Clinical significance of the BRAFV600E mutation in multifocal papillary thyroid carcinoma in Korea Hwa Young Ahn, MD,a Yun Jae Chung, MD, PhD,a Byung Seup Kim, MD,b Kyung Ho Kang, MD,b Ju Won Seok, MD, PhD,c Hee Sung Kim, MD, PhD,d Sung Jun Park, MD, PhD,b and Bo Youn Cho, MD, PhD,a Seoul, Korea
Background. We examined the frequency of the BRAFV600E mutation and compared the clinicopathologic features based on the BRAFV600E mutation status in multifocal papillary thyroid carcinoma (PTC). Methods. A total 85 patients who were diagnosed with multifocal PTC were enrolled. We confirmed the status of the BRAFV600E mutation in each tumor focus by the real-time polymerase chain reaction technique. Results. Among the 85 patients, 49 (57.6%), 34 (40.0%), and 2 (2.4%) patients were determined to have all BRAFV600E-positive, mixed BRAFV600E, and all BRAFV600E-negative in their tumor foci, respectively. When we compared clinicopathologic features according to the BRAFV600E mutation status of the dominant tumor, the BRAFV600E -positive group (n = 70) showed more extrathyroidal invasion in the dominant tumor (32.9% vs 6.7%, P = .041) and more lymph node metastasis (67.2% vs 40.0%, P = .049) than the BRAFV600E -negative group (n = 15). Considering all tumor foci, the all BRAFV600E mutation group exhibited a younger population (P = .039), showed increased extrathyroidal invasion (38.8% vs 14.7%, P = .017) and lymph node metastasis (71.4% vs 48.4%, P = .038), and received more radioactive iodine therapy (79.2% vs 52.9%, P = .012) than the mixed BRAFV600E mutation group. A larger tumor size and heavier preoperative body weight was positively correlated with the relative OOCt method. expression of BRAFV600E mutation calculated by 2 Conclusion. Most of the Korean patients with multifocal PTC had the BRAFV600E mutation in one or more tumor foci, and all BRAFV600E-positive multifocal PTC showed more aggressive features. (Surgery 2014;155:689-95.) From the Departments of Internal Medicine,a Surgery,b Nuclear Medicine,c and Pathology,d Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, Korea
PAPILLARY THYROID CARCINOMA (PTC) is the most common thyroid malignancy and accounts for approximately 80–90% of all thyroid cancer.1,2 Multifocality, defined as two or more tumor foci, is a common feature of PTC, and the incidence of multifocal PTC ranges from 18 to 87%.3,4 Studies have indicated that multifocality is correlated with poor prognostic factors.5,6 H.Y. Ahn and Y.J. Chung equally contributed to this work. Presented as a poster at the 37th Annual Meeting of the European Thyroid Association in Leiden, The Netherlands, September 7–11, 2013. Accepted for publication December 19, 2013. Reprint requests: Bo Youn Cho, MD, PhD, Department of Internal Medicine, Chung-Ang University Hospital, 224-1 Heukseok-dong, Dongjak-gu, Seoul 156-755, Korea. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2014 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2013.12.025
Mutation of B-type raf kinase (BRAF) is the most frequent genetic alteration in PTC,7 and more than 40 mutations have been identified in the BRAF gene thus far.8 The T1799 A BRAF point mutation is the most common mutation and causes an amino acid change of a valine to glutamic acid at codon 600 (V600E), designated the BRAFV600E mutation.9 Studies have indicated that the incidence of BRAF mutation in PTC is approximately 45% and ranges from 32 to 73%.7,10 Furthermore, numerous studies have reported that BRAF mutation is associated with poor clinicopathological features, tumor recurrence, and high patient mortality.11-14 Although BRAF mutation is also associated with tumor multifocality,15 most studies have examined the BRAF mutation status only with regard to the ‘‘dominant tumor,’’ even when multiple tumor foci existed. In a previous study, 39.3% of multifocal PTCs in the Korean population showed a mixed BRAF mutation status in each tumor focus.16 This result indicates that the SURGERY 689
690 Ahn et al
