Clinical implication of the BRAFV600E mutation in papillary thyroid carcinoma

Background The BRAFV600E mutation is the most common genetic alteration in papillary thyroid carcinoma (PTC). In recent studies, the BRAFV600E mutation has been associated with poor clinicopathological characteristics, such as lymph node metastasis, extrathyroidal extension, and advanced stage. However, other studies have failed to establish an association between the BRAFV600E mutation and clinicopathological features. Therefore, we investigated the relationship between the BRAFV600E mutation and its clinicopathological factors at a single institution. Methods A total of 327 consecutive patients with PTC were enrolled in this study and underwent thyroid surgery at Yeouido St. Mary’s Hospital between February 2010 and December 2011. BRAFV600E mutation analysis was performed using polymerase chain reaction (PCR)-based amplification of DNA extracted from paraffin-embedded tumour specimens. Results The BRAFV600E mutation was detected in the tumours of 241 (73.7%) patients. Lymph node metastasis, TNM stage, and multifocality were not significantly associated with the BRAFV600E mutation. However, larger tumour size, extrathyroidal extension, histologic type (classic type), and concurrent Hashimoto’s thyroiditis were associated with the BRAFV600E mutation in the univariate analysis, although no clinicopathological features were associated with the BRAFV600E mutation in the multivariate analysis. Conclusion There was no idependent prognostic factor associated with BRAFV600E mutation status in this study. The BRAFV600E mutation is unlikely to serve as a prognostic factor for PTC.


Background
Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, accounting for 85% to 90% of all thyroid malignancies [1,2]. PTC is generally treated successfully with surgery, radioiodine ablation, and levothyroxine suppression therapy. This malignancy typically has a favourable prognosis, with an average 10-year survival rate of over 90%, although up to 35% of patients suffer from disease recurrence during long-term follow up [3].
Several genetic events have been associated with PTC, and the BRAF mutation is the most common genetic alteration detected in patients with PTC [4]. The RAF protein has three isoforms, with BRAF being the most common isoform found in thyroid follicular cells and the strongest activator of mitogen-activated protein kinase signalling [5]. The most common BRAF mutation in thyroid cancer, occurring in more than 95% of cases, is the T1799A transversion mutation in exon 15. The thymidine to adenine transversion at position 1,799 results in a valine to glutamic acid substitution at residue 600 (V600E) [4,6] and occurs at a prevalence ranging from 29% to 83% [7,8].
The BRAF V600E mutation in PTC has been investigated at many institutions, but the relationship between the BRAF V600E mutation and the clinicopathological features of PTC remain controversial. Several studies have reported that the BRAF V600E mutation is associated with a poor prognosis when combined with the aggressive clinicopathological features of PTC [8,9], whereas other studies have failed to demonstrate a statistically significant association between the BRAF V600E mutation and aggressive clinicopathological features [7,10]. Therefore, the purpose of this study was to evaluate the prevalence of the BRAF V600E mutation in PTC and to analyse the association between the BRAF V600E mutation and the clinicopathological features of PTC at a single institution.

Patients
A total of 327 consecutive patients with PTC were enrolled in this study and underwent thyroid surgery at Yeouido St Mary's Hospital of the Catholic University in Seoul, Korea, between February 2010 and December 2011. We routinely performed central lymph node dissections in all patients and selectively performed lateral neck lymph node dissections if the preoperative imaging studies (ultrasound and computed tomography) raised suspicions of malignancy and if the fine needle aspiration cytology (FNAC) were found to contain atypical cells or metastatic papillary carcinomas. The patients diagnosed with a recurrence of PTC and those who refused to be enrolled were excluded. Written informed consent was obtained from all patients in advance. This study was reviewed and approved by the institutional review board at Yeouido St Mary's Hospital (SC13TISI0007).

