Papillary Thyroid Carcinoma With BRAF V600E Mutation: Sonographic Prediction

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1 Neuroradiology/Head and Neck Imaging Original Research Hwang et al. Papillary Thyroid Carcinoma Neuroradiology/Head and Neck Imaging Original Research Jiyoung Hwang 1 Jung Hee Shin 1 Boo-Kyung Han 1 Eun Young Ko 1 Seok Seon Kang 1 Jong-Won Kim 2 Jae Hoon Chung 3 Hwang J, Shin JH, Han B-K, et al. Keywords: BRAF mutation, papillary thyroid carcinoma, thyroid cancer, ultrasonography DOI: /AJR Received August 18, 2009; accepted after revision November 10, Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine 50, Irwon-dong, Gangnam-gu, Seoul , Republic of Korea. Address correspondence to J. H. Shin (jhshin11@skku.edu). 2 Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 3 Division of Endocrinology and Metabolism, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. WEB This is a Web exclusive article. AJR 2010; 194:W425 W X/10/1945 W425 American Roentgen Ray Society Papillary Thyroid Carcinoma With BRAF V600E Mutation: Sonographic Prediction OBJECTIVE. The purpose of this article is to assess the clinical and sonographic findings that can predict the presence of the BRAF V600E mutation of a papillary thyroid carcinoma. MATERIALS AND METHODS. The study included 135 consecutive patients with surgically confirmed papillary thyroid carcinoma. All patients underwent ultrasound and ultrasound-guided fine-needle aspiration. The BRAF V600E mutation was determined using allelespecific polymerase chain reaction and direct DNA sequencing from fine-needle aspiration samples. We retrospectively compared the clinical and ultrasound findings of nodules with and without the mutation, including size, margin, shape, calcifications, echogenicity, and ultrasound diagnosis. RESULTS. Of 135 patients, results were positive for the mutation for 106 patients (79%) and negative for 29 (21%). Among the patients with the BRAF V600E mutation, the ratio of men to women was 1:5.2 (p = 0.156), and the mean age was 47 years (range, years; p = 0.326). The mean tumor size was 0.9 cm (range, cm; p = 0.099). On ultrasound, papillary thyroid carcinomas with the BRAF V600E mutation tended to show a taller-than-wide shape, but this finding was not statistically significant (p = 0.055). The BRAF V600E mutation was not associated with the presence of calcifications (54% vs 71%; p = 0.032), although it was not an independent predictor. There were no significant differences in tumor echogenicity, tumor margin, and ultrasound diagnosis between the two groups. CONCLUSION. Papillary thyroid carcinoma with the BRAF V600E mutation tends to be taller than wide and is not associated with the presence of calcifications on ultrasound. However, these findings are not specific enough to predict the presence or absence of the BRAF V600E mutation of a papillary thyroid carcinoma. P apillary thyroid carcinoma is the most common endocrine malignancy, and its incidence is rapidly rising in many areas of the world [1 3]. The rising incidence of thyroid cancer is almost entirely attributed to the increased diagnosis of papillary thyroid carcinoma [1, 3]. Papillary thyroid carcinoma is frequently associated with genetic alterations leading to the activation of the mitogen-activated protein kinase signal pathway [4 8]. The genetic alterations include RET/PTC rearrangement, representing recombinant protein product of a chromosomal rearrangement with the combination of the 3 portion of the RET gene and the 5 portion of an unrelated gene, and point mutations of the Ras and the B-type Raf kinase (BRAF) genes [9, 10]. An activating mutation of the BRAF gene located on exon 15 results in a valine-to glutamic acid substitution at amino acid 600 (BRAF V600E mutation) and has been found to be the most common mutation in papillary thyroid carcinoma [11]. The BRAF V600E mutation contributes to the destabilization of the kinase encoded by the gene, which then becomes constitutively activated, promoting tumorigenesis through the action of the mitogen-activated protein kinase pathway. In Korea, the BRAF V600E mutation is present in up to 84% of papillary thyroid carcinomas [12, 13]. Numerous studies have investigated the prevalence of the BRAF V600E mutation and the association between the BRAF V600E mutation and clinicopathologic parameters in papillary thyroid carcinoma. The prevalence of the mutation is highly variable, ranging from 29% to 83% in different studies [14, 15]. Although the results are not entirely consistent, many studies have shown a significant association between the BRAF V600E AJR:194, May 2010 W425

