The Association of the BRAF V600E  Mutation with Clinicopathologic Characteristics in Chinese Population with Conventional Papillary Thyroid Carcinoma


 Objective:The aim of the study is to evaluate the association of the BRAFV600E mutation with the clinicopathologic characteristics in Chinese population with papillary thyroid carcinoma (PTC).MethodsA total of 943 PTC patients who underwent thyroidectomy from 2014 to 2016 at Henan Provincial People’s Hospital were included in the present study. The BRAFV600E mutation was examined in each resected specimen by quantitative Real-time PCR (qRT-PCR) technique. Results The PTC patients were subclassified into the overall, PTC>10mm and papillary thyroid microcarcinoma(PTMC) groups. The positive rate of BRAFV600E mutation was 85.4% in Chinese patients with PTC. In both overall PTC and PTC> 10mm groups, the BRAFV600E mutation was much more frequently detected in elderly patients and patients at T1 stage (P< 0.05). In addition, the positive rate of BRAFV600E mutation was significantly higher in PTC patients without concomitant Hashimoto’s thyroiditis in overall PTC and PTMC groups (P< 0.05). Furthermore, logistic regression analysis suggested that the risk of having a larger tumor diameter was increased by 6-fold when BRAFV600E mutation in the PTMC group. No association between the BRAFV600E mutation and other clinicopathologic factors was observed. ConclusionThe BRAFV600E mutation was significantly associated with patients age and T stage. Furthermore, the risk of having a larger tumor size was significantly increased when BRAFV600E mutation in the PTMC group. which suggests that BRAFV600E mutation might play an important role in the activation of early thyroid carcinogenesis, the effect might weaken in the progression of PTC.


Introduction
It is important to note that the global incidence of thyroid cancer has been rapidly increasing [1,2], currently ranking ninth among all cancers. In addition, the global incidence rate of thyroid cancer in women is reported to be three times than that in men [2,3]. Papillary thyroid carcinoma (PTC) is the main subtype of thyroid cancer, which accounts for 85%-90% with an early onset age [2,3]. Although ultrasonography-guided ne-needle aspiration (US-FNA) biopsy is a standard technique for diagnosing thyroid malignancies measuring >1-1.5 cm, ~15%-25% of these malignancies are classi ed as indeterminate [4], and 49% of PTCs measure ≤1 cm 2 , which increases the di culty in performing US-FNA biopsy. The World Health Organization de nes tumors ≤1 cm in diameter as papillary thyroid microcarcinoma (PTMC) [5], which challenges the diagnosis of a US-FNA biopsy. The clinical signi cance of PTMC remains unclear, although previous reports have indicated that PTMC is related to distant metastasis, mortality, and recurrence [6]; therefore, early PTMC detection and a simultaneous estimation of its clinical signi cance are essential.
In the present study, we aimed to analyze the status of the BRAF V600E mutation in 943 patients with PTC and retrospectively examine its possible association with clinicopathologic risk factors.

Patients
All patients with PTC in the present study who underwent either total thyroidectomy or near-total thyroidectomy in Henan Provincial People's Hospital, China, between October 2014 and October 2016. In total, 943 patients were recruited for this study [men: 233, mean age(average ±standard deviation), 52.0±11.6 years; women: 710, mean age, 45.4±11.6 years]. All patients received surgical treatment without radioactive iodine 131 therapy before the surgery. The patients were classi ed into three groups according to pathologic tumor diameter as follows: the overall PTC group, PTC >10 mm group and PTMC group.

Histopathologic examinations
Histopathlogic examinations of all patient tissue samples were performed by highly experienced pathologists blinded to BRAF status. Di cult cases were discussed and diagnosed by at least two pathologists. TNM stage was classi ed based on the 8th edition of the American Joint Committee on Cancer. The majority of patients with PTC (88.8%; 838/943) were diagnosed as having T1-stage tumors. Lymph node involvement was observed in 377 (54.1%; 459/848) patents. Hashimoto's thyroiditis was con rmed based on the postoperative pathologic ndings. The mean follow-up duration was 27 postoperative months (range= 11-44 months). At the time of follow-up, only two patients died (one patient of breast cancer and another of PTC recurrence).

Analyses of the BRAF V600E mutation
Hematoxylin and eosin-stained slides were reviewed by experienced pathologists. The representative tumor areas were marked to guide microdissection. The tumor areas from 3-4 pieces of 5-µm-thick sections of para n-embedded tissues were dissected for DNA extraction. The process was performed using the Puregene Tissue Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. The absorbance of the DNA samples was measured using a spectrophotometer, and only the A260/A280 values between 1.8 and 2.0 were accepted for the next step in the analysis. The extracted DNA was stored at −80°C until use. The status of the BRAF V600E mutation in each sample was examined by qRT-PCR using the AmoyDx BRAF V600E Mutation Detection Kit (Amoy Diagnostics, Xiamen, China), and the presence of the mutation was evaluated following the manufacturer's instructions. The sample was classi ed as mutation-positive when the cycle threshold (Ct) was <28 and as negative when Ct was ≥28.

Statistical analyses
All statistical analyses in present study were analyzed were conducted using SPSS v. 21 (IBM Corporation, Waltham, NY, USA). Discrete variables were presented as number and percentage. Chi-squared or Fisher's exact test was used for categorical variables when comparing frequencies between the groups.
All numerical data are expressed as means ± standard deviations, and differences between the means were compared using Student t-test and an analysis of variance for continuous variables. All signi cant factors by univariate analyses were subjected to logistic regression analysis. A probability value of <0.05 was considered statistically signi cant.

