Detection of BRAF V600E Mutation in Thyroid Tissue
Detection of BRAF V600E Mutation in Thyroid Tissue
In the current study, the detection rate of the BRAF V600E mutation was 66% with PNA-clamping PCR, 70% with Anyplex real-time PCR, and 68% with pyrosequencing. The rate of BRAF V600E mutation detection with any of the 3 methods was 77%. Previous studies used thyroid tissue to evaluate the prevalence of the BRAF V600E mutation in Korean patients with PTC. They reported a detection range of 58% to 81%. These studies used direct sequencing to detect the BRAF V600E mutation, which has been used as the reference method. However, pyrosequencing was reported to be more sensitive than direct sequencing, and direct sequencing cannot detect the presence of mutant alleles when the mutant: wild-type ratio is less than 1:5.
Pyrosequencing is a rapid and sensitive method compared with direct sequencing for quantifying the BRAF V600E mutation. This method has been used to detect the BRAF V600E mutation in FNAB specimens of thyroid nodules in prior studies. The current study found the mean pyrosequencing peak to be 8.8% in patients with the BRAF V600E mutation, which is rather low. About 30% of patients with the BRAF mutation had a pyrosequencing peak below 6%, which is lower than the detection limit of direct sequencing. These pyrosequencing results allow for an evaluation of the sensitivity and accuracy of PNA-clamping PCR and real-time PCR. PNA-clamping PCR demonstrated a better κ value and accuracy than Anyplex real-time PCR. Discrepant results between PNA-clamping PCR and pyrosequencing ranged from 2.46% to 5.41% of the pyrosequencing peak, and discrepant results between Anyplex real-time PCR and pyrosequencing ranged from 1.76% to 6.41% of the pyrosequencing peak. PNA-clamping PCR demonstrated a narrow range of pyrosequencing peak in the discrepant results compared with Anyplex real-time PCR (2.46% to 5.41% vs 1.76% to 6.41%). These results imply that PNA-clamping PCR may offer a sensitive and reliable alternative method to pyrosequencing, particularly for the detection of a small amount of mutant. The use of PNA-clamping PCR to detect the BRAF V600E mutation has not been reported in prior studies.
We used pyrosequencing and PNA-clamping PCR successfully to detect DNA concentration of 99:1 (wild-type: mutant). These results suggest that pyrosequencing and PNA-clamping PCR are more sensitive than allele-specific PCR, which detected wild-type: mutant DNA at a ratio of 98:2. Allele-specific PCR does not require special equipment such as a real-time PCR machine, sequencer, or pyrosequencer; therefore, it is easier to perform than other methods. However, the reading of PCR bands can be difficult if the bands are faint, and contamination during gel loading is possible.
Previous studies that focused on Korean patients suggested that the BRAF V600E mutation is useful for predicting clinical recurrence and that the mutation is associated with aggressiveness. We investigated the prognostic effect of the BRAF V600E mutation in PTC. We found that 61% of patients in the BRAF V600E–positive group experienced extrathyroidal extension and lymph node metastasis, whereas only 39% of patients in the BRAF V600E–negative group experienced these complications. However, this difference did not reach statistical significance (P = .063). The Fisher exact test demonstrated that the BRAF V600E mutation was significantly associated with advanced stage (P = .045).
Of the 8 patients with follicular variant papillary thyroid carcinoma studied herein, 37.5% were positive for the BRAF V600E mutation. A previous study reported that the incidence of the BRAF V600E mutation in follicular variant papillary thyroid carcinoma was 31%, which is lower than in conventional PTC. The results of the current study support this previous finding.
In conclusion, the current study demonstrates that pyrosequencing and real-time PCR are equally sensitive and that PNA-clamping PCR is a sensitive and reliable method to detect the BRAF V600E mutation. The BRAF V600E mutation was found to be associated with poor prognostic factors.
Discussion
In the current study, the detection rate of the BRAF V600E mutation was 66% with PNA-clamping PCR, 70% with Anyplex real-time PCR, and 68% with pyrosequencing. The rate of BRAF V600E mutation detection with any of the 3 methods was 77%. Previous studies used thyroid tissue to evaluate the prevalence of the BRAF V600E mutation in Korean patients with PTC. They reported a detection range of 58% to 81%. These studies used direct sequencing to detect the BRAF V600E mutation, which has been used as the reference method. However, pyrosequencing was reported to be more sensitive than direct sequencing, and direct sequencing cannot detect the presence of mutant alleles when the mutant: wild-type ratio is less than 1:5.
Pyrosequencing is a rapid and sensitive method compared with direct sequencing for quantifying the BRAF V600E mutation. This method has been used to detect the BRAF V600E mutation in FNAB specimens of thyroid nodules in prior studies. The current study found the mean pyrosequencing peak to be 8.8% in patients with the BRAF V600E mutation, which is rather low. About 30% of patients with the BRAF mutation had a pyrosequencing peak below 6%, which is lower than the detection limit of direct sequencing. These pyrosequencing results allow for an evaluation of the sensitivity and accuracy of PNA-clamping PCR and real-time PCR. PNA-clamping PCR demonstrated a better κ value and accuracy than Anyplex real-time PCR. Discrepant results between PNA-clamping PCR and pyrosequencing ranged from 2.46% to 5.41% of the pyrosequencing peak, and discrepant results between Anyplex real-time PCR and pyrosequencing ranged from 1.76% to 6.41% of the pyrosequencing peak. PNA-clamping PCR demonstrated a narrow range of pyrosequencing peak in the discrepant results compared with Anyplex real-time PCR (2.46% to 5.41% vs 1.76% to 6.41%). These results imply that PNA-clamping PCR may offer a sensitive and reliable alternative method to pyrosequencing, particularly for the detection of a small amount of mutant. The use of PNA-clamping PCR to detect the BRAF V600E mutation has not been reported in prior studies.
We used pyrosequencing and PNA-clamping PCR successfully to detect DNA concentration of 99:1 (wild-type: mutant). These results suggest that pyrosequencing and PNA-clamping PCR are more sensitive than allele-specific PCR, which detected wild-type: mutant DNA at a ratio of 98:2. Allele-specific PCR does not require special equipment such as a real-time PCR machine, sequencer, or pyrosequencer; therefore, it is easier to perform than other methods. However, the reading of PCR bands can be difficult if the bands are faint, and contamination during gel loading is possible.
Previous studies that focused on Korean patients suggested that the BRAF V600E mutation is useful for predicting clinical recurrence and that the mutation is associated with aggressiveness. We investigated the prognostic effect of the BRAF V600E mutation in PTC. We found that 61% of patients in the BRAF V600E–positive group experienced extrathyroidal extension and lymph node metastasis, whereas only 39% of patients in the BRAF V600E–negative group experienced these complications. However, this difference did not reach statistical significance (P = .063). The Fisher exact test demonstrated that the BRAF V600E mutation was significantly associated with advanced stage (P = .045).
Of the 8 patients with follicular variant papillary thyroid carcinoma studied herein, 37.5% were positive for the BRAF V600E mutation. A previous study reported that the incidence of the BRAF V600E mutation in follicular variant papillary thyroid carcinoma was 31%, which is lower than in conventional PTC. The results of the current study support this previous finding.
In conclusion, the current study demonstrates that pyrosequencing and real-time PCR are equally sensitive and that PNA-clamping PCR is a sensitive and reliable method to detect the BRAF V600E mutation. The BRAF V600E mutation was found to be associated with poor prognostic factors.
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