Original Investigation

Research Original Investigation Lack of Association of BRAF Mutation With Negative Prognostic Indicators in Papillary Thyroid Carcinoma The University of California, San Francisco, Experience Christopher Gouveia, MD; Nhu Thuy Can, MD; Alan Bostrom, PhD; James P. Grenert, MD, PhD; Annemieke van Zante, MD, PhD; Lisa A. Orloff, MD IMPORTANCE Papillary thyroid carcinoma (PTC) is the most common endocrine neoplasm. B-type raf kinase (BRAF) V600E mutation has been proposed as a negative prognostic indicator in PTC, and patients harboring it should receive more aggressive initial therapy. OBJECTIVE To assess the significance of BRAF V600E mutation in PTC in the largest US sample to date. DESIGN We identified patients from our institution s pathology archives diagnosed as having PTC and meeting criteria for BRAF mutation testing. Medical records were analyzed for BRAF status (positive or negative) and a list of standardized clinicopathologic features. PARTICIPANTS A total of 429 patients with PTC at an academic medical center. MAIN OUTCOMES AND MEASURES Clinicopathologic features in patients with PTC with and without BRAF mutation. RESULTS Of 429 cases with PTC, 314 (73.2%) were positive forthe BRAF mutation and 115 (26.8%) tested negative. BRAF mutation was significantly associated with tumor margin positivity (P =.03) and lymph node metastasis (P =.002) on univariate analysis but not on multivariate study. BRAF mutation was a predictor of male sex (odds ratio [OR], 3.2; 95% CI, 1.4-7.2), total thyroidectomy (OR, 2.6; 95% CI, 1.1-6.2), and a negative predictor of follicular variant PTC (OR, 0.1; 95% CI, 0.1-0.4). There was no significant association between BRAF positivity and tumor multicentricity, lymphovascular invasion, extranodal extension, central neck involvement, advanced stage (stage III or IV), and distant metastasis. CONCLUSIONS AND RELEVANCE BRAF V600E mutation has been extensively studied in relation to negative prognostic indicators in PTC, with no consistent relationship emerging. Two recent meta-analyses showed an overall association between BRAF status and aggressive disease features and called for tailoring treatment plans in patients accordingly. In this, the largest US study to date, BRAF status was not significantly associated with most clinicopathologic features suggestive of more aggressive disease. JAMA Otolaryngol Head Neck Surg. 2013;139(11):1164-1170. doi:10.1001/jamaoto.2013.4501 Published online September 12, 2013. Author Affiliations: Department of Otolaryngology Head and Neck Surgery, University of California, San Francisco, San Francisco (Gouveia, Bostrom, Orloff); Department of Otolaryngology Head and Neck Surgery, Northwestern University, Chicago, Illinois (Gouveia); Department of Anatomic Pathology, University of California, San Francisco, San Francisco (Can, Grenert, van Zante); Department of Pathology, University of Chicago, Chicago, Illinois (Can). Corresponding Author: Lisa A. Orloff, MD, Department of Otolaryngology Head and Neck Surgery, University of California, San Francisco, 2380 Sutter St, Second Floor, San Francisco, CA 94115 (lorloff@ohns.ucsf.edu). 1164 jamaotolaryngology.com

BRAF Mutation and Papillary Thyroid Carcinoma Original Investigation Research Papillary thyroid carcinoma (PTC) is the most common endocrine neoplasm, accounting for 88% of all thyroid malignant neoplasms. 1 Although treatment is often curative, there is a 15% recurrence rate over the course of 10 years, and approximately 10% of patients die as a result of disease progression. 2,3 Furthermore, the incidence of PTC is increasing across all demographics. 4 The association of B-type raf (BRAF) kinase V600E mutation to PTC has been extensively studied. Multiple individual publications have shown correlation of BRAF mutation with known negative prognostic clinicopathologic features, including advanced age at diagnosis, male sex, larger tumor size, extrathyroidal extension, tumor multifocality, advanced TNM stage, lymph node metastasis, and recurrence. 5-11 In addition, BRAF mutation has been reported to be associated with overall increased mortality. 12 However, the results across studies have been inconsistent, and several similar large retrospective trials have failed to corroborate these results. 