Detection of BRAF V600E mutation by pyrosequencing

INTRODUCTION: Detection of the V600E hotspot mutation in BRAF oncogene is extremely useful for the screening of hereditary non-polyposis colorectal cancer (Lynch's syndrome) and for the prediction of sensitivity to MEK inhibitors. Here we describe a method for detecting this mutation based upon pyrosequencing technology. METHODS: The efficiency of pyrosequencing for detecting BRAF V600E mutations was compared with the conventional dideoxy sequencing method in 12 tumour cell lines and in 108 colorectal tumours. RESULTS: The results from pyrosequencing were 100% concordant with those from dideoxy sequencing. This method was capable of detecting BRAF V600E mutations at a much lower ratio of mutant to wild-type alleles (1:50) than dideoxy sequencing (1:5) while being considerably faster and less expensive. CONCLUSIONS: Pyrosequencing offers a specific, sensitive, rapid and cost-effective alternative to dideoxy sequencing for the detection of BRAF V600E mutations in clinical tumour specimens.     

Effect of BRAF V600E mutation detection of fine-needle aspiration biopsy on diagnosis and treatment guidance of papillary thyroid carcinoma

ObjectiveThe aim of this study was to evaluate the diagnostic value of detection of BRAF V600E mutation in the fine-needle aspiration cytology (FNAC) specimens of thyroid nodules and the relationship between BRAF V600E mutation and the clinicopathological characteristics of papillary thyroid carcinoma (PTC).MethodsA total of 252 patients who underwent initial thyroid surgery were retrospectively analysed. All the patients underwent a preoperative FNAC at our institution, and the thyroid puncture cell fluid was used for both the cytological diagnosis and BRAF V600E mutational analysis using quantitative polymerase chain reaction. The Cochran-Mantel-Haenszel test was used to evaluate the diagnostic value of BRAF V600E mutation in FNAC fluid in diagnosing PTC. The association between BRAF V600E mutation and the clinicopathological parameters of PTC was analysed using the χ² test.ResultsThrough FNAC, 21 (8%), 60 (24%), and 171 (68%) cases were cytologically diagnosed as benign, indeterminate, and malignant, respectively. Postoperatively, 242 cases were histopathologically diagnosed as PTCs and 10 as goitre nodules. In the FNAC samples, 12 (57 %) of the 21 benign, 48 (80 %) of the 60 indeterminate, and 152 (88.9 %) of the 171 malignant cases showed BRAF V600E mutation. The histopathological diagnosis was used as the gold standard. The sensitivity and specificity of BRAF V600E mutational analysis in the FNAC samples for the diagnosis of PTC were 91.7 % and 100 % in benign, 82.8 % and 100 % in the indeterminate, and 89.4 % and 100 % in the malignant cases, respectively.ConclusionBRAF V600E mutational analysis in FNAC samples of thyroid nodules can be used an effective supplementary diagnostic method at our institution. However, BRAF V600E mutation was not associated with aggressive characteristics in PTC.     

BRAF(V600E) mutation analysis of liquid-based preparation-processed fine needle aspiration sample improves the diagnostic rate of papillary thyroid carcinoma

Early detection and diagnosis of papillary thyroid carcinoma are important for successful management of patients. Liquid-based preparations (Thinprep) of fine needle aspirations from thyroid nodules are now widely used and are replacing conventional smears because residual samples can be used for ancillary tests. Detection of the BRAF(V600E) mutation in cytology specimens could aid in the diagnosis of papillary thyroid carcinoma. We, therefore, analyzed the cytologic features and BRAF(V600E) mutation status of thyroid liquid-based preparation-fine needle aspiration samples. A total of 191 histologically confirmed thyroid liquid-based preparation-fine needle aspiration specimens were selected. We analyzed cytomorphological features and BRAF(V600E) mutation status in both liquid-based preparation-fine needle aspiration samples and the corresponding formalin-fixed, paraffin-embedded tissues. The Seeplex BRAF ACE detection kit (Seoul, Korea), melting curve analysis with SYBR green, and sequencing analysis were used to detect BRAF(V600E) mutations. Of 191 patients, 126 had histologically confirmed papillary thyroid carcinoma, whereas the remaining 65 lesions were benign lesions and carcinomas of other types. The sensitivity of liquid-based preparation alone for diagnosis of papillary thyroid carcinoma was 71.4%. When BRAF(V600E) mutation analyses results were considered in conjunction with the cytologic diagnosis, the diagnostic sensitivity for detecting papillary thyroid carcinoma increased to 84.9% regardless of the method used to detect BRAF mutations. BRAF(V600E) mutation analysis using residual liquid-based preparation cytologic samples is, therefore, a powerful additional diagnostic tool for diagnosis of papillary thyroid carcinoma.     