2 Hwang et al. mutation and one or more conventional highrisk clinicopathologic characteristics of papillary thyroid carcinoma [12, 16, 17]. These results suggest that preoperative knowledge of the BRAF V600E mutation status may be helpful to surgeons and endocrinologists to plan an appropriate surgical strategy and medical management. High-resolution sonography is becoming the method of choice for the detection and diagnosis of thyroid nodules and guidance of fineneedle aspiration. This study was designed to assess the clinical and sonographic findings that can predict the presence of the BRAF V600E mutation of a papillary thyroid carcinoma. TABLE 1: Correlation between the BRAF V600E mutation and various clinicopathologic parameters in 135 patients With papillary thyroid carcinoma Clinicopathologic Parameters Materials and Methods Patient Selection and Imaging This study was conducted with the approval of the institutional review board of our institution. Informed consent was received from patients before undergoing the ultrasound-guided fine-needle aspiration procedure and genetic analysis. A total of consecutive 135 patients (110 women and 25 men; mean age, 47 years; age range, years) with a papillary thyroid carcinoma that had been confirmed surgically were included in the study. All of the patients underwent ultrasound and ultrasound-guided fine-needle aspiration between January 2008 and December The reason for performing fine-needle aspiration in 135 patients was that 117 patients (87%) were referred for an incidentaloma at other hospitals, and 18 patients (13%) had clinical symptoms. The sonographic images were obtained using either HDI 5000 or IU22 ultrasound scanners (Philips Healthcare) equipped with a commercially available 7- to 12- MHz linear-array transducer. Four radiologists who specialized in thyroid sonography performed all of the sonographic examinations. The four radiologists had an experience of 10 years, 7 years, 6 years, and 4 years, respectively, for the performance and interpretation of thyroid ultrasound. Two radiologists retrospectively analyzed the sonographic findings, including tumor size, margin, and shape; the presence or absence of calcifications and echogenicity; and sonographic diagnosis in consensus. The shape of a nodule was categorized as follows: roundto-oval (when the anteroposterior diameter of the nodule was equal to or less than the transverse diameter), taller-than-wide (when the anteroposterior diameter of a nodule was longer than its transverse diameter), or irregular (when a nodule was neither round-to-oval nor taller-than-wide). The margin of a nodule was categorized as well defined, spiculated, or ill defined. The presence or absence of calcifications was assessed. The calcification patterns, if present on ultrasound, were classified as microcalcifications (when there were tiny, punctuate echogenic foci of 1 mm, either with or without posterior shadowing), macrocalcifications (when punctate echogenic foci were > 1 mm), and peripheral calcifications (when a nodule had a peripheral curvilinear or eggshell calcification). Echogenicity of a nodule was assessed with respect to the thyroid parenchyma and strap muscles and was classified as hyperechoic, isoechoic, or hypoechoic compared with the normal thyroid Presence of BRAF V600E Mutation Positive Negative No. (%) of patients 106 (79) 29 (21) Women:men 89:17 21: a Age (y), mean ± SD (range) 47 ± 9.2 (26 73) 45 ± 9.3 (31 64) b Multifocality 35/106 (33) 13/29 (45) a Extrathyroid extension 67/106 (63) 17/29 (59) a Lymph node metastases 47/106 (44) 18/29 (62) a Central 38/47 (81) 15/18 (83) Central and lateral 9/47 (19) 3/18 (17) Note Except where noted, data are number/total number (%). a p values were calculated using chi-square test. b p values were calculated using two-sample Student s t test. parenchyma, and as markedly hypoechoic when a nodule showed a relatively hypoechoic pattern compared with the anterior strap muscle [18]. The sonographic criteria for a malignant nodule were as follows: the presence of microcalcifications or macrocalcifications, a spiculated margin, marked hypoechogenicity, and a taller-than-wide shape, when at least one of above findings was present [18]. Lymph nodes were considered metastatic if they were round or if there was calcification, cystic change, or hyperechogenicity. TABLE 2: Correlation between the BRAF V600E mutation and sonographic findings in 135 patients With papillary thyroid carcinoma Sonographic Findings BRAF V600E mutation Present (n = 106 [79%]) Absent (n = 29 [21%]) Tumor size (cm), mean ± SD (range) 0.9 ± 0.6 ( ) 1.1 ± 0.7 ( ) a Sonographic diagnosis for malignancy 72/106 (68) 23/29 (79) b Tumor margin Circumscribed 23/106 (22) 7/29 (24) Spiculated 68/106 (64) 17/29 (59) Ill-defined 15/106 (14) 5/29 (17) Tumor shape Round-to-oval 27/106 (26) 7/29 (24) Taller-than-wide 63/106 (59) 12/29 (41) Irregular 16/106 (15) 10/29 (35) Echogenecity Isoechoic 13/106 (12) 3/29 (10) Hypoechoic 55/106 (52) 15/29 (52) Marked hypoechoic 38/106 (36) 11/29 (38) p p b b b Calcifications 57/106 (54) 22/29 (76) b Note Except where noted, data are number/total number (%). a p values were calculated using Mann-Whitney test. b p values were calculated using chi-square test. W426 AJR:194, May 2010

