VE1 Immunohistochemistry Improves the Limit of Genotyping for Detecting BRAFV600E Mutation in Papillary Thyroid Cancer (original) (raw)
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Gland Surgery
Background: Papillary thyroid carcinoma (PTC) accounts for the majority of diagnoses of thyroid carcinoma. BRAF V600E mutation is the most common genetic alteration in PTC, which has diagnostic and prognostic significance. The rate of BRAF V600E mutation in PTC from Thailand has not been reported. Our purpose was to estimate the prevalence of BRAF mutation in a large institutional series using an affordable approach, which combined mutation-specific immunohistochemistry (IHC) with VE1 antibody and tissue microarray (TMA). Methods: A total of 476 PTC cases plotted on TMA were employed for determining the mutation status in this study. The cancer tissue of initial 100 cases (pilot study) were analyzed for BRAF V600E mutation by using both direct sequencing and VE1 immunostaining. For the subsequent PTC cases, VE1 IHC was used as an alternative to direct sequencing for the detection of mutation. Univariate and multivariate analyses were done to determine the association of clinicopathological variables with BRAF V600E mutation. Results: In the pilot study, VE1 IHC showed excellent analytical performance (κ=0.884) for detecting BRAF V600E mutation in PTC TMA as compared to direct sequencing. The prevalence of BRAF V600E in the whole cohort was 60.9% by using VE1 IHC. The mutation was commonly seen in tall cell (92.9%) and classic (70.2%) variants of PTC. Multivariate analysis (P<0.05) showed association of BRAF V600E with histological type of tumor, extrathyroidal extension, and absence of Hashimoto's thyroiditis. Conclusions: In conclusion, BRAF V600E mutation was detected in 60.9% of Thai PTC and it was associated with several aggressive clinicopathological variables of thyroid cancer. VE1 IHC proved as a reliable method able to replace direct sequencing for detection of the mutation. A combination of mutation-specific IHC and TMA allows conducting large cohort studies more labor-saving and cost-efficiently.
The Journal of Clinical Endocrinology & Metabolism, 2008
Background: The BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid carcinoma (PTC). The role of BRAFV600E mutation as a poor prognostic factor has been controversially reported in series with short-term follow-ups. In this study we verified the prognostic value of the BRAFV600E mutation in PTC patients with a long-term follow-up. Methods: We studied 102 PTC patients with a median follow-up of 15 yr. The BRAFV600E mutation was analyzed by PCR-single-strand conformational polymorphism and sequencing. The correlation between the presence/absence of the BRAFV600E mutation, clinicopathological features, and outcome of PTC patients were evaluated. Results: The BRAFV600E mutation was found in 38 of 102 (37.3%) PTC patients, and was significantly more frequent in patients older than 60 yr (P = 0.02), in advanced stages (P = 0.03), and in cases with vascular invasion (P = 0.02). At univariate analysis the worst outcome for PTC patients was significantly correlate...
Surgery, 2013
Background-BRAF V600E mutation is the most common genetic alteration in papillary thyroid cancer (PTC). We utilized a mutation-specific antibody for immunohistochemical (IHC) detection of the BRAF V600E mutation and correlated expression with clinicopathological features. The study was designed to validate the accuracy and determine the significance of IHC detection of the BRAF V600E mutation in PTC. Methods-Direct sequencing and IHC for BRAF V600E mutation was performed in 37 consecutive PTCs. IHC was scored on an intensity, proportion scale. IHC positive tumors were stratified into intensity categories. The categories were assessed for clinicopathologic variables including age, extrathyroidal extension, lymphovascular invasion, and lymph node metastases. Results-25 PTCs were BRAF V600E positive and 12 were BRAF mutation negative on IHC. The BRAF V600E mutation-specific antibody showed a sensitivity of 89% and specificity of 100% for detecting the mutation. Tumors with high intensity staining were significantly more likely to have extrathyroidal extension. Conclusions-IHC is an accurate method for the detection of the BRAF V600E mutation in PTC and its ability to quantify the mutation expression may serve as a better predictor of tumor behavior than molecular sequencing. It provides a potentially rapid, easily applicable and economical alternative to current techniques.
Journal of Clinical Medicine, 2020
The detection of rare mutational targets in plasma (liquid biopsy) has emerged as a promising tool for the assessment of patients with cancer. We determined the presence of cell-free DNA containing the BRAFV600E mutations (cfBRAFV600E) in plasma samples from 57 patients with papillary thyroid cancer (PTC) with somatic BRAFV600E mutation-positive primary tumors using microfluidic digital PCR, and co-amplification at lower denaturation temperature (COLD) PCR. Mutant cfBRAFV600E alleles were detected in 24/57 (42.1%) of the examined patients. The presence of cfBRAFV600E was significantly associated with tumor size (p = 0.03), multifocal patterns of growth (p = 0.03), the presence of extrathyroidal gross extension (p = 0.02) and the presence of pulmonary micrometastases (p = 0.04). In patients with low-, intermediate- and high-risk PTCs, cfBRAFV600E was detected in 4/19 (21.0%), 8/22 (36.3%) and 12/16 (75.0%) of cases, respectively. Patients with detectable cfBRAFV600E were characterize...