13-17 The overall significance of BRAF mutation in PTC therefore remains debatable. 18 Two recent meta-analyses have shown an overall correlation of BRAF with extrathyroidal extension, lymph node metastasis, advanced TNM stage, and recurrent disease. 2,19 There remains no definitive correlation between BRAF V600E mutation and the clinicopathologic features of PTC, but these studies add support to the consensus that BRAF mutation is an overall negative prognostic indicator and call for consideration of tailoring initial treatment accordingly. The usual limitations of meta-analysis, along with wide variation in BRAF mutation rates and heterogeneity in geographic subgroups among the included studies, restrict the translation of these results. There is a continuing need for large, nation-wide studies of the relationship between BRAF mutation and PTC. The aim of this study was to summarize our institution s experience with BRAF mutation in PTC to provide the largest US study to date. To accomplish this, we analyzed a spectrum of clinical and pathologic features in PTC tumors with and without BRAF V600E mutation. Methods This retrospective study was approved by our institutional review board. The Department of Pathology at the University of California, San Francisco, began routine BRAF V600E testing of all PTC tumor specimens 0.4 cm or larger in April 2009. We identified 439 patients as having had BRAF testing of their thyroid specimens. We included all patients with PTC who underwent partial or total thyroidectomy with or without lymph node sampling or neck dissection between April 2009 and April 2012. We excluded 10 patients for the following reasons: 4 for incomplete medical records, 3 for initial pathological results that were performed at an outside hospital, 2 for unknown primary tumor, and 1 for recurrent disease. All patient medical records were reviewed for BRAF status, age, sex, type of surgery, PTC subtype, multicentricity, extrathyroidal extension, lymphovascular invasion, tumor margin status, lymph node status (including central neck Table 1. Clinicopathologic Characteristics of the 429 Patients With Papillary Thyroid Carcinoma in the Study Population Characteristic No. (%) Age at diagnosis, y Mean (SD) [range] 46.8 (14.4) [15-84] 45 243 (56.6) <45 186 (43.4) Male 102 (23.8) Female 327 (76.2) Total thyroidectomy 367 (85.5) Hemithyroidectomy 62 (14.5) Tumor size, cm a Mean (SD) [range] 1.81 (1.25) [0.15-8.50] 2 293 (68.3) >2 to <4 105 (24.5) 4 31 (7.7) Histologic subtype Classic type 370 (86.2) Follicular variant 43 (10.0) Other 16 (3.7) Multicentric 232 (54.1) Extrathyroidal extension 73 (17.0) Lymphatic invasion 42 (9.8) Vascular invasion 25 (5.8) Lymph node involvement 167 (38.9) Extranodal extension 58 (13.5) Central neck dissection Not performed 282 (65.7) Positive 99 (23.1) Negative 36 (8.4) Level not specified 12 (2.8) Margin involvement 98 (22.8) Distant metastases 11 (2.6) TNM stage b I 206 (48.0) II 20 (4.7) III 45 (10.5) IV 45 (10.5) a Largest tumor diameter based on either gross or microscopic measurement. b Based on criteria established by American Joint Cancer Committee Union Internationale Contre le Cancer, Seventh Edition, Staging System. involvement), extranodal extension, distant metastasis, and final TNM stage. BRAF Testing All specimens were formalin fixed and paraffin embedded. Areas of PTC with at least 50% tumor cells were identified and circled on hematoxylin-eosin stained slides by a pathologist. These areas were scraped from corresponding unstained slides for DNA extraction using either the QIAamp DNA Mini Kit or EZ1 automated extraction system (both, Qiagen). A portion of BRAF exon 15 encompassing codon 600 was amplified by polymerase chain reaction (PCR) with specific primers, and codon jamaotolaryngology.com JAMA Otolaryngology Head & Neck Surgery November 2013 Volume 139, Number 11 1165

