OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2015.3119 OL-0-0-3119 Articles Clinical significance of BRAF V600E mutation in 154 patients with thyroid nodules YULINGYING MALIZHEN TUQIAOFENG ZHANGYI CHENYUEMING YUDAOJUN YANGSHAOYU Department of Endocrinology, Nanjing Medical University, Affiliated Hangzhou Hospital (Hangzhou First People's Hospital), Hangzhou, Zhejiang 310006, P.R. China Correspondence to: Ms. Lizhen Ma, Department of Endocrinology, Nanjing Medical University, Affiliated Hangzhou Hospital (Hangzhou First People's Hospital), 261 Huansha Road, Hangzhou, Zhejiang 310006, P.R. China, E-mail: malizhen2006@163.com 06 2015 15 04 2015 9 6 2633 2638 07062014 27022015 Copyright © 2015, Spandidos Publications 2015

The aim of the present study was to investigate the prevalence of the BRAF V600E mutation in papillary thyroid carcinoma (PTC) patients from eastern coastal China and to determine whether it is correlated with the clinicopathological features of PTCs with or without current Hashimoto thyroiditis (HT). The BRAF V600E mutation status was analyzed in 206 thyroid nodules of 154 patients undergoing thyroidectomy using polymerase chain reaction and bi-directional sequencing. Multivariate analysis was performed to investigate the association of the BRAF V600E mutation with clinicopathological features. Thyroid nodules were classified as PTC, nodular goiter (NG), adenomatoid nodule, adenoma and HT. The BRAF V600E mutation was observed in 61.5% of PTCs analyzed; it was also detected in one normal tissue adjacent to PTC and one NG. One patient exhibited double mutations in the BRAF gene; the BRAF V600E mutation in the PTC lesion and the BRAF K601E mutation in the contralateral NG lesion. Patients harboring the BRAF V600E mutation had higher thyroid stimulating hormone levels (2.453±1.464 vs. 1.966±1.296 mIU/l), a reduced occurrence of papillary thyroid microcarcinoma (55.0 vs. 88%), and a higher occurrence of lymph node metastasis (LNM; 42.5 vs. 16.0%) compared with those with wild-type BRAF (all P<0.05). Binary logistic regression analysis revealed that the BRAF V600E mutation was associated with LNM of PTC (hazard ratio, 5.051; 95% confidence interval, 1.068–23.893; P=0.041). Conversely, no association was identified between the BRAF V600E mutation and HT (38.5 vs. 67.3%, χ2=3.656, P=0.056). Thus, in regional PTCs, the BRAF V600E mutation was prevalent, suggesting that it may be an early and phenotypically defining molecular event in PTC, and may represent an independent factor that predicts LNM.

 BRAF papillary thyroid carcinoma prognostic value Hashimoto thyroiditis Introduction

Thyroid carcinoma (TC) is the most common endocrine malignancy (1); according to data presented by the National Comprehensive Cancer Network, it is currently the sixth most common malignancy diagnosed in females (2). The incidence of TC, particularly papillary thyroid carcinoma (PTC), is increasing by 6.2% per year (2,3).

More than 70% of PTCs have genetic alterations associated with the mitogen-activated protein kinase pathway (4). BRAF mutations have been commonly observed in all three subtypes of Raf kinase, the first identified and well-characterized downstream cytosolic effector of RAS. Of the greater than 45 BRAF mutations that have been identified in human cancer types, approximately 90% are T→A transversions in exon 15 at nucleotide 1799 (T1799A), leading to a valine→glutamic acid replacement at position 600 (BRAF V600E) (4). In PTC, the prevalence of the BRAF V600E mutation varies from 29 to 83%, and occurs mainly in classic PTC (approximately 60%). In tall-cell variant PTC and PTC-derived anaplastic thyroid cancer, the BRAF V600E mutation was also reported; however, it has rarely been identified in follicular-variant PTC (FV-PTC) and never reported in follicular thyroid carcinoma (FTC) (4).

Numerous studies have demonstrated that the BRAF mutation is the main genetic event in PTC, and is associated with poor prognosis. In rat thyroid PCCL3 cells conditionally expressing oncogenic BRAF, increased invasion into Matrigel was observed compared with cells expressing RET/PTC3 in vitro, which was consistent with the biological behavior of human PTC (5). In addition, PTC without the BRAF V600E mutation mainly corresponded to FV-PTC, and maintained a thyroid differentiation expression level close to that of normal tissue and thus had a better prognosis than PTC with the gene alteration (6). Furthermore, the BRAF V600E mutation may induce genetic instability in PTC, facilitating secondary genetic alternation of members of the PI3k/Akt pathway that may induce PTC progression to a more aggressive thyroid cancer (7). However, whether the BRAF V600E mutation represents an independent risk factor for predicting thyroid cancer recurrence or metastasis is a matter of debate. In addition, although PTC is usually accompanied with Hashimoto thyroiditis (HT), which is considered a precancerous lesion of PTC by certain investigators (8), its involvement in the pathogenesis of PTC is controversial. Therefore, this study aimed to investigate the prevalence of the BRAF V600E mutation in PTCs with or without current HT in patients from eastern coastal China. Specifically, the association of the BRAF V600E mutation with the clinicopathological features of PTC patients was analyzed. These studies may provide the basis of developing individual diagnostic protocols and treatments for TC patients.

 Patients and methods <sec> <title>Patients

A total of 154 consecutive patients diagnosed with thyroid nodules (206 affected nodules in total) were enrolled from January 2012 to January 2013. All patients (31 males and 123 females, with a mean age of 50.3 years, range 22–76 years) underwent thyroidectomy at the Department of Surgical Oncology at Hangzhou First Peoples Hospital, (Hangzhou, China). Written informed consent was obtained from all study participants, and the study was approved by the Institutional Ethics Committee of Hangzhou First Peoples Hospital.

Nodules were classified according to the following diagnostic categories described by the World Health Organization and Diagnostic Histopathology of Tumors (9): PTC (73 patients, 78 nodules), nodular goiter (NG; 65 patients, 95 nodules), adenomatoid nodule (AN; seven nodules), thyroid adenoma (TA; four nodules), and HT (six nodules). HT was diagnosed according to previously described criteria (10): the presence of goiter, increased anti-thyroid antibodies (Tg-Ab, TPO-Ab) by >50%, or pathological confirmation. Fourteen adjacent normal tissues that were >1 cm distance from the PTC were also analyzed as normal controls. Fresh thyroid tissue was quickly frozen at −80°C. Of the 206 nodules, 38 were excluded due to insufficient quality of the nucleic acids, poor sequencing signal or improper storage.

 Instruments

An Ultrasonic Doppler was purchased from Esaote S.p.A. (Genova, Italy), a multi-image gel imaging system was provided by Bio-Rad Laboratories Inc. (Hercules, CA, USA) and a 2720 Thermal Cycler PCR instrument was obtained from Applied Biosystems (Life technologies Co. Ltd., Foster City, CA, USA).

 DNA extraction and direct sequencing

DNA was extracted by fresh-frozen thyroid tissue using a kit (Takara MiniBEST Universal Genomic DNA extraction kit, Ver. 4.0, Code D824A) provided by Bao-Biology Ltd. (Dalian, China) following the manufacturer's recommendations. A 5-µl sample of the DNA template amplified by polymerase chain reaction (PCR) was added in a 50-µl reaction containing 5 µl 10X buffer, 4 µl dNTPs, 1 µl each primer, 0.25 µl Taq DNA polymerase and 32.75 µl dH2O (all from Bao-Biology Ltd.). PCR amplification of the BRAF V600E mutation was carried out using the following primers obtained from Huirui Biological Technology Ltd. (Shanghai, China): sense, 5′-ACCTAAACTCTT CATAATGCTTGCT-3 and antisense, 5′-CTGATTTTTGTG AATACTGGGAACT-3′. PCR was carried out with an initial denaturation step at 95°C for 5 min; then 30 cycles at 95°C for 30 sec, 52°C for 30 sec and 72°C for 30 sec; and a final extension cycle at 72°C for 10 min. Reaction products were visualized on a 1.5% agarose gel with ethidium bromide, using standard molecular weight DNA as a control.

