the incidence of thyroid cancer has increased over the past few decades in different countries , including china [ 1 , 2 ] . among all the types of thyroid cancers , ptc , which is the most common subtype of thyroid cancer , represents approximately 80%90% of all thyroid cancers [ 35 ] . the braf 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 . several studies showed that the occurrence of the braf mutation in ptc was related to aggressive features , including extrathyroidal invasion and lymph node metastases [ 810 ] . 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 . preoperative medical checkups did not identify all metastases , including some small metastases in the lymph node . this common clinical dilemma is troubling , although some authors reported that the braf mutation is an independent predictor of central node metastasis [ 1720 ] . therefore , it is necessary to determine if the braf mutation is a predictor of lnm in chinese ptc patients and 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 [ 2124 ] , 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 [ 2629 ] , 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 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 10-fold higher rate than most developed countries [ 3234 ] . the incidence of the braf mutation in ptc also has a wide range of frequencies , from 25 to 90% [ 8 , 12 , 3538 ] . jeong et al .   reported that the frequency of the braf mutation in korean ptc patients was 90% . there is a clear need to determine the frequency of the braf mutation , prior to administering any targeted clinical therapies into chinese ptc patients . in this study , we investigated the status of the braf mutation in primary tumors from 543 chinese ptc patients to identify correlations between this genetic event and clinicopathological factors . 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 . a total of 28 sections , each with a thickness of 5  m and a surface area of approximately 200  mm in total , were used for dna extraction . a qiaamp dna ffpe tissue kit ( qiagen cat.56404 ) air - dried tissue was resuspended in atl buffer with proteinase k and incubated overnight . after a 1 hour incubation at 90c , 200  l of al was added to the sample . then , the sample was washed with aw1 and aw2 wash buffer , and the genomic dna was eluted in 50  l of ate buffer . the polymerase chain reaction ( pcr ) primers used were the forward primer , 5-tgcttgctctgataggaaaatg-3 , and reverse primer , 5-agcctcaattcttaccatcca-3. the thermocycler program was set as follows : 94c 3  min , 35  ( 94c 30  sec , 60c 30  sec , and 72c 30  sec ) , and 72c for 5  min . pcr products ( 191  bp ) were subjected to automated sequencing using an abi prism 3500 ( applied biosystems , foster city , ca , usa ) . pearson  test and fisher 's exact test were used to calculate the bivariate analysis of the relationship between the braf 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 . the information for the 543 patients diagnosed with ptc included in this retrospective study is summarized in table 1 . a total of 409 females ( 75.3% ) and 134 males ( 24.7% ) with a mean age of 42.10  12.12 years at the time of diagnosis were included . lnm was discovered in 321 patients ( 59.1% ) . of all ptc patients , 170 patients ( 31.3% ) harbored braf . for tumor stage , 434 patients ( 79.9% ) were at stage i , 6 patients ( 1.1% ) were at stage ii , 70 patients ( 12.9% ) were at stage iii , and 33 patients ( 6.1% ) were at stage iv . bivariate analysis showed that the tumor size , bilaterality , extrathyroidal invasion and lnm are associated with braf ( table 2 ) . there was no significant association of the braf mutation with gender , age at diagnosis , multifocality , and ajcc stage in our study . the mean tumor size of the 170 patients with braf was 1.47  0.99  cm , whereas the mean tumor size of the wild type patients was 0.98  0.87  cm ( t = 5.80 , p < 0.001 ) . additionally , multivariate analysis ( binary logistic regression ) showed that braf was positively associated only with tumor size ( > 1  cm ) and extrathyroidal invasion ( table 3 ) . we then performed a binary logistic regression analysis to determine whether braf 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 mutation , we found that the braf 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 . sanger sequencing , pcr , and immunohistochemistry are three primary methods for detecting the braf mutation . 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 , braf , braf , braf , braf , braf , braf , braf , and braf [ 4244 ] . however , in our study , we only identified the common type of braf mutation variant , braf . 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 rate for braf 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 . furthermore , the sample size was not large enough to identify all reported braf genetic changes some studies reported that the braf mutation is related to male gender , older age , tumor size , thyroid capsular invasion , extrathyroidal extension , and lnm ( table 6 ) [ 18 , 4549 ] . in our study , the braf 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 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 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 . 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 . the braf mutation may render thyroid cells susceptible to transforming growth factor beta - induced emt . the aberrant methylation of tumor suppressor genes , leading to the increased aggressiveness of thyroid tumor cells , is also related to the braf mutation . several authors determined whether the braf mutation is a predictive factor for lnm to help surgeons decide which ptc patients should have lymph node dissection [ 1719 ] . in our study , we did not find that the braf 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 . our study shows that the occurrence of the braf mutation in chinese patients is approximately the same as the other countries . the braf mutation is positively associated with worse prognostic factors , including larger tumor size and tumors extending to the thyroid capsule or extrathyroidal region . however , in our study , the braf mutation does not show predictive value for lnm .
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 .