Research Original Investigation BRAF Mutation and Papillary Thyroid Carcinoma Table 2. Clinicopathologic Features of Papillary Thyroid Carcinoma With and Without the BRAF V600E Mutation in 429 Patients Mutation, No. (%) Positive Negative Characteristic (n = 314) (n = 115) Age at diagnosis, y 45 177 (72.8) 66 (27.2) <45 137 (73.7) 49 (26.3) Male 82 (80.4) 20 (19.6) Female 232 (70.9) 95 (29.1) Total thyroidectomy 283 (77.1) 84 (22.9) Hemithyroidectomy 31 (50.0) 31 (5.0) Tumor size, cm a 0 to 2 224 (76.5) 69 (23.5) >2 to <4 72 (68.6) 33 (31.4) 4 18 (58.1) 13 (41.9) Histologic subtype Classic type 297 (80.3) 73 (19.7) Follicular variant 11 (25.6) 32 (74.4) Other 6 (37.5) 10 (62.5) Multicentric Yes 167 (72.0) 65 (28.0) No 147 (74.5) 50 (25.5) Extrathyroidal extension Yes 60 (82.2) 13 (17.8) No 254 (71.3) 102 (28.7) Lymphatic invasion Yes 34 (81.0) 8 (19.0) No 280 (72.4) 107 (27.6) Vascular invasion Yes 17 (68.0) 8 (32.0) No 297 (73.5) 107 (26.6) Lymph node involvement Yes 136 (81.4) 31 (18.6) No 94 (65.7) 49 (34.3) Extranodal extension Yes 46 (79.3) 12 (20.7) No 181 (72.7) 68 (27.3) Central neck dissection Not performed 202 (71.6) 80 (28.4) Performed 100 (74.1) 35 (25.9) Positive 73 (73.7) 26 (26.3) Negative 27 (75.0) 9 (25.0) Margin involvement Yes 79 (80.6) 19 (19.4) No 235 (71.0) 96 (29.0) Distant metastases Yes 10 (90.9) 1 (9.1) No 304 (72.7) 114 (27.3) Advanced stage (III and IV) b Yes 71 (78.9) 19 (21.1) No 162 (71.7) 64 (28.3) P Value.91.07 <.001.02 <.001.59.06.27.64.002.32.72.63.30.30.21 a Largest tumor diameter based on either gross or microscopic measurement. b Based on criteria established by American Joint Cancer Committee Union Internationale Contre le Cancer, Seventh Edition, Staging System. 1166 JAMA Otolaryngology Head & Neck Surgery November 2013 Volume 139, Number 11 jamaotolaryngology.com

BRAF Mutation and Papillary Thyroid Carcinoma Original Investigation Research 600 was analyzed with fluorescence-labeled hybridization probes in a real-time LightCycler 480 PCR (Roche Applied Science) melting curve assay following the protocol of Rowe et al. 20 A melting temperature of approximately 65 C corresponds with the wild-type sequence, while melting at approximately 60 C indicates the T to A transversion at nucleotide 1799 that results in the V600E mutation. This assay was validated to have sensitivity for V600E mutation detection down to a minimum of at least 25% tumor cells in the specimen. Statistical Analysis Associations of demographic and clinical variables with BRAF were analyzed using Fisher exact test for nominal variables and a Mann-Whitney nonparametric test for ordinal and continuous variables. All P values were 2-tailed, and P <.05 was considered statistically significant. Variables associated with BRAF V600E mutation at P <.10 were used in a multivariate logistic regression model for BRAF mutation positivity. All computations were performed using SAS statistical software (version 9.2). Results A total of 429 patients with PTC from our institution met inclusion criteria. A total of 314 (73.2%) were positive for BRAF, and 115 (26.8%) tested negative. Results of demographic features (Table 1), univariate analysis (Table 2), and multivariate analysis (Table 3) are shown. Demographics There was no significant association of BRAF mutation with patientage (P =.91). Ahigherproportionofmalepatientswasfound to be BRAF positive compared with female patients (80.4% vs 70.9%), but this was not significant (P =.07). On multivariate analysis, male sex was found to be a predictor of positive BRAF status (odds ratio [OR], 3.2; 95% CI, 1.4-7.2). Patients who underwent total thyroidectomy were more likely to test positive for BRAF than those who received hemithyroidectomy (77.1% v. 50.0%; P <.001), and total thyroidectomy was a significant predictor of BRAF mutation (OR, 2.6; 95% CI, 1.1-6.2). Tumor Characteristics Classic-type PTC was significantly associated with BRAF mutation (P <.001) vs follicular variant and other, less common subtypes. Follicular variant was a predictor of negative status (OR, 0.1; 95% CI, 0.1-0.4). When examining patients with classic-type PTC in our population, smaller tumor size was the only clinicopathologic feature significantly associated with BRAF V600E mutation (Table 4). There was no correlation between BRAF mutation and tumor size (P =.15). The BRAF mutation-positive group had a significant association with tumor margin positivity (P =.03) and a trend toward increased extrathyroidal extension (P =.06). In multivariate models, these were not significantly associated with BRAF status. There was no significant association between tumor multicentricity (P =.59) or lymphovascular invasion with BRAF V600E mutation (P =.27 and P =.64, respectively). Table 3. Multivariate