Bi-directional sequencing was used to detect the presence of the BRAF V600E point mutation by Inhuaiweiji Biological Technology Ltd. (Shanghai, China). DNA sequences were compared with those of the normal BRAF gene exon 15 in the GenBank database using sequence assembly software (Sequencher 4.8; Gene Codes Corporation, Ann Arbor, MI, USA).

 Statistical analysis

Statistical analysis was performed using SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL, USA). The association between categorical and continuous variables was evaluated using the two-tailed Fisher's exact test and Student's t-test, respectively. Binary logistic regression analysis was used to assess the correlation between the BRAF V600E mutation and clinicopathological features. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Electrophoresis and sequencing results

Representative agarose gel electrophoresis results of the PCR products are shown in Fig. 1, lanes 2–8. Sequencing results are shown in Fig. 2. In one patient, two different BRAF mutations were noted; the BRAF V600E mutation was identified in the PTC lesion, and the BRAF K601E mutation was detected in the contralateral NG lesion. An A→G transversion at exon 15 nucleotide 1801 (A1801G) of the BRAF gene resulted in the replacement of lysine with glutamic acid at position 601 (BRAF K601E) as shown in Fig. 3.

 Detection of thyroid function and <italic>BRAF</italic> V600E mutation

Table I shows the levels of T3 and T4 thyroid hormone, free triiodothyronine, free thyroxine and thyroid stimulating hormone (TSH), as well as the prevalence of the BRAF V600E mutation for each group. No significant difference was noted in thyroid function among the groups (P>0.05). The prevalence of the BRAF V600E mutation in PTCs was 61.5%, which was higher than that observed in patients with benign lesions (χ2=75.732, P<0.001).

Comparisons between 17 NGs and their contralateral PTCs, five cases of bilateral PTCs and 15 cases of bilateral NGs were also conducted. Among the five bilateral PTC patients, two cases had the BRAF V600E mutations in both bilateral PTC lesions, and two cases had wild-type BRAF in both bilateral PTC lesions. The remaining PTC patient carried the BRAF V600E mutation in one PTC lesion, but was negative in the contralateral PTC lesion. Notably, the BRAF V600E mutation was identified in two benign lesions; in normal tissue adjacent to the PTC harboring the BRAF V600E and in NG tissue. The PTC lesion offside to NG tissue carrying the BRAF V600E was also observed to be mutated. In addition, immunohistochemistry analysis revealed that both galectin-3 and HBME-1 were partially expressed in the mutated NG tissue. Conversely, none of the other benign lesions, including 14 bilateral NGs, 38 unilateral NGs, five ANs and three TAs, had the BRAF V600E mutation.

 Correlation of the <italic>BRAF</italic> V600E mutation and HT

After the NG, AN and TA groups were merged into a benign tissue (BT) group, this group was compared with the PTC group; there was no significant difference in the prevalence of patients with concurrent HT between the two groups (20.0 vs. 20.2%, χ2=0.001, P=0.973; Table II). The BRAF V600E mutation was detected mainly in PTCs (χ2=70.186, P<0.001). Comparative analysis using layered HT from BT and PTC indicated that the prevalence of the BRAF V600E mutation was lower in PTCs accompanied with HT than in those without HT (38.5 vs. 67.3%), but it was not statistically significant (χ2=3.656, P=0.056).

