BRAF and TERT promoter mutations: clinical application in thyroid cancer

Abstract

Given the long-term survival of most patients with thyroid cancer, it is very important to distinguish patients who need aggressive treatment from those who do not. Conventional clinicopathological prognostic parameters could not completely predict the final outcome of each patient. Recently, molecular marker-based risk stratification of thyroid cancer has been proposed to better estimate the cancer risk. Although BRAF mutation has drawn much attention based on its high prevalence, its association with recurrence or mortality is not clear. Recently, telomerase reverse transcriptase (TERT) promoter mutation has been identified in thyroid cancer. It increases telomerase activity, which allows cancer cells to immortalize. It was found in 10 to 20% of differentiated thyroid carcinoma and 40% of dedifferentiated thyroid carcinoma. It is highly prevalent in old age, large tumor, aggressive histology, advanced stages, and distant metastasis. It is associated with increased recurrence and mortality. Concomitant BRAF and TERT promoter mutations worsen the survival rate. Inclusion of TERT promoter mutation analysis with conventional clinicopathological evaluation can lead to better prognostication and management for individual patients.

Introduction

Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) referred together as differentiated thyroid carcinoma (DTC). It accounts for more than 90% in all thyroid cancers, and has a favorable prognosis [1-3]. Although the incidence of thyroid cancer has increased with time, there has been no increase in mortality rate and still remains low [4-7]. However, DTC patients who present with advanced stage or distant metastasis and patients with poorly-differentiated thyroid carcinoma (PDTC) or anaplastic thyroid carcinoma (ATC) have poor prognoses, although the number of these patients are limited [8-12]. Considering the long-term survival of most patients with DTC, it is very important to distinguish patients who need aggressive treatment from those who do not. Conventional clinicopathological parameters, such as age, histological type, tumor size, local invasion, lymph node metastasis and distant metastasis, have been traditionally used to predict the prognosis [13, 14]. However, they could not completely predict the final outcome of each patient [15]. Therefore, more precise parameters for estimating the final outcome should be required and they can make an optimal therapeutic strategy in each patient.

Two signaling pathways are involved in the development of thyroid cancer: mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases & protein kinase B (PI3K–AKT) pathways. Both signaling pathways regulate cellular proliferation, differentiation and survival. The MAPK pathway has been known as the Ras-Raf-MEK-ERK pathway. It is frequently activated in thyroid cancer through BRAF mutation, RAS mutation, and RET/PTC rearrangement, which are the common initiating events in DTC [16, 17]. A few of mutations in the PI3K–AKT pathway are detected more frequently in FTC, PDTC, and ATC [18, 19].

Molecular tests for genetic alterations in thyroid cancer may enhance the diagnostic value of cytologic examination and predictability of clinical outcomes [20]. Molecular changes precede histological changes. Gupta et al. described heterogeneous histological changes in a tumor: microfollicular areas with well-developed nuclear features of PTC, large follicles with borderline nuclear features of PTC, and large follicles lacking nuclear features of PTC. When tested separately, all three areas were positive for the same NRAS61 mutation [21]. Much progress has been made in elucidating the molecular mechanisms involved in the pathogenesis of thyroid cancer after the detection of RAS mutation and RET/PTC rearrangement. Newly identified oncogenes have provided the basic information upon which new diagnostic tools, new prognostic markers, and new targeted agents have been developed. The Cancer Genome Atlas (TCGA) study presented integrated genomic characterization of PTC in 2014 [22]. It described the low frequency of somatic alterations in 496 PTCs compared to other cancers. It reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Genetic alterations in thyroid cancer included point mutations (75%), gene fusions (15%), and copy number variations (7%). In patients with advanced stage, tumors may have more than one mutation, therefore, the overall mutation burden exceeds 100%. The frequency of the main drivers (BRAF, RAS, and RET) sums to less than 100% because in some cases the drivers are not known or they are lower frequency events [23].

In this article, I would like to update a review article published in 2018 by presenting the findings of some additional studies that have been published subsequently [24].

BRAF Mutation in Thyroid Cancer

BRAF mutations were firstly detected in human cancer in 2002 [25]. Thereafter, more than 40 mutations of BRAF gene have been identified. Most cases activating BRAF mutation involve codon 600 and result in the V600E mutation, and a few cases of other BRAF mutations occur as K601E mutation, small in-frame insertions, deletions, or rearrangement [16, 26]. The BRAF mutations were frequently detected in thyroid cancer [16, 17, 27-30]. They have been reported in PTC and PDTC or ATC arising from PTC, but not in FTC, medullary thyroid cancer (MTC) and benign thyroid tumor [31, 32]. Exceptionally, there was a report that one case with FTC carried a BRAF^K601E mutation [33]. The thymine-to-adenine transversion at nucleotide position 1799 in exon 15 of the BRAF gene results in a valine-to-glutamate substitution at residue 600, which leads to constitutive activation of MAPK signaling downstream resulting in tumor development [17, 25, 34]. Small PTCs, less than 1 cm in diameter, have also been shown to harbor BRAF mutations, which are considered to be the early stage of PTCs or an inducing factor of oncogenesis [35-37]. The prevalence of BRAF mutation in PTC ranges from 30% to more than 80%, depending on the iodine intake and geographic location [36, 38]. BRAF mutation is known to be highly specific to PTC, but false-positive results have been rarely reported [39]. In Korea, approximately 97% of newly diagnosed thyroid cancer is PTC and more than 80% of PTC cases harbor BRAF mutation [3, 40, 41]. Therefore, molecular testing for the BRAF mutation in cytological specimen increases diagnostic sensitivity & accuracy in a BRAF mutation-prevalent population [40, 41].