Analysis of the Association of BRAF V600E Mutation and Clinicopathologic Features of Papillary Thyroid Cancer Characteristic Odds Ratio (95% CI) P Value Male 3.2 (1.4-7.2) Female.06 Total thyroidectomy 2.6 (1.1-6.2) Hemithyroidectomy.32 Tumor size, cm a 0.8 (0.7-1.1).15 Histologic subtype Classic type Follicular variant 0.1 (0.1-0.4) <.001 Other 0.3 (0.1-1.3).10 Extrathyroidal extension Yes 0.9 (0.4-2.1) No.82 Lymph node involvement Yes 1.4 (0.7-2.6) No.36 Margin involvement Yes 2.0 (0.9-4.4) No.10 a Largest tumor diameter based on either gross or microscopic measurement. Lymph Node Features and Metastasis BRAF status had no association with central neck dissection (P =.72) or with positive central compartment lymph nodes (P =.63). Lymph node metastasis was significantly associated with BRAF mutation (P =.002). This association did not remain significant in multivariate analysis (OR, 1.4; 95% CI, 0.7-2.6). There was no significant relationship with BRAF status and extranodal extension (P =.32). There was no significant association between distant metastasis or advanced-stage disease (stage III and IV) and BRAF positivity. Discussion The significance of BRAF mutation in the management of PTC remains unclear. Several articles have shown a significant association between poor prognostic variables and BRAF status, 5-12 but the results are inconsistent with some studies showing a lack thereof. 13-17 Despite this, 2 recent meta-analyses 2,19 that showed an overall relationship between BRAF mutation and high-risk features concluded that steps should be taken to modify initial treatments in BRAF-positive patients with PTC. Given this controversy, the purpose of our study was to provide a large, single-institution sampling to evaluate the association of BRAF mutation and poor prognostic variables in PTC. To our knowledge, this is the largest US study examining this relationship. Our results showed a lack of association between BRAF mutation status and most clinicopathologic risk jamaotolaryngology.com JAMA Otolaryngology Head & Neck Surgery November 2013 Volume 139, Number 11 1167

Research Original Investigation BRAF Mutation and Papillary Thyroid Carcinoma Table 4. Clinicopathologic Features of Classic-Type Papillary Thyroid Carcinoma With and Without the BRAF V600E Mutation Mutation, No. (%) Positive Negative Characteristic (n = 297) (n = 73) Age at diagnosis, y 45 165 (79.7) 42 (20.3) <45 132 (81.0) 31 (19.0) Male 80 (26.9) 13 (17.8) Female 217 (73.1) 60 (82.2) Total thyroidectomy 268 (81.5) 61 (18.5) Hemithyroidectomy 29 (70.7) 12 (29.3) Tumor size, cm a 0 to 2 213 (82.9) 44 (17.1) >2 to <4 69 (78.4) 19 (21.6) 4 15 (60.0) 10 (40.0) Multicentric Yes 154 (76.6) 47 (23.4) No 143 (84.5) 26 (15.5) Extrathyroidal extension Yes 59 (85.5) 10 (14.5) No 238 (79.1) 63 (20.9) Lymphatic invasion Yes 33 (84.6) 6 (15.4) No 265 (79.8) 67 (20.2) Vascular invasion Yes 15 (78.9) 4 (21.1) No 282 (80.3) 69 (19.7) Lymph node involvement Yes 132 (83.5) 26 (16.5) No 85 (75.9) 27 (24.1) Extranodal extension Yes 44 (80.0) 11 (20.0) No 171 (80.3) 42 (19.7) Central neck dissection Not performed 195 (80.9) 46 (19.1) Performed 97 (78.2) 27 (21.8) Positive 72 (76.6) 22 (23.4) Negative 25 (83.3) 5 (16.7) Margin involvement Yes 76 (84.4) 14 (15.6) No 221 (78.9) 59 (21.1) Distant metastases Yes 8 (88.9) 1 (11.1) No 289 (81.4) 66 (18.6) Advanced stage (III and IV) b Yes 61 (81.3) 14 (18.7) No 236 (80.0) 59 (20.0) P Value.79.13.09.02.07.31.67.78.12 <.99.58.61.22 <.99.87 a Largest tumor diameter based on either gross or microscopic measurement. b Based on criteria established by American Joint Cancer Committee Union Internationale Contre le Cancer, Seventh Edition, Staging System. factors studied. Only male sex, increased rate of total thyroidectomy, and classic-type PTC were significantly associated with positive BRAF status on multivariate analysis. Male sex is an established poor prognostic factor in PTC 19 and was found to be a predictor of positive BRAF status on multivariate analysis in our study. This association is inconsistent with findings in prior studies, with many articles unable to establish a relationship between patient sex and the presence of BRAF mutation. 6,10,12,14,15,21-23 There was a significant association between BRAF V600E positivity and receiving a 1168 JAMA Otolaryngology Head & Neck Surgery November 2013 Volume 139, Number 11 jamaotolaryngology.com

