Associations of the BRAFV600E Mutation with Sonographic Features and Clinicopathologic Characteristics in a Large Population with Conventional Papillary Thyroid Carcinoma

Objective

To evaluate the association of the BRAF^V600E mutation with sonographic features and clinicopathologic characteristics in a large population with conventional papillary thyroid carcinoma (PTC).

Methods

We retrospectively reviewed the sonographic features, clinicopathologic characteristics, and presence of the BRAF^V600E mutation in 688 patients who underwent thyroidectomy for conventional PTC between January and July 2010 at a single institution. The incidence of the BRAF^V600E mutation was calculated. The sonographic features and clinicopathologic characteristics were compared between BRAF-positive and BRAF-negative patients. BRAF-positive patients were subdivided into those with papillary thyroid microcarcinoma (the PTMC group) and those with PTC larger than 10 mm (the PTC>10 mm group), and their sonographic features were compared.

Results

The BRAF^V600E mutation was detected in 69.2% of patients (476 of 688). Sonographic features were not significantly different between BRAF-positive and BRAF-negative PTC, nor between PTMC and PTC>10 mm groups. The BRAF^V600E mutation was associated with male sex (P = 0.028), large tumor size, extrathyroidal extension, central and lateral lymph node metastasis, and advanced tumor stage (P<0.0001).

Conclusion

The BRAF^V600E mutation was significantly associated with several poor clinicopathologic characteristics, but was not associated with sonographic features, regardless of tumor size. We recommend that patients with a thyroid nodule with any suspicious sonographic feature undergo preoperative BRAF^V600E testing for risk stratification and to guide the initial surgical approach in PTC.     

Bisphenol A at a human exposed level can promote epithelial-mesenchymal transition in papillary thyroid carcinoma harbouring BRAFV600E mutation

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, alters the function of endocrine system and enhances the susceptibility to tumorigenesis in several hormone-dependent tumours as thyroid carcinoma. About 50% of papillary thyroid cancers (PTC), the most common type of thyroid malignancy, harbours the BRAF^V600E mutation. This study aimed to investigate a potential combined effect of BPA exposure and BRAF^V600E mutation on epithelial-mesenchymal transition (EMT) in PTC. Firstly, the level of BPA in plasma, the evaluation of BRAF^V600E mutation and the level of EMT-related proteins in PTC samples were individually determined. Additionally, the migration, invasion, colony formation capacity and the expression of EMT-related proteins after exposure to BPA were precisely analysed in vitro thyroid cells genetically modified by the introduction of BRAF^V600E mutation. Moreover, ERK-Cox2 signalling pathway was also introduced to explore the possible mechanism in PTC development. As expected, whether the clinical investigation or cultured thyroid cells demonstrated that BPA at a concentration compatible with human exposed levels (10^-7 M) synergized with the BRAF^V600E mutation promoted EMT via the activation of ERK-Cox2 signalling pathway. Our findings offer some evidence that BPA as an environmental risk factor can facilitate the progression of PTC harbouring BRAF^V600E mutation.     

Tumorigenesis of Papillary Thyroid Cancer Is Not BRAF-Dependent in Patients with Acromegaly

Introduction

Several studies have reported a high frequency of papillary thyroid cancer (PTC) in patients with acromegaly. The aim of this study was to determine the prevalence and predictors of thyroid cancer in patients with acromegaly and to investigate the frequency of the BRAF ^V600E mutation in PTC patients with and without acromegaly.

Materials and Methods

We conducted a retrospective study of 60 patients with acromegaly. Thyroid ultrasonography (US) and US-guided fine needle aspiration were performed on nodules with sonographic features of malignancy. We selected 16 patients with non-acromegalic PTC as a control group. The BRAF ^V600E mutation was analyzed in paraffin-embedded surgical specimens of PTC by real-time polymerase chain reaction, and tumor specimens from patients with PTC were stained immunohistochemically with an antibody against insulin-like growth factor-1 receptor β (IGF-1Rβ).

