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Oxidative stress (OS) is a state of excessive free radicals and reactive metabolites among which the most important class is reactive oxygen species (ROS) – radicals derived from oxygen – as represented by the superoxide anion radical ( ) and its reactive metabolites, hydroxyl radical (^•OH) and hydrogen peroxide (H₂O₂). In essence, OS represents an imbalance between the production of oxidants – ROS – and their elimination by antioxidative systems in the body. Many studies have linked OS to thyroid cancer by showing its association with abnormally regulated oxidative or antioxidative molecules. The study by Wang et al. in the December 2011 issue of Endocrine-Related Cancer (18, 773–782) further supports this relationship by demonstrating a high total oxidant status and OS index in thyroid cancer patients. The origin of ROS in thyroid cancer patients has not been defined, but thyroid cancer itself can be one since inflammation, a major event in it, is a classical source of ROS. ROS may in turn enhance the mitogen-activated protein (MAP) kinase and phosphatidylinositol-3-kinase (PI3K) pathways, forming a vicious cycle propelling thyroid tumorigenesis. Regardless of the mechanism, the clinical implication of the association of OS with thyroid cancer is severalfold: one, OS is a new risk factor for thyroid cancer; two, OS confers thyroid cancer patients an increased risk for cardiovascular diseases, degenerative neurological disorders, and other cancers that are classically associated with OS; and three, interference with OS may reduce this risk and be therapeutically beneficial to thyroid cancer itself in thyroid cancer patients. These interesting possibilities deserve further studies.

The 2013 discovery of Telomerase reverse transcriptase (TERT) promoter mutations chr5, 1,295,228 C>T (C228T) and 1,295,250 C>T (C250T) in thyroid cancer represents an important event in the thyroid cancer field and much progress has occurred since then. This article provides a comprehensive review of this exciting new thyroid cancer field. The oncogenic role of TERT promoter mutations involves their creation of consensus binding sites for E-twenty-six transcriptional factors. TERT C228T is far more common than TERT C250T and their collective prevalence is, on average, 0, 11.3, 17.1, 43.2 and 40.1% in benign thyroid tumors, papillary thyroid cancer (PTC), follicular thyroid cancer, poorly differentiated thyroid cancer and anaplastic thyroid cancer, respectively, displaying an association with aggressive types of thyroid cancer. TERT promoter mutations are associated with aggressive thyroid tumor characteristics, tumor recurrence and patient mortality as well as BRAF V600E mutation. Coexisting BRAF V600E and TERT promoter mutations have a robust synergistic impact on the aggressiveness of PTC, including a sharply increased tumor recurrence and patient mortality, while either mutation alone has a modest impact. Thus, TERT with promoter mutations represents a prominent new oncogene in thyroid cancer and the mutations are promising new diagnostic and prognostic genetic markers for thyroid cancer, which, in combination with BRAF V600E mutation or other genetic markers (e.g. RAS mutations), are proving to be clinically useful for the management of thyroid cancer. Future studies will specifically define such clinical utilities, elucidate the biological mechanisms and explore the potential as therapeutic targets of TERT promoter mutations in thyroid cancer.

Abstract

The incidence rate of thyroid cancer has been rising rapidly in recent decades; however, its trend remains unclear. To investigate this, we analyzed the database of the Surveillance, Epidemiology and End Results (SEER) 13, 1992–2012 in the USA, particularly focusing on conventional papillary thyroid cancer (CPTC) and follicular variant of PTC (FVPTC). Of the 75,992 thyroid cancers, 61.3% were CPTC and 25.7% were FVPTC, and their incidence rates (IRs) were significantly increased from 1992 to 2012 (P all < 0.001), with CPTC being 2.4 times of FVPTC (P < 0.001) and the overall average annual percent change (AAPC) of incidence being 6.3% in the former and 5.3% in the latter. IRs were increased in all thyroid cancers, albeit most dramatically in PTC, in virtually all ethnic/demographic groups in recent two decades; however, the incidence trends varied among different thyroid cancers, particularly differentiable between CPTC and FVPTC. For example, Joinpoint analyses revealed that the APC of CPTC before 1996 was 1.5% (P > 0.05), which jumped to 6.8% (P < 0.05) after 1996, whereas the APC of FVPTC before 2000 was 6.6% (P < 0.05), which dropped to 4.8% (P < 0.05) after 2000. IRs and incidence trends of PTC were uneven among different ethnic/demographic groups, as exemplified by the lower IRs of both PTC variants in the Black females than in non-Hispanic White females but higher AAPCs of incidence in the former than in the latter. Interestingly, the data also suggest that the rise in the IRs of PTC is becoming plateaued in the most recent 2 years. These novel observations are helpful in understanding the incidence and incidence trends of thyroid cancer.

