Inhibitory effects of the mitogen-activated protein kinase kinase inhibitor CI-1040 on the proliferation and tumor growth of thyroid cancer cells with BRAF or RAS mutations

CONTEXT: Targeting MAPK kinase (MEK) in the MAPK pathway is a potentially effective therapeutic strategy for thyroid cancer. OBJECTIVE: The objective of the study was to investigate genotype-dependent therapeutic potential of the MEK inhibitor CI-1040 for thyroid cancer. EXPERIMENTAL DESIGN: We examined the effects of CI-1040 on proliferation, apoptosis, transformation, thyroid gene reexpression, and xenograft tumor growth with respect to genotypes in 10 thyroid tumor cell lines. RESULTS: Cell proliferation was potently inhibited by CI-1040 in cells harboring BRAF or RAS mutations but not in cells harboring RET/PTC rearrangement or wild-type alleles. For example, the IC50 values for BRAF mutation-harboring KAT10 cells and DRO cells and H-RAS mutation-harboring C643 cells were 0.365, 0.031, and 0.429 microm, respectively, whereas the IC50 values for RET/PTC1-harboring TPC1 cells and the wild-type MRO and WRO cells were 44, 46, and 278 microm, respectively. Proapoptotic effect of CI-1040 was seen in DRO cells, and cytostatic effect was seen in other cells. Down-regulation of cyclin D1 and reexpression of some thyroid genes were induced by CI-1040 in some BRAF mutation-harboring cells, and transformation was inhibited in all cells. CI-1040 also inhibited the growth of xenograft tumors in nude mice derived from KAT10 or C643 cells but not that derived from MRO cells. CONCLUSIONS: We for the first time demonstrated potent inhibitory effects of a MEK inhibitor, CI-1040, on thyroid cancer cells, some of which, particularly cell proliferation and tumor growth, seemed to be BRAF mutation or RAS mutation selective. Our data encourage a clinical trial on CI-1040 in thyroid cancer patients.     

BRAF(V600E) mutation in Turkish patients with papillary thyroid cancer: strong correlation with indicators of tumor aggressiveness

Papillary thyroid cancer (PTC) constitutes more than 90?% of the thyroid cancers. MAP kinase/ERK pathway plays an important role in the development of several cancers. BRAF which is a member of Raf-kinase family activates this way. BRAF gene activating mutations lead to neoplastic transformation in thyroid follicle cells. In PTC, this mutation itself is a poor prognostic sign independent of other clinicopathological characteristics. We evaluated BRAF(V600E) mutation and clinical-pathological characteristics in Turkish population with PTC. We assessed 109 patients with PTC (88 female, 21 male). The average age was 38.7?±?9.9 (17-71). BRAF(V600E) mutation was detected using polymerase chain reaction and fluorescent melting curve analysis. The results show that BRAF(V600E) mutation rate was found in 39.45?% of our patients. We observed that BRAF(V600E) mutation was significantly higher in men, in tumors larger than 1?cm in size, and in patients with classical PTC. Moreover, statistically significant correlations of BRAF(V600E) with indicators of tumor aggressiveness such as thyroid capsular invasion, multifocality, lymph node metastasis, and extrathyroidal spread were found. Patient groups below and over the age of 45 did not differ in mutation frequency. Patients with micro-PTC were evaluated separately, it was found that BRAF(V600E) mutation was more frequent in the classic type and that lymph node metastasis rate significantly increased when the mutation was present. We concluded that BRAF(V600E) was correlated with indicators of tumor aggressiveness in our study population. This fact is taken into consideration in treatment and follow-up of our patients with PTC and positive BRAF(V600E) mutation.     

Genome-wide alterations in gene methylation by the BRAF V600E mutation in papillary thyroid cancer cells

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.     