Molecular marker-based risk stratification of thyroid cancer has been recently proposed for estimation of cancer risk [42]. The BRAF mutation has drawn much attention based on its high prevalence [40, 41, 43, 44]. Many studies have demonstrated that BRAF mutation is significantly associated with the aggressive clinicopathological characteristics of PTC, such as extrathyroidal extension, lymph node metastasis and advanced stages [29, 31, 44, 45]. Lee et al. performed a meta-analysis from 12 studies including 1,168 PTC patients [46]. They reported that BRAF mutation was associated with histologic subtype, the presence of extrathyroidal extension, and higher clinical stage, but not with age, sex, race, or tumor size. Tufano et al. published a meta-analysis from 14 studies including 2,470 PTC patients [47]. They demonstrated that BRAF mutation in PTC was significantly associated with the recurrence, lymph node metastasis, extrathyroidal extension, and advanced stages. Even in Korea, the same results were initially published with the meta-analysis of Tufano et al. [44, 45]. Xing et al. reported the association of BRAF mutation with high recurrence and mortality rate in a large multicenter study [48, 49]. Recently, Shen et al. also reported that there was a linear association between patient age and mortality in patients with BRAF^V600E mutation, but not in patients with wild-type BRAF [50]. They concluded that age was a strong, continuous, and independent mortality risk factor in patients with BRAF^V600E mutation. However, it was no longer significant after the adjustment of risk factors, such as lymph node metastasis, extrathyroidal invasion and distant metastasis. In addition, many studies from East Asian countries including Taiwan, Korea, and Japan demonstrated that the BRAF mutation was not associated with disease-free survival as well as poor prognostic factors [51-53]. Many physicians wonder why thyroid cancer-related mortality is still low, although the BRAF mutation is highly prevalent. These findings suggest that the isolated BRAF mutation may be a sensitive, but not specific marker of tumor recurrence and tumor-related mortality. Recently, more specific markers predictive of aggressive behavior have emerged. In patients with advanced stage, tumors may have more than one mutation. Next generation sequencing-based analysis revealed that some PTCs had more than one mutation, and these have aggressive behavior [54].

TERT Promoter Mutation in Thyroid Cancer

Telomeres are located at the ends of linear chromosomes. They are composed of hundreds to thousands of tandem DNA repeat sequences: hexameric TTAGGG in the leading strand and CCCTAA in the lagging strand in humans [55]. They were coated by protective proteins termed shelterin [56]. The 3' end of the telomeric leading strand terminates as a single-stranded overhang, which folds back and invades the double-stranded telomeric helix, forming the T loop. Telomeres can be directly visualized under the microscope at the ends of metaphase chromosomes by fluorescence in situ hybridization (FISH). Average telomere length can be measured by several methods: a technique that combines flow cytometry and FISH, Southern blotting, and a quantitative polymerase-chain-reaction assay. The average length of telomeres in human leukocytes varies, ranging from approximately 11 kb at birth (in umbilical-cord blood) to 6 kb at 90 years of age. Telomere loss is most rapid early in life, and over a life span [57]. The normal cell will divide 50 to 70 times before cell death (Hayflick limit). As the cell divides, the telomeres get smaller. Because of the telomeres shortening through each division, the telomeres will eventually no longer be present on the chromosome. The cells finally become senescent and cell division stops. Shortened telomeres impair immune function that might also increase cancer susceptibility [58]. Therefore, telomere shortening can be related to the risk of cancer. Telomerase is a reverse transcriptase that utilizes an RNA template to add telomeric repeats to the 3' ends of chromosomes. Active telomerase enzyme complex is composed solely of two protein components, telomerase reverse transcriptase (TERT) and dyskerin, and the telomerase RNA component. It protects the telomere repeats from erosion, such as DNA damage or fusion with neighboring chromosomes. Therefore, it reverses telomere shortening. Telomerase is not expressed in normal cells, but is frequently activated in most cancer cells as well as stem cells [59, 60].

Cancer cells are immortal because activated telomerase allows them to survive much longer than normal cell. Therefore, maintenance of telomere length is very important for immortalization of cancer cells. In 2013, point mutations of TERT promoter were firstly found in melanoma, and these somatic mutations enhanced promoter activity by two- to four-fold, which could immortalize cancer cells by maintaining telomere length [61-63]. Somatic mutations in the TERT promoter have been identified in over 50 human cancers including thyroid cancer [64]. TERT promoter mutation has been shown to increase telomerase activity, which protects the telomere repeats from erosion and maintain telomere length. Two mutations in the TERT promoter (chr5: 1295228C>T, termed C228T, and chr5: 1295250C>T, termed C250T) were found in melanoma [61, 62].

The C228T and C250T mutations were detected in follicular cell-derived thyroid cancer but were absent in benign tumors and MTC [64-68]. These two mutations occurred in a mutually exclusive manner. C228T is far more dominant than C250T. The mutual exclusivity of the two mutations suggests that either may function sufficiently to play an important role in thyroid tumorigenesis although which one is more powerful oncogenically has not been established [66, 69, 70]. In meta-analysis, TERT promoter mutation was found in approximately 10% of PTC, 17% of FTC, and 40% of PDTC/ATC [70, 71]. Among PTC, it was more prevalent in tall cell variant than conventional or follicular variants. The TERT promoter mutation has a significantly higher prevalence in old age, large tumor, aggressive histology, advanced stages, and distant metastasis [70, 71]. Why it is more prevalent in old age? Thyroid epithelial cells from young age are telomerase proficient with longer telomere, whereas those from later are telomerase deficient with shorter telomere. When it is attacked by oncogenic events, it undergoes active proliferation with further erosion of its telomere. In that case, telomere dysfunction or even telomere crisis occurs in old age because of its initial shorter telomere and deficient telomerase. Telomere crisis triggers genomic instability and finally induces telomerase activation. TERT promoter mutation is thus the consequence of genomic instability, whereas in turn contributes to derepressing TERT transcription and telomerase activation [67]. TERT promoter mutation has not been found in childhood thyroid cancer, except one in Saudi Arabia [72-75]. TERT promoter mutations were rarely found in small-sized thyroid cancer. de Biase et al. reported that TERT promoter mutations were found in 4.7% of the analyzed 431 papillary thyroid microcarcinomas [76]. Yang et al. suggested that TERT promoter mutation closely associated with non-radioiodine avidity in distant metastatic DTC [77]. BRAF mutation has been known to be associated with iodine intake, but TERT promoter mutation is not [78]. In a multivariate comparison between the PTC with and without anaplastic transformation, TERT promoter mutation was independently associated with anaplastic transformation [79]. Collectively, PTC-derived ATCs are characterized by BRAF and TERT promoter mutations, and these mutations occur prior to anaplastic transformation. Of note, a PTC harboring TERT promoter mutation is at higher risk for anaplastic transformation. Interestingly, TERT mutations in some studies were found to be more common in tumors with BRAF mutation, which may suggest a possible synergistic interplay between MAPK pathway activation and telomerase activation to promote aggressive tumor behavior [65, 66]. TERT promoter mutation as well as BRAF mutation is known to be associated with tumor recurrence, but second recurrence rate is significantly high in patients with TERT promoter mutation, not BRAF mutation [80]. In a meta-analysis including 11 studies with 3,911 patients, PTC with concurrent BRAF and TERT promoter mutations were associated with increased tumor aggressiveness and had worst prognosis in comparison with PTCs harboring BRAF or TERT promoter mutation alone [81]. Recently, Song et al. suggested the potential mechanism of synergistic effect of BRAF and TERT promoter mutations on cancer progression [82]. They explained that BRAF mutation activated MAPK pathway, and it upregulated E-twenty six (ETS) transcription factors. ETS factors bound to mutant TERT promoter, and it increased TERT promoter expression. The genotype of primary tumors has high concordance with the genotype of lymph node metastasis. However, distant metastases show enrichment in TERT promoter mutations and a decrease in BRAF mutations. Therefore, TERT promoter mutations may play a role in distant metastases [83]. The TERT promoter mutation is also associated with increased mortality in PTC as well as other thyroid cancer. Liu et al. reported that PTC with concurrent BRAF and TERT promoter mutations were associated with increased cancer-specific mortality in comparison with PTCs harboring BRAF or TERT promoter mutation alone (TERT/BRAF 22.7% vs. TERT 6.3% vs. BRAF 2.4% vs. wild type 0.6%) [84]. Kim et al. evaluated the association of TERT promoter mutation with survival of 409 thyroid cancer patients followed for median 13 years [71]. They reported that the TERT promoter mutation was independently associated with poorer overall survival in patients with DTC (10-year survival, TERT promoter mutation 66% vs. wild type 98%) and in patients with PTC (74% vs. 99%). Concomitant BRAF and TERT promoter mutations worsened the survival rate of patients with PTC (10-year survival, both mutations 83% vs. wild type 99%; HR 5.62). Recently, Liu et al. reported that coexisting BRAF and TERT promoter mutations are strongly associated with the loss of RAI avidity in recurrent PTC, showing a robust predictive value for failure of RAI treatment of PTC [85]. Thereafter, several studies have supported the synergistic effects of concomitant BRAF and TERT promoter mutations [78, 86-88].

Clinical Application of BRAF and TERT Promoter Mutations in Thyroid Cancer

A few of studies investigated the association of BRAF and TERT promoter mutations with ultrasonography (USG) findings. Hahn et al. suggested that PTC with no mutation, with BRAF mutation alone, and with both BRAF and TERT promoter mutations linearly increased in the probability of displaying malignant USG features [89]. They also reported that PTC with BRAF mutation tended to show a nonparallel orientation (taller-than-wide) shape, but this finding was marginally significant (p = 0.055). There were no significant differences in tumor echogenicity, tumor margin, and calcification between BRAF mutation and wild type. Kim et al. reported that nonparallel orientation and microlobulated margin were independent USG findings for predicting TERT promoter mutation in PTC, especially patients over 50 years [90]. Therefore, they suggested that tests for TERT promoter mutation should be done when physicians met the unique USG findings (nonparallel orientation and microlobulated margin) in thyroid nodular patients, especially over 50 years.

Wang et al. reported an interesting observation in 2014 [91]. They identified TERT promoter mutation (C228T) in 1 of 58 follicular adenoma postoperatively. The patient with C228T mutated follicular adenoma later developed a scar recurrence and died of FTC, whereas none of the remaining 57 patients with follicular adenoma had recurrence. Therefore, they concluded that TERT promoter mutation might occur as an early genetic event in thyroid follicular tumors that have not developed malignant features on routine histopathological workup. According to this finding, it is advisable to develop a more aggressive treatment strategy when a physician sees a patient diagnosed with follicular neoplasm after histological examination but has a TERT promoter mutation. In my personal opinion, at least total thyroidectomy should be done in this patient with TERT promoter mutation, even though they were diagnosed with follicular neoplasm by histologic examination.

Xu et al. also reported an interesting finding in 2017 [92]. They observed 15 cases with low-risk histology with distant metastases at present. The majority was encapsulated follicular variant of PTC with capsular invasion only. Among 8 tumors that were subjected to next-generation sequencing analysis, TERT promoter mutation occurred at a higher rate than that seen in PTC in general and may help explain their aggressive behavior. DTC with low-risk histologic features and distant metastasis was a rare occurrence, accounting for less than 3% of metastatic non-ATC. When meet DTC cases with low-risk histology together with distant metastasis, distant metastasis is almost always found at presentation. These tumors might have a high probability to have TERT promoter mutations.

In 2015, the American Thyroid Association introduced new therapeutic guidelines for DTC included the reclassification of cancer recurrence risk after initial treatment [93]. The new dynamic risk stratification (DRS) system can predict the structural recurrence with higher accuracy than traditional predictive system based on clinicopathologic information [94]. Kim et al. aimed to refine risk prediction for structural recurrence and cancer-specific mortality using the TERT promoter mutation in 357 DTC patients without initial distant metastasis with long-term follow-up (median 14 years) [95]. They developed a new integrative prognostic system that incorporates TERT promoter mutation into the recently proposed DRS system after initial therapy to better categorize and predict outcomes. Cox regression was used to calculate adjusted hazard ratios (AHRs) to derive AHR groups. AHR was adjusted by age, sex, histologic type, multifocality, size, extrathyroidal invasion, node metastasis & RAI treatment. They compared AHR group with pre-existing DRS system and TNM classification using proportion of variance explained (PVE). Larger numbers of PVE suggest better predictability. Patients in higher AHR groups were significantly at higher risk of recurrence and cancer-related death. They suggested that the PVE of new AHR system to predict recurrence was higher than the pre-existing DRS system (22.4% vs. 18.5%). PVE of new AHR system to predict cancer-related death was also higher than pre-existing TNM system (11.5% vs. 7.4%). Therefore, they concluded that inclusion of TERT promoter mutation analysis with conventional clinicopathological evaluation could lead to better prognostication and management for individual patients.

Conclusion

Although the BRAF mutation has drawn much attention, it cannot predict the clinical outcome of each patient. It was no longer significant after the adjustment with other prognostic factors. The isolated BRAF mutation may be a sensitive, but not specific marker of recurrence and mortality. TERT promoter mutation has been found in 10–20% of DTC and 40% of dedifferentiated thyroid cancer. Although it is highly prevalent in old age, large tumor, aggressive histology, advanced stages, and distant metastasis, it is strongly associated with tumor recurrence and mortality in thyroid cancer. Therefore, inclusion of TERT promoter mutation analysis with conventional clinicopathological evaluation could lead to better prognostication and management for individual patients.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

References

• 1   Hundahl  SA,  Cady  B,  Cunningham  MP,  Mazzaferri  E,  McKee  RF, et al. (2000) Initial results from a prospective cohort study of 5583 cases of thyroid carcinoma treated in the United States during 1996. U.S. and German Thyroid Cancer Study Group. An American College of Surgeons Commission on Cancer Patient Care Evaluation study. Cancer 89: 202–217.
• 2   Lim  H,  Devesa  SS,  Sosa  JA,  Check  D,  Kitahara  CM (2017) Trends in thyroid cancer incidence and mortality in the United States, 1974–2013. JAMA 317: 1338–1348.
• 3   Oh  CM,  Kong  HJ,  Kim  E,  Kim  H,  Jung  KW, et al. (2018) National epidemiologic survey of thyroid cancer (NEST) in Korea. Epidemiol Health 40: e2018052.
• 4   Davies  L,  Welch  HG (2006) Increasing incidence of thyroid cancer in the United States, 1973–2002. JAMA 295: 2164–2167.
• 5   Cho  BY,  Choi  HS,  Park  YJ,  Lim  JA,  Ahn  HY, et al. (2013) Changes in the clinicopathological characteristics and outcomes of thyroid cancer in Korea over the past four decades. Thyroid 23: 797–804.
• 6   La Vecchia  C,  Malvezzi  M,  Bosetti  C,  Garavello  W,  Bertuccio  P, et al. (2015) Thyroid cancer mortality and incidence: a global overview. Int J Cancer 136: 2187–2195.
• 7   Jung  KW,  Won  YJ,  Kong  HJ,  Lee  ES (2019) Prediction of cancer incidence and mortality in Korea, 2019. Cancer Res Treat 51: 431–437.
• 8   Jung  TS,  Kim  TY,  Kim  KW,  Oh  YL,  Park  DJ, et al. (2007) Clinical features and prognostic factors for survival in patients with poorly differentiated thyroid carcinoma and comparison to the patients with the aggressive variants of papillary thyroid carcinoma. Endocr J 54: 265–274.
• 9   Ito  Y.,  Higashiyama  T.,  Takamura  Y,  Kobayashi  K,  Miya  A, et al. (2010) Clinical outcomes of patients with papillary thyroid carcinoma after the detection of distant recurrence. World J Surg 34: 2333–2337.
• 10   Nixon  IJ,  Whitcher  MM,  Palmer  FL,  Tuttle  RM,  Shaha  AR, et al. (2012) The impact of distant metastases at presentation on prognosis in patients with differentiated carcinoma of the thyroid gland. Thyroid 22: 884–889.
• 11   Xu  B,  Ghossein  R (2016) Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma. Endocr Pathol 27: 205–212.
• 12   Sohn  SY,  Kim  HI,  Kim  YN,  Kim  TH,  Kim  SW, et al. (2018) Prognostic indicators of outcomes in patients with lung metastases from differentiated thyroid carcinoma during long-term follow-up. Clin Endocrinol 88: 318–326.
• 13   DeGroot  LJ,  Kaplan  EL,  McCormick  M,  Straus  FH (1990) Natural history, treatment, and course of papillary thyroid carcinoma. J Clin Endocrinol Metab 71: 414–424.
• 14   Mazzaferri  EL,  Kloos  RT (2001) Clinical review 128: current approaches to primary therapy for papillary and follicular thyroid cancer. J Clin Endocrinol Metab 86: 1447–1463.
• 15   Hay  ID,  Bergstralh  EJ,  Goellner  JR,  Ebersold  JR,  Grant  CS (1993) Predicting outcome in papillary thyroid carcinoma: development of a reliable prognostic scoring system in a cohort of 1779 patients surgically treated at one institution during 1940 through 1989. Surgery 114: 1050–1057.
• 16   Soares  P,  Trovisco  V,  Rocha  AS,  Lima  J,  Castro  P, et al. (2003) BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC. Oncogene 22: 4578–4580.
• 17   Kimura  ET,  Nikiforova  MN,  Zhu  Z,  Knauf  JA,  Nikiforov  YE, et al. (2003) High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 63: 1454–1457.
• 18   Saji  M,  Ringel  MD (2010) The PI3K-Akt-mTOR pathway in initiation and progression of thyroid tumors. Mol Cell Endocrinol 321: 20–28.
• 19  Xing M (2010) Genetic alterations in the phosphatidylinositol-​3 kinase/Akt pathway in thyroid cancer. Thyroid 20: 697–706.
• 20   Schmitt  FC,  Longatto-Filho  A,  Valent  A,  Vielh  P (2008) Molecular techniques in cytopathology practice. J Clin Pathol 61: 258–267.
• 21   Gupta  N,  Dasyam  AK,  Carty  SE,  Nikiforova  MN,  Ohori  NP, et al. (2013) RAS mutations in thyroid FNA specimens are highly predictive of predominantly low-risk follicular-pattern cancers. J Clin Endocrinol Metab 98: E914–E922.
• 22   The Cancer Genome Atlas Research  Network (2014) Integrated genomic characterization of papillary thyroid carcinoma. Cell 159: 676–690.
• 23   Fagin  JA,  Wells SA  Jr (2016) Biologic and clinical perspectives on thyroid cancer. N Engl J Med 375: 1054–1067.
• 24  Chung JH (2018) Telomerase reverse transcriptase promoter mutation and its clinical implication in thyroid cancer. Precis Future Med 2: 8–17.
• 25   Davies  H,  Bignell  GR,  Cox  C,  Stephens  P,  Edkins  S, et al. (2002) Mutations of the BRAF gene in human cancer. Nature 417: 949–954.
• 26   Garnett  MJ,  Marais  R (2004) Guilty as charged: B-RAF is a human oncogene. Cancer Cell 6: 313–319.
• 27   Cohen  Y,  Xing  M,  Mambo  E,  Guo  Z,  Wu  G, et al. (2003) BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 95: 625–627.
• 28   Fukushima  T,  Suzuki  S,  Mashiko  M,  Ohtake  T,  Endo  Y, et al. (2003) BRAF mutations in papillary carcinomas of the thyroid. Oncogene 22: 6455–6457.
• 29   Namba  H,  Nakashima  M,  Hayashi  T,  Hayashida  N,  Maeda  S, et al. (2003) Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrinol Metab 88: 4393–4397.
• 30   Xu  X,  Quiros  RM,  Gattuso  P,  Ain  KB,  Prinz  RA (2003) High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines. Cancer Res 63: 4561–4567.
• 31   Nikiforova  MN,  Kimura  ET,  Gandhi  M,  Biddinger  PW,  Knauf  JA, et al. (2003) BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 88: 5399–5404.
• 32  Xing M (2005) BRAF mutation in thyroid cancer. Endocr Relat Cancer 12: 245–262.
• 33   Pennelli  G,  Vianello  F,  Barollo  S,  Pezzani  R,  Merante Boschin  I, et al. (2011) BRAF(K601E) mutation in a patient with a follicular thyroid carcinoma. Thyroid 21: 1393–1396.
• 34   Melillo  RM,  Castellone  MD,  Guarino  V,  De Falco  V,  Cirafici  AM, et al. (2005) The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells. J Clin Invest 115: 1068–1081.
• 35   Park  YJ,  Kim  YA,  Lee  YJ,  Kim  SH,  Park  SY, et al. (2010) Papillary microcarcinoma in comparison with larger papillary thyroid carcinoma in BRAF(V600E) mutation, clinicopathological features, and immunohistochemical findings. Head Neck 32: 38–45.
• 36   Jung  CK,  Im  SY,  Kang  YJ,  Lee  H,  Jung  ES, et al. (2012) Mutational patterns and novel mutations of the BRAF gene in a large cohort of Korean patients with papillary thyroid carcinoma. Thyroid 22: 791–797.
• 37   de Biase  D,  Cesari  V,  Visani  M,  Casadei  GP,  Cremonini  N, et al. (2014) High-sensitivity BRAF mutation analysis: BRAFV600E is acquired early during tumor development but is heterogeneously distributed in a subset of papillary thyroid carcinomas. J Clin Endocrinol Metab 99: E1530–E1538.
• 38   Guan  H,  Ji  M,  Bao  R,  Yu  H,  Wang  Y, et al. (2009) Association of high iodine intake with the T1799A BRAF mutation in papillary thyroid cancer. J Clin Endocrinol Metab 94: 1612–1617.
• 39   Nikiforov  YE,  Steward  DL,  Robinson-Smith  TM,  Haugen  BR,  Klopper  JP, et al. (2009) Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab 94: 2092–2098.
• 40   Kim  SK,  Kim  DL,  Han  HS,  Kim  WS,  Kim  SJ, et al. (2008) Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules. Diagn Mol Pathol 17: 118–125.
• 41   Kim  SW,  Lee  JI,  Kim  JW,  Ki  CS,  Oh  YL, et al. (2010) BRAFV600E mutation analysis in fine-needle aspiration cytology specimens for evaluation of thyroid nodule: a large series in a BRAFV600E-prevalent population. J Clin Endocrinol Metab 95: 3693–3700.
• 42   Xing  M,  Haugen  BR,  Schlumberger  M (2013) Progress in molecular based management of differentiated thyroid cancer. Lancet 381: 1058–1069.
• 43   Xing  M (2007) BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 28: 742–762.
• 44   Kim  TH,  Park  YJ,  Lim  JA,  Ahn  HY,  Lee  EK, et al. (2012) The association of the BRAF(V600E) mutation with prognostic factors and poor clinical outcome in papillary thyroid cancer: a meta-analysis. Cancer 118: 1764–1773.
• 45   Lee  JI,  Jang  HW,  Kim  SW,  Kim  JW,  Oh  YL, et al. (2013) BRAFV600E mutation in fine-needle aspiration aspirate is associated with poorer prognostic factors in larger papillary thyroid carcinoma. Head Neck 35: 548–553.
• 46   Lee  JH,  Lee  ES,  Kim  YS (2007) Clinicopathologic significance of BRAF V600E mutation in papillary carcinomas of the thyroid: a meta-analysis. Cancer 110: 38–46.
• 47   Tufano  RP,  Teixeira  GV,  Bishop  J,  Carson  KA,  Xing  M (2012) BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine (Baltimore) 91: 274–286.
• 48   Xing  M,  Alzahrani  AS,  Carson  KA,  Viola  D,  Elisei  R, et al. (2013) Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA 309: 1493–1501.
• 49   Xing  M,  Alzahrani  AS,  Carson  KA,  Shong  YK,  Kim  TY, et al. (2015) Association between BRAFV600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol 33: 42–50.
• 50   Shen  X,  Zhu  G,  Liu  R,  Viola  D,  Elisei  R, et al. (2018) Patient age-associated mortality risk is differentiated by BRAF V600E status in papillary thyroid cancer. J Clin Oncol 36: 438–445.