分化型甲状腺癌中BRAF V600E突变分析

目的探讨分化型甲状腺癌中BRAF V600E突变与临床病理特征的关系。方法收集甲状腺乳头状癌(papillary thyroid carcinoma,PTC)80例(其中经典型67例、滤泡亚型8例、嗜酸细胞亚型3例、高细胞亚型2例)、滤泡癌5例,其中30例PTC取相应癌旁组织,全部送基因检测室检测BRAF V600E突变情况。结果 80例PTC中BRAF V600E突变率为65.0%,5例滤泡癌及30例癌旁组织中未发现BRAF V600E突变;BRAF V600E突变与患者年龄、肿瘤包膜侵犯、淋巴结转移及TNM分期有关。PTC亚型中,经典型和高细胞亚型的BRAF V600E突变率较高(70.1%、100.0%),滤泡亚型的突变率较低(33.3%)。结论PTC中BRAF V600E突变可能与患者年龄有一定相关性,还与包膜侵犯、淋巴结转移及TNM分期有关,经典型和高细胞亚型的BRAF V600E突变率较高,明显高于滤泡亚型。     

BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma

The serine/threonine‐protein kinase B‐raf (BRAF) is an oncogene mutated in various neoplasms, including 5–15% of colorectal carcinomas. The T1799A point mutation, responsible for a large majority of these alterations, results in an amino acid substitution (V600E) causing the constitutive activation of a protein kinase cascade. BRAF V600E in MLH1 deficient tumors implicates somatic tumor‐only methylation of the MLH1 promoter region instead of a germline MLH1 mutation. BRAF V600E also predicts poor prognosis in microsatellite stable colorectal cancers and may be a marker of resistance to anti‐EGFR therapy in metastatic disease. Currently, only molecular methods are available for assessing BRAF mutational status. An immunohistochemical approach is evaluated here. Colon cancers from 2008 to 2012 tested by pyrosequencing for BRAF V600E mutation were selected. A total of 31 tumors with (n = 14) and without (n = 17) the BRAF V600E mutation were analyzed by immunohistochemistry using a commercially available antibody specific to the V600E‐mutated protein. All 14 colorectal carcinomas with the BRAF V600E mutation demonstrated cytoplasmic positivity in tumor cells with the anti‐BRAF antibody. In a minority of cases, staining intensity for the mutated tumor samples was weak (n = 2) or heterogeneous (n = 4); however, the majority of cases showed diffuse, strong cytoplasmic positivity (8 of 14 cases). None of the 17 BRAF wild‐type colorectal cancers showed immunoreactivity to the antibody. The overall sensitivity and specificity of the immunohistochemical BRAF V600E assay was 100%. Detection of the BRAF V600E mutation in colorectal cancer by immunohistochemistry is a viable alternative to molecular methods. © 2013 Wiley Periodicals, Inc.     

Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis

Background

A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence of 29–83%.

Aims

To use a PCR assay to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings.

Methods

Formalin‐fixed paraffin‐embedded (FFPE) sections were evaluated for the BRAF V600E mutation using LightCycler PCR with allele‐specific fluorescent probe melting curve analysis (LCPCR).

Results

42 (37 PTC; 5 benign) surgical tissue samples were analysed for the BRAF V600E activating point mutation. Using LCPCR and direct DNA sequencing, the BRAF mutation was identified in 23/37 (62.2%) PTC FFPE samples. DNA sequencing results demonstrated confirmation of the mutation.

