38

Clinicopathologic Significance of BRAF V600E Mutation in Papillary Carcinomas of the Thyroid

A Meta-analysis

Ju-Han Lee, MD, PhD Eung-Seok Lee, MD, PhD Young-Sik Kim, MD, PhD
Department of Pathology, Bioinformatics Interest Group, Korea University Ansan Hospital, Ansan, Republic of Korea.

BACKGROUND. Numerous studies have investigated the clinical significance of

BRAF mutation in papillary thyroid carcinoma (PTC). However, there have been conflicting data on the usefulness of BRAF mutation as a prognostic marker of PTC. To address this controversy, the frequency of the BRAF mutation and the associations between BRAF mutation and clinicopathologic parameters in PTC were evaluated by meta-analysis.

METHODS. The relevant published studies were reviewed according to the defined
selection criteria. The effect sizes of outcome parameters were estimated by odds ratio or weighted mean difference.

RESULTS. The current meta-analysis included 12 studies with a total of 1168

patients. The frequency of the BRAF mutation was 49%. The BRAF mutation was associated with histologic subtype, the presence of extrathyroidal extension, and higher clinical stage, but not with age, sex, race, or tumor size.

CONCLUSIONS. The effect of the BRAF mutation on the poor prognosis of PTC
patients was evident from the current meta-analysis. The detection of the BRAF mutation may be used as an important prognostic marker of patients with PTC. Cancer 2007;110:3846. 2007 American Cancer Society.

KEYWORDS: thyroid, papillary carcinoma, BRAF, meta-analysis.

Address for reprints: Young-Sik Kim, MD, Department of Pathology, Korea University Ansan Hospital, 516, Gojan-1 Dong, Danwon-Gu, Ansan-Si, Gyeonggi-Do 425-707, Republic of Korea; Fax: (011) 8231-412-5324; E-mail: apysk@korea.ac.kr Received January 4, 2007; revision received March 13, 2007; accepted March 14, 2007.

apillary carcinoma is the most common type of thyroid malignancy. The overall 10-year survival rate for middle-aged adults with thyroid carcinomas is about 80% to 95%.1 Local or regional tumor recurrences occur in 5% to 20% of papillary thyroid carcinoma (PTC) patients.1 The recurrences may result from either incomplete initial treatment or the presence of an aggressive tumor component.1 Distant metastasis, usually in the lungs, occurs in 10% to 15% of PTC patients. The overall 10-year survival rate after the discovery of distant metastases is about 40%.1 Poor prognostic factors of PTC include older age, male, large tumor size, the presence of extrathyroidal extension, and metastasis. However, these factors are not entirely reliable in predicting the tumor recurrence, metastasis, or cancer-related death.2 The classical oncogenic genetic alterations commonly seen in thyroid cancer include Ras mutation, RET/PTC rearrangement, and PAX8-peroxisome proliferator-activated receptor g1 fusion.35 Recently, much attention in the study of thyroid molecular carcinogenesis has been paid to the RAF family genes, which encode the RAS-regulated kinase. RAF kinase is a component of the RAS/RAF/ MAPK kinase/MAPK signaling pathway, which plays a central role in the regulation of cell growth, division, and proliferation.6,7 Among

2007 American Cancer Society

DOI 10.1002/cncr.22754 Published online 22 May 2007 in Wiley InterScience (www.interscience.wiley.com).

Meta-analysis of BRAF Mutation/Lee et al.

