Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules

BACKGROUND: Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method. METHODS: We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line. RESULTS: The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%). CONCLUSIONS: The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.

Can Ultrasound Be as a Surrogate Marker for Diagnosing a Papillary Thyroid Cancer? Comparison with BRAF Mutation Analysis

PURPOSE: We investigated the merit of ultrasound (US) features and BRAF(V600E) mutation as an additional study of cytology and compared the diagnostic performances of cytology alone, cytology with US correlation, cytology with BRAF(V600E) mutation, and a combination of cytology, US, and BRAF(V600E) mutation all together. MATERIALS AND METHODS: This study included 185 patients (mean age, 48.4 years; range 20-77 years) with 191 thyroid nodules who underwent US-guided fine-needle aspiration (FNA) with an additional BRAF(V600E) mutation test. Three radiologists highly experienced in thyroid imaging retrospectively reviewed US images and classified each nodule into two categories (positive for malignancy or negative for malignancy). Interobserver variability (IOV) of US assessment between the three readers was estimated using the generalized kappa statistic of Landis and Koch. We also calculated the diagnostic performances of these studies. RESULTS: There were 131 cases of malignancy (131/191, 68.6%) and 60 cases of benign nodules (60/191, 31.4%). In terms of IOV of US assessment, the generalized kappa value was 0.242, indicating fair agreement was reached. The combination of cytology with BRAF(V600E) showed higher specificity (100%) and positive predictive value (PPV) (100%) compared to the combination of cytology, BRAF(V600E), and US (specificity 28.3%, 66.7%, 68.3%; PPV 74.6%, 86.6%, 86.8%, respectively; p<0.001). However, cytology with BRAF(V600E) showed lower sensitivity (84.7%) than cytology with BRAF(V600E) and US (96.2%, 98.5%, 95.4%, respectively; p<0.001). CONCLUSION: Considering the diagnostic performance and low reproducibility of US, the combination of FNA with BRAF(V600E) is the most reliable and objective method for diagnosing thyroid malignancy.

Can Ultrasound Be as a Surrogate Marker for Diagnosing a Papillary Thyroid Cancer? Comparison with BRAF Mutation Analysis

PURPOSE: We investigated the merit of ultrasound (US) features and BRAF(V600E) mutation as an additional study of cytology and compared the diagnostic performances of cytology alone, cytology with US correlation, cytology with BRAF(V600E) mutation, and a combination of cytology, US, and BRAF(V600E) mutation all together. MATERIALS AND METHODS: This study included 185 patients (mean age, 48.4 years; range 20-77 years) with 191 thyroid nodules who underwent US-guided fine-needle aspiration (FNA) with an additional BRAF(V600E) mutation test. Three radiologists highly experienced in thyroid imaging retrospectively reviewed US images and classified each nodule into two categories (positive for malignancy or negative for malignancy). Interobserver variability (IOV) of US assessment between the three readers was estimated using the generalized kappa statistic of Landis and Koch. We also calculated the diagnostic performances of these studies. RESULTS: There were 131 cases of malignancy (131/191, 68.6%) and 60 cases of benign nodules (60/191, 31.4%). In terms of IOV of US assessment, the generalized kappa value was 0.242, indicating fair agreement was reached. The combination of cytology with BRAF(V600E) showed higher specificity (100%) and positive predictive value (PPV) (100%) compared to the combination of cytology, BRAF(V600E), and US (specificity 28.3%, 66.7%, 68.3%; PPV 74.6%, 86.6%, 86.8%, respectively; p<0.001). However, cytology with BRAF(V600E) showed lower sensitivity (84.7%) than cytology with BRAF(V600E) and US (96.2%, 98.5%, 95.4%, respectively; p<0.001). CONCLUSION: Considering the diagnostic performance and low reproducibility of US, the combination of FNA with BRAF(V600E) is the most reliable and objective method for diagnosing thyroid malignancy.

Identification of a Rare 3 bp BRAF Gene Deletion in a Thyroid Nodule by Mutant Enrichment with 3'-Modified Oligonucleotides Polymerase Chain Reaction

Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor, and 36-69% of PTC cases are caused by mutations in the BRAF gene. The substitution of a valine for a glutamic acid (V600E) comprises up to 95-100% of BRAF mutations; therefore, most diagnostic methods, including allele-specific PCR and real-time PCR, are designed to detect this mutation. Nevertheless, other mutations can also comprise the genetic background of PTC. Recently, a novel and sensitive technique called mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR has been introduced. When we applied allelespecific PCR and MEMO-PCR for the detection of the BRAF V600E mutation, we found an unusual 3' bp deletion mutation (c.1799_1801delTGA) only when using MEMO-PCR. This deletion results in the introduction of a glutamic acid into the B-Raf activation segment (p.V600_K601delinsE), leading to an elevated basal kinase activity of BRAF. This is the first report of a rare 3 bp BRAF deletion in a PTC patient that could not be detected by allele-specific PCR.

Funçäo tireoidian e produçäo de AMP cíclico em nódulos autônomos da tiróide / Thyroid function and cyclic AMP production in autonomous thyroid nodules

Foram estudadas cinco pacientes eutiroidianas portadores de nódulos autônomos à cintilografia tireiodiana, com diàmetro superio a 3cm e näo supressíveis pela administraçäo de T, (100µg/dia durante 10 dias). Quatro das pacientes apresentaram exames laboratoriais (T4 livre, T3 TSH basal e captaçäo de 131I em 24h) normais. Fatias de tecido tireoidiano adenomatoso e paranodular obtidas pela tiroidectomia foram incubadas com concentraçöes crescentes de TSH e na presença ou näo de iodeto de potássio (KI)ou triiodotironina (T3). O incremento da produçäo de AMP cíclico (cAMP) provocado pelo TSH no tecido adenomatoso foi igual ao encontrado do tecido adjacente paranodular. No tecido paranodular a adiçäo de T3 ou KI inibiu significativamente a produçäo de cAMP, enquanto que, no tecido adenomatoso, a açäo inibitória ocorreu apenas com KI. Estes resultados sugerem: 1) a inibiçäo da secreçäo pode ocorrer na presença de níveis hormonais tiroidianos circulantes normais; 2) nâo há evidência de sensibilidade aumentada ao TSH no tecido autônomo quando avaliada pela produçäo de cAMP; 3) a ausência inibitória do T, na resposta do TSH no tecido nodular sugere alteraçäo celular ao nível de outros reguladores da geraçäo de cAMP.