The Clinicopathological Features of BRAF Mutated Papillary Thyroid Cancers in Chinese Patients

The BRAFV600E mutation is commonly found in papillary thyroid cancers (PTCs) at different frequencies in different regions. However, the association between the BRAFV600E mutation and clinicopathological features in Chinese PTC patients is unknown. A total of 543 Chinese patients with histologically confirmed PTC were enrolled in this study. For the BRAF mutation assay, the target fragments were amplified and sequenced with an ABI 3500 gene analyzer. In 170 of 543 samples (31.3%), the BRAFV600E mutation was detected. In the bivariate analysis, the BRAFV600E mutation showed an association with bilaterality, tumor size, extrathyroidal invasion, and lymph node metastases (LNM). However, in the multivariate analysis, the BRAFV600E mutation was positively related to only tumor size (>1 cm) and extrathyroidal invasion. In addition, the multivariate analysis also showed that the age at diagnosis (<45 y) and tumor size (>1 cm) were independent predictors for LNM. In this study, the BRAFV600E mutation is positively associated with worse prognostic factors, including larger tumor size and the tumor extending to the thyroid capsule or extrathyroidal region; however, it is not an independent predictor for LNM.


Introduction
The incidence of thyroid cancer has increased over the past few decades in different countries [1], including China [1,2]. Among all the types of thyroid cancers, the increased rate of papillary thyroid cancer (PTC) is particularly high [3]. PTC, which is the most common subtype of thyroid cancer, represents approximately 80%-90% of all thyroid cancers [3][4][5].
The BRAF V600E mutation leads to uncontrolled activation of the MAPK pathway, which is crucial for both tumor initiation and the progression of PTC [6,7]. Gene mutations and/or chromosome changes in certain effectors of the MAPK pathway, such as BRAF mutations, RAS mutations, and RET/PTC rearrangements, lead to constitutive activation of cell signaling, resulting in uncontrolled proliferation and carcinogenesis. However, the association between BRAF V600E mutation and clinicopathological features remains controversial. Several studies showed that the occurrence of the BRAF V600E mutation in PTC was related to aggressive features, including extrathyroidal invasion and lymph node metastases [8][9][10]. However, other authors did not find this association [11,12]. Lymph node metastasis (LNM) is an aggressive factor and is associated with recurrence and cancer-related mortality [13,14]. Patients who underwent central lymph node dissection suffered a greater risk of permanent hypoparathyroidism or permanent nerve injury [15]. Preoperative medical checkups did not identify all metastases, including some small metastases in the lymph node [16]. This common clinical dilemma is troubling, although some authors reported that the BRAF V600E mutation is an independent predictor of central node metastasis [17][18][19][20]. Therefore, it is necessary to determine if the BRAF V600E mutation is a predictor of LNM in Chinese PTC patients and 2 International Journal of Endocrinology if it may help surgeons decide whether to perform lymph node dissection.
In addition, the BRAF mutation may be involved in the decreased expression of iodine metabolism genes, such as the sodium/iodide symporter and TSH receptor [21][22][23][24], leading to iodine resistance. Target inhibition of BRAF activity is currently the alternative therapeutic approach for iodine-refractory PTC [25,26], and several small molecule inhibitors of BRAF have been developed, including selumetinib, sorafenib, BAY 43-9006, PLX4032, RAF265, and PLX4720 [26][27][28][29], each with different selectivities [30,31]. Furthermore, some of these drugs are currently in phase II and phase III clinical trial studies for the treatment of thyroid cancer [26,31]. By contrast, the incidence of thyroid cancer is different depending on race and geographic regions. The countries with a high reported incidence of thyroid cancer are Polynesia, Iceland, Italy, Israel, Finland, Hong Kong, China, Canada, and United States; the highest rate is found in New Caledonia, which has reported an approximately 10fold higher rate than most developed countries [32][33][34]. The incidence of the BRAF V600E mutation in PTC also has a wide range of frequencies, from 25 to 90% [8,12,[35][36][37][38]. Jeong et al. [8] reported that the frequency of the BRAF mutation in Korean PTC patients was 90%. Currently, this is the highest frequency of the BRAF mutation reported. The different geographic region is the most likely explanation for this phenomenon [39]. There is a clear need to determine the frequency of the BRAF V600E mutation, prior to administering any targeted clinical therapies into Chinese PTC patients.
In this study, we investigated the status of the BRAF V600E mutation in primary tumors from 543 Chinese PTC patients to identify correlations between this genetic event and clinicopathological factors.

Patients and Samples.
This study was a retrospective study and all data were analyzed anonymously. The Institutional Ethics Committee of the West China Hospital approved this study. Patients with histologically confirmed PTC from January 2013 to December 2014 were assessed for this study. A total of 597 primary cases that underwent thyroidectomy and routine central lymph node dissection at the West China Hospital were selected for further study. Of these, 54 patients were excluded because of incomplete patient information or an inadequate tumor sample ( Figure 1). Finally, we included 543 formalin-fixed paraffin-embedded (FFPE) PTC specimens in this study. Some patients selectively underwent lateral neck lymph node dissection if the preoperative medical checkups, such as ultrasound, computed tomography, fine needle aspiration cytology, or cervical lymph node biopsy, suggested metastatic papillary cancer. Demographic and clinicopathological features, including gender, age of the patient at diagnosis, multifocality, bilaterality, tumor size, extrathyroidal invasion, and lymph node status, were collected from the patient medical histories and pathology reports. Specifically, "extrathyroidal invasion" in this study indicates that the tumor invaded the thyroid capsule or grew into the extrathyroidal region. The International  Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) tumor node metastasis (TNM) classification system was used for tumor staging [40]. All information regarding these samples is presented in Table 1.  of the relationship between the BRAF V600E mutation status and clinicopathological features, including gender, age of the patient at diagnosis, multifocality, bilaterality, tumor size, extrathyroidal invasion, lymph node status, and AJCC stage. A multivariate analysis was performed using binary logistic regression analysis for variables, which were significant in the bivariate analysis. Binary logistic regression analysis was also conducted to identify the variables associated with LNM independently. These variables were assessed using preoperative examination and intraoperative frozen-section examination, including gender, age of the patient at diagnosis, multifocality, bilaterality, tumor size, and extrathyroidal invasion. Continuous variables are presented as the mean ± standard deviation. Independent samples -tests were used for analysis. The two-sided significance level was set at < 0.05. Additionally, multivariate analysis (binary logistic regression) showed that BRAF V600E was positively associated only with tumor size (>1 cm) and extrathyroidal invasion ( Table 3).