• 51   Liu  RT,  Chen  YJ,  Chou  FF,  Li  CL,  Wu  WL, et al. (2005) No correlation between BRAFV600E mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan. Clin Endocrinol (Oxf) 63: 461–466.
• 52   Kim  TY,  Kim  WB,  Song  JY,  Rhee  YS,  Gong  G, et al. (2005) The BRAF mutation is not associated with poor prognostic factors in Korean patients with conventional papillary thyroid microcarcinoma. Clin Endocrinol (Oxf) 63: 588–593.
• 53   Ito  Y,  Yoshida  H,  Maruo  R,  Morita  S,  Takano  T, et al. (2009) BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients. Endocr J 56: 89–97.
• 54   Nikiforova  MN,  Wald  AI,  Roy  S,  Durso  MB,  Nikiforov  YE (2013) Targeted next-generation sequencing panel (ThyroSeq) for detection of mutations in thyroid cancer. J Clin Endocrinol Metab 98: E1852–E1860.
• 55   Meyne  J,  Ratliff  RL,  Moyzis  RK (1989) Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci U S A 86: 7049–7053.
• 56   Martínez  P,  Blasco  MA (2010) Role of shelterin in cancer and aging. Aging Cell 9: 653–666.
• 57   Calado  RT,  Young  NS (2009) Telomere diseases. N Engl J Med 361: 2353–2365.
• 58  Eisenberg DTA (2011) An evolutionary review of human telomere biology: the thrifty telomere hypothesis and notes on potential adaptive paternal effects. Am J Hum Biol 23: 149–167.
• 59   Cohen  SB,  Graham  ME,  Lovrecz  GO,  Bache  N,  Robinson  PJ, et al. (2007) Protein composition of catalytically active human telomerase from immortal cells. Science 315: 1850–1853.
• 60   Shay  JW,  Bacchetti  S (1997) A survey of telomerase activity in human cancer. Eur J Cancer 33: 787–791.
• 61   Huang  FW,  Hodis  E,  Xu  MJ,  Kryukov  GV,  Chin  L, et al. (2013) Highly recurrent TERT promoter mutations in human melanoma. Science 339: 957–959.
• 62   Horn  S,  Figl  A,  Rachakonda  PS,  Fischer  C,  Sucker  A, et al. (2013) TERT promoter mutations in familial and sporadic melanoma. Science 339: 959–961.
• 63   Vinagre  J,  Pinto  V,  Celestino  R,  Reis  M,  Pópulo  H, et al. (2014) Telomerase promoter mutations in cancer: an emerging molecular biomarker? Virchows Arch 465: 119–133.
• 64   Vinagre  J,  Almeida  A,  Populo  H,  Batista  R,  Lyra  J, et al. (2013) Frequency of TERT promoter mutations in human cancers. Nat Commun 4: 2185.
• 65   Landa  I,  Ganly  I,  Chan  TA,  Mitsutake  N,  Matsuse  M, et al. (2013) Frequent somatic TERT promoter mutations in thyroid cancer: higher prevalence in advanced forms of the disease. J Clin Endocrinol Metab 98: E1562–E1566.
• 66   Liu  X,  Bishop  J,  Shan  Y,  Pai  S,  Liu  D, et al. (2013) Highly prevalent TERT promoter mutations in aggressive thyroid cancers. Endocr Relat Cancer 20: 603–610.
• 67   Liu  T,  Wang  N,  Cao  J,  Sofiadis  A,  Dinets  A, et al. (2014) The age- and shorter telomere-dependent TERT promoter mutation in follicular thyroid cell-derived carcinomas. Oncogene 33: 4978–4984.
• 68   Melo  M,  da Rocha  AG,  Vinagre  J,  Batista  R,  Peixoto  J, et al. (2014) TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas. J Clin Endocrinol Metab 99: E754–E765.
• 69   Liu  R,  Xing  M (2016) TERT promoter mutations in thyroid cancer. Endocr Relat Cancer 23: R143–R155.
• 70   Alzahrani  AS,  Alsaadi  R,  Murugan  AK, Sadiq BB (2016) TERT promoter mutations in thyroid cancer. Horm Cancer 7: 165–177.
• 71   Kim  TH,  Kim  YE,  Ahn  S,  Kim  JY,  Ki  CS, et al. (2016) TERT promoter mutations and long-term survival in patients with thyroid cancer. Endocr Relat Cancer 23: 813–823.
• 72   Alzahrani  AS,  Qasem  E,  Murugan  AK,  Al-Hindi  HN,  AlKhafaji  D, et al. (2016) Uncommon TERT promoter mutations in pediatric thyroid cancer. Thyroid 26: 235–241.
• 73   Ballester  LY,  Sarabia  SF,  Sayeed  H,  Patel  N,  Baalwa  J, et al. (2016) Integrating molecular testing in the diagnosis and management of children with thyroid lesions. Pediatr Dev Pathol 19: 94–100.
• 74   Onder  S,  Ozturk Sari  S,  Yegen  G,  Sormaz  IC,  Yilmaz  I, et al. (2016) Classic architecture with multicentricity and local recurrence, and absence of TERT promoter mutations are correlates of BRAF (V600E) harboring pediatric papillary thyroid carcinomas. Endocr Pathol 27: 153–161.
• 75   Oishi  N,  Kondo  T,  Nakazawa  T,  Mochizuki  K,  Inoue  T, et al. (2017) Frequent BRAF V600E and absence of TERT promoter mutations characterize sporadic pediatric papillary thyroid carcinomas in Japan. Endocr Pathol 28: 103–111.
