Evaluation of molecular diagnostic approaches for the detection of BRAF p.V600E mutations in papillary thyroid cancer: Clinical implications.

Differentiated papillary thyroid cancer (PTC) is the most common cancer of the endocrine system. PTC has a very good prognosis and a high 5 year survival rate; however, some patients are unresponsive to treatment, and their diagnosis eventually results in death. Recent efforts have focused on searching for prognostic and predictive factors that may enable treatment personalization and monitoring across the course of the disease. The presence of the BRAF mutation is considered to contribute to the risk of poor clinical course, according to American Thyroid Association (ATA) recommendations. The method used for genotyping can impact the predicted mutation frequency; however, ATA recommendations do not address this issue. We evaluated the molecular diagnostic (BRAF p.V600E mutation) results of 410 patients treated for PTC. We thoroughly analyzed the impact of three different BRAF mutation detection methods, Sanger Sequencing (Seq), allele-specific amplification PCR (ASA-PCR), and quantitative PCR (qPCR), on the frequency of mutation detection in 399 patients. Using Seq, we detected the BRAF mutation in 37% of patients; however, we were able to detect BRAF mutations in 57% and 60% of patients using the more sensitive ASA-PCR and qPCR technologies, respectively. Differences between methods were particularly marked in the thyroid papillary microcarcinoma group; BRAF p.V600E mutations were found in 37% of patients using Seq and 63% and 66% of patients using ASA-PCR and qPCR, respectively. We also evaluated how these different diagnostic methods were impacted by DNA quality. Applying methods with different sensitivities to the detection of BRAF p.V600E mutations may result in different results for the same patient; such data can influence stratification of patients into different risk groups, leading to alteration of treatment and follow-up schemes.

The usefulness of determining the presence of BRAF V600E mutation in fine-needle aspiration cytology in indeterminate cytological results.

INTRODUCTION: Fine-needle aspiration biopsy (FNAB) is regarded as the gold standard method for the diagnosis of thyroid nodules, but it has its limitations. Additional methods that would improve sensitivity and specificity in the diagnosis of thyroid cancer (TC), especially in indeterminate lesions. Molecular tests seem to be such a tool. BRAF V600E mutation (the most common in TC) can be detected in FNAB and can be potentially a very useful ancillary marker for FNAB practice. The aim of our study was to evaluate the usefulness of the detection of the BRAF V600E mutation in FNAC in the early diagnosis of TC in patients with indeterminate cytology. MATERIAL AND METHOD: 2290 FNAB were performed and 147 indeterminate results (group 3, 4, and 5 of the Bethesda system) were obtained. Material from these groups was submitted for molecular tests for the occurrence of BRAF V600E mutation. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the tests were calculated. RESULTS: Determining the presence of BRAF V600E mutation in FNAC material in groups 3 and 4 together and in group 5 is associated with sensitivity of TC diagnosis of 37.5% and 81.8%, respectively. In all cases the detection of BRAF V600E mutation was associated with histopathologically proving the presence of TC (specificity of the test - 100%). CONCLUSIONS: The presence of BRAF V600E mutation in FNAC material is always associated with the presence of TC. The usefulness of determining the presence of BRAF V600E in FNAC in cytological groups 3 and 4 is associated with low sensitivity in the diagnosis of thyroid cancer. Due to its high specificity BRAF V600E study may be useful in determining the scope of surgery in patients in cytological group 5.

The BRAF(V600E) mutation in papillary thyroid microcarcinoma: does the mutation have an impact on clinical outcome?

CONTEXT: An activating mutation in the gene BRAF has been correlated with poorer prognosis and more aggressive clinical course in papillary thyroid carcinoma (PTC). We therefore hypothesized that the good prognosis, high 5-year disease-free rate and high survival rate of patients with less aggressive papillary thyroid microcarcinoma (pT1aNo-x) would be associated with a lower incidence of the BRAF(V600E) mutation. OBJECTIVES: To evaluate the frequency of the activating mutation BRAF(V600E) in low-risk papillary thyroid microcarcinoma (pT1aNo-x at the moment of diagnosis) and the association of the mutation with the clinical outcome in a retrospective analysis. STUDY DESIGN: BRAF(V600E) was characterized in 113 PTC patients diagnosed with pT1aNo-x (one PTC focus with a diameter <1 cm, without lymph node or distant metastases according to IUCC/AJCC TNM staging system 2010). Genotyping was performed on DNA extracted from thyroid tumour tissue using direct capillary sequencing, and allele-specific amplification PCR was used to resolve equivocal results. Retrospective analysis of the clinical course of PTC was then correlated with BRAF status in the primary tumour tissue. RESULTS: The BRAF(V600E) mutation was detected in 78 of the 113 pT1aNo-x patients (69·0%). We observed no persistence, locoregional recurrence, lymph node or distant metastases or deaths in the study group during the 12-year study (January 2001 to December 2012). CONCLUSIONS: The presence of the activating BRAF(V) (600E) mutation in a significant percentage of papillary thyroid microcarcinoma indicates that further analyses are required to verify its usefulness as a predictor of clinical outcome in PTC. In this study, there was no correlation between BRAF-positive primary focus of papillary microcarcinoma and more aggressive or recurrent disease.