Predictive Factors of LNM.
We then performed a binary logistic regression analysis to determine whether BRAF V600E is an independent predictive factor for central LNM (Table 4) or lateral LNM (Table 5). After controlling for gender, age at diagnosis, multifocality, bilaterality, extrathyroidal invasion, tumor size, and BRAF V600E mutation, we found that the BRAF V600E mutation was not an independent predictor for central LNM or lateral LNM. However, age at diagnosis (<45 y) and tumor size (>1 cm) were independent predictors for both central LNM and lateral LNM. Gender (female) was an independent predictor for only central LNM, whereas bilateral tumor and extrathyroidal invasion were independent predictors for only lateral LNM.

Discussion
Sanger sequencing, PCR, and immunohistochemistry are three primary methods for detecting the BRAF mutation. There were no significant differences among these methods [41]. However, PCR is commonly used because of its high-efficiency; therefore, we only used this method to detect mutations in this study. There are more than ten types of BRAF mutation variants reported for malignant tumors such as bladder, melanoma, and PTC. These variants include BRAF V600E , BRAF V600D , BRAF V600M , BRAF V600V , BRAF V600R , BRAF V600E2 , BRAF V600Q , BRAF V600L , and BRAF V600K [42][43][44]. However, in our study, we only identified the common type of BRAF mutation variant, BRAF V600E . Therefore, theoretically, any drugs that specifically inhibit B-type RAF kinase should at least target the mutant type    to inhibit the MAPK signaling pathway. Several studies reported probability of using a BRAF mutation inhibitor in iodine-refractory PTC [25,26]. This requires additional large-scale prospective trail to determine the availability and safety in patients.
The frequency of BRAF V600E in Chinese PTC patients is 31.3% (170/543). The rate for BRAF V600E mutation is approximately the same as reported in other countries [6,7,31]. We did not identify any other BRAF mutation variant types as described in other studies. This may occur in a racedependent manner. Furthermore, the sample size was not large enough to identify all reported BRAF genetic changes.
Additionally, we analyzed the BRAF V600E mutation and its clinical and pathological characteristics. Some studies reported that the BRAF V600E mutation is related to male gender, older age, tumor size, thyroid capsular invasion, extrathyroidal extension, and LNM (Table 6) [18,[45][46][47][48][49]. These features indicated a poor outcome in PTC patients [50][51][52]. In our study, the BRAF V600E mutation had a relationship with the tumor size, bilaterality, extrathyroidal invasion, and LNM based on the bivariate analysis. Multivariate analysis showed that tumor size (>1 cm) and extrathyroidal invasion had significant positive associations with the BRAF V600E mutation, after controlling for the tumor size, bilaterality, extrathyroidal invasion, and LNM. This indicates that patients with a larger tumor size (>1 cm) or tumors extending to the thyroid capsule and extrathyroidal region are more likely to have BRAF V600E mutation. In this study, we regard thyroid capsular invasion and extrathyroidal extension as "extrathyroidal invasion" to reduce errors, because the manner by which to distinguish extrathyroidal extension remains controversial. Although the publishers of the UICC/AJCC 7th edition TNM classification system have taken the degree of extrathyroidal extension into consideration, the criteria for defining extrathyroid extension are subjective and problematic because of the discontinuous capsule of the thyroid gland [53].
Several studies further defined the relationship between the BRAF mutation and the aggressiveness of thyroid tumor cells. Epithelial mesenchymal transition (EMT) is common in PTC invasion and is associated with LNM [54]. The BRAF mutation may render thyroid cells susceptible to transforming growth factor beta-induced EMT [55]. The aberrant methylation of tumor suppressor genes, leading to the increased aggressiveness of thyroid tumor cells, is also related to the BRAF mutation [56]. These studies help us understand the importance of this type of mutation in LNM. Several authors determined whether the BRAF V600E mutation is a predictive factor for LNM to help surgeons decide which PTC patients should have lymph node dissection [17][18][19]. In our study, we did not find that the BRAF V600E mutation was an independent predictor for LNM in Chinese PTC patients. However, the binary logistic regression analysis revealed that being female, younger age at diagnosis (<45 y), and tumor size >1 cm had a more significant association with central LNM. For lateral LNM, younger age at diagnosis (<45 y), bilateral tumor, extrathyroidal invasion, and larger tumor size (>1 cm) were related. Therefore, Chinese PTC patients who are young (<45 y) and have a larger tumor size (>1 cm) tend to have both central LNM and lateral LNM.

Conclusion
Our study shows that the occurrence of the BRAF V600E mutation in Chinese patients is approximately the same as the other countries. The BRAF V600E mutation is positively associated with worse prognostic factors, including larger 6 International Journal of Endocrinology tumor size and tumors extending to the thyroid capsule or extrathyroidal region. However, in our study, the BRAF V600E mutation does not show predictive value for LNM.