Detection of BRAF V600E mutation in thyroid fine-needle aspiration specimens by High Resolution Melting (HRM) analysis.
OBJECTIVE
The aim of our study was to test the feasibility of High Resolution Melting (HRM) analysis for detection of BRAF V600E mutation in various types of fine-needle aspiration (FNA) specimens from patients with papillary thyroid carcinoma (PTC).


METHODS
We analyzed fresh thyroid aspirates and smears from eight cases of PTC: three classic PTCs (CPTC), three follicular variant of PTCs (FVPTC), one tall cell, and one oncocytic variant of PTC. DNA extraction was performed using a MasterPure purification kit. The isolated DNA quantity was assessed using a NanoDrop spectrophotometer and the DNA quality was tested by PCR amplification of β-globin gene and by native DNA electrophoresis. HRM was performed on a LightCycler 480 (Roche). We amplified the 15th exon of BRAF gene, using selected primers to flank the BRAF V600E mutation point.


RESULTS
For all types of cytological specimens, the quantity of isolated DNA was adequate and allowed amplification. Similarly, the DNA quality control did not show signs of DNA degradation and the DNA was amplifiable for β-globin gene. Four cases revealed the BRAF V600E mutation: two CPTCs, one oncocytic PTC, one tall cell PTC. None of the three cases of FVPTC had this mutation.


CONCLUSIONS
HRM analysis represents a feasible and reproducible molecular technique, offering new perspectives for detecting BRAF mutation in various FNA specimens. In our study, BRAF V600E mutation revealed a strong association with specific histological variants of PTC: highly specific for CPTC, tall cell or oncocytic PTC, but negative in all cases of FVPTC.
Department of Histology, University of Medicine and Pharmacy of Targu Mures, 540000 Târgu Mureş, Romania. adelanechifor@gmail.com
Adela
A
Nechifor-Boilă
Myriam
M
Decaussin-Petrucci
Sophie
S
Gazzo
Ionela
I
Paşcanu
Angela
A
Borda
D000328:Adult; D000368:Aged; D044963:Biopsy, Fine-Needle / Q000379:methods; D002291:Carcinoma, Papillary / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D013964:Thyroid Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology
C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf