Abstract
Background: The BRAF<sup>V600E</sup> mutation is common in papillary thyroid cancer (PTC). Lymph node metastasis (LNM) may be associated with poor prognosis. However, the LNM mechanism remains unclear. Objectives: Our aim was to evaluate the prevalence of the BRAF<sup>V600E</sup> mutation in primary tumors and accompanying LNM at the time of diagnosis. Methods: This retrospective study included 51 PTC patients (40 women, 11 men; mean age 40.0 ± 16.5 years; range 6-81) who underwent total thyroidectomy accompanied by a lateral neck dissection due to preoperatively detected LNM. Real-time PCR was used for the detection of the BRAF<sup>V600E</sup> mutation in specimens from primary thyroid tumors and metastatic lymph node tumors. Results: The prevalence of the BRAF<sup>V600E</sup> mutation was 64.7% (n = 33) in primary tumors and 47.1% (n = 24) in metastatic lymph nodes. Of 33 patients with BRAF<sup>V600E</sup>-positive primary tumors, 18 (54.5%) had BRAF<sup>V600E</sup>-positive metastatic lymph nodes. Of 18 patients with BRAF<sup>V600E</sup>-negative primary tumors, 6 (33.3%) had BRAF<sup>V600E</sup>-positive metastatic lymph nodes. The presence of the BRAF<sup>V600E</sup> mutation in the primary tumor did not affect the tumor size, but the diameter of metastatic lymph nodes significantly increased (by nearly 3 mm) with the presence of BRAF<sup>V600E</sup> in LNM (p = 0.01). Conclusions: In our study, the BRAF<sup>V600E</sup> mutation did not show a one-to-one correspondence. This indicates that the presence of BRAF<sup>V600E</sup> in the primary tumor is not clonal and addresses the role of intratumor heterogeneity in PTC tumorigenesis. This supports the theses that mutations occur in the later stages of tumorigenesis, might be subclonal, and develop de novo, or that some other factors may be involved in the development of metastasis.
Introduction
Papillary thyroid cancer (PTC) is the most common type of endocrine cancer [1]. It originates from follicular epithelial cells and usually has a slow progression with a good prognosis. However, of note, it presents with a high rate of lymph node metastasis (LNM; 45%) at the time of diagnosis [2]. The basic mechanism underlying PTC tumorigenesis is the MAP kinase/ERK pathway involved in cell division, proliferation, differentiation, adhesion, migration, and apoptosis. Somatic point mutations that occur in the BRAF gene, which encodes serine/threonine protein kinase involved in this cascade, represent a significant genetic event in PTC [3]. Among these, the most common BRAF alteration is the V600E mutation (BRAFV600E), which involves transversion of thymine to adenine at position 1799 in exon 15 and, subsequently, valine(V)-to-glutamate(E) substitution at residue 600 [4]. This mutation turns BRAF into an oncogene that strongly activates the MAPK pathway. It is known that the presence of BRAFV600E results in the suppression of tumor suppressor genes and increases the number of tumor-facilitating molecules [5]. Controversy exists in the literature about the prognostic nature of the BRAFV600E mutation, with some studies having reported an association with poor prognostic characteristics [6,7] and some having failed to show such an association [8]. Discrepancies in the results of these studies may be due to variations in the evaluation of clinical data or to genetic and geographic diversity.
Initially, the BRAFV600E mutation was thought to occur in the early stages of thyroid carcinogenesis. However, thanks to the molecular dissection of the genome, intratumor heterogeneity has been increasingly understood and shown to take part in thyroid tumorigenesis; however, the question of how this heterogeneity develops has yet to be clarified. It has been suggested that heterogeneity might originate from certain groups of tumor cells rather than from clonal growth, and thus mutations might exhibit intratumor heterogeneity [9].Tumor heterogeneity may be associated with thyroid tumor microenvironment, thus some cells provide a basis for mutation while others do not harbor mutations. This may also be associated with other genetic factors or defects in DNA repair mechanisms [10,11]. Moreover, despite our current knowledge of BRAFV600E having a role in PTC tumorigenesis, it is still to be clarified why this mutation is not present in all cases [12,13]. It is likely that the broad spectrum of PTC encompassing the latent microcarcinoma classic or aggressive variants is based on intratumor heterogeneity. The BRAFV600E mutation and tumor heterogeneity have been thought to have a particular role in the development of LNM detected even in the early stages of tumor growth. The present study was designed to evaluate the prevalence of the BRAFV600E mutation in primary tumors and accompanying metastases in PTC patients who were detected to have cervical LNM at the time of diagnosis.
Materials and Methods
This retrospective study included 51 patients (40 women, 11 men) who presented to our thyroid disease department between January 2012 and December 2014 and were diagnosed as having PTC. All the patients underwent total thyroidectomy accompanied by a lateral neck dissection due to preoperatively detected LNM. The mean age of the patients was 40.0 ± 16.5 years (range 6-81), with 39 patients (76.5%) being younger than 45 years. The clinicopathological characteristics of the patients are shown in table 1. All the patients were operated on by the same surgical team. Tissue samples were processed by the same group of pathologists. Samples from primary tumors and metastatic lymph nodes were fixed in paraffin for molecular pathology examination. Histological diagnoses were made based on the criteria of the UICC/AJCC (Union for International Cancer Control/American Joint Committee on Cancer) TNM staging system [14]. The presence of the BRAFV600E mutation was sought in primary tumors and accompanying neck lymph node metastases. From paraffin-fixed and hematoxylin-eosin-stained specimens of the primary thyroid tumors and metastatic lymph node tumors, morphologically well-preserved areas with adequate numbers of tumor cells were marked for real-time PCR analysis. Five 5-μm-thick sections were then obtained from these areas and treated with paraffin. Then, DNA isolation was performed using the Cobas DNA Sample Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany), and DNA quantification was made on a NanoDrop 2000 spectrophotometer (ThermoScientıfıc, Wilmington, N.C., USA). All the samples studied were greater than 2 ng/ml. V600E mutation analysis of the BRAF gene was performed using the Light Cycler 480 Instrument and the Cobas 4800 BRAF V600 Mutation Test (Roche Diagnostics GmbH). Validation of each measurement was checked using mutation-positive and mutation-negative controls.
BRAFV600E mutation and clinicopathological features in primary tumors and LNM
Statistical Analysis
Data are presented as frequency, percentage, and arithmetic mean. Statistical analyses were made with the use of the χ2, Mann-Whitney U, and Spearman correlation tests. A p value of less than 0.05 was considered statistically significant.
Results
The mean primary tumor size was 18.2 ± 12.2 mm (range 5-75), and the mean metastatic lymph node diameter was 14.2 ± 9.7 mm (range 1-50).The prevalence of the BRAFV600E mutation was 64.7% (n = 33) in primary tumors. This prevalence was 39.4% in our previous study [6]. We think that the reason for high prevalence in the current study is the fact that most of the patients had LNM. We have actually previously shown that the risk of LNM increases in the presence of BRAF mutation. The prevalence was 47.1% (n = 24) in metastatic lymph nodes. The presence of the BRAFV600E mutation in primary tumors did not directly match its presence in metastatic lymph nodes (p = 0.14). The relationship of BRAFV600E between primary tumors and LNM is summarized in table 2. Of 33 patients with BRAFV600E-positive primary tumors, 18 (54.5%) had BRAFV600E-positive metastatic lymph nodes. In the remaining 15 patients without BRAFV600E-positive metastatic lymph nodes, there were 7 unifocal and 8 multifocal primary tumors. Eighteen patients had BRAFV600E-negative primary tumors. In this group, BRAFV600E-positive metastatic lymph nodes were detected in 6 patients (33.3%) whose primary tumors were multifocal in 5 patients and unifocal in 1 patient.