39

several isoforms of RAF kinase, BRAF is the strongest activator of downstream MAPK signaling. When this pathway is constitutively activated, it leads to tumorigenesis. Mutations of the BRAF gene have been found in a variety of human cancers, most notably in melanomas.8 The most common BRAF mutation is the T1799A transversion mutation (formerly referred to as the BRAF T1796A mutation) in exon 15 of the gene, which causes a V600E (formerly termed V599E) amino acid substitution in the protein and consequent constitutive activation of the kinase.8 In thyroid tumors, the BRAF mutation occurs only in papillary carcinomas and a subset of anaplastic thyroid carcinomas; it has not been found in benign thyroid neoplasms or in follicular or medullary carcinomas. Several groups have reported that the prevalence of a distinct BRAF mutation ranged from 29% to 83% in different series of PTCs.920 There is no general consensus about a wide range of prevalence. Moreover, the BRAF mutation and its correlation with clinical outcome has been the subject of controversy. Some investigators have suggested that the BRAF mutation is associated with poor clinicopathologic outcomes, such as the high incidence of extrathyroidal extension and metastasis of the tumor.9,14,18,19 In contrast, others have found that the BRAF mutation is not associated with age, gender, multicentricity, clinical stage, or recurrence rate.16,17 Meta-analysis is a powerful tool for summarizing the results of different studies by producing a single estimate of the major effect with enhanced precision. One of the major advantages of meta-analysis is to increase the sample size. It may reduce the probability that random error will produce false-positive or false-negative associations. In this meta-analysis, we examined whether the BRAF mutation has an effect on the prognosis of PTC.

data were reported in the article; and 3) single case reports.

Collection of Published Studies A literature search was carried out using the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed). The keywords BRAF 1 thyroid, BRAF 1 papillary carcinoma, and BRAF 1 thyroid carcinoma hit 113, 70, and 104 citations, respectively. We selected relevant studies on the basis of the summary analysis. We carefully avoided duplication of data by examining the names of all authors and different medical centers involved in each publication. Overlapping articles and articles that were unrelated to our questions were excluded. Data Pooling and Statistics An effect size for each of the studies was calculated by the odds ratio (OR) using the Mantel-Haenszel method or the weighted mean difference (WMD) according to the Cohen method. The choice of each individual statistical method depended on whether the measured event was dichotomous or continuous, whereas the choice of a random or fixed effect model for analysis depended on the Q statistics. The effect sizes of all trials were tested for heterogeneity using the Q statistics, which were an adaptation of the chisquare goodness-of-fit test. The OR was the ratio across different groups for the odds that the event would occur. A 95% confidence interval (CI) was constructed around the effect size to establish its significance. If the 95% CI of an OR included 1, the 2 groups were not considered statistically different. The WMD was calculated as the difference in the means of 2 groups divided by the pooled standard deviation. For studies in which the standard deviation of outcome data was not reported, the effect size was estimated by using the reported P-values. If the 95% CI of a WMD included the null point (zero), the 2 groups were not considered statistically different. For the WMD, if the 95% CI crossed the null point (zero), the possibility that the difference should be attributed to chance could not be excluded. When the null point fell outside the 95% CI of a WMD, the observed difference was considered statistically significant. Statistical analysis was performed using the Comprehensive Meta-analysis software (v. 2.0, Biostat, Englewood, NJ). A P-value less than 0.05 was considered statistically significant.

MATERIALS AND METHODS

Eligibility Criteria for Meta-analysis We extensively searched for articles that examined the associations of BRAF mutation and clinicopathologic characteristics. The following articles were included: 1) BRAF mutation data of primary PTC tissue only were included from the articles that dealt with different types of carcinoma, such as follicular, anaplastic, and medullary carcinomas or the cell lines. 2) Articles published before July 2006 in English. 3) When multiple articles were published by the same authors or groups, the newest or most informative single article was selected. The following were excluded: 1) review articles without original data; 2) an absence of or inappropriate clinicopathologic

RESULTS
Twelve articles fulfilled the eligibility criteria.920 Our data search using PubMed retrieved a total of 97 arti-