DNA extraction
DNA was extracted from five paraffin sections with a thickness of 10 μm that contained a representative portion of the tumour tissue obtained after thyroidectomy. DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue was conducted using the QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

Direct sequencing
The DNA templates were processed for the DNA sequencing reaction using the ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster, CA, USA) with both forward and reverse sequence-specific primers. Twenty nanograms of purified PCR products were used in a 10-μl sequencing reaction solution containing 1 μl of BigDye Terminator v3.1 and 0.1 μM of the same PCR primer. Sequencing reactions were performed using 25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 minutes at 60°C. The sequence data were generated with the ABI PRISM 3730 DNA Analyzer (Applied Biosystems). The sequences were analysed and compared using Sequencing Analysis 5.1.1. software (Applied Biosystems).

Statistical analysis
Data analyses were performed with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). The chi-square test and Fisher's exact test were used for categorical variables, and Student's t-test for independent samples was used for continuous variables. Continuous variables were presented as the mean ± standard deviation. A multivariate analysis was performed using logistic regression analysis. Differences were considered statistically significant when P was <0.05.

Results
The clinicopathological characteristics of the 327 patients, 268 female (82%) and 59 male (18%), with a mean age of 47.79 ± 11.03 years at the time of surgery, are summarised in Table 1.
Total thyroidectomy was performed in 270 patients (82.6%). Other thyroidectomies, including hemithyroidectomies and completion thyroidectomies, were performed in 57 (17.4%) patients. Of the 327 patients with PTC, 56 (17.1%) demonstrated pathological features of Hashimoto's thyroiditis. In total, 137 patients (41.9%) had stage I disease, 40 (12.2%) had stage II disease, 121 (37.0%) had stage III disease, and 18 (8.9%) had stage IVA disease. The BRAF V600E mutation was detected in the tumours of 241 patients (73.7%). Table 2 shows the association between BRAF V600E mutation status and the clinicopathological characteristics of the patients. Lymph node metastasis, TNM stage, and multifocality were not significantly associated with the BRAF V600E mutation. However, larger tumour size, extrathyroidal extensions, and histologic type (classic type) concurrent with Hashimoto's thyroiditis were associated with the BRAF V600E mutation.
The results of the multivariate analysis are shown in Table 3. The BRAF V600E mutation was not significantly associated with tumour size, extrathyroidal extension, or the histologic type concurrent with Hashimoto's thyroiditis.
Of the 327 patients with PTC, 247 (75.5%) received postoperative radioiodine-131 treatment. There was no I-131 avidity in the foci of lateral lymph node metastases.