analysis of BRAF mutation only in the dominant tumor may fail to identify all of the tumors with BRAF mutation. One question that has not been addressed is with regard to the BRAF mutation status of the dominant tumor and whether this status represents the clinicopathologic features of multifocal PTC. Indeed, only one study has investigated whether there are any differences in the clinicopathologic features between all BRAF and mixed BRAF mutationpositive groups in multifocal PTC.16 However, the detection method employed for the BRAFV600E mutation in that study was polymerase chain reaction (PCR) restriction fragment length polymorphism, which has a low sensitivity for detecting the presence or absence of the BRAFV600E mutation. The aim of the present study was to evaluate the frequency of the BRAFV600E mutation by the use of a highly sensitive real-time PCR technique and to compare the clinicopathologic features based on BRAFV600E mutation status in multifocal PTC. PATIENTS AND METHODS Patients. We recruited patients who underwent a total thyroidectomy and were diagnosed with PTC at Chung-Ang University Hospital (Seoul, Korea) from December 2011 to June 2012. Among these patients, 85 were determined to have two or more tumor foci (Fig), and 199 tumor foci were studied. We defined the largest tumor as the dominant tumor, and the remaining tumor foci were designated as the minor tumor(s). To test the status of the BRAFV600E mutation, written informed consent was obtained from all the patients before surgery, and this study was approved by the Institutional Review Board of Chung-Ang University Hospital. An 18F-fluorodeoxyglucose-positron emission tomography/computed tomography scan was performed on those patients who were suspected of having multiple tumors. The patients were divided into two groups according to the BRAFV600E status of the dominant tumor. As a result, 70 patients were assigned to the BRAFpositive dominant tumor group, and the remaining 15 patients were assigned to the BRAF-negative dominant tumor group. We also constructed groups according to the BRAFV600E mutation heterogeneity of each tumor focus. As shown in Fig, patients were included in the all BRAF-positive group if all of the tumor foci contained BRAFV600E, whereas patients with both BRAFV600E and BRAFWild-type tumor foci were assigned to the mixed BRAF group. The patients underwent central compartment neck dissection when either biopsy-proven lymph node metastasis or suspicious findings were found
Surgery April 2014
by preoperative neck ultrasonography or when advanced primary tumors (T3 or T4) were noted. Lateral neck compartmental lymph node dissection was performed in patients with biopsy-proven metastatic lateral cervical lymphadenopathy. As a result, 83 of the 85 patients underwent central and/or lateral compartmental lymph node dissection. Three months after surgery, each patient who showed regional lymph node metastasis and/or extrathyroidal invasion on the basis of pathologic reports received a 3.7 GBq (100 mCi) dose of 131I after thyroid hormone withdrawal and a low-iodine diet for 2 weeks. An increased serum thyroidstimulating hormone level (>30 mU/L) was confirmed in all patients who underwent radioactive iodine (RAI) therapy. DNA extraction and BRAFV600E mutation analysis. Genomic DNA was extracted from formalin-fixed, paraffin-embedded operative tissue resections from each tumor focus using a QIAamp DNA FFPE Tissue Kit (QIAGEN, Valencia, CA). The BRAFV600E mutation was detected by use of the Anyplex BRAF V600E Real-Time Detection system (Seegene Inc, Seoul, Korea). The reaction mixture contained 2 mL of 10 3 BRAF Oligo Mix, which included amplification and detection reagents, 3 mL of 8-methoxypsoralen solution to prevent carryover contamination, and 10 mL of 2 3 Anyplex PCR Master Mix (Seegene Inc), which included DNA polymerase and a buffer with deoxynucleoside triphosphates. A total of 15 mL of the reaction mixture was dispensed into 0.2-mL PCR tubes, and 5 mL of nucleic acid from each sample was added to the reaction mixture tube to reach a total reaction volume of 20 mL. A real-time PCR was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) with the following cycle conditions: 15 minutes at 948C, followed