Associations between BRAFV600E and prognostic factors and poor outcomes in papillary thyroid carcinoma: a meta-analysis

Background The objective of this study is to perform a meta-analysis to evaluate the associations between the BRAFV600E mutation status and aggressive clinicopathological features and poor prognostic factors in papillary thyroid cancer. Methods A literature search was performed within the PubMed, MEDLINE, Web of Science databases, and EMBASE databases using the Medical Subject Headings and keywords from January 2003 to July 2015. Individual study-specific odds ratios and confidence intervals were calculated, as were the Mantel-Haenszel pooled odds ratios for the combined studies. Results Sixty-three studies of 20,764 patients were included in the final analysis. Compared with wild-type BRAF, the BRAFV600E mutation was associated with aggressive clinicopathological factors, including extrathyroidal extension, higher TNM stage, lymph node metastasis, and recurrence, and was associated with reduced overall survival; however, there was no significant association between the presence of BRAF mutation and distant metastasis. Conclusions BRAF mutations are closely associated with aggressive clinicopathological characteristics and poorer prognosis in papillary thyroid cancer. Accordingly, aggressive treatment should be considered for papillary thyroid cancer patients with BRAF mutation.


Background
Papillary thyroid carcinoma (PTC) is the most common malignant thyroid neoplasm, accounting for 80 % of all thyroid cancers [1]. The incidence of PTC has increased dramatically in the past decades [2], and Chen et al. demonstrated that the increasing rate of PTC resulted from an actual increase in disease incidence rather than improvements in the diagnosis such as by high-resolution ultrasound [3]. PTCs, especially papillary thyroid microcarcinomas, tend to have a good prognosis; however, even for TNM stage I patients, approximately 15 % of patients experienced recurrence during 10 years of follow-up in one previous study [4]. Therefore, the classification and targeted therapies for PTC still need more investigation.
The BRAF V600E mutation is a common mutation in PTC, with a reported frequency of 25-82.3 %. Many authors have reported that the BRAF V600E mutation is associated with aggressive clinical features and poor prognosis; however, others have suggested that there is no association between the BRAF V600E mutation and clinicopathologic features in PTCs [5][6][7]. Thus, it remains controversial whether the BRAF V600E mutation closely correlates with poor clinical characteristics in PTC, and it is necessary to confirm the association between BRAF V600E and the clinical prognosis of PTC using meta-analysis.
Accordingly, the present meta-analysis was designed to determine whether the BRAF V600E mutation is associated with high-risk clinicopathological factors and poor prognostic outcomes in PTC patients. Additionally, we performed subgroup analyses to assess the effects of factors that might modify these associations. The results of our meta-analysis are considered very helpful for clinical surgeons to choose the optimal surgical managements, such as whether prophylactic central neck dissection is needed, and for postoperative risk stratification of PTCs.

Methods
A comprehensive search was conducted in the PubMed, MEDLINE, and Web of Science databases from January 2003 to July 2015. The search terms and keywords used included "papillary thyroid carcinoma," "BRAF," "mutation," "V600E," "T1799A," and "PTC." All studies that included BRAF V600E mutation data from primary PTC tissues were further evaluated. In addition, the reference lists from single case reports and relevant reviews articles were also inspected for further eligible studies by two independent reviewers. Clinical characteristics such as extrathyroidal invasion, lymph node metastasis, TNM stage, cancer recurrence, and rates of cancer-specific death at the last follow-up were included in the present analysis. The search was restricted to English language publications only. Duplicate articles and studies with no clinicopathologic data were excluded. To avoid falsenegative results, studies that determined the BRAF V600E mutation status by preoperative fine needle aspiration biopsies only were also excluded. Further, studies confined to only low-risk groups or papillary thyroid microcarcinomas were excluded, as were unpublished data that were presented at international meetings. In instances where the same study cohort was used in multiple articles, either the most recent or the most appropriately informative single article was included. For example, Fugazzola et al. [6] included only data from a single institute and contained a cohort overlapping with Fugazzola et al. [8]; therefore, we used the latter study for our analysis.