 Correlation of the <italic>BRAF</italic> V600E mutation with clinicopathological features

As shown in Table III, no significant difference in gender (22.8 vs. 16.7%, χ2=1.068, P=0.301) or age (49.0±11.1 vs. 51.1±10.1 years, t=1.134, P=0.259) was noted between patients with the mutated and non-mutated nodules. However, patients harboring the BRAF V600E mutation had higher TSH levels compared with the patients with wild-type BRAF (2.453±1.464 vs. 1.966±1.296 mIU/l, respectively; t=2.019; P=0.045). Among the PTCs, 44 (67.7%) were papillary thyroid microcarcinomas (PTMCs) with a diameter of 1 cm or less. Compared with non-mutated PTCs, PTCs harboring the BRAF V600E mutation were less likely to be a PTMC (55.0 vs. 88%, P<0.05), and they had a higher occurrence of lymph node metastasis (LNM; 42.5 vs. 16.0%, P<0.05). Similarly, PTMC tumors harboring the BRAF V600E mutation had a higher incidence of LNM, although the difference was not significant (36.4 vs. 13.6%, χ2=3.030, P=0.162).

Next, correlation analysis of the BRAF V600E mutation and clinicopathological features was conducted. Binary logistic regression analysis revealed that LNM of PTC was associated with the BRAF V600E mutation, gender and PTMC, but not with age, TSH or HT (Table IV). In addition, the BRAF V600E mutation [hazard ratio (HR), 5.051; 95% confidence interval (CI), 1.068–23.893; P=0.041) and female gender (HR, 0.147; 95% CI, 0.031–0.697; P=0.016) were independent factors that predicted LNM.

 Discussion

The prevalence of the BRAF V600E mutation in PTCs ranges from 29 to 83% and is reported only in malignant thyroid tumors (4,11,12); similarly, the incidence of the BRAF V600E mutation was 61.5% in PTC patients from China's eastern coast in this study. All of the BRAF V600E mutations identified in these PTCs were heterozygous mutations. Similar to the results of previous studies, most of the benign lesions, including 14 bilateral NGs, 38 unilateral NGs, five ANs and three TAs, carried the wild-type BRAF gene. In contrast to other studies that failed to identify BRAF mutations in benign thyroid disease, two benign tissues carried the BRAF V600E mutation in the present study. In the first case, the mutation was detected in normal tissue adjacent to PTC that also carried the BRAF V600E mutation, suggesting the presence of infiltrative growth. In the second case, the BRAF V600E mutation was detected in the NG tissue, on the contralateral PTC lesion also carrying the mutation, suggesting that this mutation may represent an early event in PTC progression. Moreover, immunohistochemistry analysis of galectin-3 and HBME-1 expression in the BRAF V600E-positive NG revealed that both were weakly expressed. Combined immunohistochemistry for galectin-3 and HBME-1 was useful for the differentiation of benign and malignant thyroid tumors (13); therefore, the mutation in the NG tissue may be indicative of a trend toward malignant biological progression. In summary, the BRAF V600E mutation may represent a critical step in tumor progression.

Another notable finding in the present study was that one patient exhibited double mutations in the BRAF gene; the BRAF V600E mutation was identified in the PTC lesion, and the BRAF K601E mutation was identified in the contralateral NG lesion. An A→G transversion at exon 15 nucleotide 1801 (A1801G) of the BRAF gene resulted in the replacement of lysine with glutamic acid at position 601 (BRAF K601E), which was also a heterozygous mutation in this patient. Trovisco et al (14) first reported the BRAF K601E mutation in a PTC patient in 2005, and 18 additional cases have been subsequently described (15–24). Meta-analysis demonstrated that the BRAF K601E mutation was predominantly identified in FV-PTC, followed by FTC and PTC, and only one case of TA had the BRAF K601E mutation. Although it may affect the PI3k/Akt pathway, this mutation exhibited relatively inactive biological characteristics in the pathogenesis of TC compared with the BRAF V600E mutation (15–24). Moreover, in multifocal papillary thyroid carcinoma (mPTC), individual tumor foci may be identical and are frequently composed of various histological types, and individual tumor foci also harbor different mutations. For example, Kim et al (23) described a case of mPTC consisting of one FV-PTC harboring the BRAF K601E mutation and three conventional foci harboring the BRAF V600E mutation. Similarly, we present a patient with both the BRAF V600E and BRAF K601E mutations although the BRAF K601E mutation was detected in a NG. To our knowledge, this is the first study of a BRAF K601E mutation in NG tissue. We suspect that the BRAF K601E mutation in the NG tissue may be an indication of oncogene activation that is believed to take place early in the course of tumorigenesis. However, determining the exact mechanism requires further research.

