Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA).
OBJECTIVE
The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF(V600E)), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker.


METHODS
We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC.


RESULTS
The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF(V600E) significantly correlated with absence of node metastasis.


CONCLUSIONS
BRAF(V600E) is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.
Sapio
Maria Rosaria
MR
Department of Endocrinologia ed Oncologia Molecolare e Clinica, Università Federico II, Via S. Pansini, 5 Napoli, 803131 Italy.
Posca
Daniela
D
Troncone
Giancarlo
G
Pettinato
Guido
G
Palombini
Lucio
L
Rossi
Guido
G
Fenzi
Gianfranco
G
Vitale
Mario
M
D000328:Adult; D000483:Alleles; D002291:Carcinoma, Papillary / Q000235:genetics*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA; D013964:Thyroid Neoplasms / Q000235:genetics*
D004273:DNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf