BRAF (V600E) mutation analysis in papillary thyroid carcinomas by peptide nucleic acid clamp real-time PCR.

BACKGROUND: Activating somatic mutation of the BRAF (V600E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC) with a variable frequency (32-87%) in different series by different methods. The BRAF (V600E) mutation is associated with various clinicopathological parameters. The mutation is an important factor for the management of the PTC patients. The objective of this study was to detect the BRAF (V600E) mutation in PTCs by peptide nucleic acid (PNA) clamp real-time PCR and to analyze the results with clinicopathological parameters. METHODS: We performed genetic analysis of BRAF (V600E) by PNA clamp real-time PCR in 211 PTCs in Korea, stratified by clinicopathological parameters. RESULTS: The BRAF (V600E) mutation was detected in 90% of PTC cases, and it occurred significantly more often in female patients than in male patients (p = 0.001). The clinicopathological parameters of age, tumor size, and disease stage were not associated with the BRAF (V600E) mutation, while extrathyroid invasion (p = 0.031), lymph nodal metastasis (p = 0.002), and tumor multiplicity (p = 0.020) were. CONCLUSIONS: The prevalence (90%) of the BRAF (V600E) mutation in this study is the highest ever reported, confirming the key role of this mutation in PTC tumorigenesis. The BRAF (V600E) mutation was associated with aggressive clinical behaviors including extrathyroid invasion, lymph nodal metastasis and tumor multifocality. The PNA clamp real-time PCR method for the BRAF (V600E) mutation detection is sensitive and is applicable in a clinical setting.

Klotho inhibits the capacity of cell migration and invasion in cervical cancer.

Aberrant activation of the Wnt/ß-catenin signaling pathway is common in human cervical cancers. However, the mechanisms of Wnt activation in cervical cancer remain largely unknown. In the present study, we demonstrate that Klotho, a Wnt antagonist, is downregulated in invasive human cervical tumors and in a cell line we analyzed. Our data demonstrated that in vivo Klotho expression was not observed in invasive cervical carcinoma. In vitro restoration of Klotho expression in SiHa cells resulted in a decreased cell motility and invasiveness through upregulation of E-cadherin, downregulation of N-cadherin and reduced expression of MMP7 and -9. Ectopic expression of Klotho also reduced the expression of the epithelial-to-mesenchymal transition (EMT) transcription factors Slug and Twist. Furthermore, Klotho causes a significant inhibition of the Wnt/ß-catenin pathway in cervical cancer cells, as supported by the expression of Wnt/ß-catenin transcriptional target genes such as c-Myc and cyclin D1. Consequently, our findings demonstrate for the first time that Klotho regulates tumor invasion through the EMT process and provide novel mechanistic insights into the role of Klotho in cervical cancer progression and contribute to treatment for metastatic cervical cancer patients.

Detection of BRAF(V600E) Mutations in Papillary Thyroid Carcinomas by Peptide Nucleic Acid Clamp Real-Time PCR: A Comparison with Direct Sequencing.

BACKGROUND: Papillary thyroid carcinoma (PTC) of the thyroid is the most common endocrine malignancy. High prevalence of an activating point mutation of BRAF gene, BRAF(V600E), has been reported in PTC. We assessed the efficiency of peptide nucleic acid clamp real-time polymerase chain reaction (PNAcqPCR) for the detection of BRAF(V600E) mutation in PTC in comparison with direct sequencing (DS). METHODS: A total of 265 thyroid lesions including 200 PTCs, 5 follicular carcinomas, 60 benign lesions and 10 normal thyroid tissues were tested for BRAF(V600E) mutation by PNAcqPCR and DS. RESULTS: The sensitivity and accuracy of the PNAcqPCR method were both higher than those of DS for the detection of the BRAF(V600E) mutation. In clinical samples, 89% of PTCs harbored the BRAF(V600E) mutation, whereas 5 follicular carcinomas, 50 benign lesions and 10 normal thyroid tissues lacked the mutation. The mutation was associated with aggressive clinical behaviors as extrathyroid invasion (p=0.015), lymph node metastasis (p=0.002) and multiple tumor numbers (p=0.016) with statistical significance. CONCLUSIONS: The PNAcqPCR method is efficiently applicable for the detection of the BRAF(V600E) mutation in PTCs in a clinical setting.