Numerous studies have demonstrated an association between the BRAF V600E mutation with aggressive clinicopathological characteristics of PTC, including extrathyroidal invasion, LNM, advanced tumor-node-metastasis stage, loss of radioiodine avidity and disease recurrence (5–7). BRAF mutation presents a low positive predictive value (28%) and a high negative predictive value (87%) for PTC recurrence, suggesting that BRAF mutational status in clinical management of PTC should be used with caution. However, it is still controversial to use BRAF mutation as an independent predictive risk factor for PTC due to the fact that the BRAF V600E mutation is identified in approximately half of PTCs, among which less than 10–15% of the tumors exhibit aggressive behavior (25). In accordance with previous studies, a higher rate of LNM was noted in BRAF V600E-positive PTCs than in negative ones at the time of surgery in this study. In addition, binary logistic regression analysis indicated that this mutation was an independent predictive factor of LNM in PTC patients. The proportion of PTMC was lower in PTCs with the BRAF V600E mutation than in those with the wild-type gene. Taken together, this mutation may promote tumor growth and LNM; therefore, the BRAF V600E mutation was correlated with a more aggressive behavior of PTC.

Even though tumor-related mortality is as low as 0.5% for PTMC, microcarcinoma harboring the BRAF V600E mutation is also associated with features predictive of a high risk of recurrence and metastasis (26–28). In our study, 67.7% (44/65) of PTCs were PTMCs, among which 50% (22/44) of the patients had the BRAF V600E mutation. Consistent with previous studies, the presence of the BRAF V600E mutation in PTMC further suggests that the mutation is an early event in thyroid carcinogenesis. Similarly, patients with PTMCs harboring the BRAF V600E mutation had a higher incidence of LNM although this was not significant (P=0.162). However, we expect that this correlation will be confirmed in further studies with larger sample sizes.

A number of studies (29,30) have reported a weak association between serum TSH concentration and thyroid cancer, with certain investigators suggesting that higher preoperative TSH serum concentration may be associated with advanced tumor stage and poor prognosis (29); however, the correlation between TSH and PTC remains controversial. Although TSH suppressive therapy is a well-known adjuvant therapy to prevent recurrence in differentiated thyroid cancer, no significant difference in TSH level was identified among different pathological types in the present study, which may be due to the insufficient sample size. In the present study, compared with tumors with wild-type BRAF, patients with PTCs harboring the BRAF V600E mutation had higher TSH levels. Whether the mutation is involved in the activation of TSH receptor remains unclear and requires further analysis.

PTC with HT is frequently observed; however, the association between HT and PTC is controversial. When compared with benign lesions, there was no significant difference in the rate of PTCs with concurrent HT; therefore, the correlation between HT and TCs remains unclear. However, Kim et al (30) reported that in Korean patients with PTC, the BRAF V600E mutation was associated with a low frequency of background HT and a high frequency of LNM. Similarly, PTCs accompanied with HT had a lower frequency of the BRAF V600E mutation in our study, although this was not significant (P=0.056). However, additional analysis in an expanded sample size may yield significant differences.