by 15 cycles of 15 seconds at 948C and 30 seconds at 608C and 35 cycles of 30 seconds at 948C and 32 seconds at 608C. In these assays, the cycle threshold (Ct) was defined as the number of cycles required for the fluorescent signal to cross the threshold for the presence of the BRAF mutation. To evaluate the correlations between the amount of BRAFV600E gene in BRAFV600E -positive dominant tumor and clinicopathologic parameters, we calculated the relative expression of BRAFV600E by using Ct value. To standardize the Ct levels of BRAFV600E gene, the Ct levels of BRAFV600E gene were normalized by the Ct levels of human b2-microglobulin (housekeeping gene); OCt = Ct of BRAFV600E Ct of human b2-microglobulin. The OCt is inversely proportional to the BRAFV600E mRNA level, with
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Fig. Flow chart of enrollment and grouping according to BRAF mutation. Among multifocal tumor foci, the largest tumor was defined as a dominant tumor and other small foci were designated as minor tumor(s). Patients were included in the all BRAF-positive group if all of the tumor foci contained BRAFV600E, whereas patients with both BRAFV600E and BRAFWild-type tumor foci were assigned to the mixed BRAF group.
high OCt value corresponding to a lower mRNA level. So we designated a patient who showed the greatest OCt value as a ‘‘standard patient.’’ After that, the relative expression levels of BRAFV600E of comparable patient to standard patient were OOCt calculated by 2 method; OOCt = [(Ct V600E of BRAF Ct of human b2microglobulin)comparable patient (Ct of BRAFV600E Ct of human b2-microglobulin)standard patient]. Statistical analysis. All the statistical analyses were performed with SPSS version 20 software (IBM Inc, Armonk, NY). To compare the clinicopathologic features, we used a chi-square test for the categorical variables or a Student t test for the continuous variables. The Kolmogorov-Smirnov test was used to determine the normal distribution of all the variables. Non-normally distributed variables were log-transformed before the analysis. A Pearson or Spearman correlation and multivariate stepwise linear regression were used. We considered a P value of
Background. We examined the frequency of the BRAFV600E mutation and compared the clinicopathologic features based on the BRAFV600E mutation status in multifocal papillary thyroid carcinoma (PTC). Methods. A total 85 patients who were diagnosed with multifocal PTC were enrolled. We confirmed the status of the BRAFV600E mutation in each tumor focus by the real-time polymerase chain reaction technique. Results. Among the 85 patients, 49 (57.6%), 34 (40.0%), and 2 (2.4%) patients were determined to have all BRAFV600E-positive, mixed BRAFV600E, and all BRAFV600E-negative in their tumor foci, respectively. When we compared clinicopathologic features according to the BRAFV600E mutation status of the dominant tumor, the BRAFV600E -positive group (n = 70) showed more extrathyroidal invasion in the dominant tumor (32.9% vs 6.7%, P = .041) and more lymph node metastasis (67.2% vs 40.0%, P = .049) than the BRAFV600E -negative group (n = 15). Considering all tumor foci, the all BRAFV600E mutation group exhibited a younger population (P = .039), showed increased extrathyroidal invasion (38.8% vs 14.7%, P = .017) and lymph node metastasis (71.4% vs 48.4%, P = .038), and received more radioactive iodine therapy (79.2% vs 52.9%, P = .012) than the mixed BRAFV600E mutation group. A larger tumor size and heavier preoperative body weight was positively correlated with the relative OOCt method. expression of BRAFV600E mutation calculated by 2 Conclusion. Most of the Korean patients with multifocal PTC had the BRAFV600E mutation in one or more tumor foci, and all BRAFV600E-positive multifocal PTC showed more aggressive features. (Surgery 2014;155:689-95.) From the Departments of Internal Medicine,a Surgery,b Nuclear Medicine,c and Pathology,d Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, Korea