Results

Thyroid cancer was found in 15 (25.0%) patients. No differences in age, sex, initial growth hormone (GH) and IGF-1 percentage of the upper limit of normal values or treatment modalities were observed between patients with and without PTC. Acromegaly was active in 12 of 15 patients at the time of PTC diagnosis; uncontrolled acromegaly had a significantly higher frequency in the PTC group (60%) than in the non-PTC group (28.9%) (p = 0.030). The BRAF ^V600E mutation was present in only 9.1% (1/11) of PTC patients with acromegaly, although 62.5% (10/16) of control patients with PTC had the mutation (p = 0.007). IGF-1Rβ immunostaining showed moderate-to-strong staining in all malignant PTC cells in patients with and without acromegaly. Significantly less staining for IGF-1Rβ was observed in normal adjacent thyroid tissues of PTC patients with acromegaly compared with those without (p = 0.014).

Conclusion

The prevalence of PTC in acromegalic patients was high (25%). An uncontrolled hyperactive GH-IGF-1 axis may play a dominant role in the development of PTC rather than the BRAF ^V600E mutation in patients with acromegaly.     

Expression levels of HMGA2 and CD9 and its clinicopathological significances in the benign and malignant lesions of the gallbladder

Background

Papillary thyroid carcinoma (PTC) is one of the most frequent endocrine malignancies. In most cases, it often presents as multifocal tumor. It has been reported that multifocal tumors are associated with elevated risk of lymph node and distant metastases. Multifocality is also one of the factors predicting prognosis. Recent studies show that BRAF^V600E mutation occurs more frequently in aggressive PTC. The purpose of this study was to evaluate BRAF^V600E status and clinicopathological features in multiple and solitary PTC.

Methods

We performed a retrospective study to analyze 512 PTC cases who received surgery, including 376 solitary PTCs and 136 multiple PTCs.

Results

Multiple PTC is more related to lymph node metastasis and vascular invasion than solitary PTC. However, the distant metastasis rate and 10-year survival rate showed no difference between these two groups. BRAF^V600E mutation status was more frequent in multiple PTC patients with lymph node metastasis and late stage at diagnosis.

Conclusion

BRAF^V600E mutation is most commonly associated with extra-thyroidal extension and lymph node metastasis in PTC. Multiple PTC patients with young age, large tumors and BRAF^V600E mutation should be followed carefully. Our study provides useful information for PTC patients?? followup and treatment.     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.     

Prognostic Value of Lymph Node Uptake on Pretreatment F-18 FDG PET/CT in Patients with N1B Papillary Thyroid Carcinoma

Objective: The aim of this study was to investigate the prognostic value of metabolic characteristics of metastatic lymph node (LN) using pretreatment F-18 fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) for patients with papillary thyroid carcinoma (PTC) and metastatic lateral LN (N1b).Methods: Ninety-six PTC patients (female:male = 72:24; median age, 44.5 years) with pathologic N1b who underwent pretreatment FDG PET/CT, total thyroidectomy, and radioactive iodine ablation were retrospectively reviewed. To predict responses to initial therapy and recurrence, clinicopathologic factors and metabolic parameters were reviewed, such as sex, age, tumor size, extranodal extension, number and ratio of metastatic LNs, serum thyroglobulin, and maximum standardized uptake value (SUVmax).Results: Among the 96 PTC patients, 81 (84.4%) were classified into the acceptable response (58 excellent; 23 indeterminate) and 15 (15.6%) into the incomplete response (8 biochemical incomplete; 7 structural incomplete) by the 2015 American Thyroid Association management guideline for differentiated thyroid carcinoma. The multivariate analysis showed that SUVmax of N1b (P = .018), pre-ablation stimulated thyroglobulin level (P = .006), and the ratio of metastatic LNs (P = .018) were related to incomplete response. The cutoff value of each variable was determined by receiver operating characteristic analysis. Nine (9.4%) patients experienced recurrences (median follow-up: 50 months). The Kaplan-Meier analysis revealed that SUVmax of N1b (cutoff value: 2.3; P = .025) and ratio of metastatic LNs (cutoff value: 0.218; P = .037) were significant prognostic factors for recurrence.Conclusion: High SUVmax of N1b cervical LN on pretreatment FDG PET/CT could predict incomplete responses to initial therapy and recurrence in patients with N1b PTC.Abbreviations: ATA = American Thyroid Association; DTC = well-differentiated thyroid carcinoma; FDG = F-18 fluorodeoxyglucose; IQR = interquartile range; LN = lymph node; N1b = metastatic lateral cervical lymph node; PET/CT = positron emission tomography/computed tomography; PTC = papillary thyroid carcinoma; RAI = radioactive iodine; ROC = receiver operating characteristic; SUVmax = maximum standardized uptake value; Tg = thyroglobulin; USG = ultrasonography     

False-Positive FNA Due to Highly Sensitive BRAF Assay

ObjectiveFalse-positive BRAF analysis on fine-needle aspiration (FNA) has rarely been reported in the literature but may become more common with the advent of assays that can detect the BRAF V600E mutation in only 2% of otherwise wild-type thyroid cells. We present the case of an indeterminate BRAF-positive FNA that showed no evidence of cancer on final surgical pathology.MethodsCase report and literature review.ResultsAn 87-year-old female with an indeterminate 1.7-cm nodule but BRAF-positive cytology underwent thyroid lobectomy. Final pathology revealed a benign adenomatoid nodule. An area rich in tumor cells from the nodule was identified, labeled, and microdissected for molecular testing, which demonstrated only wild-type BRAF, at the analytical limit of the assay.ConclusionIncreasingly sensitive BRAF assays using dual-priming oligonucleotide-based multiplex polymerase chain reaction analysis can detect the BRAF V600E mutation when present in only 2% of a population of wild-type cells. This increases the risk of false-positive results, particularly in cases of indeterminate FNA. Clinicians must caution patients in these circumstances that BRAF molecular testing may not have a 100% positive predictive value. (Endocr Pract. 2014;20:e8-e10)     

Intra- and inter-tumor heterogeneity of BRAF(V600E))mutations in primary and metastatic melanoma

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAF^V600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAF^V600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAF^V600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAF^V600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAF^V600Eand BRAF^wild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAF^V600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAF^V600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.     

Combined tazemetostat and MAPKi enhances differentiation of papillary thyroid cancer cells harbouring BRAFV600E by synergistically decreasing global trimethylation of H3K27

Clinical efficacy of differentiation therapy with mitogen-activated protein kinase inhibitors (MAPKi) for lethal radioiodine-refractory papillary thyroid cancer (RR-PTC) urgently needs to be improved and the aberrant trimethylation of histone H3 lysine 27 (H3K27) plays a vital role in BRAF^V600E-MAPK-induced cancer dedifferentiation and drug resistance. Therefore, dual inhibition of MAPK and histone methyltransferase (EZH2) may produce more favourable treatment effects. In this study, BRAF^V600E-mutant (BCPAP and K1) and BRAF-wild-type (TPC-1) PTC cells were treated with MAPKi (dabrafenib or selumetinib) or EZH2 inhibitor (tazemetostat), or in combination, and the expression of iodine-metabolizing genes, radioiodine uptake, and toxicity were tested. We found that tazemetostat alone slightly increased iodine-metabolizing gene expression and promoted radioiodine uptake and toxicity, irrespective of the BRAF status. However, MAPKi induced these effects preferentially in BRAF^V600E mutant cells, which was robustly strengthened by tazemetostat incorporation. Mechanically, MAPKi-induced decrease of trimethylation of H3K27 was evidently intensified by tazemetostat in BRAF^V600E-mutant cells. In conclusion, tazemetostat combined with MAPKi enhances differentiation of PTC cells harbouring BRAF^V600E through synergistically decreasing global trimethylation of H3K27, representing a novel differentiation strategy.     

The BRAF mutation confers sensitivity of thyroid cancer cells to the BRAF inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF^V600E, as a result of the BRAF^T1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF^V600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF^T1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF^T1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC₅₀ values (0.115–1.156 μM) in BRAF^V600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC₅₀ values (56.674–1349.788 μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF^T1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF^T1799A mutation-selective therapeutic agent for thyroid cancer.     

Unfavorable Responses to Radioiodine Therapy in N1b Papillary Thyroid Cancer: A Propensity Score Matching Study

Objective: Regional nodal metastases carry prognostic significance in papillary thyroid cancer (PTC). However, whether different locational nodal metastases correlate with different therapeutic responses remains controversial. We innovatively applied the response to therapy restratification system to evaluate the dynamic disease status after surgery and radioactive iodine (RAI) therapy in PTC patients with different locational nodal metastases.Methods: A total of 585 nondistant-metastatic PTC patients who underwent total thyroidectomy and RAI therapy were retrospectively enrolled. Patients with nodal metastases were categorized into N1a and N1b groups. Propensity score matching was used to balance the bias between the 2 groups. Therapeutic responses were dynamically evaluated, and responses to RAI therapy were classified into excellent (ER), indeterminate (IDR), biochemical incomplete (BIR) and structural incomplete response (SIR).Results: N1b group patients showed a significantly higher pre-ablation stimulated thyroglobulin (Ps-Tg) level than N1a group patients (7.4 ng/mL versus 3.2ng/mL, P<.001). After RAI therapy, N1b group patients took a longer time to achieve ER (9.86 months versus 3.29 months, P<.001) and exhibited a higher proportion of non-ER (IDR, BIR, and SIR) (39.15% versus 17.46%, P<.001) compared to N1a group patients. In logistic regression, N1b and Ps-Tg ≥10 ng/mL were confirmed to be independent factors predicting non-ER (odds ratio: 2.591, and 9.196, respectively). In Cox regression, patients with N1b disease and Ps-Tg ≥10 ng/mL showed significantly lower hazards for achieving ER (hazard ratio: 0.564, and 0.223, respectively).Conclusion: N1b PTC patients showed inferior responses to surgery and RAI therapy compared to N1a patients. N1b was confirmed to be an independent factor predicting unfavorable responses to RAI therapy.Abbreviations: AJCC = American Joint Committee on Cancer; ATA = American Thyroid Association; BIR = biochemical incomplete response; BRAF^V600E = proto-oncogene B-Raf V600E mutation; CI = confidence interval; CT = computed tomography; DNA = deoxyribonucleic acid; DTC = differentiated thyroid cancer; ER = excellent response; ETE = extrathyroidal extension; HR = hazard ratio; IDR = indeterminate response; LNM = lymph node metastasis; N1a = central cervical LNM; N1b = lateral cervical LNM; OR = odds ratio; PSM = propensity score matching; Ps-Tg = pre-ablation stimulated thyroglobulin; PTC = papillary thyroid cancer; RAI = radioactive iodine; SIR = structural incomplete response; Tg = thyroglobulin; TgAb = thyroglobulin antibody; TSH = thyroid-stimulating hormone     

Is Follow-up BRAFV600E Mutation Analysis Helpful in the Differential Diagnosis of Thyroid Nodules with Negative Results on Initial Analysis?

Background

We evaluated the usefulness of follow-up BRAF^V600E mutation analysis using ultrasonography-guided fine-needle aspiration (US-FNA) in diagnosis of thyroid nodules showing negative BRAFV600E mutation on prior analysis.

Methodology/Principal Finding

A total of 49 patients (men: 6, women: 43, mean age: 50.4 years) with 49 thyroid nodules were included. Patients had undergone initial and follow-up US-FNA and subsequent BRAF^V600E mutation analysis from US-FNA aspirates. All patients had negative results on initial BRAF^V600E mutation analysis. Clinicopathologic findings, US assessment, and BRAF^V600E mutation results were analyzed according to the final pathology. Of the 49 nodules, 12 (24.5%) were malignant and 37 (75.5%) were benign. Seven (58.3%) of the 12 malignant nodules were positive for BRAF^V600E mutation on follow-up, all showing suspicious US features. Initial US-FNA cytology of the 7 nodules were non-diagnostic (n = 2), benign (n = 2), or atypia (n = 3), while follow-up were benign (n = 1), indeterminate (n = 1), suspicious for malignancy (n = 4), and malignancy (n = 1).

Conclusions/Significance

Follow-up BRAF^V600E mutation analysis may be helpful in the diagnosis of selected thyroid nodules negative for BRAF^V600E mutation on initial analysis, which are assessed as suspicious malignant on US, diagnosed as non-diagnostic, benign or atypia on follow-up US-FNA.     

Identification and synthesis of N-(thiophen-2-yl) benzamide derivatives as BRAFV600E inhibitors

The V600E BRAF kinase mutation, which activates the downstream MAPK signaling pathway, commonly occurs in about 8% of all human malignancies and about 50% of all melanomas. In this study, we employed virtual screening and chemical synthesis to identify a series of N-(thiophen-2-yl) benzamide derivatives as potent BRAF^V600E inhibitors. Structure–activity relationship studies of these derivatives revealed that compounds b40 and b47 are the two most potent BRAF^V600E inhibitors in this series.     

Downregulation of uPAR inhibits migration,invasion, proliferation,FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells

Papillary thyroid carcinoma (PTC) is the most common endocrine and thyroid malignancy. The urokinase plasminogen activator receptor (uPAR) plays an important role in cancer pathogenesis, including breakdown of the extracellular matrix, invasion and metastasis. Additionally, there is increasing evidence that uPAR also promotes tumorigenesis via the modulation of multiple signaling pathways. BRAF^V600E, the most common initial genetic mutation in PTC, leads to ERK1/2 hyperphosphorylation, which has been shown in numerous cancers to induce uPAR. Treatment of the BRAF^V600E-positive PTC cell line, BCPAP, with the MEK/ERK inhibitor U0126 reduced uPAR RNA levels by 90%. siRNA-mediated downregulation of uPAR in BCPAP cells resulted in greatly decreased activity in the focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. This phenomenon was concurrent with drastically reduced proliferation rates and decreased clonigenic survival, as well as demonstrated senescence-associated nuclear morphology and induction of β-galactosidase activity. uPAR-knockdown BCPAP cells also displayed greatly reduced migration and invasion rates, as well as a complete loss of the cells'' ability to augment their invasiveness following plasminogen supplementation. Taken together, these data provide new evidence of a novel role for uPAR induction (as a consequence of constitutive ERK1/2 activation) as a central component in PTC pathogenesis, and highlight the potential of uPAR as a therapeutic target.Key words: urokinase plasminogen activator receptor (uPAR), papillary thyroid carcinoma, invasion, migration, proliferation, senescence, FAK, PI3K, Akt     

Analysis of the Association between CIMP and BRAFV600E in Colorectal Cancer by DNA Methylation Profiling

A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAF^V600E) is tightly associated with CIMP, raising the question of whether BRAF^V600E plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAF^V600E. We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAF^V600E causes DNA hypermethylation by stably expressing BRAF^V600E in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAF^V600E is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAF^V600E and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAF^V600E-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAF^V600E-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAF^V600E in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding CIMP in colorectal cancer and gaining insights into the role of aberrant DNA hypermethylation in colorectal tumorigenesis.