In conclusion, in PTC patients from China's eastern coast, the BRAF V600E mutation was prevalent (61.5%). Similar to findings in previous studies, our results supported the notion that the mutation was an early and phenotypically defining molecular event in PTC, associated with features predictive of a high risk of LNM even in microcarcinomas. In addition, one patient in our study exhibited two mutations in the BRAF gene; BRAF V600E and BRAF K601E, with the latter identified in NG tissue.

 Acknowledgements

This study was supported by grants from the Medical Science Research Foundation of Zhejiang Province (2010KYA161) and the Key Project of Scientific and Technological Innovation in Hangzhou (20131813A08).

 References DaviesLWelchHGIncreasing incidence of thyroid cancer in the United StatesJAMA29521642167200610.1001/jama.295.18.216416684987 TuttleRMBallDWByrdDThyroid carcinomaJ Natl Compr Canc Netw812281274201021081783 VeigaLHNetaGAschebrook-KilfoyBRonEDevesaSSThyroid cancer incidence patterns in Sao Paulo, Brazil and the US SEER ProgramThyroid23748757201310.1089/thy.2012.053223410185 TangKTLeeCHBRAF mutation in papillary thyroid carcinoma: pathogenic role and clinical implicationsJ Chin Med Assoc73113128201010.1016/S1726-4901(10)70025-320230995 MesaCJrMirzaMMitsutakeNConditional activation of RET/PTC3 and BRAFV600E in thyroid cells is associated with gene expression profiles that predict a preferential role of BRAF in extracellular matrix remodelingCancer Res6665216529200610.1158/0008-5472.CAN-06-073916818623 DurandSFerraro-PeyretCJoufreMChaveABorson-ChazotFSelmi-RubySRoussetBMolecular characteristics of papillary thyroid carcinomas without BRAF mutation or RET/PTC rearrangement: relationship with clinico-pathological featuresEndocr Relat Cancer16467481200910.1677/ERC-08-008119208736 XingMGenetic alterations in the phosphatidylinositol-3 kinase/Akt pathway in thyroid cancerThyroid20697706201010.1089/thy.2010.164620578891 KonturekABarczynskiMWierzchowskiWStopaMNowakWCoexistence of papillary thyroid cancer with Hashimoto thyroiditisLangenbecks Arch Surg398389394201310.1007/s00423-012-1021-x23099542 ZhouGYLiuHQZhangQHThyroid and parathyroid gland tumorsDiagnostic Histopathology of TumorsFletcherCD1stShandong Science and Technology PressJinan5992001 LiaoEYChaoCSChronic lymphocytic thyroiditisEndocrinologyZhangHDaiRCLiaoEY1stPeople's Medical PublisherBeijing7152001 GouveiaCCanNTBostromAGrenertJPvan ZanteAOrloffLALack of association of BRAF mutation with negative prognostic indicators in papillary thyroid carcinoma: the University of California, San Francisco, experienceJAMA Otolaryngol Head Neck Surg13911641170201310.1001/jamaoto.2013.450124030686 WittRLFerrisRLPribitkinEAShermanSIStewardDLNikiforovYEDiagnosis and management of differentiated thyroid cancer using molecular biologyLaryngoscope12310591064201310.1002/lary.2383823404751 YuLYZhangCXuRJZhouHMaLZDiagnostic value of TPO, CK-19, Gal-3 and HBME-1 in fine needle aspiration cytological specimens for thyroid carcinoma with Hashimoto's diseaseZhejiang Medical321007101010132010 TroviscoVSoaresPSoaresRMagalhãesJSa-CoutoPSobrinho-SimoesMA new BRAF gene mutation detected in a case of a solid variant of papillary thyroid carcinomaHum Pathol36694697200510.1016/j.humpath.2005.04.01116021577 SchmidtJDerrVHeinrichMCCrumCPFletcherJACorlessCLNoseVBRAF in papillary thyroid carcinoma of ovary (struma ovarii)Am J Surg Pathol3113371343200710.1097/PAS.0b013e31802f540417721188 