Data analyses and statistical methods
We used RevMan (version 5) to calculate the summary odds ratios (ORs) with 95 % confidence intervals (CIs), using a random-effects model when p value (chi-square test of homogeneity) <0.1 and a fixed-effects model when p value (chi-square test of homogeneity) ≥0.1. We assessed the heterogeneity of the studies using the chisquare test of heterogeneity and the I 2 measure of inconsistency. Significant heterogeneity was defined as a chisquare test p value of <0.10 or as an I 2 measure >50 % (according to a statement from Cochrane Handbook). The extent to which the combined metarisk or heterogeneity were affected by individual studies was assessed further by sequentially excluding every study from the meta-analysis. We investigated potential sources of the identified heterogeneity among the studies using a stratification process according to the country or ethnics in which the research was conducted. The potential for publication bias was assessed using a funnel plot analysis.

Results
Results of the literature search and study characteristics Figure 1 shows the study selection process. A total of 997 abstracts and titles of full-text papers obtained through electronic searches were deemed relevant and examined in detail. Following this detailed review, 63 studies met our inclusion/exclusion criteria; these studies contributed 20,764 patients with PTC to this metaanalysis [6,. The main features of the 63 eligible studies that investigated prognostic factors are summarized in Table 1. The funnel plots for each outcome did not suggest the presence of publication bias (data not shown).

Meta-analyses of BRAF mutation effects on clinicopathological and prognostic features BRAF mutation and extrathyroidal extension
Fifty-two studies contained data on extrathyroidal invasion. Extrathyroidal invasion was present in 5306 (51.5 %) of 10,301 patients with BRAF mutations and in 1910 (28.7 %) of 6655 patients without BRAF mutations (Fig. 2). The pooled odds ratio (OR) from these 52 studies was 2.04 (95 % confidence interval [CI], 1.75-2.37). A random-effects model was adopted because the heterogeneity of the data was significant (p < 0.00001), and the I 2 estimate of the variance between the studies was 69 %. According to our analysis, the association between the occurrence of extrathyroidal invasion and BRAF mutation was significant (p < 0.00001).

BRAF mutation and TNM stage
TNM stage was reported for patients in 51 studies (Fig. 3). The advanced TNM stage (III/IV) was present in 2913 (37.5 %) of 7765 patients with BRAF mutations and in 1364 (23.3 %) of 5842 patients without BRAF mutations (Fig. 3). The pooled odds ratio (OR) from these 51 studies was 1.82 (95 % CI, 1.53-2.17). A randomeffects model was adopted because the heterogeneity of the data was significant (p < 0.00001), and the I 2 estimate of the variance between the studies was 70 %. According to our analysis, the association between the occurrence of advanced TNM stage (III/IV) and BRAF mutations was significant (p < 0.00001).

BRAF mutation and lymph node metastasis
Lymph node metastasis was reported for patients in 51 studies (Fig. 4). Lymph node metastasis was present in 4575 (43.4 %) of 10,542 patients with BRAF mutations and in 2405 (32.5 %) of 7394 patients without BRAF mutations (Fig. 4). The pooled odds ratio (OR) from these 51 studies was 1.45 (95 % CI, 1.24-1.69). A randomeffects model was adopted because the heterogeneity of the data was significant (p < 0.00001), and the I 2 estimate of the variance between the studies was 73 %. According to our analysis, the association between the occurrence of lymph node metastasis and BRAF mutations was significant (p < 0.00001).