Two promoter mutations, chr5:1 295 228C>T and chr5:1 295 250C>T, in the gene for telomerase reverse transcriptase (TERT) have been recently identified in thyroid cancers and shown to be important in thyroid tumor pathogenesis. The diagnostic and prognostic potentials of testing for these mutations on thyroid fine-needle aspiration biopsy (FNAB) have not been investigated. Herein, we examined the two TERT promoter mutations along with the BRAF V600E mutation by direct DNA sequencing on 308 FNAB specimens preoperatively obtained from thyroid nodules with postoperatively confirmed pathological diagnoses. We found TERT promoter mutations in 0.0% (0/179) of benign thyroid nodules and 7.0% (9/129) of thyroid nodules of differentiated thyroid cancer, representing a 100% diagnostic specificity and 7.0% sensitivity, with the latter rising to 38.0% (49/129) when combined with BRAF V600E testing. Several TERT-promoter-mutation-positive thyroid nodules were cytologically indeterminate on FNAB. Approximately 80% of the TERT promoter mutation-positive thyroid nodules were thyroid cancers with aggressive clinicopathological behaviors, such as extrathyroidal invasion, lymph node metastases, distant metastases, disease recurrence or patient death. Thus, a positive TERT promoter mutation test not only definitively diagnoses a thyroid nodule as cancerous but also preoperatively identifies a cancer with aggressive potential. This is the first study, to our knowledge, of TERT promoter mutations on thyroid FNAB, demonstrating the value of this novel molecular testing in the diagnosis of thyroid nodules and preoperative risk stratification of thyroid cancer. Thus, testing of TERT promoter mutations on FNAB will enhance and improve the current molecular-based approaches to the management of thyroid nodules and thyroid cancer.

A unique prognostic role of the genetic duet of BRAF V600E and TERT promoter mutations in papillary thyroid cancer (PTC) has been recently established, but the role of RAS mutation in this genetic interplay remains to be established. Using The Cancer Genome Atlas (TCGA) data of patients with PTC from 19 medical centers, we investigated the interactions among the three mutations in clinical outcomes of PTC. We found that BRAF and RAS mutations were mutually exclusive, but both were associated with TERT promoter mutations, with the genetic duet of BRAF/RAS and TERT mutations occurring in 34/388 (8.76%) patients. BRAF/RAS or TERT mutation had no or minimal effect alone, whereas coexisting BRAF/RAS and TERT mutations had a robust synergistic effect on poor clinicopathologic outcomes of PTC, including disease recurrence and patient mortality. For example, PTC recurrence rate was 52% with coexisting BRAF V600E/RAS and TERT promoter mutations vs 6.9% with no mutation, corresponding to a HR of 8.17 (95% CI 3.09–21.58), which remained significant at 14.71 (95% CI 2.79–77.61) after adjustment for clinicopathologic factors and institution. BRAF/RAS mutation or TERT mutation alone minimally affected Kaplan–Meier patient survival curves, whereas the genetic duet was associated with a sharp curve decline. Thus, by confirming and expanding previous findings in single-institution studies, this multicenter data analysis establishes a six-genotype genetic prognostic model for poor outcomes of PTC with a risk order of genetic duet of BRAF V600E/RAS mutation and TERT mutation >>>>BRAF V600E = TERT mutation alone >RAS mutation alone = wild-type genes.

The BRAF V600E mutation plays an important role in the tumorigenesis of papillary thyroid cancer (PTC). To explore an epigenetic mechanism involved in this process, we performed a genome-wide DNA methylation analysis using a methylated CpG island amplification (MCA)/CpG island microarray system to examine gene methylation alterations after shRNA knockdown of BRAF V600E in thyroid cancer cells. Our results revealed numerous methylation targets of BRAF V600E mutation with a large cohort of hyper- or hypo-methylated genes in thyroid cancer cells, which are known to have important metabolic and cellular functions. As hypomethylation of numerous genes by BRAF V600E was particularly a striking finding, we took a further step to examine the selected 59 genes that became hypermethylated in both cell lines upon BRAF V600E knockdown and found them to be mostly correspondingly under-expressed (i.e. they were normally maintained hypomethylated and over-expressed by BRAF V600E in thyroid cancer cells). We confirmed the methylation status of selected genes revealed on MCA/CpG microarray analysis by performing methylation-specific PCR. To provide proof of concept that some of the genes uncovered here may play a direct oncogenic role, we selected six of them to perform shRNA knockdown and examined its effect on cellular functions. Our results demonstrated that the HMGB2 gene played a role in PTC cell proliferation and the FDG1 gene in cell invasion. Thus, this study uncovered a prominent epigenetic mechanism through which BRAF V600E can promote PTC tumorigenesis by altering the methylation and hence the expression of numerous important genes.

The impact of metastasized cervical lymph nodes (CLN) identified on central neck dissection (CND) on the recurrence/persistence of papillary thyroid cancer (PTC) and the extent of CND needed to reduce recurrence/persistence have not been firmly established. To assess the impact of CLN metastasis and BRAF mutation on the recurrence/persistence of PTC and the potential of BRAF mutation in assisting CND. Analyses of 379 consecutive patients with PTC who underwent thyroidectomy with (n=243) or without CND (n=136) at a tertiary-care academic hospital during the period 2001–2010 for their clinicopathological outcomes and BRAF mutation status. Increasingly aggressive tumor characteristics were found as the extent of CND was advanced following conventional risk criteria from non-CND to limited CND to formal CND. Disease recurrence/persistence rate also sharply rose from 4.7% to 15.7% and 40.5% in these CND settings respectively (P<0.0001). CLN metastasis rate rose from 18.0 to 77.3% from limited CND to formal CND (P<0.0001). An increasing rate of BRAF mutation was also found from less to more extensive CND. A strong association of CLN metastasis and BRAF mutation with disease recurrence/persistence was revealed on Kaplan–Meier analysis and BRAF mutation strongly predicted CLN metastasis. CLN metastases found on CND are closely associated with disease recurrence/persistence of PTC, which are both strongly predicted by BRAF mutation. Current selection of PTC patients for CND is appropriate but higher extent of the procedure, once selected, is needed to reduce disease recurrence, which may be defined by combination use of preoperative BRAF mutation testing and conventional risk factors of PTC.

The BRAF V600E mutation causes impaired expression of sodium iodide symporter (NIS) and radioiodine refractoriness of thyroid cancer, but the underlying mechanism remains undefined. In this study, we hypothesized that histone deacetylation at the NIS (SLC5A5) promoter was the mechanism. Using the chromatin immunoprecipitation approach, we examined histone acetylation status on the lysine residues H3K9/14, H3K18, total H4, and H4K16 at the NIS promoter under the influence of BRAF V600E. We found that expression of stably or transiently transfected BRAF V600E inhibited NIS expression while the deacetylase inhibitor SAHA stimulated NIS expression in PCCL3 rat thyroid cells. Although BRAF V600E enhanced global histone acetylation, it caused histone deacetylation at the NIS promoter while SAHA caused acetylation in the cells. In human thyroid cancer BCPAP cells harboring homozygous BRAF V600E mutation, BRAF V600E inhibitor, PLX4032, and MEK inhibitor, AZD6244, increased histone acetylation of the NIS promoter, suggesting that BRAF V600E normally maintained histone in a deacetylated state at the NIS promoter. The regions most commonly affected with deacetylation by BRAF V600E were the transcriptionally active areas upstream of the translation start that contained important transcription factor binding sites, including nucleotides −297/−107 in the rat NIS promoter and −692/−370 in the human NIS promoter. Our findings not only reveal an epigenetic mechanism for BRAF V600E-promoted NIS silencing involving histone deacetylation at critical regulatory regions of the NIS promoter but also provide further support for our previously proposed combination therapy targeting major signaling pathways and histone deacetylase to restore thyroid gene expression for radioiodine treatment of thyroid cancer.

Genetic alterations in the PIK3CA gene of the phosphoinositide 3-kinase (PI3K)/AKT pathway have been found in many human tumors, but they have not been explored in pituitary tumors. We undertook the present study to explore mutations and amplifications of the PIK3CA gene in pituitary tumors. DNA sequencing and real-time quantitative PCR were used to examine mutations and amplifications respectively, on genomic DNA samples isolated from 353 cases of pituitary tumors, and immunohistostaining was used to assess PIK3CA expression. About 8 out of 91 (9%) invasive pituitary tumors versus 0 out of 262 (0%) noninvasive tumors were found to harbor somatic mutations in exons 9 and 20 of the PIK3CA gene (P<0.001), and the mutation was associated with increased disease recurrence. Genomic PIK3CA amplifications (defined as ≥4 copies) were observed in both invasive and noninvasive tumors, with a prevalence of around 20–40% in various types of pituitary tumors. PIK3CA protein overexpression was observed in cases with high PIK3CA copy number. RAS mutations were also examined and found in 6 out of the 91 (7%) invasive tumors. PIK3CA amplifications were mutually exclusive with PIK3CA or RAS mutations (P<0.001). This study demonstrated for the first time relatively common PIK3CA mutations and amplifications as well as RAS mutations and their tendency of mutual exclusivity in pituitary tumors. The data provide strong genetic evidence supporting a role of the PI3K/AKT signaling pathway in the tumorigenesis of pituitary tumors, particularly the invasive types.

Mutations 1 295 228 C>T and 1 295 250 C>T (termed C228T and C250T respectively), corresponding to −124 C>T and −146 C>T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene, have recently been reported in human cancers, but not in thyroid cancers yet. We explored these mutations in thyroid cancers by genomic sequencing of a large number of primary tumor samples. We found the C228T mutation in 0 of 85 (0.0%) benign thyroid tumors, 30 of 257 (11.7%) papillary thyroid cancers (PTC), 9 of 79 (11.4%) follicular thyroid cancers (FTC), 3 of 8 (37.5%) poorly differentiated thyroid cancers (PDTC), 23 of 54 (42.6%) anaplastic thyroid cancers (ATC), and 8 of 12 (66.7%) thyroid cancer cell lines. The C250T mutation was uncommon, but mutually exclusive with the C228T mutation, and the two mutations were collectively found in 11 of 79 (13.9%) FTC, 25 of 54 (46.3%) ATC, and 11 of 12 (91.7%) thyroid cancer cell lines. Among PTC variants, the C228T mutation was found in 4 of 13 (30.8%) tall-cell PTC (TCPTC), 23 of 187 (12.3%) conventional PTC, and 2 of 56 (3.6%) follicular variant PTC samples. No TERT mutation was found in 16 medullary thyroid cancer samples. The C228T mutation was associated with the BRAF V600E mutation in PTC, being present in 19 of 104 (18.3%) BRAF mutation-positive PTC vs 11 of 153 (7.2%) the BRAF mutation-negative PTC samples (P=0.0094). Conversely, BRAF mutation was found in 19 of 30 (63.3%) C228T mutation-positive PTC vs 85 of 227 (37.4%) C228T mutation-negative PTC samples (P=0.0094). We thus for the first time, to our knowledge, demonstrate TERT promoter mutations in thyroid cancers, that are particularly prevalent in the aggressive thyroid cancers TCPTC, PDTC, ATC and BRAF mutation-positive PTC, revealing a novel genetic background for thyroid cancers.