LupiCGianniniRUgoliniCAssociation of BRAF V600E mutation with poor clinicopathological outcomes in 500 consecutive cases of papillary thyroid carcinomaJ Clin Endocrinol Metab9240854090200710.1210/jc.2007-117917785355 FrauDVLaiMLCariaPTrisomy 17 as a marker for a subset of noninvasive thyroid nodules with focal features of papillary carcinoma: cytogenetic and molecular analysis of 62 cases and correlation with histological findingsJ Clin Endocrinol Metab93177181200810.1210/jc.2007-097017956956 WolffEFHughesMMerinoMJReynoldsJCDavisJLCochranCSCeliFSExpression of benign and malignant thyroid tissue in ovarian teratomas and the importance of multimodal management as illustrated by a BRAF-positive follicular variant of papillary thyroid cancerThyroid20981987201010.1089/thy.2009.045820718682 PennelliGVianelloFBarolloSBRAF (K601E) mutation in a patient with a follicular thyroid carcinomaThyroid2113931396201110.1089/thy.2011.012022136270 OhoriNPSinghalRNikiforovaMNBRAF mutation detection in indeterminate thyroid cytology specimens: underlying cytologic, molecular and pathologic characteristics of papillary thyroid carcinomaCancer Cytopathol121197205201310.1002/cncy.2122922887810 SchultenHJSalamaSAl-MansouriZBRAF mutations in thyroid tumors from an ethnically diverse groupHered Cancer Clin Pract101011201210.1186/1897-4287-10-1022925390 ParkJYKimWYHwangTSBRAF and RAS mutations in follicular variants of papillary thyroid carcinomaEndocr Pathol246976201310.1007/s12022-013-9244-023625203 KimWYKoYSHwangTSA case of multifocal papillary thyroid carcinoma consisting of one encapsulated follicular variant with BRAF K601E mutation and three conventional types with BRAF V600E mutationKorean J Pathol47293298201310.4132/KoreanJPathol.2013.47.3.29323837025 BarolloSPezzaniRCristianiAPrevalence, tumorigenic role and biochemical implications of rare BRAF alterationsThyroid24809819201410.1089/thy.2013.040324295088 SoaresPCelestinoRMeloMFonsecaESobrinho-SimoesMPrognostic biomarkers in thyroid cancerVirchows Archiv464333346201410.1007/s00428-013-1521-224487783 RotiEDegliUberti ECBondanelliMBravermanLEThyroid papillary microcarcinoma: a descriptive and meta-analysis studyEur J Endocrinol159659673200810.1530/EJE-07-089618713843 LeeXGaoMJiYAnalysis of differential BRAF (V600E) mutational status in high aggressive papillary thyroid microcarcinomaAnn Surg Oncol16240245200910.1245/s10434-008-0233-319034577 VirkRKVan DykeALFinkelsteinABRAFV600E mutation in papillary thyroid microcarcinoma: a genotype-phenotype correlationMod Pathol266270201310.1038/modpathol.2012.15222918165 KimDParkJWClinical implications of preoperative thyrotropin serum concentrations in patients who underwent thyroidectomy for nonfunctioning nodule(s)J Korean Surg Soc851519201310.4174/jkss.2013.85.1.1523833755 KimSKSongKHLimSDLimYCYooYBKimJSHwangTSClinical and pathological features and the BRAF (V600E) mutation in patients with papillary thyroid carcinoma with and without concurrent Hashimoto thyroiditisThyroid19137141200910.1089/thy.2008.014419014278

Agarose gel (1.5%) electrophoresis for thyroid tissue polymerase chain reaction products. Lane 1, DL2000 DNA marker (Dingguo Biology Ltd., Beijing, China). Lanes 2–8, representative positive cases.

(A) Representative sequencing traces from one of the positive replicates indicating the BRAF V600E heterozygous mutation, a T to A transversion at exon 15 nucleotide 1799 (T1799A) of the BRAF gene; (B) the wild type.