BRAF mutation and distant metastasis
Distant metastasis was reported for patients in 19 studies (Fig. 5). Distant metastasis was present in 77 (5.4 %) of 1415 patients with BRAF mutations and in 98 (5.2 %) of 1868 patients without BRAF mutations (Fig. 5). The pooled odds ratio (OR) from these 19 studies was 1.05 (95 % CI, 0.65-1.68). A random-effects model was adopted because the heterogeneity of the data was significant (p = 0.05), and the I 2 estimate of the variance between the studies was 37 %. According to our analysis, the association between the occurrence of distant metastasis and BRAF mutations was not significant (p = 0.85).

BRAF mutation and recurrence
Recurrence was reported in 24 studies (Fig. 6). Recurrence was present in 608 (24.8 %) of 2450 patients with BRAF mutations and in 407 (14.1 %) of 2877 patients without BRAF mutations (Fig. 6). The pooled OR from these 24 studies was 2.20 (95 % CI, 1.57-3.09). A random-effects model was adopted because the heterogeneity of the data was significant (p < 0.00001), and the I 2 estimate of the variance between the studies was 74 %. According to our analysis, the association between the occurrence of recurrence and BRAF mutations was significant (p < 0.00001).

BRAF mutation and OS
Overall survival (OS) was reported in five studies (Fig. 7). OS was present in 65 (5.1 %) of 1264 patients with BRAF mutations and in 17 (1.1 %) of 1605 patients without BRAF mutations (Fig. 7). The pooled OR from these five studies was 4.61 (95 % CI, 2.69-7.90). A fixedeffects model was adopted because the heterogeneity of the data was not significant (p = 0.77), and the I 2 estimate of the variance between the studies was 0 %. According to our analysis, the association between the occurrence of death and BRAF mutations was significant (p = <0.00001).