40

CANCER July 1, 2007 / Volume 110 / Number 1

TABLE 1 Characteristics of Individual Studies Included in the Meta-analysis

BRAF mutation Study Lee et al.9 Jo et al.10 Riesco-Eizaguirre et al.11 Kim et al.12 Liu et al.13 Xing et al.14 Kim et al.15 Fugazzola et al.16 Puxeddu et al.17 Nikiforova et al.18 Namba et al.19 Xu et al.20 Total No. of cases 100 161 67 60 101 219 70 47 57 104 126 56 1168 Country (Ethnicity) Korea (A) Korea (A) Spain (C) Korea (A) Taiwan (A) USA (C) Korea (A) Italy (C) Italy (C) USA (C) Japan (A) USA (C) Method Sq Sq Sq Sq Sq Sq Sq Sq SSCP,Sq FMCA,Sq Sq MASA,Sq No. (%) 58 (58) 102 (63) 28 (42) 31 (52) 47 (47) 107 (49) 58 (83) 18 (38) 24 (42) 38 (37) 38 (30) 21 (38) 570 (49) Extrathyroidal (%) Absent 54 (54) 62 (39) 38 (57) 34 (57) 49 (49) 157 (72) 35 (50) 20 (43) 31*(54) 75 (72) 88 (70) 45 (80) 688 (59) Present 46 (46) 99 (61) 29 (43) 26 (43) 52 (51) 62 (28) 35 (50) 27 (57) 20*(35) 29 (28) 38 (30) 11 (20) 474 (41) LN metastasis (%) Absent 76 (76) 85 (53) 49 (73) 24 (40) 60 (59) 137 (63) 27 (39) 14*(30) 32*(56) 52 (50) 51 (40) 43 (77) 650 (56) Present 24 (24) 76 (47) 18 (27) 36 (60) 41 (41) 82 (37) 43 (61) 25*(53) 24*(42) 52 (50) 75 (60) 13 (23) 509 (44) I, II 74 (74) 127 (79) 28 (42) 36 (60) 65 (64) 170*(78) NA 31 (66) 21*(37) 82 (79) 54 (43) NA 688 (59) Stage (%) III, IV 26 (26) 34 (21) 39 (58) 24 (40) 36 (36) 47*(21) NA 16 (34) 17*(30) 22 (21) 72 (57) NA 333 (29)

No. indicates number; Extrathyroidal, extrathyroidal extension; LN, lymph node; A, Asian; C, Caucasian; Sq, sequencing; SSCP, single strand conformational polymorphism; FMCA, fluorescence melting curve analysis; MASA, mutant allele-specific amplification; NA, not available. * The cases have insufficient clinical information.

cles related to BRAF mutation and thyroid carcinoma. Among these, 16 articles dealt with different types of carcinoma including follicular carcinoma, anaplastic carcinoma, and medullary carcinoma or thyroid cancer cell lines. Ten articles were not published in English. Twenty-six articles were published by the same authors or groups. Thirteen articles were review articles without original data. Sixteen articles were excluded because of the absence of data or inclusion of inappropriate clinicopathologic data. Four articles were single case reports. The main features of the eligible studies are summarized in Table 1. The sample size of the studies ranged from 47 to 219 patients, for a total of 1168 patients. In this meta-analysis, BRAF mutation was found in 570 (49%) of 1168 PTCs. The insufficient clinicopathologic data in the individual study were excluded. The excluded cases were composed of 4 cases of Liu et al.,13 9 of Fugazzola et al.,16 3 of Puxeddu et al.,17 15 of Nikiforova et al.,18 and 44 of Namba et al.19

No statistical heterogeneity was detected among the studies (Q 5 0.819, df 5 11, P 5 1.000).

Age Six studies presented clinical data including mean age.9,10,14,15,17,18 The mean ages of patients with a BRAF mutation ranged from 39 to 49.3 years, whereas the mean ages of patients showing no BRAF mutation ranged from 35 to 46.7 years. Significant statistical heterogeneity was found among the studies (Q 5 12.168, df 5 5, P 5 .033). No association was found between mean age and BRAF mutation (WMD 5 0.216; 95% CI: 0.0380.469; P 5 .096) (Fig. 2). Sex These 12 studies comprise 259 male and 909 female patients. The BRAF mutation was detected in 129 (50%) of 259 male and 441 (49%) of 909 female PTC patients. No association was found between sex and BRAF mutation (OR 5 1.293; 95% CI: 0.9411.778; P 5 .113) (Fig. 3). There was no statistical heterogeneity among the studies (Q 5 14.254, df 5 11, P 5 .219). Mean Tumor Size Six studies presented clinical data including mean tumor size.9,10,14,15,17,18 The mean tumor sizes of BRAF mutation ranged from 2.3 to 2.9 cm, whereas the mean tumor sizes with no BRAF mutation from 1.8 to 2.7 cm. No association was found between mean tumor size and BRAF mutation (WMD 5