3 Papillary Thyroid Carcinoma Clinicopathologic parameters, including sex, age at the time of diagnosis, multifocality, extrathyroid extension, and the presence of a lymph node metastasis, were also reviewed for each patient. Detection of the BRAF V600E Mutation All patients underwent an ultrasound-guided fine-needle aspiration. Fine-needle aspiration was performed using 23-gauge needles attached to a 2-mL syringe with or without administration of local anesthesia. The washout samples obtained from aspiration were sent to laboratory medicine department to evaluate BRAF V600E mutation status. Genomic DNA was extracted from aspirated thyroid cells using the QIAamp DNA Mini kit (QIAGEN) according to the manufacturer s instructions. Both direct DNA sequencing and allele-specific polymerase chain reaction were used to detect the BRAF V600E mutation. Scoring for a mutation was positive even if only one of the two methods identified the presence of the mutation. Statistical Analysis We used the Fisher s exact test, the two-sample Student s t test, Mann-Whitney test, and chisquare test to analyze the clinicopathologic and radiologic data of the patients with and those without the BRAF V600E mutation. The multiple logistical regression test was used to determine independent parameters of the BRAF V600E mutation. A p value of less than 0.05 was regarded as statistically significant. Results The 135 patients were distinguished according to the presence or absence of the BRAF V600E mutation in fine-needle aspiration samples: 106 (79%) of the patients had the mutation, and 29 (21%) of the patients did not have the mutation. Clinicopathologic and radiologic features were compared between the two groups (Tables 1 and 2). Among the BRAF V600E mutation positive patients, the ratio of men to women was 1:5.2 (p = ), and the mean age was 47 years (age range, years; p = ). The mean tumor size was 0.9 cm (range, cm; p = ). Seventy-five (56%) of the 135 papillary thyroid carcinomas were microcarcinomas ( 1 cm). All patients underwent total thyroidectomy with routine central neck dissection. Twelve patients with lateral neck node metastases underwent modified lateral neck dissection because of suspected lateral neck metastasis seen on preoperative images. None of these patients was positive for distant metastases except for lung metastasis in one of the mutation-positive patients. On the basis of surgical pathologic examinations, multifocality was seen in 33% (35/106) of the mutation-positive patients and in 45% (13/29) of the mutation-negative patients (p = ). Extrathyroid extension and the presence of a lymph node metastasis was seen in 63% (67/106) and 44% (47/106) of the mutation-positive patients and in 59% (17/29) and 62% (18/29) of the mutation-negative patients, respectively (p = and p = , respectively). All three pathologic findings showed no statistically significant difference between the two groups. Lateral node metastases were more common for the mutation-positive patients than for the mutationnegative patients, but there was no statistical difference (19% [9/47] vs 17% [3/18]) (p = ). Histologic examinations of the 135 patients revealed that 130 (96%) had conventional papillary thyroid carcinomas, four (3%) had follicular variants, and one (1%) had tall cell variant. The mean follow-up period after surgery was 13 months (range, 6 18 months). The sonographic diagnosis was malignant in 68% of the mutation-positive patients and in 79% of the mutation-negative patients. No statistical difference between the two groups was seen for the sonographic diagnosis (p = ). On ultrasound, papillary thyroid Fig year-old woman with papillary thyroid carcinoma that was positive for BRAF V600E mutation. On transverse sonogram, 0.7-cm hypoechoic nodule (arrow) with taller-than-wide shape is noted in left thyroid gland. There is no calcification within nodule. BRAF V600E mutation from aspiration sample was identified using allele-specific polymerase chain reaction and direct DNA sequencing. Cytologic analysis and surgical pathologic analysis showed papillary thyroid carcinoma. Fig year-old woman with papillary thyroid carcinoma that was negative for BRAF V600E mutation. On transverse sonogram, 1.4-cm taller-than-wide hypoechoic nodule (arrows) with microcalcifications is noted in right thyroid gland. Sonography diagnosed nodule as malignancy. Sonography-guided fine-needle aspiration and total thyroidectomy confirmed papillary thyroid carcinoma. AJR:194, May 2010 W427