Influence of the BRAF V600E mutation on expression of vascular endothelial growth factor in papillary thyroid cancer

CONTEXT: The BRAF mutation may influence the expression patterns of molecular markers that are related to the development and progression of thyroid cancer. OBJECTIVE: The objective of the study was to investigate the effects of the BRAF V600E mutation on expression of galectin-3, cyclooxygenase-2, cyclin D1, p53, and vascular endothelial growth factor (VEGF) in papillary thyroid cancer (PTC). DESIGN, SETTING, AND SUBJECTS: One hundred sixty-three PTC and 28 nodular hyperplasia patients were selected retrospectively. The presence of the BRAF V600E mutation and the level of expression of the molecular markers were determined. RESULTS: Of 161 PTC patients, 102 patients (63.4%) were BRAF V600E(+), and these cases had significantly larger tumor sizes (P = 0.01), compared with V600E(-) cases (n = 59, 36.6%). Although PTC tissues had higher expression levels of the selected molecular markers than nodular hyperplasia tissues, expression levels of several molecular markers in BRAF V600E(+) PTC were not significantly different from those of BRAF V600E(-) PTC. But VEGF was significantly up-regulated in BRAF V600E(+) PTC, compared with BRAF V600E(-) PTC. VEGF expression levels were strongly positively correlated to tumor size (P < 0.001), extrathyroidal invasion (P = 0.02), and tumor stage (P = 0.04). Multivariate analysis clearly showed that VEGF expression was up-regulated in BRAF V600E(+) PTC (odds ratio 2.5, confidence interval 1.1-5.6; P = 0.03). CONCLUSIONS: BRAF V600E(+) PTC tended to have larger tumor volumes and higher expression of VEGF. The level of VEGF expression was closely correlated with tumor size, extrathyroidal invasion, and stage. The relatively high levels of VEGF expression may be related to poorer clinical outcomes and recurrences in BRAF V600E(+) PTC.     

The influence of the BRAF V600E mutation in thyroid cancer cell lines on the anticancer effects of 5-aminoimidazole-4-carboxamide-ribonucleoside

5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) is an activator of 5'-AMP-activated protein kinase (AMPK), which plays a role in the maintenance of cellular energy homeostasis. Activated AMPK inhibits the protein kinase mechanistic target of rapamycin, thereby reducing the extent of protein translation and suppressing both cell growth and cell cycle entry. Recent reports indicate that AMPK-mediated growth inhibition is achieved via an action of the RAF-MEK-ERK mitogen-activated protein kinase pathway in melanoma cells harboring the V600E mutant form of the BRAF oncogene. In this study, we investigated the anti-cancer efficacy of AICAR by measuring its effects on proliferation, apoptosis, and cell cycle progression of BRAF wild-type and V600E-mutant thyroid cancer cell lines. We also explored the mechanism underlying these effects. AICAR inhibited the proliferation of BRAF V600E-mutant thyroid cancer cell lines more strongly than was the case with wild-type cell lines. The suppressive effect of AICAR on cell proliferation was associated with increased S-phase cell cycle arrest and apoptosis. Interestingly, AICAR suppressed phosphorylation of ERK and p70S6K in BRAF V600E-mutant thyroid cancer cells, but rather increased phosphorylation in wild-type cells. Together, the results indicate that AICAR-induced AMPK activation in BRAF V600E-mutant thyroid cancer cell lines resulted in increases in apoptosis and S-phase arrest via downregulation of ERK and p70S6K activity. Thus, regulation of AMPK activity may be potentially useful as a therapy for thyroid cancer if the cancer harbors a BRAF V600E mutation.     

BRAF mutations are not a major event in post-Chernobyl childhood thyroid carcinomas

The BRAF gene has been shown to be a major target for mutations in papillary thyroid carcinoma (PTC) (36-69%), which forms almost all of the over 2000 cases of thyroid carcinoma that have occurred in Chernobyl. BRAF is activated by point mutation, and were it to occur at a high frequency in Chernobyl-related tumors, it would challenge the dominant role of double-strand breaks in radiation-induced PTC. In a previous study, we detected the BRAF V600E mutation in 46% (23 of 50) of sporadic adult PTC. Using the same methodology, we have analyzed 34 post-Chernobyl PTC and detected RET/PTC rearrangements in 14 (41%) and BRAF mutations (V600E) in four (12%). These two alterations did not coexist in any PTCs. The mean age at exposure of patients with PTC showing BRAF mutation was higher than that of patients with tumors without BRAF mutation irrespective of their RET status. We have also analyzed 17 sporadic cases of childhood PTC and found that only one (6%) harbored the BRAF V600E mutation. We conclude that the frequency of BRAF mutations is significantly lower (P = 0.0008) in post-Chernobyl PTC than in adult sporadic PTC, whereas no significant difference was found between post-Chernobyl and sporadic childhood PTCs.     

BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy

BRAF(V600E) is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAF(V600E) mutation, TIMP-1 expression, and NF-κB activation. We found that BRAF(V600E) mutation occurs selectively in PTC nodules and is associated with hyperactivation of NF-κB and upregulation of both TIMP-1 and its receptor CD63. To assess the functional relationship among these factors, we first silenced BRAF gene in BCPAP cells, harboring BRAF(V600E) mutation. We found that silencing causes a marked decrease in TIMP-1 expression and NF-κB binding activity, as well as decreased invasiveness. After treatment with specific inhibitors of MAPK pathway, we found that only sorafenib was able to increase IκB-α and reduce both TIMP-1 expression and Akt phosphorylation in BCPAP cells, indicating that BRAF(V600E) activates NF-κB and this pathway is MEK-independent. Taken together, our findings demonstrate that BRAF(V600E) causes upregulation of TIMP-1 via NF-κB. TIMP-1 binds then its surface receptor CD63, leading eventually to Akt activation, which in turn confers antiapoptotic behavior and promotion of cell invasion. The recognition of this functional trilogy provides insight on how BRAF(V600E) determines cancer initiation, progression, and invasiveness in PTC, also identifying new therapeutic targets for the treatment of highly aggressive forms.     

Clinical significance of immunohistochemistry to detect BRAF V600E mutant protein in thyroid tissues

This study investigated the feasibility of using immunohistochemistry (IHC) instead of PCR to detect BRAF V600E mutant protein in papillary thyroid carcinoma (PTC), and to determine the value of using preoperative BRAF V600E mutant protein by IHC to assist in the diagnosis of thyroid nodule patients with Hashimoto''s thyroiditis (HT).The expression of BRAFV600E mutant protein was measured in 23 cases of HT+PTC, 31 cases of PTC, and 28 cases of HT by IHC, followed by PCR in the same samples for validation. SPSS 19.0 software was used for statistical analysis.The sensitivity and specificity of IHC to detect BRAF V600E mutation were 100% and 42.86%, respectively. In addition, the mutation rate of BRAF V600E protein in the HT+PTC group (34.78%, 8/23) was lower than that in the PTC group (80.65%, 25/31).The application of IHC to detect BRAF V600E mutant protein has good sensitivity but not specificity to diagnose PTC. IHC can be used as a preliminary screening method to detect BRAF V600E mutation. The strongly positive (+++) staining of IHC potently indicated BRAF V600E gene mutation. For suspicious thyroid nodules combined with HT, the detection of BRAF V600E mutant protein with IHC alone is not of great significance for differentiating benign and malignant nodules.     

Association of BRAF V600E mutation with poor clinicopathological outcomes in 500 consecutive cases of papillary thyroid carcinoma

CONTEXT: Because very few studies have examined the correlation between BRAF mutations and clinicopathological features of papillary thyroid carcinoma (PTC), we analyzed here a large and homogeneous cohort of patients with PTC for the presence of the BRAF mutation. OBJECTIVE: We examined BRAF mutations in a consecutive series of 500 PTC patients who underwent surgery in the Department of Surgery of the University of Pisa, and we correlated the presence of the mutation with clinicopathological parameters of the patients: age, gender, tumor size, presence of tumor capsule, extrathyroidal invasion, multicentricity, presence of node metastases, and tumor class. DESIGN: BRAF (exon 15) mutation was examined by PCR-single strand conformational polymorphism followed by DNA sequencing in laser-capture microdissected tissue samples. RESULTS: In this study, BRAF mutation was found in 219 of 500 cases (43.8%). In particular, we found the most common BRAF V600E mutation in 214 cases (42.8%), BRAF K601E mutation in three cases (0.6%), BRAF VK600-1E (0.2%) in one case, whereas in one case we found a new 14-bp deletion with concomitant 2-bp insertion, VKSR600-3del and T599I, respectively. BRAF V600E was associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001), in particular in follicular- and micro-PTC variants. By multivariate analysis, the absence of tumor capsule remained the only parameter associated (P = 0.0005) with BRAF V600E mutation. CONCLUSIONS: Our data suggest that BRAF V600E mutation is associated with high-risk PTC and in particular in follicular variant with invasive tumor growth.     

Detection of BRAFV600E mutation on fine needle aspiration specimens of thyroid nodule refines cyto-pathology diagnosis, especially in BRAF600E mutation-prevalent area

BACKGROUND: Between 10 and 30% of the fine needle aspiration biopsies (FNABs) of thyroid nodules are diagnosed as 'indeterminate'. A molecular diagnostic method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is the classic papillary type and the BRAF(V600E) mutation is highly prevalent. AIM: To evaluate the role of pre-operative detection of BRAF(V600E) mutation in the FNAB specimens of thyroid nodules in a BRAF(V600E) mutation-prevalent geographical area. PATIENTS AND METHODS: In 137 specimens of FNAB (107 papillary thyroid carcinomas (PTC); 3 follicular thyroid carcinomas (FTC); 2 undifferentiated thyroid carcinomas; 25 benign lesions), both direct DNA sequencing and PCR-RFLP were used for detecting the BRAF(V600E) mutation. The sensitivity and specificity were calculated. We analysed the association between BRAF(V600E) mutation and the clinico-pathological parameters. RESULTS: The BRAF(V600E) mutation was present in 93 (83%) of 112 thyroid cancers. Direct DNA sequencing showed a sensitivity of 83.0% and a specificity of 96.0%. The sensitivity and specificity of PCR-RFLP were 78.6% and 80.0%, respectively. Among 25 cases with indeterminate FNAB cytology, 8 patients had malignant lesions (5 PTC and 3 FTC). Three (60%) of 5 PTCs and 1 out of 17 benign lesions had BRAF(V600E) mutation (only one false positive case and the definitive pathology showed atypical nodular hyperplasia that could be a premalignant lesion). The diagnostic accuracy of this molecular method in only the 25 indeterminate nodules was 76% (19/25). No mutation was found in 3 FTCs. Among 107 PTCs, there was no significant association of the BRAF(V600E) mutation with the known risk factors. CONCLUSION: Detection of the BRAF(V600E) mutation in FNAB specimens refines the FNAB-cytology diagnosis, especially in a BRAF(V600E) mutation-prevalent area. Direct DNA sequencing was a more reliable method than PCR-RFLP for detecting the BRAF(V600E) mutation with a high sensitivity and specificity.     

The BRAF mutation is useful for prediction of clinical recurrence in low-risk patients with conventional papillary thyroid carcinoma

BACKGROUND: The activating BRAF(V600E) mutation is the most common genetic alteration reported in papillary thyroid carcinoma (PTC). While some reports suggest the BRAF(V600E) mutation is associated with factors predicting a poor prognosis and recurrence, this remains a controversial issue. AIM: To determine whether the presence of the BRAF(V600E) mutation is a prognostic indicator for clinical recurrence in low-risk patients with conventional PTC. PATIENTS AND METHODS: The study involved 203 conventional PTC patients who underwent total or near-total thyroidectomy followed by immediate 131I ablation of the remnants. Patients with antithyroglobulin antibodies and those with extracervical metastases at presentation were excluded. DNA was extracted from paraffin-embedded tumour specimens, and the presence of the BRAF(V600E) mutation was evaluated using PCR amplification and direct sequencing. RESULTS: The BRAF(V600E) mutation was found to be present in 149 (73.4%) of 203 patients. The BRAF(V600E) mutation was correlated with male gender (P = 0.006) and with tumour size (P = 0.005). While there appeared to be an association between the BRAF(V600E) mutation and extrathyroid extension, this did not reach statistical significance (P = 0.062). During follow-up of the 203 patients (median 7.3 years; range 0.7-10.0 years), 36 (18%) patients experienced recurrence. While univariate analysis showed the BRAF(V600E) mutation was associated with tumour recurrence (21% with mutation vs 7% without mutation; P = 0.037), this association was not shown following multivariate analyses adjusting for the clinicopathological prognostic factors of age, gender, tumour size, extrathyroid extension, multifocality and lymph node metastasis. CONCLUSIONS: Although the BRAF(V600E) mutation was found to be associated with a higher clinical recurrence of disease in low-risk conventional PTC patients, it was not an independent predictor.     

Prediction Table and Nomogram as Tools for Diagnosis of Papillary Thyroid Carcinoma: Combined Analysis of Ultrasonography,Fine-Needle Aspiration Biopsy,and BRAF V600E Mutation

Although ultrasonography (US)-guided fine-needle aspiration biopsy (FNAB) is the most reliable diagnostic modality for evaluating thyroid nodules, 10% to 40% of FNAB samples yield indeterminate findings. The BRAF V600E mutation, a highly specific molecular marker for papillary thyroid carcinoma (PTC), well known for its prognostic value, has dubious diagnostic value because of its low sensitivity. Novel strategies are clearly needed to distinguish PTC, which represents the majority of thyroid malignancies, from other thyroid nodules.The records of 3297 patients with surgically proven PTC were retrospectively reviewed. A prediction table and nomogram were designed using a combination of diagnostic parameters for US, FNAB, and the BRAF V600E mutation. For the nomogram, parameters were proportionally assigned 0 to 100 points according to their regression coefficient for PTC.The probability of PTC for thyroid nodules with intermediate-risk (IR) US and atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) FNAB was significantly dependent on BRAF V600E mutation status based on our prediction table (negative, 29.2% vs positive, 87.5%; P < 0.001). By our nomogram, the probability of PTC for thyroid nodules with IR US, AUS/FLUS FNAB, and positive BRAF V600E mutation was approximately 85% to 90%.We strongly recommend preoperative evaluation of the BRAF V600E mutation in indeterminate thyroid nodules. The prediction table and nomogram developed in this study could help clinicians and patients easily assess the probability of PTC in the preoperative period.     

Mutational and clinico-pathological analysis of papillary thyroid carcinoma in Serbia

Molecular pathogenesis of papillary thyroid carcinoma (PTC) is largely associated with mutational changes in the BRAF, RAS family and RET genes. Our aim was to assess clinico-pathological and prognostic correlations of these PTC-specific gene alterations, with a particular emphasis on the BRAF mutation, in a group of 266 Serbian PTC patients, for the first time. The reference center-based retrospective cohort included 201 (75.6%) females and 65 (24.4%) males aged 48.0±16.1 years (8-83 years old, range) diagnosed and treated for PTC during 1993-2008. Follow-up period was 53.1±41.6 months (7-187 months, range). BRAF and RAS mutations were determined by direct sequencing of genomic DNA. RET/PTC rearrangements were analyzed by RT-PCR/Southern blotting. Genetic alterations were detected in 150/266 tumors (56.4%). One tumor displayed two genetic alterations. The BRAF(V600E) was found in 84/266 (31.6%) cases, RAS mutations in 11/266 (4.1%) and RET/PTC in 55/266 (20.7%; 42/266 (15.8%) RET/PTC1 and 13/266 (4.9%) RET/PTC3). On multivariate analysis BRAF(V600E) was associated with the classical papillary morphology (P = 0.05), the higher pT category (P = 0.05) and advanced clinical stage (P = 0.03). In a proportional hazard model, BRAF(V600E) did not appear to be an independent risk factor for the faster recurrence (P = 0.784). We conclude that under the extensive thyroid surgery and limited application of radioiodine ablation BRAF(V600E) may not be an indicator of poorer disease-free survival during the short to middle follow-up period. However, it has a potential to contribute to patients stratification into high- and low-risk groups.     

