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Summary Context Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAFV600E mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi-quantitative methods, such as dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR), have the potential to generate false-positive (FP) results. Objectives To eliminate the possibility of FP results, we generated a receiver operating characteristic (ROC) curve to investigate the diagnostic accuracy of pyrosequencing using quantitative data. Design Cytological diagnoses of 983 thyroid nodules were made according to the Bethesda System 2007. The BRAFV600E mutation was analysed by pyrosequencing, and statistical analyses were performed. Results Of the 983 nodules, 902 were adopted to evaluate the diagnostic value of pyrosequencing. The number of pathologically confirmed malignancies was 192, of which 182 were papillary thyroid cancer (PTC). By generating an ROC curve, we defined the optimal cut-off value of the mutant allele peak as 5·95% (area under the curve, 0·849; sensitivity, 0·55; 1-specificity, 0). When we applied this selective cut-off value, the number of PTCs positive for BRAFV600E was 99 (54·4% of the total number of PTCs). With cytology alone, the diagnostic sensitivity and specificity of detecting malignancy were 71·2% and 100%, respectively. Pyrosequencing improved the diagnostic sensitivity from 71·2% to 78·5% (McNemar's test, P < 0·001), without any change in the diagnostic specificity. When 'suspicious for malignancy' was considered a positive cytological outcome, pyrosequencing increased the diagnostic sensitivity of cytology from 95·8% to 96·9%; however, this improvement did not show statistical significance (McNemar's test, P > 0·05). Conclusions Pyrosequencing is an effective method for detecting the BRAFV600E mutation in FNAB samples. By allowing the optimal cut-off value to be determined, pyrosequencing improves the diagnostic sensitivity while eliminating the possibility of FP results. [ABSTRACT FROM AUTHOR]

Background: The BRAFV600E mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAFV600E and MST1,which activates Foxo3,has not been investigated. Methodology/Principal Findings: The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAFV600E positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAFV600Emarkedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAFV600Eis strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAFV600Ewas able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereasaddition of BRAFV600E inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAFV600Ein the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAFV600Etransgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. Conclusions/Significance: The results of this study revealed that the oncogenic effect of BRAFV600E is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAFV600E tumors. [ABSTRACT FROM AUTHOR]

Context Dideoxy sequencing is the most commonly used method for detecting the BRAFV600E mutation in thyroid cancer and melanoma. However, this gold standard method often makes less definite results in detecting the BRAF V600E mutation when there are relatively low amounts of the mutant template in biopsy specimens, which are invariably contaminated with normal tissues. Pyrosequencing, which measures the incorporation of each of the four nucleotides at each template position and indicates the amounts of mutant template present, may be more useful in such situations. Objective To investigate the diagnostic efficiency of pyrosequencing for the mutant BRAF allele in ultrasound (US)-guided fine needle aspiration biopsies (FNABs) of thyroid incidentalomas. Design, setting and subjects A total of 101 thyroid incidentaloma cases were included prospectively. Cytological diagnoses of the FNAB samples were made according to the American Thyroid Association (ATA) guidelines, 2006. The presence of the BRAF V600E mutation was investigated by pyrosequencing and dideoxy sequencing. Results On the basis of cytological analysis, the thyroid incidentalomas were classified into benign ( n = 43), malignant ( n = 30), indeterminate or suspicious neoplasm ( n = 24), and nondiagnostic ( n = 4) categories. Pyrosequencing detected the BRAF V600E mutation in 30 cases: 22 malignant cases, 7 indeterminate cases, and 1 nondiagnostic case. Dideoxy sequencing also detected the BRAF V600E mutation in 28 of the same cases but failed to clearly distinguish the mutant allele from the wild-type allele in one indeterminate case and one nondiagnostic case. Histopathological analysis ascertained that all BRAF V600E-positive cases were papillary thyroid carcinomas. Conclusions Pyrosequencing may be suitable for detecting the BRAF V600E mutation in thyroid incidentaloma and may be superior to dideoxy sequencing when low amounts of the mutant template are present in the biopsy. [ABSTRACT FROM AUTHOR]

Metabolic syndrome is a cluster of cardiovascular risk factors and it is closely related with insulin resistance. So, improvement of insulin resistance is important target of the treatment of metabolic syndrome and diabetes. D-chiroinositol (DCI) is a phospholipid mediating intracellular insulin action. Recently, we reported that DCI had the glucose-lowering effect through the improvement insulin resistance in clinical study. To investigate the mechanism of the glucose-lowering effect of DCI, we explored the effect on glucose uptake, gene expression of adipocytokines and AMPK pathway in 3T3-L1 cells. 2-deoxyglucose uptake increased in DCI-treated cells by about 1.2-fold (relative to the control) and was inhibited by phosphoinositide 3-kinase (PI3kinase) inhibitors (Wortmanin, LY294002) and AMPK inhibitor (STO-609). In Western blot analysis, DCI did not show the difference of phosphorylation of Akt and AMPK compared with control. However, it decreased the gene expression of leptin and resistin in 3T3-L1 cells in a dose-dependent manner. These results suggest that DCI may involve other pathway of glucose metabolism, but not PI3 Kinase and AMPK signaling pathways and it may be useful in management of metabolic syndrome by improving insulin resistance through increasing glucose uptake and decreasing adipocytokines related with insulin resistance. [ABSTRACT FROM AUTHOR]