Results
The frequency of the BRAF V600E mutation in patients with PTC Of the 943 patients with PTC, there were 570 (60.4%) PTMC patients and 373 (39.6%) patients with PTC >10 mm. The rate of the BRAF V600E mutation in all patients with PTC was 85.4% (806/943) ( Table 1). Although the incidence of the BRAF V600E mutation in the PTMC group (86.8%; 495/570) was higher than that in the PTC >10 mm group (83.4%; 311/373), there was no signi cant difference between the two groups (P = 0.140; Table 1).
The association between the BRAF V600E mutation and clinicopathologic characteristics is detailed in Table 1. The mean age of patients positive of BRAF V600E mutation was (46.3±11.8) years, which was elder than patients negative of BRAF V600E mutation, logistic regression analyses estimated that advanced age was an independent predictive factor for the BRAF V600E mutation [P= 0.03; odds ration(OR)= 1.02; 95% con dence interval(CI)= 1.00-1.03] (

Discussion
The present study showed an 85.4% frequency rate of the BRAF V600E mutation in conventional PTC, the proportion was slightly higher in the PTMC group.
The present study showed that the BRAF V600E mutation was much more frequent in elderly patients in both the overall PTC group and PTC>10 mm group.
Previous studies have also revealed an association between the BRAF V600E mutation and elderly patients [23,27,28]. The youngest patients in our cohort were 15 years of age. The positive rate of the BRAF V600E mutation was 60% (3/5) in patients <20 years of age, and it is interesting that this rate increased to more than 80% in those aged ≥20 years. The possible reason for this phenomenon is that the BRAF V600E mutation might occur early in thyroid carcinogenesis, in addition, the effect might become more remarkable along with the development and progress of PTC. However, no such relationship was noted in the results of other studies [24,25,29].
Furthermore, in the present study, the BRAF V600E mutation was more likely to be present in patients with a T1-stage tumor than in those with a T2-or T3-stage tumor in both overall and PTC >10 mm groups. This phenomenon also indicates that PTC patients having the BRAF V600E mutation might be susceptible to an early T stage, and that the mutation might play an important role in the early stages of PTC, but it might also slow PTC progression; however, no signi cant difference was found among TNM stages, perhaps because all patients enrolled in the present study were in the early stages of the cancer (0 and 1). Previous studies have also found no relationship between the BRAF V600E mutation and TNM stage [27][28][29][30].
Moreover, our data showed that the risk of having a large tumor was increased about 6-fold when BRAF V600E mutation in the PTMC group, however, no in uence was found in the PTC>10 mm group. This occurrence also indicated that the BRAF V600E mutation might be an initiating factor in PTC; however, the probable effect decreased along withing. The reported studies have found no association between the mutation and tumor in PTMC [19,29].
In addition, our results showed that the BRAF V600E mutation was more frequent in PTC patients without concomitant Hashimoto's thyroiditis in both overall PTC and PTMC groups. The meta-analysis also suggested that patients negative for the BRAF V600E mutation were signi cantly more likely to have Hashimoto's thyroiditis [22]. Other previous studies found no association between BRAF V600E mutation and PTC concomitant Hashimoto's thyroiditis [17][18][19][20][21][22]. The possible reason of such a discrepancy might be the result of the study population that was different between our study and the above-mentioned study.
There was no relationship between the BRAF V600E mutation and both lymph node metastasis and tumor stage in patients with PTC in the present study. Similar phenomena were also reported in previous studies [17][18][19][20][21][22]30]; however, several studies have reported that the high-risk clinicopathologic parameters, such as lymph node metastasis and tumor stage, were associated with the BRAF V600E mutation [10][11][12][13][14][15][16][25][26][27][28][29]. The inconsistent results might be because of the following factors: geography or ethnicity, sample size, tumor subtypes, or the method used to test the BRAF V600E mutation. In addition, 88.5% of patients with PTC (838/947) enrolled in the present study were at early stages of the disease, limiting the effect of the BRAF V600E mutation on progressive clinical characteristics.
The results of our study found no association between the BRAF V600E mutation and disease-free survival. Similar results were found in studies on both Korean and Japanese populations [10,13,20]; however, the BRAF V600E mutation was reported as an independent prognostic factor in patients with PTC [19,24].
Numerous reports have indicated that the BRAF V600E mutation is associated with disease-free survival even in patients in the early stages of the disease [25][26][27][28]. The discrepancy among the results of the published studies might be associated with sample size, geographic and ethnic differences, duration of followup, and treatment regimen. Most patients with PTC are diagnosed at early stages of the disease and have a low mortality rate. Majority of the patients enrolled in the present study were at T1 or T2 stage at the time of diagnosis. The intensive initial treatment might be a potential reason for nding no association between the BRAF V600E mutation and the disease because patients received all scheduled treatments [13,30].
Strengths of the present study. Firstly, we analyzed the relationship between BRAF V600E mutation and clinicopathological features in both PTMC and PTC> 10mm groups and found that there were different features in each group. Secondly, subjects enrolled in the present study were only conventional PTC to exclude heterogeneity and variations in tumor subtypes. Limitation of our study. Majority of patients enrolled in the present study were at T1or T2 stage when diagnosis and the limited time of follow-up were constrict the effect of clarifying the relationship of BRAF V600E mutation and prognosis.

Conclusions
This study found that an 85.4% frequency in the BRAF V600E mutation in patients with PTC. The BRAF V600E mutation was associated with elderly patients and early tumor T stages in both overall PTC group and PTC >10 mm groupF, BRAF V600E mutation was also related to the large tumor diameter in the PTMC group patients. which suggests that BRAF V600E mutation might play an important role in the activation of early thyroid carcinogenesis, the effect might weaken in the progression of PTC.  *Numbers are presented as mean ± standard deviation.