The relationship of BRAFV600E in primary tumors and accompanying metastatic lymph nodes
The presence of the BRAFV600E mutation was not correlated with age and gender. Lymphocytic infiltration in nontumoral tissue was detected in 32 patients (62.7%). Despite not being statistically significant, this rate was considered to be high. The majority of the patients (n = 45, 88.2%) had conventional papillary thyroid carcinoma, but this did not show a statistically significant difference. The presence of the BRAFV600E mutation in the primary tumor was not correlated with tumor size or with the diameter of the metastatic lymph node. Conversely, metastatic lymph nodes showed a significant relationship between BRAFV600E positivity and the diameter of metastatic lymph nodes (p = 0.01). BRAFV600E-positive metastatic lymph nodes were nearly 3 mm greater in diameter than those without the BRAFV600E mutation.
Discussion
Despite considerable improvements in our understanding of the molecular mechanism of PTC, its LNM mechanism remains unclear. The frequent detection of the BRAFV600E mutation in PTC, along with its absence in other thyroid cancers, suggests that this mutation may play a role in the early stages of PTC tumorigenesis and thus may exist in tumor metastases [15]. Recent studies have shown that wild-type BRAF and mutated BRAF (BRAFV600E) genetic alterations coexist in PTC, forming intratumor heterogeneity. Mutational differences between primary tumors and accompanying lymph node metastases are thought to be a result of tumor heterogeneity [9,10,16]. Therefore, a BRAF-mutated primary tumor may cause LNM to have either a mutant or wild-type variation. On the other hand, a primary tumor with wild-type BRAF may give rise to LNM of mutated BRAF [17,18]. However, it is clear that if LNM was characterized by BRAF mutation, mutation-bearing cells would form a prevailing majority over wild-type BRAF in the primary tumor and LNM. On the other hand, Jovanovic et al. [19] evaluated microsatellite allelic imbalance in multifocal PTC and demonstrated that not all foci harbored the BRAFV600E mutation. This suggests that the BRAFV600E mutation may be a subclonal event in the development of PTC, providing further support to the view that the BRAFV600E mutation may not be the initiator of PTC tumorigenesis.
Our study group was composed of PTC patients with LNM, which enabled us to make a more refined evaluation to understand the relationship of the BRAFV600E mutation with primary tumors and accompanying LNM. Primary tumors of 33 patients (64.7%) were BRAFV600E positive. The overall BRAFV600E positivity for LNM (with and without primary tumor positivity) was 47.1% (n = 24). Our finding that the rate of accompanying LNM mutations was lower than that seen in primary tumors seems to contradict the view advocated by the clonal evolution model that most tumor cells would harbor mutations. Rather, it provides support for the theses that mutations occur in the later stages of tumorigenesis, might be subclonal, or develop de novo, or that some other factors may be involved in the development of metastasis [9,17]. Specifically, in those with primary tumor mutations, the rate of accompanying LNM mutations was 54.5%. This shows that primary tumor mutations do not necessarily correspond to LNM mutations and that other factors may take part in LNM. Of note, some reported rates of accompanying LNM mutations are remarkably higher than that found in our study, being 73.9% [20], 81.0% [17], and even 96.0% [21] in three studies. Discrepancies in the reported rates of LNM mutations may be related to tumoral multifocality. The natural environment of lymph nodes and growth factors may provide a favorable settlement and harbor for PTC cells carrying BRAFV600E. Moreover, access of mutated cells to lymph nodes may bring about some biological changes, with eventual proliferation of cancer cells [22]. The latter may account for how even small tumor foci may cause larger LNM. In particular, multifocal PVC may present as a heterogeneous BRAFV600E distribution, which is thought to result from independent clonal growth in diverse tumor foci [23]. On the other hand, it is not clear whether multifocal tumors develop from one tumor focus by intratumoral spread or from diverse tumor foci. Intratumor heterogeneity of mutations may be associated with this, as well. It is not known whether multifocal tumors develop as independent foci (resulting from different progenitor cells) or arise from intrathyroidal metastasis. The dense lymphatic network in the thyroid suggests a higher likelihood of intratumoral spread. Some studies demonstrated increases in both intrathyroidal multifocality and LNM with increased intratumoral lymphogenesis [24,25]. It is important to know how multifocality occurs, because if this condition occurs through intrathyroidal metastasis, then there is an increased likelihood of extrathyroidal metastasis. Park et al. [26] reported in patients with multifocal PTC that BRAFV600E showed heterogeneity in 39.3% of the cases, that mutations were not present in all foci, and that mutations did not demonstrate clonal development. Some studies reported polyclonality as an uncommon entity for multifocal tumors [27]. Guerra et al. [9] assessed intratumor heterogeneity with the allelic percentage of the BRAFV600E mutation and found mutations in less than 50% of the alleles, with the majority having wild-type BRAF. This intratumor heterogeneity raises the view that BRAFV600E initiates tumorigenesis, but progression is mainly due to secondary genetic events. On the other hand, BRAFV600E can be secondary in PTC tumorigenesis. In this case two questions remain unanswered: what initiates tumorigenesis if BRAFV600E occurs secondarily, and what is the role of BRAFV600E at this stage? Guerra et al. [16] reported that an allele percentage of more than 30% pointed to a poor prognosis and recommended the measurement of the allelic percentage of BRAFV600E to evaluate the prognosis or the possibility of response to treatment with kinase inhibitors.