PAPILLARY THYROID CARCINOMA (PTC) is the most common thyroid malignancy and accounts for approximately 80–90% of all thyroid cancer.1,2 Multifocality, defined as two or more tumor foci, is a common feature of PTC, and the incidence of multifocal PTC ranges from 18 to 87%.3,4 Studies have indicated that multifocality is correlated with poor prognostic factors.5,6 H.Y. Ahn and Y.J. Chung equally contributed to this work. Presented as a poster at the 37th Annual Meeting of the European Thyroid Association in Leiden, The Netherlands, September 7–11, 2013. Accepted for publication December 19, 2013. Reprint requests: Bo Youn Cho, MD, PhD, Department of Internal Medicine, Chung-Ang University Hospital, 224-1 Heukseok-dong, Dongjak-gu, Seoul 156-755, Korea. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2014 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2013.12.025
Mutation of B-type raf kinase (BRAF) is the most frequent genetic alteration in PTC,7 and more than 40 mutations have been identified in the BRAF gene thus far.8 The T1799 A BRAF point mutation is the most common mutation and causes an amino acid change of a valine to glutamic acid at codon 600 (V600E), designated the BRAFV600E mutation.9 Studies have indicated that the incidence of BRAF mutation in PTC is approximately 45% and ranges from 32 to 73%.7,10 Furthermore, numerous studies have reported that BRAF mutation is associated with poor clinicopathological features, tumor recurrence, and high patient mortality.11-14 Although BRAF mutation is also associated with tumor multifocality,15 most studies have examined the BRAF mutation status only with regard to the ‘‘dominant tumor,’’ even when multiple tumor foci existed. In a previous study, 39.3% of multifocal PTCs in the Korean population showed a mixed BRAF mutation status in each tumor focus.16 This result indicates that the SURGERY 689
690 Ahn et al
analysis of BRAF mutation only in the dominant tumor may fail to identify all of the tumors with BRAF mutation. One question that has not been addressed is with regard to the BRAF mutation status of the dominant tumor and whether this status represents the clinicopathologic features of multifocal PTC. Indeed, only one study has investigated whether there are any differences in the clinicopathologic features between all BRAF and mixed BRAF mutationpositive groups in multifocal PTC.16 However, the detection method employed for the BRAFV600E mutation in that study was polymerase chain reaction (PCR) restriction fragment length polymorphism, which has a low sensitivity for detecting the presence or absence of the BRAFV600E mutation. The aim of the present study was to evaluate the frequency of the BRAFV600E mutation by the use of a highly sensitive real-time PCR technique and to compare the clinicopathologic features based on BRAFV600E mutation status in multifocal PTC. PATIENTS AND METHODS Patients. We recruited patients who underwent a total thyroidectomy and were diagnosed with PTC at Chung-Ang University Hospital (Seoul, Korea) from December 2011 to June 2012. Among these patients, 85 were determined to have two or more tumor foci (Fig), and 199 tumor foci were studied. We defined the largest tumor as the dominant tumor, and the remaining tumor foci were designated as the minor tumor(s). To test the status of the BRAFV600E mutation, written informed consent was obtained from all the patients before surgery, and this study was approved by the Institutional Review Board of Chung-Ang University Hospital. An 18F-fluorodeoxyglucose-positron emission tomography/computed tomography scan was performed on those patients who were suspected of having multiple tumors. The patients were divided into two groups according to the BRAFV600E status of the dominant tumor. As a result, 70 patients were assigned to the BRAFpositive dominant tumor group, and the remaining 15 patients were assigned to the BRAF-negative dominant tumor group. We also constructed groups according to the BRAFV600E mutation heterogeneity of each tumor focus. As shown in Fig, patients were included in the all BRAF-positive group if all of the tumor foci contained BRAFV600E, whereas patients with both BRAFV600E and BRAFWild-type tumor foci were assigned to the mixed BRAF group. The patients underwent central compartment neck dissection when either biopsy-proven lymph node metastasis or suspicious findings were found