Presence of BRAF V600E in very early stages of papillary thyroid carcinoma.

OBJECTIVE: Extremely small (often 0.5-4 mm) incidental thyroid malignancies (incidental microcarcinomas, IM) might be discovered at histological examination after surgery in patients who were operated on for benign thyroid conditions. Morphologically, these malignancies have virtually always features of differentiated papillary thyroid carcinoma (PTC). Although IM are in general considered of little clinical significance their potential malignant behavior cannot be ignored. BRAF(V600E) mutation has emerged as the most prevalent genetic alteration in PTC. DESIGN: The incidence of BRAF(V600E) was studied in 85 microdissected cases of IM detected in two series of 334 and 398 patients operated on for benign thyroid disease in 2005 and in 2006, respectively. IM ranged in size from 0.5 to 4 mm and all featured cytology consistent with the diagnosis of PTC. Microdissected lesions were also evaluated for galectin-3 expression by immunohistochemistry. MAIN OUTCOME: BRAF(V600E) was identified in 15 out of 85 (17.6%) IM compared to 45% clinically evident PTCs (n = 91) and 38.3% preoperatively identified microcarcinomas (n = 47). CONCLUSION: These data suggest that it is possible to find BRAF(V600E) in IM despite their extremely small (<1 mm) size. This is not a formal demonstration that IM can evolve into clinical PTC, but on these bases, patients with BRAF(V600E) IM may need to be managed more carefully.     

High prevalence and possible de novo formation of BRAF mutation in metastasized papillary thyroid cancer in lymph nodes

CONTEXT: The role of the T1799A BRAF mutation in lymph node metastasis of papillary thyroid cancer (PTC) is not clear. OBJECTIVE: Our objective was to explore the relationship between BRAF mutation and lymph node metastasis of PTC by examining the mutation in both the primary tumors and their paired lymph node metastases. DESIGN: We isolated genomic DNA from primary thyroid tumors and paired lymph node metastases and performed direct sequencing of exon 15 of the BRAF gene mutation that carries the T1799A mutation. RESULTS: In a series of 33 cases, 21 harbored the T1799A mutation in the primary tumors, and 17 (81%) of them harbored the same mutation also in the paired lymph node metastases. Twelve cases did not harbor the T1799A mutation in the primary tumors, among which nine cases also did not harbor BRAF mutation in the lymph node-metastasized tumors, whereas the other three did harbor the T1799A mutation in lymph node-metastasized tumor tissues. A novel tandem TG1799-1800AA mutation within one allele was found in a lymph node-metastasized tumor but not in the primary tumor. This mutation results in the change of codon 600 (GTG) of the gene to GAA with the consequent amino acid change (V600E) in the B-type Raf (BRAF) protein, same as that caused by the T1799A mutation alone. CONCLUSION: The high prevalence of BRAF mutation in lymph node-metastasized PTC tissues from BRAF mutation-positive primary tumors and the possible de novo formation of BRAF mutation in lymph node-metastasized PTC were consistent with a role of BRAF mutation in facilitating the metastasis and progression of PTC in lymph nodes.     

Modifications in the papillary thyroid cancer gene profile over the last 15 years

Background: Evidence for an increased prevalence of BRAF(V600E) mutations has been documented in recent decades. The aim of this study was to evaluate the prevalence of both RET/PTC rearrangements and BRAF(V600E) mutations in an Italian cohort of papillary thyroid carcinoma (PTC) patients followed at the Endocrine Units of Pisa, Milano, and Perugia from 1996-2010. Patients and Methods: In total, 401 PTC patients were examined and grouped according to the time of surgery: group 1, 1996-2000; group 2, 2001-2005; and group 3, 2006-2010. Patients were analyzed for clinical, pathological, and molecular features. In parallel, the molecular characteristics of 459 PTC from Sicily were studied. Results: The genetic profiles of the three groups were significantly different (P < 0.0001). In particular, the frequency of RET/PTC rearrangements decreased from 1996-2010, occurring in 33 of 100 (33%) of the patients in group 1, 26 of 148 (17%) in group 2, and 15 of 153 (9.8%) in group 3. The incidence of BRAF(V600E) mutations increased over the same period, from 28% in group 1 (28 of 100) to 48.9% in group 2 (73 of 148) and 58.1% in group 3 (89 of 153). A consistent increase in BRAF(V600E) prevalence was observed in the Sicilian group (P < 0.0001). Moreover, a statistically significant increase in the mean age at diagnosis and decrease in tumor size over the study period was observed. Conclusion: The genetic profile of PTC changed over the last 15 yr, with a significant decrease in the prevalence of RET/PTC rearrangements and an increase in BRAF(V600E) mutations. In addition, the mean age at diagnosis increased and tumor size decreased over the study period.     