In our study, of 18 patients with BRAFV600E-positive primary tumors, 14 had mutation-positive LNM despite multifocal primary tumors. It is likely that mutations in these patients were homogeneously transported. On the other hand, 15 patients had mutation-negative LNM despite primary tumor mutations. In 8 of these patients with multifocal tumors, metastasis spread was thought to develop from tumoral foci without mutations. Conversely, as reported in the study of Guerra et al. [9], these tumor foci consisted of numerous wild-type BRAF and a small number of mutant BRAFV600E, and because the mutations were not clonal, mutant BRAFV600E could not have a chance to proceed from the primary tumor to LNM.
Quantitative studies have demonstrated that BRAFV600E is a subclonal or oligoclonal event rather than being clonal. Oncogen, distributed in some cells or groups of cells within the tumor cells, constitutes intratumor heterogeneity. All these data suggest that the BRAFV600E mutation is not an early event in PTC tumorigenesis, as previously thought, but rather an event occurring later, and that other genetic events are likely to take part in tumorigenesis and the metastatic process [9,10].
Of 18 patients with BRAFV600E-negative primary tumors, 12 also had BRAFV600E-negative LNM, suggesting that non-BRAFV600E mechanisms were involved in the metastatic process. Interestingly, in the remaining 6 patients, BRAFV600E-positive metastatic lymph nodes were detected despite BRAFV600E-negative primary tumors. This was at odds with the view that mutations initiate PTC tumorigenesis. Mutations in these patients either developed at late stages of tumorigenesis or were simply de novo. One of these patients had a unifocal primary tumor, suggesting a de novo mutation in the metastatic lymph node. Vasko et al. [17] also pointed to the possibility of de novo mutations in LNM. The detection of mutated LNM in 5 patients with multifocal primary tumors raises the possibility that mutations could be carried to LNM from a focus other than the dominant tumor. Apparently, this finding may challenge the approach of choosing the dominant focus for mutation analysis used in many studies.
Another important finding of our study is that mutation-positive LNM was significantly associated with a greater diameter of metastatic lymph nodes (p = 0.01). This is either because mutation itself accelerates cell growth in the lymph nodes or because lymph node microenvironment paves the way for the settlement of mutation through the growth of some nodes [22].
Although BRAFV600E is considered to be a high-risk factor for aggressive behavior of tumors, it is not an independent risk factor for negative results. There are also some reports which suggest that it may not be used as a poor prognostic factor [28]. The mechanism of BRAFV600E and metastasis-based distribution characteristics of mutations in PTC will affect pharmaceutical mutation-directed studies. The subclonal nature of mutations may cause variations in metastasis-induced responses to BRAF inhibitors [29].