Ethnicity To evaluate the effect of BRAF mutation on race, we divided the population into Caucasian and Asian. These studies consisted of 6 articles on Caucasian populations (550 patients) and 6 articles on Asian populations (618 patients). The BRAF mutation was detected in 236 (43%) of 550 Caucasian and 334 (54%) of 618 Asian PTC patients. There was no association between ethnicity and BRAF mutation (OR 5 1.359; 95% CI: 0.4354.246; P 5 .598) (Fig. 1).

Meta-analysis of BRAF Mutation/Lee et al.

41

FIGURE 1. Odd ratios with corresponding 95% confidence intervals of individual studies and pooled data for the association of BRAF mutation and ethnicity.
The graph demonstrates the effect sizes and 95% confidence intervals for each study and overall.

FIGURE 2. Pooled estimates of the association of BRAF mutation and age.

0.097; 95% CI, 0.0570.250; P 5 .217) (Fig. 4). There was no statistical heterogeneity among the studies (Q 5 11.095, df 5 5, P 5 .050). ventional type and tall cell variant were 3.521 (95% CI, 2.5064.948; P 5 .000) (Fig. 5) and 4.319 (95% CI, 1.9709.469; P 5 .000) (Fig. 6), respectively. There was no statistical heterogeneity (Q 5 5.894, df 5 8, P 5 .659, Q 5 3.221, df 5 3, P 5 .359) among the studies, respectively. In contrast, the overall OR for BRAF mutation in follicular variant was 0.153 (95% CI, 0.0970.242; P 5 .000) (Fig. 7). There was no statistical heterogeneity among the studies (Q 5 11.476, df 5 6, P 5 .075). Therefore, the BRAF mutation was significantly related to the conventional PTC and tall cell variant, but not to the follicular variant.

Histologic Subtype Ten studies addressed BRAF mutation according to the histologic subtype.918 The frequency of BRAF mutation of conventional type, tall cell variant, and follicular variant were compared in 9,9,1118 4,11,13,14,18 and 7 studies,911,13,14,17,18, respectively. The BRAF mutation was present in 337 (59%) of 570 conventional PTCs, in 29 (17%) of 172 follicular variants, and in 34 (79%) of 43 tall cell variants. The BRAF mutation was closely associated with histologic subtype. The overall ORs for BRAF mutation in con-

Extrathyroidal Extension These 12 studies were composed of 474 cases with extrathyroidal extension and 688 cases without extra-

42

CANCER July 1, 2007 / Volume 110 / Number 1

FIGURE 3. Pooled estimates of the association of BRAF mutation and sex.

FIGURE 4. Pooled estimates of the association of BRAF mutation and tumor size.

FIGURE 5. Pooled estimates of the association of BRAF mutation and conventional type papillary carcinoma.

Meta-analysis of BRAF Mutation/Lee et al.

43

FIGURE 6. Pooled estimates of the association of BRAF mutation and tall cell variants of papillary carcinoma.

FIGURE 7. Pooled estimates of the association of BRAF mutation and follicular variants of papillary carcinoma.
thyroidal extension. The BRAF mutation was detected in 283 (41%) of 688 cases of intrathyroidal PTC and in 285 (60%) of 474 cases of extrathyroidal extension of PTC. There was a statistically significant association between extrathyroidal extension and BRAF mutation (OR 5 1.958; 95% CI, 1.5152.530; P 5 .000) (Fig. 8). There was no statistical heterogeneity among the studies (Q 5 14.685, df 5 11, P 5 .197). df 5 11, P 5 .005). There was no significant association between lymph node metastasis and BRAF mutation (Fig. 9) (OR 5 1.500; 95% CI, 0.9922.268; P 5 .055).