Fine-needle aspiration molecular analysis for the diagnosis of papillary thyroid carcinoma through BRAF V600E mutation and RET/PTC rearrangement.

OBJECTIVE: To evaluate BRAF(V600E) mutation on consecutive fine-needle aspiration biopsy (FNAB) specimens in order to assess FNAB's usefulness in preoperative papillary thyroid carcinoma (PTC) diagnosis with the contemporaneous analysis of RET/PTC1 and RET/PTC3 rearrangements obtained from ex vivo thyroid nodules. DESIGN: Thyroid FNABs from 156 subjects with nodules and 49 corresponding surgical samples were examined for the presence of BRAF mutation by real-time allele-specific polymerase chain reaction, confirmed with the use of a laser pressure catapulting system. Samples were also examined for RET/PTC rearrangements. The results were compared with the cytological diagnosis and histopathology. MAIN OUTCOMES: 13/156 cytological examinations were diagnostic for PTC and 19/156 showed suspicious/indeterminate FNAB (12.2%). FNAB-BRAF(V600E) mutation was detected in 11/16 (69%) cases with histological confirmation of PTC. In our series, RET/PTC rearrangement was detected in only one case of PTC, whereas it was not present in any case of adenoma, goiter, or Hashimoto's thyroiditis. No PTC case was found positive at the same time for BRAF mutation and RET/PTC rearrangements. CONCLUSION: BRAF(V600E) mutation detected on FNAB specimens, more than RET/PTC rearrangements, is highly specific for PTC and its routine research might well be an adjunctive and integrative diagnostic tool for the preoperative diagnostic iter.     

The oncogene BRAF V600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na+/I- targeting to the membrane

The oncogene BRAF(V600E) is the most frequent genetic event in papillary thyroid carcinoma (PTC) but its prognostic impact still remains to be elucidated. We evaluated a representative series of 67 individuals with PTC who underwent total thyroidectomy. BRAF-positive tumours correlated with early recurrences (32% vs 7.6%; P=0.02) during a median postoperative follow-up period of 3 years. Interestingly, within the recurrences, a significant majority had negative radioiodine ((131)I) total body scans, predicting a poorer outcome as treatment with (131)I is not effective. This last observation led us to investigate the role of BRAF(V600E) and the MEK-ERK pathway in thyroid dedifferentiation, particularly in Na(+)/I(-) symporter (NIS) impairment, as this thyroid-specific plasma membrane glycoprotein mediates active transport of I(-) into the thyroid follicular cells. A subset of 60 PTC samples was evaluated for NIS immunoreactivity and, accordingly, we confirmed a significant low NIS expression and impaired targeting to membranes in BRAF-positive samples (3.5% vs 30%; P=0.005). Furthermore, experiments with differentiated PCCl3 thyroid cells demonstrated that transient expression of BRAF(V600E) sharply impaired both NIS expression and targeting to membrane and, surprisingly, this impairment was not totally dependent on the MEK-ERK pathway. We have concluded that BRAF(V600E) is a new prognostic factor in PTC that correlates with a high risk of recurrences and less differentiated tumours due to the loss of NIS-mediated (131)I uptake.     

Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA)

OBJECTIVE: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF(V600E)), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. DESIGN AND METHODS: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. RESULTS: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF(V600E) significantly correlated with absence of node metastasis. CONCLUSIONS: BRAF(V600E) is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.