Clinical significance of BRAF V600E mutation in 154 patients with thyroid nodules
- Authors:
- Published online on: April 15, 2015 https://doi.org/10.3892/ol.2015.3119
- Pages: 2633-2638
Abstract
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
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
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 BRAF 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).
 | Table I.Correlation of BRAF V600E mutation and thyroid function in thyroid nodules [n(%), (mean ± SD)]. |
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 BRAF 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).
| Table II.Prevalence of BRAF V600E mutation in thyroid nodules in relation to Hashimoto thyroiditis [n(%)]. |
Correlation of the BRAF 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).
| Table III.Correlation of BRAF V600E mutation and clinicopathological characteristics in thyroid nodules [n(%), (mean ± SD)]. |
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.
| Table IV.Associations between clinicopathological features and lymph node metastasis of papillary thyroid carcinoma. |
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).
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