Detection of Circulating BRAFV600E in Patients with Papillary Thyroid Carcinoma
The Journal of Molecular Diagnostics, 2015
BRAF V600E is a common mutation in papillary thyroid carcinoma (PTC) correlated with aggressive features. Our objective was to assess the feasibility and accuracy of a novel RNA-based blood assay to identify individuals with a high-risk tumor mutation in patients with PTC. Patients with benign or malignant thyroid disorders were included between September 2013 and July 2014 before either thyroidectomy (n Z 62) or treatment of recurrent or metastatic PTC (n Z 8). RNA was isolated from peripheral blood lymphocytes and reverse transcribed and followed by two rounds of nested PCR amplification with a restriction digest specific for wild-type BRAF. BRAF V600E levels were quantified with standardization curves. Circulating BRAF V600E levels were compared with BRAF mutation status from surgical pathologic DNA-based tissue assays. Testing characteristics and receiving-operator curve using tissue results as the gold standard were assessed. Matched blood and tissue assays for BRAF V600E were performed on 70 patients with PTC (stages I to IV, n Z 48) or other (n Z 22) thyroid tumors. Sixty-three percent of PTC patients tested positive for BRAF V600E with conventional tissue assays on surgical specimens. The correlation between the RNA-based blood assay and tissue BRAF status was 0.71. PTC patients harbor detectable BRAF V600E circulating tumor cells. This blood assay is feasible and has potential as a biomarker for prognosis, surveillance, clinical decision making, and assessment of treatment response to BRAF-targeted therapies.
A High Percentage of BRAF V600E Alleles in Papillary Thyroid Carcinoma Predicts a Poorer Outcome
The Journal of Clinical Endocrinology & Metabolism, 2012
Context: BRAFV600E is considered a negative prognostic marker in papillary thyroid carcinoma (PTC), but unexplained conflicting results are present in the literature. In light of the new finding that most PTC consist of a mixture of tumor cells with wild-type and mutant BRAF, we examined the associations between the percentage of BRAFV600E alleles and both the clinicopathological parameters at time of diagnosis and the disease outcome in a large series of PTCs. Study Design: Tumors from 168 patients with PTC were genotyped for BRAFV600E using BigDye Terminator sequencing and pyrosequencing, and the clinical parameters were analyzed. The associations between clinicopathological characteristics, including disease recurrence at follow-up (median 5.1 yr) and the percentage of mutant BRAF alleles were assessed. Results: The observed prevalence of BRAFV600E was higher when using pyrosequencing then when using BigDye Terminator sequencing (53.6 vs. 36.9%). In the PTC positive for BRAFV600E...
Pathology - Research and Practice, 2012
Literature has consistently shown associations of BRAFV600E mutation with papillary thyroid cancer clinical features. However, the clinical utility of BRAF expression has not been clinically explored so far. We studied 67 thyroid nodules (32 benign nodules and 35 PTC cases). BRAF mRNA expression levels measured by a quantitative real-time PCR and a PCR-RFLP were used to identify BRAFV600E mutation. BRAF mRNA expression was significantly higher in malignant (198.2 ± 373.9 AU) than in benign (4.1 ± 6.9 AU) nodules (p < 0.0001). BRAF expression identified malignancy with a sensitivity of 80.6%, specificity of 77.1%, positive predictive value of 75.8%, and negative predictive value of 81.8%. A cut-point of 4.712, identified by the ROC curve, was able to sort out malignant nodules with an accuracy of 78.8%. Although we did not find any correlation between the presence of BRAF V600E mutation and clinical or tumor features such as age (p = 0.309), gender (p = 0.5453), ethnicity (p = 0.9820), tumor size (p = 1.000), multifocality (p = 0.2530) or mRNA levels (p = 0.7510), the study power for BRAF expression and diagnosis (99%; FPRP = 0.85) indicated that data is noteworthy despite the relative small number of patients investigated. We concluded that BRAF mRNA expression may help to identify PTC among thyroid nodules independently of the presence of BRAFV600E mutation.
Medicinski glasnik : official publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina, 2020
Aim BRAF mutation inhibits many tumour suppressor genes, increases pro-angiogenic molecules and reduces radioactive iodine uptake of tumour in papillary thyroid cancer (PTC), giving it more aggressive clinical characteristics. In this study, we aimed to evaluate the effect of BRAF V600E mutation on the clinicopathological features in patients with PTC. Methods The laboratory and clinical findings of 256 PTC patients who were referred to our clinic between 2007 and 2017 were assessed. Subjects involved in the study were divided into two groups depending on the presence of BRAF V600E mutation. Results BRAF V600E mutation testing gave positive results for 65 (25.4%) out of 256 patients. No significant correlation between BRAF V600E mutation, age and gender was detected. There was no difference between the groups in terms of tumour variant, tumour localization, tumour focality, and perineural invasion. In terms of histopathologic characteristics, presence of tumour capsular invasion (p=...
Annals of Surgical Oncology, 2009
Papillary thyroid cancers often occur as microcarcinoma. Some papillary thyroid microcarcinoma (PTMC) have been considered to be high aggressive according to advanced disease stages, extrathyroidal extension, and severe cervical lymph node metastasis. Although several factors are thought to predict the occurrence of aggressiveness from PTMCs, the origin of aggressiveness has been rarely studied. To answer this question, the correlation between BRAF V600E mutation and high aggressive PTMCs was investigated. The clinicopathological characteristic of totally 64 cases of PTMCs was investigated and the BRAF V600E mutational status of them was identified. BRAF V600E mutation was exclusively detected in PTMCs (37.5%). The data provided no correlation between the occurrence of BRAF V600E mutations and clinicopathological parameters, such as sex, age, and tumorlike lesions combination. The prevalence of BRAF V600E mutation of PTMCs with high aggressiveness (advanced disease stages, extrathyroidal extension, and nodal metastasis) was significantly higher (p \ 0.05) than that of PTMCs without aggressive behavior. The BRAF V600E mutated PTMCs exhibited signs of higher aggressiveness than PTMCs without the mutation. BRAF V600E mutation may be a marker of high aggressiveness in PTMCs.
European Journal of Endocrinology, 2006
Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF V600E ), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. Design and methods: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. Results: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF V600E significantly correlated with absence of node metastasis. Conclusions: BRAF V600E is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.