Sequencing traces from one patient indicating the BRAF K601E heterozygous mutation, an A to G transversion at exon 15 nucleotide 1801 (A1801G) of the BRAF gene.

Correlation of BRAF V600E mutation and thyroid function in thyroid nodules [n(%), (mean ± SD)].

BRAF V600E mutation
Group n – + T3 (µg/l) T4 (µg/l) FT3 (pmol/l) FT4 (pmol/l) TSH (mIU/l)
NT 14 13 (92.9) 1 (7.1) 1.04±0.26 97.58±22.02 4.66±0.43 16.02±2.44 2.15±1.21
NG 77 76 (98.7) 1 (1.3) 1.07±0.40 93.29±17.05 4.75±0.46 16.53±7.79 2.02±1.55
AN 5 5 (100.0) 0 (0.0) 1.08±0.29 86.45±26.00 4.51±0.93 13.29±3.79 2.65±2.87
TA 3 3 (100.0) 0 (0.0) 1.16±0.40 106.83±29.53 4.78±0.59 15.80±1.07 1.65±0.92
TC 65 25 (36.9) 40 (61.5)a 1.07±0.56 90.72±21.78 4.59±0.73 19.00±15.56 2.34±1.37
HT 4 4 (100.0) 0 (0.0) 1.61±1.66 60.87±36.17 4.31±0.82 13.19±7.64 3.04±1.71

Compared with other groups, χ2=75.732, P<0.001. NT, normal tissue adjacent to carcinoma; NG, nodular goiter; AN, adenomatoid nodule; TA, thyroid adenoma; TC, thyroid carcinoma; HT, Hashimoto thyroiditis; -, negative; +, positive; FT3, free triiodothyronine; FT4, free thyroxine; TSH, thyroid stimulating hormone.

Prevalence of BRAF V600E mutation in thyroid nodules in relation to Hashimoto thyroiditis [n(%)].

BRAF V600E mutation
Group HT n – + χ2 P-value
BT – 71 70 (98.6) 1 (1.4) 0.256 0.613
+ 18 18 (100.0) 0 (0.0)
PTC – 52 17 (32.7) 35 (67.3) 3.656 0.056
+ 13 8 (61.5) 5 (38.5)

BT, benign tissue; PTC, papillary thyroid carcinoma, HT, Hashimoto thyroiditis; -, negative; +, positive.

Correlation of BRAF V600E mutation and clinicopathological characteristics in thyroid nodules [n(%), (mean ± SD)].

Gender PTMC Lymph node metastasis
Male Female Age (years) TSH (mIU/l) – + – +
BRAF V600E − 21 (16.7) 105 (83.3) 51.1±10.1 1.966±1.296 3 (12.0) 22 (88.0) 21 (84.0) 4 (16.0)
BRAF V600E + 10 (23.8) 32 (76.2) 49.0±11.1 2.453±1.464 18 (45.0) 22 (55.0) 23 (57.5) 17 (42.5)
Total 31 (18.5) 137 (81.5) 50.6±10.4 2.198±1.514 21 (32.3) 44 (67.7) 44 (67.7) 21 (32.3)
χ2=1.068 t=1.134 t=2.019 χ2=7.661 χ2=4.940
P-value 0.301 0.259 0.045 0.006 0.026

PTMC, papillary thyroid microcarcinoma; TSH, thyroid stimulating hormone; -, negative; +, positive.

Associations between clinicopathological features and lymph node metastasis of papillary thyroid carcinoma.

Clinical features P-value HR 95% CI
Gender 0.016 0.147 0.031–0.697
Age, years 0.279
TSH level 0.374
PTMC 0.181
HT 0.121
BRAF mutation 0.041 5.051 1.068–23.893

TSH, thyroid stimulating hormone; PTMC, papillary thyroid microcarcinoma; HT, Hashimoto thyroiditis; HR, hazard ratio; CI, confidence interval.