Lymph Node Metastasis These 12 studies were composed of 509 cases with lymph node metastasis and 650 cases without lymph node metastasis. The BRAF mutation was detected in 278 (55%) of 509 cases with lymph node metastasis, and was detected in 289 (45%) of 650 cases without lymph node metastasis. Significant statistical heterogeneity was found among the studies (Q 5 26.697,

Clinical Stage Ten studies presented BRAF mutation according to the clinical stage.914,1619 These studies described 688 cases with stages I or II, and 333 with stages III or IV. The BRAF mutation was detected in 295 (43%) of 688 cases classified in the lower stages, and 186 (56%) of 333 cases in the higher stages. There was a significant association between advanced clinical stage and BRAF mutation (Fig. 10) (OR 5 2.143; 95% CI, 1.5972.875; P 5 .000). There was no statistical heterogeneity among the studies (Q 5 15.669, df 5 9, P 5 .074).

44

CANCER July 1, 2007 / Volume 110 / Number 1

FIGURE 8. Pooled estimates of the association of BRAF mutation and extrathyroidal extension.

FIGURE 9. Pooled estimates of the association of BRAF mutation and lymph node metastasis.

DISCUSSION
This meta-analysis showed that BRAF mutation is correlated with histologic subtype, presence of extrathyroidal extension, and advanced clinical stages, but not with age, sex, race, or tumor size of PTC patients.

Interestingly, the BRAF mutation is most frequent in tall cell variant PTC (79.1%) and is second in conventional PTC (59.1%). The BRAF mutation is closely related to the presence of extrathyroidal extension and advanced clinical stages, each with 2-fold ORs.

Meta-analysis of BRAF Mutation/Lee et al.

45

FIGURE 10. Pooled estimates of the association of BRAF mutation and clinical stages.
In this meta-analysis, no significant association was found between ethnicity and BRAF mutation. To determine whether the ethnic background may affect the frequency of BRAF mutation, we divided the population into 2 groups, Caucasian and Asian. The frequency of BRAF mutation ranged from 30% to 83%.920 It was originally reported that the BRAF mutation was detected in 49 (28.8%) of 170 PTCs in a Japanese population.19 Kim et al.15 reported that the BRAF mutation was found in 58 (83%) of 70 PTCs in a Korean population. Our study found that the BRAF mutation is not associated with the age, sex, or tumor size of patients with PTC. The association between the age or sex of patient and BRAF mutation has been the subject of considerable controversy. Nikiforova et al.18 reported that the BRAF mutation was associated with older age. Xu et al.20 reported that the BRAF mutation occurred in PTCs at a significantly higher frequency in male patients. However, other studies failed to show significant relations between age or sex and the BRAF mutation.917,19 Jo et al.10 reported that BRAFmutated PTCs had a larger tumor size. In contrast, Xing et al.14 suggested that tumor sizes in BRAF mutation-positive patients were significantly smaller than those of the mutation-negative group. This study shows that the BRAF mutation is most frequent in tall cell variant PTC (79.1%), second in conventional PTC (59.1%), and lowest in follicular variant PTC (16.9%). The overall ORs for the BRAF mutation in tall cell variant, conventional type, and follicular variant were 4.3, 3.5, and 0.2, respectively. This indicates that the BRAF mutation plays more important roles in the tumorigenesis of tall cell variant and conventional type than in that of other types. However, the diagnostic significance of BRAF mutation may be limited because diagnostic difficulty can arise, especially in follicular variant PTC. Morphologically, the differential diagnosis between follicular variant PTC and follicular tumor is often difficult. Because the prevalence of the BRAF mutation is low in follicular variant PTC, its usefulness as a diagnostic marker may be restricted. This meta-analysis reveals that the BRAF mutation is significantly associated with extrathyroidal extension and advanced clinical stages. Xing et al.14 reported that the BRAF mutation was significantly associated with extrathyroidal extension, lymph node metastasis, and advanced clinical stages. However, several lines of evidence showed that the BRAF mutation was not associated with extrathyroidal extension, lymph node metastasis, or clinical stages.13,15,17 The ORs of extrathyroidal extension and advanced clinical stages were each approximately 2-fold. The results for lymph node metastasis were not clearcut. The OR of lymph node metastasis was 1.500 (95% CI: 0.9922.268; P 5 .055). However, in a fixed model the OR of lymph node metastasis was 1.544 (95% CI: 1.1981.991), and was statistically significant (P 5 .001). In particular, the association between the BRAF mutation and lymph node metastasis is important, because regional lymph node involvement is the main clinical presentation of PTC recurrence.