In conclusion, despite the high frequency rates of BRAFV600E in PTC patients, the mutation does not show a one-to-one correspondence in terms of LNM mutations. This unmatched pattern of the mutation provides support for the view that intratumor heterogeneity plays a significant role in PTC tumorigenesis and that the mutation comes into effect at any stage rather than at the beginning of tumorigenesis. Another important point is that the metastatic lymph node harboring BRAFV600E is enlarged most likely due to mutationrelated causes, which may denote progression and thus should be borne in mind during treatment and follow-up of PTC patients with BRAFV600E-positive metastatic lymph nodes. Additionally, knowing that the BRAFV600E mutation presents as a subclonal or oligoclonal event resulting in intratumor heterogeneity can increase the response rates of novel mutation-directed drug therapies.
Acknowledgments
We thank S. Pinar Barut (molecular biology and genetic specialist) and Meltem Algan (biologist) for technical support in the BRAFV600E mutation analysis.
Disclosure Statement
The authors declare that they have no conflicts of interest.
Footnotes
verified
References
- 2↑
Popadich A, Levin O, Lee JC, Smooke-Praw S, Ro K, Fazel M, Arora A, Tolley NS, Palazzo F, Learoyd DL, Sidhu S, Delbridge L, Sywak M, Yeh MW: A multicenter cohort study of total thyroidectomy and routine central lymph node dissection for cN0 papillary thyroid cancer. Surgery 2011;150:1048-1057.
- 3↑
Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, Westra WH, Ladenson PW, Sidransky D: BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 2003;95:625-627.
- 4↑
Ciampi R, Nikifarov YE: RET/PTC rearrangements and BRAF mutations in thyroid tumorigenesis. Endocrinology 2007;148:936-941.
- 5↑
Tufano RP, Teixeira GV, Bishop J, Carson KA, Xing M: BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine (Baltimore) 2012;91:274-286.
- 6↑
Kurtulmus N, Duren M, Ince U, Cengiz Yakicier M, Peker O, Aydin O, Altiok E, Giray S, Azizlerli H: BRAFV600E mutation in Turkish patients with papillary thyroid cancer: strong correlation with indicators of tumor aggressiveness. Endocrine 2012;42:404-410.
- 7↑
Lee JH, Lee ES, Kim YS: Clinicopathologic significance of BRAF V600E mutation in papillary carcinomas of the thyroid: a meta-analysis. Cancer 2007;110:38-46.
- 8↑
Trovisco V, Couto JP, Cameselle-Teijeiro J, de Castro IV, Fonseca E, Soares P, Sobrinho-Simoes M: Acquisition of BRAF gene mutations is not a requirement for nodal metastasis of papillary thyroid carcinoma. Clin Endocrinol (Oxf) 2008;69:683-685.
- 9↑
Guerra A, Sapio MR, Marotta V, Campanile E, Rossi S, Forno I, Fugazzola L, Budillon A, Moccia T, Fenzi G, Vitale M: The primary occurrence of BRAFV600E is a rare clonal event in papillary thyroid carcinoma. J Clin Endocrinol Metab 2012;97:517-524.
- 10↑
Walts AE, Pao A, Sacks W, Bose S: BRAF genetic heterogeneity in papillary thyroid carcinoma and its metastasis. Hum Pathol 2014;45:935-941.
- 11↑
Takano T: Fetal cell carcinogenesis of the thyroid: a modified theory based on recent evidence. Endocr J 2014;61:311-320.
- 12↑
Xing M: BRAFV600E mutation and papillary thyroid cancer: chicken or egg? J Clin Endocrinol Metab 2012;97:2295-2298.
- 13↑
Unger K, Zitzelsberger H, Salvatore G, Santoro M, Bogdanova T, Braselmann H, Kastner P, Zurnadzhy L, Tronko N, Hutzler P, Thomas G: Heterogeneity in the distribution of RET/PTC rearrangements within individual post-Chernobyl papillary thyroid carcinomas. J Clin Endocrinol Metab 2004;89:4272-4279.
- 14↑
UICC; Sobin LH, Wittekind CH (eds): TNM Classification of Malignant Tumors, ed 6. New York, Wiles-Liss, 2002.
- 15↑
Xing M: BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 2007;28:742-762.
