Many studies demonstrated that somatic BRAF gene mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma (PTC), even from very small samples. The gold standard for point mutations research is direct sequencing, that implies DNA extraction, PCR with specific primers, sequencing reaction and run on an automatic sequencer. This is an expensive and time consuming method, and the possible contamination with wild-type DNA not coming from the nodule significantly reduces sensitivity. Allelic discrimination is a real-time PCR application that can discriminate between two alleles differing for the insertion, substitution or deletion of a single base, due to the presence of two real time Taqman probes, each labelled with a different fluorochrome (FAM for the mutated allele and VIC for the wild-type allele).
The aim of our study is to verify whether allelic discrimination can be useful in the diagnosis of BRAF somatic mutation, starting from fine needle aspiration biopsies (FNAB). In each allelic discrimination reactions three positive controls are present, one for each possible genotype; omozygous controls are oligonucleotides containining the target sequence (wild-type or mutated), whereas the eterozygous control is a 4:1 mix of wild-type omozygous control and mutated omozygous control, respectively, in order to mimic wild-type contamination. To evaluate the method sensitivity, mutated DNA has been diluted in wild-type DNA at variable concentrations (1:2, 1:4, 1:10, 1:20, 1:100) and analyzed both by direct sequencing and allelic discrimination. Allelic discrimination was more sensitive since it detected the presence of mutated DNA in all dilutions, while direct sequencing detected the mutation until 1:20 dilution.
Five hundred FNAB have been analyzed with both methods; allelic discrimination identified 55 V600E mutated samples, while direct sequencing identified only 50 V600E mutated samples. Post surgical histological examination confirmed 54 PTC and one anaplastic carcinoma. In conclusion, allelic discrimination is more sensitive (P<0.05) and more accurate (P<0.05) than direct sequencing, and its use in diagnostic procedures is very useful, even when samples (i.e. FNAB) are contaminated with wild-type DNA.