4 Hwang et al. Fig year-old man with papillary thyroid carcinoma that was negative for BRAF V600E mutation from fine-needle aspiration samples. On transverse sonogram, 1.2-cm irregular-shaped, markedly hypoechoic nodule (arrows) with microcalcifications and macrocalcifications is noted in left thyroid gland. Sonography diagnosed nodule as malignancy. Sonography-guided fine-needle aspiration and total thyroidectomy confirmed papillary thyroid carcinoma with extracapsular invasion. carcinomas with a circumscribed margin were seen in 22% of the mutation-positive patients and in 24% of the mutation-negative patients. Papillary thyroid carcinomas with a spiculated margin were seen in 64% of the mutation-positive patients and 59% of the mutation-negative patients. Papillary thyroid carcinomas with an ill-defined margin were seen in 14% of the mutation-positive patients and 17% of the mutation-negative patients. No statistically significant difference was found for the tumor margin between the two groups (p = ). The shape of a papillary thyroid carcinoma was round-to-oval in 26% of the mutationpositive patients and in 24% of the mutationnegative patients. A taller-than-wide shape was found in 59% of the mutation-positive patients and in 41% of the mutation-negative patients, and irregular shape was found in 15% of the mutation-positive patients and in 35% of the mutation-negative patients. Papillary thyroid carcinomas with the BRAF V600E mutation tended to show a taller-than-wide shape, but this finding was not statistically significant (p = ) (Figs. 1 and 2). For echogenicity of a papillary thyroid carcinoma, the mutation-positive patients showed isoechogenicity in 12%, hypoechogenicity in 52%, and marked hypoechogenicity in 36%. Among the mutation-negative patients, papillary thyroid carcinomas were isoechoic in 10%, hypoechoic in 52%, and markedly hypoechoic in 38%. There was no significant Fig year-old woman with papillary thyroid carcinoma that had BRAF V600E mutation. On transverse sonogram, 1.2-cm irregular-shaped, markedly hypoechoic nodule (arrows) with peripheral calcification is noted in isthmic portion of thyroid gland. Sonography diagnosed nodule as malignancy. Sonography-guided fineneedle aspiration and total thyroidectomy confirmed papillary thyroid carcinoma with extracapsular invasion. difference in echogenicity between the two groups (p = ). The BRAF V600E mutation was not associated with the presence of calcifications (54% with the mutation vs 71% without the mutation; p = ), which showed a significantly statistical difference on univariate analysis. However, this association was lost on multivariate analysis adjusting for the other sonographic predictors of tumor size, margin, shape, and echogenicity (p = ) (Figs. 3 and 4) (Table 3). For the calcification type, among mutationpositive patients, microcalcifications were present in 39% (41/106), macrocalcifications were present in 20% (21/106), and peripheral calcifications were present in 8% (9/106). Among the mutation-negative patients, microcalcifications were present in 45% (13/29), macrocalcifications were present in 24% (7/29), and peripheral calcifications were present in 7% (2/29). There was no statistically significant difference for the calcification type (p = , p = , and p = 1.000, respectively). Discussion Our results indicate that papillary thyroid carcinomas with the BRAF V600E mutation tended to have a taller-than-wide shape as seen on ultrasound, although the finding did not reach statistical significance (p = TABLE 3: Multivariate analyses of the association of the sonographic findings with the presence of the BRAF V600E mutation in 135 patients With papillary thyroid carcinoma Parameter Odds Ratio (95% CI) p Tumor size ( ) Margin Circumscribed vs ill-defined ( ) Spiculated vs ill-defined ( ) Shape Round-to-oval vs irregular ( ) Taller-than-wide vs irregular ( ) Echogenicity Isoechoic vs marked hypoechoic ( ) Hypoechoic vs marked hypoechoic ( ) Calcifications ( ) W428 AJR:194, May 2010

5 Papillary Thyroid Carcinoma 0.055). A taller-than-wide shape indicates that a nodule is greater in the anteroposterior dimension than in either the sagittal or transverse dimension. Kim et al. [19] found that a solid thyroid nodule that is taller-than-wide has 93% specificity for malignancy. In addition, it has been documented that nodules that are taller-than-wide are likely to be malignant in breast cancer [20, 21]. The growth of most benign nodules has been found to remain within normal tissue planes, whereas malignant nodules grow across normal tissue planes. Therefore, although the statistical significance is lacking, we suggest that a taller-than-wide shape is indicative of an aggressive tendency in tumor growth, which is associated with a papillary thyroid carcinoma that has the BRAF V600E mutation. We also found a significant correlation between the BRAF V600E mutation and a lower frequency of calcifications detected by sonography on univariate analysis, although this correlation was lost on multivariate analysis when we adjusted for the other sonographic predictors of tumor size, margin, shape, and echogenicity. Formation of calcifications is attributed partly to the degeneration and necrosis of tumor cells [22]. This finding within a tumor may represent less aggressiveness and slower growth of the tumor. In terms of the ultrasound features, this finding might indicate that a papillary thyroid carcinoma with the BRAF V600E mutation frequently