BRAF Mutation and Papillary Thyroid Carcinoma Original Investigation Research total vs subtotal thyroidectomy in our patient population. The cause for this association is unclear but may be due to BRAF mutation being associated with higher-risk fine-needle aspiration features or neck ultrasonographic findings that would sway a surgeon toward definitive surgery; we did not examine these factors in our study. We also found a significant association between BRAF mutation and classic-type PTC, whereas follicular variant PTC was found to be a negative predictor of BRAF mutation status on multivariate analysis. This association between BRAF mutation and PTC variants is well documented and highlights the importance of classifying subtype in the study and management of PTC. 7,10,13-15,22,23 We conducted a separate analysis of patients with classic-type PTC only and found no significant differences from our overall study population. Our study included 59 patients with nonclassic PTC. While this could contribute to our study s overall findings, it is a small percentage of our sample and is accounted for in the multivariate analysis. There are several possible explanations for the difference between our findings and those of prior studies showing significant association of BRAF mutation and negative prognostic factors in PTC. We analyzed data for 429 patients, the largest study in the United States and one of the largest internationally, which could show differences vs smaller individual studies with less power. BRAF-positive patients accounted for 74% of our study population. While this is within the range of prior studies, it is on the higher end of the spectrum and likely represents a heterogeneous population. Our sample is from 1 region of the United States, in contrast with prior studies, most of which are international. However, the patient population in our study is ethnically diverse. Genetic, dietary, and other environmental differences have been hypothesized as accounting for differences in incidence of BRAF mutation, although we did not analyze specific ethnic factors in this study. In the metaanalysis by Kim et al, 2 subgroup analysis by country and BRAF mutation rates showed the association between BRAF mutation and negative prognostic variables remained true overall but could be significantly affected by each, strengthening our study s findings. Our study did not evaluate disease recurrence and mortality association. Papillary thyroid carcinoma is a slowly progressive, often curable disease, so obtaining long-term follow-up data is an area in need of further study. The role and value of central neck dissection in PTC is still being investigated, and its relationship to BRAF mutation has been inconsistent. 11,24,25 Our study found no significant association between central neck dissection rates or positive central neck lymph nodes with BRAF mutation status. In conclusion, the results of the present study show little correlation between BRAF V600E mutation and negative prognostic indicators in PTC. If anything, the absence of BRAF mutation may prove to be a favorable prognostic indicator and a basis for deescalation of therapy, such as more selective use of radioactive iodine or lymph node dissection, in patients with PTC. Such data have not yet been adequately studied. Certainly, efforts to recommend more aggressive initial therapies to BRAF-positive patients with PTC should be tempered until more long-term data related to outcome are available. ARTICLE INFORMATION Submitted for Publication: February 27, 2013; final revision received May 16, 2013; accepted June 21, 2013. Published Online: September 12, 2013. doi:10.1001/jamaoto.2013.4501. Author Contributions: Dr Orloff had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Gouveia, Can, van Zante, Orloff. Acquisition of data: Gouveia, Can, Grenert, van Zante, Orloff. Analysis and interpretation of data: Gouveia, Can, Bostrom, Grenert, Orloff. Drafting of the manuscript: Gouveia, Can, Bostrom. Critical revision of the manuscript for important intellectual content: Gouveia, Grenert, van Zante, Orloff. Statistical analysis: Gouveia, Bostrom. Administrative, technical, or material support: Grenert, Orloff. Study supervision: van Zante, Orloff. Conflict of Interest Disclosures: None reported. Previous Presentation: This study was presented at the American Head and Neck Society 2013 Annual Meeting; April 11, 2013; Orlando, Florida. REFERENCES 1. Davies L, Welch HG. Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA. 2006;295(18):2164-2167. 2. Kim TH, Park YJ, Lim JA, et al. The association of thebraf (V600E) mutation with prognostic factors and poor clinical outcome in papillary thyroid cancer: a meta-analysis. Cancer. 2012;118(7):1764-1773. 3. Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. Cancer. 1998;83(12):2638-2648. 4. Chen AY, Jemal A, Ward EM. Increasing incidence of differentiated thyroid cancer in the United States, 1988-2005. Cancer. 2009;115(16):3801-3807. 5. XingM,WestraWH,TufanoRP,etal.BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab. 2005;90(12):6373-6379. 6. Yip L, Nikiforova MN, Carty SE, et al. Optimizing surgical treatment of papillary thyroid carcinoma associated with BRAF mutation. Surgery. 2009;146(6):1215-1223. 7. Xing M. BRAF mutation in thyroid cancer. Endocr Relat Cancer. 2005;12(2):245-262. 8. Lin KL, Wang OC, Zhang XH, Dai XX, Hu XQ, Qu JM. The BRAF mutation is predictive of aggressive clinicopathological characteristics in papillary thyroid microcarcinoma. Ann Surg Oncol. 2010;17(12):3294-3300. 9. Chakraborty A, Narkar A, Mukhopadhyaya R, Kane S, D Cruz A, Rajan MG. BRAF V600E mutation in papillary thyroid carcinoma: significant association with node metastases and extra thyroidal invasion. Endocr Pathol. 2012;23(2):83-93. 10. Kim SJ, Lee KE, Myong JP, et al. BRAF V600E mutation is associated with tumor aggressiveness in papillary thyroid cancer. World J Surg. 2012;36(2):310-317. 11. Howell GM, Nikiforova MN, Carty SE, et al. BRAF V600E mutation independently predicts central compartment lymph node metastasis in patients with papillary thyroid cancer. Ann Surg Oncol. 2013;20(1):47-52. 12. Elisei R, Ugolini C, Viola D, et al. BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median follow-up study. J Clin Endocrinol Metab. 2008;93(10): 3943-3949. 13. 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Research Original Investigation BRAF Mutation and Papillary Thyroid Carcinoma 15. 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 (Oxf). 2005;63(5):588-593. 16. Brzeziańska E, Pastuszak-Lewandoska D, Wojciechowska K, et al. Investigation of V600E BRAF mutation in papillary thyroid carcinoma in the Polish population. Neuro Endocrinol Lett. 2007;28(4):351-359. 17. Ito Y, Yoshida H, Maruo R, et al. BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients.endocr J. 2009;56(1):89-97. 18. Sipos JA, Mazzaferri EL. Thyroid cancer epidemiology and prognostic variables. Clin Oncol (R Coll Radiol). 2010;22(6):395-404. 19. Tufano RP, Teixeira GV, Bishop J, Carson KA, Xing M. BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine (Baltimore). 2012;91(5):274-286. 20. Rowe LR, Bentz BG, Bentz JS. Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis. J Clin Pathol. 2007;60(11):1211-1215. 21. Rivera M, Ricarte-Filho J, Tuttle RM, et al. Molecular, morphologic, and outcome analysis of thyroid carcinomas according to degree of extrathyroid extension. Thyroid. 2010;20(10): 1085-1093. 22. Lupi C, Giannini R, Ugolini C, et al. Association of BRAF V600E mutation with poor clinicopathological outcomes in 500 consecutive cases of papillary thyroid carcinoma. J Clin Endocrinol Metab. 2007;92(11):4085-4090. 23. Ricarte-Filho J, Ganly I, Rivera M, et al. Papillary thyroid carcinomas with cervical lymph node metastases can be stratified into clinically relevant prognostic categories using oncogenic BRAF,the number of nodal metastases, and extra-nodal extension. Thyroid. 2012;22(6):575-584. 24. Paulson L, Shindo M, Schuff K, Corless C. The role of molecular markers and tumor histological type in central lymph node metastasis of papillary thyroid carcinoma. Arch Otolaryngol Head Neck Surg. 2012;138(1):44-49. 25. Shen WT, Ogawa L, Ruan D, et al. Central neck lymph node dissection for papillary thyroid cancer: comparison of complication and recurrence rates in 295 initial dissections and reoperations. Arch Surg. 2010;145(3):272-275. 1170 JAMA Otolaryngology Head & Neck Surgery November 2013 Volume 139, Number 11 jamaotolaryngology.com