- 16↑
Guerra A, Fugazzola L, Marotta V, Cirillo M, Rossi S, Cirello V, Forno I, Moccia T, Budillon A, Vitale M: A high percentage of BRAFV600E alleles in papillary thyroid carcinoma predicts a poorer outcome. J Clin Endocrinol Metab 2012;97:2333-2340.
- 17↑
Vasko V, Hu S, Wu G, Xing JC, Larin A, Savchenko V, Trink B, Xing M: High prevalence and possible de novo formation of BRAF mutation in metastasized papillary thyroid cancer in lymph nodes. J Clin Endocrinol Metab 2005;90:5265-5269.
- 18↑
Costa AM, Herrero A, Fresno MF, Heymann J, Alvarez JA, Cameselle-Teijeiro J, Garcia-Rostan G: BRAF mutation associated with other genetic events identifies a subset of aggressive papillary thyroid carcinoma. Clin Endocrinol (Oxf) 2008;68:618-634.
- 19↑
Jovanovic L, Delahunt B, McIver B, Eberhardt NL, Grebe SK: Most multifocal papillary thyroid carcinomas acquire genetic and morphotype diversity through subclonal evolution following the intra-glandular spread of the initial neoplastic clone. J Pathol 2008;215:145-154.
- 20↑
Rodolico V, Cabibi D, Pizzolanti G, Richiusa P, Gebbia N, Martorana A, Russo A, Amato MC, Galluzzo A, Giardano C: BRAFV600E mutation and p27kip1 expression in papillary carcinomas of the thyroid ≤1 cm and their paired lymph node metastases. Cancer 2007;110:1218-1226.
- 21↑
Lu J, Gao J, Zhang J, Sun J, Wun H, Shi X, Teng L, Liang Z: Association between BRAFV600E mutation and regional lymph node metastasis in papillary thyroid carcinoma. Int J Clin Exp Pathol 2015;8:793-799.
- 22↑
Ito Y, Yoshida H, Kihara M, Kobayashi K, Miya A, Miyauchi A: BRAFV600E mutation analysis in papillary thyroid carcinoma: is it useful for all patients? World J Surg 2014;38:679-687.
- 23↑
Giannini R, Ugolini C, Lupi C, Proietti A, Elisei R, Salvatore G, Berti P, Materazzi G, Miccoli P, Santoro M, Basolo F: The heterogeneous distribution of BRAF mutation supports the independent clonal origin of distinct tumor foci in multifocal papillary thyroid carcinoma. J Clin Endocrinol Metab 2007;92:3511-3516.
- 24↑
Choi Y, Park KJ, Ryu S, Kim DH, Yun J, Kang DK, Chun M: Papillary thyroid carcinoma involving cervical neck lymph nodes: correlations with lymphangiogenesis and ultrasound features. Endocr J 2012;59:941-948.
- 25↑
Hall FT, Freeman JL, Asa SL, Jackson DG, Beasley NJ: Intratumoral lymphatics and lymph node metastases in papillary thyroid carcinoma. Arch Otolaryngol Head Neck Surg 2003;129:716-719.
- 26↑
Park SY, Park YJ, Lee YJ, Lee HS, Choi SH, Choe G, Jang HC, Park SH, Park DJ, Cho BY: Analysis of differential BRAFV600E mutational status in multifocal papillary thyroid carcinoma: evidence of independent clonal origin in distinct tumor foci. Cancer 2006;107:1831-1838.
- 27↑
Abrosimov A, Saenko V, Rogounovitch T, Namba H, Lushnikov E, Mitsutake N, Yamashita S: Different structural components of conventional papillary carcinoma display mostly identical BRAF status. Int J Cancer 2007;120:196-200.
- 28↑
Russo M, Malandrino P, Nicolosi ML, Manusia M, Marturano I, Trovato MA, Pellegriti G, Frasca F, Vigneri R: The BRAFV600E mutation influences the short- and medium-term outcomes of classic papillary thyroid cancer, but is not an independent predictor of unfavorable outcome. Thyroid 2014;24:1267-1274.
- 29↑
Vitale M: Intratumor BRAFV600E heterogeneity and kinase inhibitors in the treatment of thyroid cancer: a call for participation. Thyroid 2013;23:517-519.