grows and proliferates only without undergoing regression or degeneration. Moon et al. [18] reported that the frequency of calcifications detected on sonography was found to significantly drop in lesions smaller than 1 cm. This finding can be another explanation of the low frequency of calcification detected for the BRAF V600E mutation because most (56%) papillary thyroid carcinomas in our study were made of microcarcinomas. Future work with a large series, where the confounding factor of tumor size can be considered, will be needed for appreciating the additional value of calcifications in the BRAF V600E mutation. However, in daily practice, we encounter more frequently papillary thyroid carcinomas smaller than 1 cm than large papillary thyroid carcinomas. One of the problems that we should solve at this point is to decide on the management and the stratification of microcarcinomas, rather than confining ourselves to diagnosing and treating large carcinomas. Therefore, it is likely that our data are very representative of daily encountered carcinomas. Many studies support the association of the BRAF V600E mutation with aggressive features of papillary thyroid carcinoma [12, 16, 17]. For example, in a large series of papillary thyroid carcinoma cases consisting of mainly American patients, Nikiforova et al. [17] reported a significant association between the BRAF V600E mutation and extrathyroidal invasion and advanced disease (stages III and IV). In a study performed in Japan by Namba et al. [23], an association of the BRAF V600E mutation with advanced disease stages of papillary thyroid carcinoma was observed. Three studies performed in South Korea reported a significant association of the BRAF V600E mutation with the presence of a lymph node metastasis or extrathyroidal invasion [12, 24, 25]. In a large comprehensive international multicenter study, Xing et al. [16] reported a close association of the BRAF V600E mutation with extrathyroidal invasion, lymph node metastasis, and advanced disease stages. A recent short metaanalysis showed a significant association of the BRAF V600E mutation with extrathyroidal invasion, lymph node metastasis, and advanced stages of papillary thyroid carcinoma [26]. Elisei et al. [27] showed that the BRAF V600E mutation is a poor prognostic factor independent from other negative prognostic features. Several studies have showed that BRAF V600E mutation harboring anaplastic thyroid cancer often has a papillary thyroid carcinoma component, which suggests that the BRAF V600E mutation may play a role in promoting the progression of papillary thyroid carcinoma to anaplastic thyroid cancer [17]. However, our study showed no significant correlation between the presence of the BRAF V600E mutation and any clinicopathologic factors. We consider that this finding occurred because 56% of our series included microcarcinomas. Actually, these microcarcinomas are frequently encountered in daily practice for thyroid ultrasound or fine-needle aspiration. The management of thyroid papillary microcarcinoma is flexible, according to the preference of the patient or surgeon. However, a papillary thyroid carcinoma with the BRAF V600E mutation that is associated with poor prognosis would benefit from surgery that is more extensive or with no delay, even in a microcarcinoma. Thus, sonographic prediction for the presence of the BRAF V600E mutation could have a significant impact on clinical care. However, we are afraid that a meaningful ultrasound finding in our results is not specific for the presence of the BRAF V600E mutation of a papillary thyroid carcinoma, because calcifications detected on ultrasound are an important diagnostic clue to thyroid malignancy, especially papillary thyroid carcinomas. This study has several limitations. First, this study could have a selection bias because the study group included all types of papillary thyroid carcinomas, such as conventional papillary thyroid carcinoma or variants. However, we did not exclude variants that were rare and had a very small role in the contribution to the statistical analysis. Second, we used two methods to detect the BRAF V600E mutation. Discordant cases between the allele-specific polymerase chain reaction and the direct DNA sequencing method were present, but the false-positive cases could not be differentiated because higher sensitivity was favored. Third, ultrasound is dependent on the operator and facilitates detailed lesion descriptors under real time. Retrospective review of images can have limitations for more exact use of ultrasound descriptors. Last, with lack of a longterm follow-up, the study has a limitation for the analysis of recurrence or prognosis. Further follow-up will be necessary to compare the prognosis between the positive and negative mutation groups and to identify the clinically aggressive features of papillary thyroid carcinomas with the BRAF V600E mutation. 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