTC). In this retrospective study, paraffin-embedded surgical samples were used to detect BRAF mutations and validate the molecular methods, because FNA smears cannot be scraped for DNA extraction as it is a destructive method, cell block of the thyroid aspirate materials have low cellularity, so only 10 cases of fine needle aspiration cytology (FNAC) cell block of PTC case were detected for BRAF V600E mutation. Altogether 85 cases including 50 PTC, 14 follicular carcinoma (FC), 1 medullary thyroid cancer (MTC), 10 follicular adenomas (FA) and 10 benign thyroid nodule cases using paraffin-embedded surgical samples were detected for BRAF mutation. The BRAF mutations found in various human cancers are mainly localized to exons 11 and 15. BRAF gene exon 15 mutation was identified by polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP) using the restriction endonuclease TspRl and Single-strand conformational polymorphism (SSCP)-Polyacrylamide gel electrophoresis (PAGE). BRAF gene exon 11 was performed by SSCP-PAGE. The sensitivity of both detection methods were 48% and 50%, respectively. The specificity of both detection methods were 100%, so the result of these two detection methods were comparable. Many studies reported that BRAF gene exon 11 has been found in other human cancers but not in thyroid cancer, so it acts as a negative control of this study. The SSCP-PAGE result, showed different banding patterns in different kinds of thyroid tumours (FC, FA and MTC), 17 cases of different kinds of thyroid tumours were selected to perform DNA sequencing showing the same DNA sequence. No BRAF mutation was found in exon 11. There were 23 PTC cases of BRAF exon 15 selected to perform DNA sequencing method. BRAF mutation was found in 17 of 23 cases of cases of PTC samples, all mutations were identical and involved a T -> A transversion at nucleotide 1799. This mutation results in a valine-to-glutamic substitution at amino acid V600E in the BRAF protein, but no mutation was detected in follicular carcinomas and adenomas, medullary thyroid carcinomas, and benign thyroid nodules. The BRAF mutation (V600E) was detected in 24 out of 50 cases (48%) of the PTC samples on histopathology; 10 PTC cases of FNA cell block materials were detected for BRAF V600E mutation, no positive result were found by PCR-RFLP and SSCP analysis of BRAF exon 15, 4 out of 10 PTC cases were showed the same BRAF mutation after DNA sequencing, so if the FNA material is suspicious for PTC but not diagnostic of PTC, detection of BRAF V600E mutation in FNAC cell block using DNA sequencing is applicable. Any positive result could confirm the diagnosis of PTC, and then total thyroidectomy can be performed, to avoid any treatment delay, repeat FNA, second operation of the thyroid. BRAF mutation is a molecular marker of PTC that could be applied to thyroid aspirates.

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