• 76   de Biase  D,  Gandolfi  G,  Ragazzi  M,  Eszlinger  M,  Sancisi  V, et al. (2015) TERT promoter mutations in papillary thyroid microcarcinomas. Thyroid 25: 1013–1019.
• 77   Yang  X,  Li  J,  Li  X,  Liang  Z,  Gao  W, et al. (2017) TERT promoter mutation predicts radioiodine-refractory character in distant metastatic differentiated thyroid cancer. J Nucl Med 58: 258–265.
• 78   Liu  X,  Qu  S,  Liu  R,  Sheng  C,  Shi  X, et al. (2014) TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer. J Clin Endocrinol Metab 99: E1130–E1136.
• 79   Oishi  N,  Kondo  T,  Ebina  A,  Sato  Y,  Akaishi  J, et al. (2017) Molecular alterations of coexisting thyroid papillary carcinoma and anaplastic carcinoma: identification of TERT mutation as an independent risk factor for transformation. Mod Pathol 30: 1527–1537.
• 80   George  JR,  Henderson  YC,  Williams  MD,  Roberts  DB,  Hei  H, et al. (2015) Association of TERT promoter mutation, but not BRAF mutation, with increased mortality in PTC. J Clin Endocrinol Metab 100: E1550–E1559.
• 81   Vuong  HG,  Altibi  AMA,  Duong  UNP,  Hassell  L (2017) Prognostic implication of BRAF and TERT promoter mutation combination in papillary thyroid carcinoma-A meta-analysis. Clin Endocrinol (Oxf) 87: 411–417.
• 82   Song  YS,  Yoo  SK,  Kim  HH,  Jung  G,  Oh  AR, et al. (2019) Interaction of BRAF-induced ETS factors with mutant TERT promoter in papillary thyroid cancer. Endocr Relat Cancer 26: 629–641.
• 83   Melo  M,  Gaspar da Rocha  A,  Batista  R,  Vinagre  J,  Martins  MJ, et al. (2017) TERT, BRAF, and NRAS in primary thyroid cancer and metastatic disease. J Clin Endocrinol Metab 102: 1898–1907.
• 84   Liu  R,  Bishop  J,  Zhu  G,  Zhang  T,  Ladenson  PW, et al. (2017) Mortality risk stratification by combining BRAF V600E and TERT promoter mutations in papillary thyroid cancer genetic duet of BRAF and TERT promoter mutations in thyroid cancer mortality. JAMA Oncol 3: 202–208.
• 85   Liu  J,  Liu  R,  Shen  X,  Zhu  G,  Li  B, et al. (2020) The Genetic duet of BRAF V600E and TERT promoter mutations robustly predicts the loss of radioiodine avidity in recurrent papillary thyroid cancer. J Nucl Med 61: 177–182.
• 86   Xing  M,  Liu  R,  Liu  X,  Murugan  AK,  Zhu  G, et al. (2014) BRAF V600E and TERT promoter mutations cooperatively identify the most aggressive papillary thyroid cancer with highest recurrence. J Clin Oncol 32: 2718–2726.
• 87   Moon  S,  Song  YS,  Kim  YA,  Lim  JA,  Cho  SW, et al. (2017) Effects of coexistent BRAFV600E and TERT promoter mutations on poor clinical outcomes in papillary thyroid cancer: a meta-analysis. Thyroid 27: 651–660.
• 88   Trybek  T,  Walczyk  A,  Gąsior-Perczak  D,  Pałyga  I,  Mikina  E, et al. (2019) Impact of BRAF V600E and TERT promoter mutations on response to therapy in papillary thyroid cancer. Endocrinology 160: 2328–2338.
• 89   Hahn  SY,  Kim  TH,  Ki  CS,  Kim  SW,  Ahn  SH, et al. (2017) Ultrasound and clinicopathological features of papillary thyroid carcinomas with BRAF and TERT promoter mutations. Oncotarget 8: 108946–108957.
• 90   Kim  TH,  Ki  CS,  Hahn  SY,  Oh  YL,  Jang  HW, et al. (2017) Ultrasonographic prediction of highly aggressive telomerase reverse transcriptase (TERT) promoter-mutated papillary thyroid cancer. Endocrine 57: 234–240.
• 91   Wang  N,  Liu  T,  Sofiadis  A,  Juhlin  CC,  Zedenius  J, et al. (2014) TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA. Cancer 120: 2965–2979.
• 92   Xu  B,  Tuttle  RM,  Sabra  MM,  Ganly  I,  Ghossein  R (2017) Primary thyroid carcinoma with low-risk histology and distant metastases: clinicopathologic and molecular characteristics. Thyroid 27: 632–640.
• 93   Haugen  BR,  Alexander  EK,  Bible  KC,  Doherty  GM,  Mandel  SJ, et al. (2016) 2015 American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American Thyroid Association Guidelines Task Force on thyroid nodules and differentiated thyroid cancer. Thyroid 26: 1–133.
• 94   Tuttle  RM,  Tala  H,  Shah  J,  Leboeuf  R,  Ghossein  R, et al. (2010) Estimating risk of recurrence in differentiated thyroid cancer after total thyroidectomy and radioactive iodine remnant ablation: using response to therapy variables to modify the initial risk estimates predicted by the new American Thyroid Association staging system. Thyroid 20: 1341–1349.
• 95   Kim  TH,  Ki  CS,  Kim  HS,  Kim  K,  Choe  JH, et al. (2017) Refining dynamic risk stratification and prognostic groups for differentiated thyroid cancer with TERT promoter mutations. J Clin Endocrinol Metab 102: 1757–1764.