Subgroup analyses of the BRAF mutation effects on aggressive clinicopathological features and poor prognostic factors
Subgroup analysis was conducted according to the ethnicities of the study subjects in order to investigate the potential sources of heterogeneity and to assess whether the effects of the BRAF mutation status on aggressive clinicopathological features and poor prognosis of PTC patients were associated with geographic regions ( Table 2). The heterogeneity was decreased in the subgroup analyses of extrathyroidal invasion, TNM stage, lymph node metastasis, and recurrence. Thus, the results  [70,71]. Since its initial discovery, the BRAF V600E mutation has been debatable as a diagnostic as well as prognostic indicator of PTC, as many of the studies on the topic have reported contradictory outcomes of the association between the BRAF V600E mutation and clinical characteristics and Fig. 2 The odds ratios (ORs) with 95 % confidence intervals (CIs) for the association between BRAF mutation and extrathyroidal extension in patients with PTC prognosis [37,40]. Therefore, the present meta-analysis to illustrate the role of BRAF mutation in PTC was considered highly necessary.
Various clinicopathological risk factors have been reported to be related to recurrence and cancer death in PTC [72]; according to most authors' results, we chose extrathyroidal invasion, lymph node metastasis, advanced TNM stage, and distant metastasis as reliable predictors for poor prognosis in the present analysis, as these factors not only represent aggressive cancer behavior but also poor prognosis [72,73].
Extrathyroidal extension is associated with an increased risk of invasion into cervical structures such as the trachea, which requires more aggressive treatment. Accordingly, extrathyroidal extension is an important factor related to PTC prognosis, contributing to an increased risk of local recurrence/persistence of the disease, independent of the tumor size [74]. In our meta-analysis, the risk of extrathyroidal extension was increased by 2.04-fold in cases with positive BRAF mutation status compared to in wild-type cases. In fact, out of all included studies, only six studies showed that patients with BRAF mutation presented less frequent extrathyroidal extension than those without BRAF mutation.
The American Joint Committee on Cancer (AJCC) staging system comprises the sum of several tumor characteristics, such as the tumor size, lymph node status, and distant metastasis, which are generally considered aggressive features of cancer. AJCC stage III/IV cancers are associated with a poorer prognosis in terms of both recurrence and overall survival than stage I/II tumors [75]. According to our results, there was a significant association between high AJCC stage and BRAF mutation, Fig. 4 The odds ratios (ORs) with 95 % confidence intervals (CIs) for the association between BRAF mutation and lymph node metastasis in patients with PTC with positive BRAF mutation status being associated with a 1.82-fold increased risk of stage III/IV cancers compared to wild-type cases. Out of all included studies, nine studies suggested that the risk of a high AJCC stage was lower in patients with BRAF mutation than in those without BRAF mutation; however, the other 42 studies demonstrated the opposite results, especially for patients aged >45 years, in whom positive BRAF mutation status was associated with more frequent cases of stage III/IV cancers compared to stage I/II [20,76]. Howell et al. also reported that older age and positive mutation status were more frequently associated with TNM stage III/IV and recurrence [14].
Further, according to the present meta-analysis, the prevalence of lymph node metastasis was increased in patients with BRAF mutation, with an odds ratio of 1.45; increased risk was observed in 39 of 60 studies. Lymph node metastasis is an important risk factor for recurrence and/or persistent disease, as well as overall survival [75]. Based on these results, we suggest that the presence of BRAF mutation is a prognostic factor in PTCs. However, it is worth mentioning that the different surgical approaches and treatments used in these 60 studies probably influenced the outcome in terms of the real incidence of nodal metastasis; therefore, evaluation of the BRAF mutation status as a prognostic indicator for lymph node metastasis in PTCs should be more cautious [74].
Recurrence and persistent disease demand additional therapy and can affect the PTC patients' quality of life. For example, recurrence increases the risk of reoperations and the exposure to a high cumulative radioiodine dose. Recently, Xing et al., in their large multicenter study, demonstrated that the BRAF V600E mutation was an independent prognostic factor for PTC recurrence in various clinicopathologic categories [77]. However, these outcomes were questioned by Bal & Ballal and Yarchoan et al. [78,79], as the median follow-up was only 36 months and the recurrence rate was very high, which might have resulted from persistent disease rather than true recurrence. However, our meta-analysis indicated that the association between the occurrence of recurrence and BRAF mutations was significant, and these results were obtained from 24 studies including a total of 2450 patients; hence, our findings are considered more reliable.
Our present meta-analysis encompassing a total of 20,764 patients suggested that the BRAF V600E mutation is associated with several of the high-risk clinical variables used in prognostic staging systems, including extrathyroidal invasion, high TNM stage, lymph node metastasis, recurrence, and overall survival. Especially, PTCs with positive BRAF V600E mutation exhibited a 4.61-fold increased risk of death compared with cases with the wildtype form of the BRAF gene. On the other hand, one interesting finding in the present meta-analysis was that the BRAF V600E mutation was not related to distant metastasis. One potential reason for this result may be the fact that the diagnosis of distant metastasis differs between different countries and medical centers.
There were several limitations regarding our study design, especially in terms of the studies included in our analysis. Additionally, our study could not determine a causal relationship between BRAF mutations and outcomes, and BRAF mutations may correlate with some other mutations or confounders, which may turn out to be even more useful prognostic indicators. Importantly, our analysis should be interpreted with caution because of the heterogeneity in the data. Possible explanations for this heterogeneity include differences in the patient demographics and ethnicities, as well as in the thyroidectomy, approaches to lymph node dissection, pathology reporting, radioactive iodine treatment, and the time of follow-up. Therefore, our conclusions should be interpreted with caution. Fig. 6 The odds ratios (ORs) with 95 % confidence intervals (CIs) for the association between BRAF mutation and recurrence with PTC Fig. 7 The odds ratios (ORs) with 95 % confidence intervals (CIs) for the association between BRAF mutation and OS with PTC

Conclusions
In conclusion, our meta-analysis illustrated that the BRAF V600E mutation correlates with high-risk clinicopathological factors and poor clinical outcome. These results provide a valuable reference for physicians when assessing the prognosis of PTCs by BRAF V600E mutation analysis, and our findings may be of great value in evidence-based clinical decision-making for PTC patients.