Discussion
In 2003, Kimura et al. initially reported the BRAF V600E mutation in thyroid carcinomas. Since this discovery, additional research has been conducted to understand the tumorigenic role and clinical importance of this mutation [11][12][13]. The BRAF V600E mutation has been reported in PTC with a frequency ranging from 29% to 83% in various studies [14], and recent studies have reported differences in the incidence of the BRAF V600E mutation according to region; for example, the incidence of the BRAF V600E mutation was shown to be higher in Korea (range 52% to 87%) in comparison to other countries (range 36% to 65%) [7,8]. The reason for this variation in frequency remains unclear but may be due to geographic factors. For example, some studies have reported higher rates of the BRAF V600E mutation in high iodine intake areas, which may indicate the significance  of iodine as a risk factor for the BRAF V600E mutation in PTC [8,15]. Iodine intake is very high in Korea as compared to Western countries, which may explain the high prevalence of the BRAF V600E mutation among Koreans with PTC. The prevalence of the BRAF V600E mutation in PTC patients in our study was 73.7% (241/327), and further studies will be needed to clarify differences in the prevalence of the BRAF V600E mutation between geographic regions. Many studies have reported the relationship between the BRAF V600E mutation and the clinicopathological characteristics of PTC. However, recent clinical studies have reported controversial results regarding whether the BRAF V600E mutation serves as a prognostic or predictive marker of PTC. Xing et al. [16] first reported that the BRAF V600E mutation was associated with a poorer clinicopathological outcome and independently predicted recurrence. These authors therefore concluded that the BRAF V600E mutation may be a useful marker for risk stratification in PTC. Many studies have found that extrathyroidal extensions, lymph node metastases, and advanced stage are the three most common risk factors consistently associated with the BRAF V600E mutation [17][18][19]. In addition, several studies have reported significant associations between the BRAF V600E mutation and clinicopathological characteristics, such as older age, male sex, tumour size, and an aggressive subtype [17][18][19][20][21].
Although all of the above-mentioned studies were conducted in Western countries, some results from studies conducted in Eastern countries have not been consistent, especially those performed in the Korean population. Ito et al. [22] investigated the BRAF V600E mutation in 631 patients with PTC and found that the BRAF V600E mutation was not significantly associated with high-risk biological features, such as lymph node metastasis, extrathyroidal extension, advanced age, distant metastasis at surgery, and advanced stage. Furthermore, the disease-free survival of patients with the BRAF V600E mutation did not differ from that of patients without the BRAF V600E mutation. In addition, other study groups have reported that age distribution, tumour size, extrathyroid extension, multifocality, and staging do not differ significantly between patients with and without the BRAF V600E mutation [7]. Our study found that large tumour size (odds ratio (OR) 2.131, 95% CI 1.266, 3.588) and extrathyroidal extensions (OR 1.964, 95% CI 1.194, 3.232) were associated with the BRAF V600E mutation in a univariate analysis, although no clinicopathological features were associated with the BRAF V600E mutation in a multivariate analysis. Therefore, our results indicate that there may be differences between PTC subjects in Eastern and Western countries.
The prevalence of the BRAF V600E mutation in PTC is distinctly different between subtypes. For example, this mutation is specific to the classic and tall-cell variants of PTC and is rarely found in other types of differentiated thyroid cancer, including follicular variant PTC [13]. We also observed a significant prevalence of the BRAF V600E mutation among patients with classic type PTC311 (95.1%), whereas only 16 patients (4.9%) with the follicular variant PTC harboured the BRAF V600E mutation.
In this study, we found that the BRAF V600E mutation was associated with a lower frequency of concurrent Hashimoto's thyroiditis in a univariate analysis (P <0.05). The BRAF V600E mutation was present in 21 (24.4%) of 86 patients with PTC without Hashimoto's thyroiditis and in 35 (14.5%) of 241 patients with both PTC and Hashimoto's thyroiditis. In a previous Korean study [23], the BRAF V600E mutation was detected at a higher frequency among patients with PTC but without Hashimoto's thyroiditis (90%) as compared to those with PTC and Hashimoto's thyroiditis (64%). Together, these results suggest that the BRAF V600E mutation is associated with the tumourigenesis of most classic type PTCs and that the somatic BRAF V600E mutation likely acts on a component of Hashimoto's thyroiditis following PTC as cells age. However, the current study had some inherent limitation. First, the rate of mirocarcinoma was higher (65.1%) in this study, and this condition is known to be associated with low aggressiveness and a more favourable prognosis in most cases. Second, we were not able to examine disease recurrence and describe the long-term follow up results, and the time period of the present study was too short to identify the prognostic value of the BRAF V600E mutation. Thus, a long-term follow up study is needed to identify the association between the prognosis of PTC and the BRAF V600E mutation.

Conclusions
The prevalence of the BRAF V600E mutation in this study was in accordance with that observed in previous studies. This prevalence was related to several clinicopathological factors, including extrathyroidal extension, large tumour size, classic histologic type, and a low frequency of concurrent Hashimoto's thyroiditis. However, we failed to establish a correlation with regard to lymph node metastasis, advanced stage, sex, and age. Moreover, there were no independent factors associated with BRAF V600E mutation status. However, it will be necessary to perform a longterm follow up study to identify the clinical usefulness of the BRAF V600E mutation in PTC.