46

CANCER July 1, 2007 / Volume 110 / Number 1

11. Riesco-Eizaguirre G, Gutierrez-Martinez P, Garcia-Cabezas MA, Nistal M, Santisteban P. The oncogene BRAF V600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na1/I- targeting to the membrane. Endocr Relat Cancer. 2006;13:257269. 12. Kim TY, Kim WB, Song JY, et al. The BRAF mutation is not associated with poor prognostic factors in Korean patients with conventional papillary thyroid microcarcinoma. Clin Endocrinol. 2005;63:588593. 13. Liu RT, Chen YJ, Chou FF, et al. No correlation between BRAFV600E mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan. Clin Endocrinol. 2005;63:461466. 14. Xing M, Westra WH, Tufano RP , et al. BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab. 2005;90:63736379. 15. Kim KH, Kang DW, Kim SH, Seong IO, Kang DY. Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population. Yonsei Med J. 2004;45:818821. 16. Fugazzola L, Mannavola D, Cirello V, et al. BRAF mutations in an Italian cohort of thyroid cancers. Clin Endocrinol. 2004;61:239243. 17. Puxeddu E, Moretti S, Elisei R, et al. BRAF(V599E) mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas. J Clin Endocrinol Metab. 2004;89:2414 2420. 18. Nikiforova MN, Kimura ET, Gandhi M, et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab. 2003;88:53995404. 19. Namba H, Nakashima M, Hayashi T, et al. Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrinol Metab. 2003;88:43934397. 20. Xu X, Quiros RM, Gattuso P, Ain KB, Prinz RA. High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines. Cancer Res. 2003;63: 45614567.

In summary, our study indicates that the BRAF mutation of PTC is associated with histologic subtype, extrathyroidal extension, and advanced clinical stage, but is not correlated with race, patient age, sex, or tumor size of PTC patients. These data suggest that the BRAF mutation is an important and useful prognostic molecular marker, and may add a new dimension to the traditional risk evaluation of PTCs.

REFERENCES
Schlumberger MJ. Papillary and follicular thyroid carcinoma. N Engl J Med. 1998;338:297306. 2. Dean DS, Hay ID. Prognostic indicators in differentiated thyroid carcinoma. Cancer Control. 2000;7:229239. 3. Fagin JA. Minireview: branded from the startdistinct oncogenic initiating events may determine tumor fate in the thyroid. Mol Endocrinol. 2002;16:903911. 4. Nikiforov YE. RET/PTC rearrangement in thyroid tumors. Endocr Pathol. 2002;13:316. 5. Kroll TG, Sarraf P, Pecciarini L, et al. PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma. Science. 2000;289:13571360. 6. Mercer KE, Pritchard CA. Raf proteins and cancer: B-Raf is identified as a mutational target. Biochim Biophys Acta. 2003;1653:2540. 7. Hilger RA, Scheulen ME, Strumberg D. The Ras-Raf-MEKERK pathway in the treatment of cancer. Onkologie. 2002;25:511518. 8. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949954. 9. Lee JH, Lee ES, Kim YS, Won NH, Chae YS. BRAF mutation and AKAP9 expression in sporadic papillary thyroid carcinomas. Pathology. 2006;38:201204. 10. Jo YS, Li S, Song JH, et al. Influence of the BRAF V600E mutation on expression of VEGF in papillary thyroid cancer. J Clin Endocrinol Metab. 2006;91:36673670. 1.