Surgery April 2014
by preoperative neck ultrasonography or when advanced primary tumors (T3 or T4) were noted. Lateral neck compartmental lymph node dissection was performed in patients with biopsy-proven metastatic lateral cervical lymphadenopathy. As a result, 83 of the 85 patients underwent central and/or lateral compartmental lymph node dissection. Three months after surgery, each patient who showed regional lymph node metastasis and/or extrathyroidal invasion on the basis of pathologic reports received a 3.7 GBq (100 mCi) dose of 131I after thyroid hormone withdrawal and a low-iodine diet for 2 weeks. An increased serum thyroidstimulating hormone level (>30 mU/L) was confirmed in all patients who underwent radioactive iodine (RAI) therapy. DNA extraction and BRAFV600E mutation analysis. Genomic DNA was extracted from formalin-fixed, paraffin-embedded operative tissue resections from each tumor focus using a QIAamp DNA FFPE Tissue Kit (QIAGEN, Valencia, CA). The BRAFV600E mutation was detected by use of the Anyplex BRAF V600E Real-Time Detection system (Seegene Inc, Seoul, Korea). The reaction mixture contained 2 mL of 10 3 BRAF Oligo Mix, which included amplification and detection reagents, 3 mL of 8-methoxypsoralen solution to prevent carryover contamination, and 10 mL of 2 3 Anyplex PCR Master Mix (Seegene Inc), which included DNA polymerase and a buffer with deoxynucleoside triphosphates. A total of 15 mL of the reaction mixture was dispensed into 0.2-mL PCR tubes, and 5 mL of nucleic acid from each sample was added to the reaction mixture tube to reach a total reaction volume of 20 mL. A real-time PCR was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) with the following cycle conditions: 15 minutes at 948C, followed by 15 cycles of 15 seconds at 948C and 30 seconds at 608C and 35 cycles of 30 seconds at 948C and 32 seconds at 608C. In these assays, the cycle threshold (Ct) was defined as the number of cycles required for the fluorescent signal to cross the threshold for the presence of the BRAF mutation. To evaluate the correlations between the amount of BRAFV600E gene in BRAFV600E -positive dominant tumor and clinicopathologic parameters, we calculated the relative expression of BRAFV600E by using Ct value. To standardize the Ct levels of BRAFV600E gene, the Ct levels of BRAFV600E gene were normalized by the Ct levels of human b2-microglobulin (housekeeping gene); OCt = Ct of BRAFV600E Ct of human b2-microglobulin. The OCt is inversely proportional to the BRAFV600E mRNA level, with
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Fig. Flow chart of enrollment and grouping according to BRAF mutation. Among multifocal tumor foci, the largest tumor was defined as a dominant tumor and other small foci were designated as minor tumor(s). Patients were included in the all BRAF-positive group if all of the tumor foci contained BRAFV600E, whereas patients with both BRAFV600E and BRAFWild-type tumor foci were assigned to the mixed BRAF group.
high OCt value corresponding to a lower mRNA level. So we designated a patient who showed the greatest OCt value as a ‘‘standard patient.’’ After that, the relative expression levels of BRAFV600E of comparable patient to standard patient were OOCt calculated by 2 method; OOCt = [(Ct V600E of BRAF Ct of human b2microglobulin)comparable patient (Ct of BRAFV600E Ct of human b2-microglobulin)standard patient]. Statistical analysis. All the statistical analyses were performed with SPSS version 20 software (IBM Inc, Armonk, NY). To compare the clinicopathologic features, we used a chi-square test for the categorical variables or a Student t test for the continuous variables. The Kolmogorov-Smirnov test was used to determine the normal distribution of all the variables. Non-normally distributed variables were log-transformed before the analysis. A Pearson or Spearman correlation and multivariate stepwise linear regression were used. We considered a P value of