Conclusions

Detection of BRAF‐activating mutations in PTC suggests new approaches to management and treatment of this disease that may prove worthwhile. Identification of the BRAF V600E activating mutation in routine FFPE pathology samples by a rapid laboratory method such as LCPCR could have significant value.Although thyroid cancer represents only 1% of all human malignancies, it accounts for more than 90% of all endocrine cancers.¹ The incidence of thyroid cancer has risen in the United States, from a rate of 3.6 per 100 000 in 1973 to 8.7 per 100 000 in 2002.² The increase in thyroid cancer incidence can be attributed primarily to an increase in the incidence of papillary thyroid carcinoma (PTC). For the time period 1973 to 2002, the incidence of PTC increased from 2.7 to 7.7 per 100 000, a 2.9‐fold increase.² In a report of 15 700 patients in the United States, overall survival rates, corrected for age and sex, were 98% for PTC, 92% for follicular carcinoma, 80% for medullary carcinoma, and 13% for anaplastic carcinoma.³ Among the most curable of cancers, PTC tends to remain localised in the thyroid gland, but in time it may metastasise to regional lymph nodes and, less commonly, to the lungs. Peak incidence of PTC is in the fifth decade of life and it occurs nearly three times more frequently in women than in men.⁴A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in PTC, with a prevalence of 29–83%.^{5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24} Activating BRAF mutations may be an important event in the development of PTC. This mutation had been formerly termed T1796A, based on the NCBI GenBank nucleotide sequence NM 004333, which missed a codon (three nucleotides) in exon 1 of the BRAF gene. With the correct version of the NCBI GenBank nucleotide sequence NT 007914 available, this BRAF mutation is now designated T1799A.²⁵ This thymine (T) to adenine (A) transversion mutation (T→A) results in the substitution of valine with glutamate in codon 600 (V600E, formerly V599E) and converts BRAF into a dominant transforming protein that causes constitutive activation of the mitogen‐activated protein kinase (MAPK) pathway, independent of RAS activation.²⁶ Amino acid 600 lies in the kinase domain of BRAF, and the V600E mutation makes the enzyme more active than wild‐type BRAF. The resulting protein shows increased kinase activity that can transform NIH3T3 cells.²⁶ This suggests that therapy with RAF kinase inhibitors may be of use in this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of PTC for the presence or absence of mutations.The BRAF V600E activating point mutation appears to be highly specific for PTC, with no benign or other well‐differentiated thyroid neoplasm having been found to harbour this mutation. Moreover, some studies have suggested that BRAF mutation may serve as a novel prognostic biomarker that predicts poor clinicopathological outcomes, helping to identify patients who should undergo more aggressive clinical follow‐up.^7,9,27 While these preliminary reports are somewhat controversial^{17,18,21,28,29,30} and additional studies are required to establish the efficacy of this marker, these findings suggest that BRAF mutation detection may serve as a useful tool for diagnosis, management and treatment of PTC.LightCycler PCR with allele specific fluorescent probe melting curve analysis (LCPCR) has been used successfully to detect BRAF activating point mutations in PTC.⁷ Recently, we reported on the clinical utility of this method for detecting BRAF mutations in a series of indeterminate thyroid fine needle aspirate (FNA) cytology samples.³¹ Based on the detection of either a fluorescent reporter probe or double‐stranded DNA‐binding dyes, “real‐time” PCR instruments, such as the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) can provide quantitative information regarding target nucleic acid sequences. The LightCycler is a microvolume fluorimeter integrated with a thermal cycler. Using post‐amplification melting curve analysis, the LightCycler instrument can also be used to differentiate alleles for the purpose of determining sequence variations or point mutations. In this report, we used LCPCR to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings.     

High frequency of BRAF V600E mutations in ameloblastoma

Ameloblastoma is a benign but locally infiltrative odontogenic neoplasm. Although ameloblastomas rarely metastasise, recurrences together with radical surgery often result in facial deformity and significant morbidity. Development of non‐invasive therapies has been precluded by a lack of understanding of the molecular background of ameloblastoma pathogenesis. When addressing the role of ERBB receptors as potential new targets for ameloblastoma, we discovered significant EGFR over‐expression in clinical samples using real‐time RT–PCR, but observed variable sensitivity of novel primary ameloblastoma cells to EGFR‐targeted drugs in vitro. In the quest for mutations downstream of EGFR that could explain this apparent discrepancy, Sanger sequencing revealed an oncogenic BRAF V600E mutation in the cell line resistant to EGFR inhibition. Further analysis of the clinical samples by Sanger sequencing and BRAF V600E‐specific immunohistochemistry demonstrated a high frequency of BRAF V600E mutations (15 of 24 samples, 63%). These data provide novel insight into the poorly understood molecular pathogenesis of ameloblastoma and offer a rationale to test drugs targeting EGFR or mutant BRAF as novel therapies for ameloblastoma. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk     

Morphology predicts BRAF V600E mutation in papillary thyroid carcinoma: an interobserver reproducibility study

Papillary thyroid carcinomas (PTC) with BRAF ^V600E mutation are morphologically distinctive. They are typically classic or tall cell variants, show infiltrative borders, and are associated with desmoplasia/fibrosis, psammoma bodies, and well-developed nuclear features of papillary carcinoma. We hypothesize that morphologic features of PTC can help in the prediction of BRAF ^V600E mutation, and we evaluate the accuracy and the interobserver reproducibility of such prediction. Hematoxylin and eosin-stained sections from 50 PTCs comprising of 26 mutation-positive and 24 mutation-negative tumors were examined. BRAF ^V600E mutation was predicted correctly in 42/50 tumors (accuracy, 84 %) with 96 % sensitivity, 71 % specificity, and 78 % positive and 94 % negative predictive values (NPV). Subtle nuclear features of PTC (n?=?10) had the highest (100 %) negative predictive value followed by well-circumscribed non-infiltrative tumor borders (17/22 mutation-negative tumors, 95 % NPV). The positive predictive value of infiltrative tumor borders (21/28 [75 %] mutation-positive), desmoplasia/fibrosis (23/31 [74 %] mutation-positive), and psammoma bodies (13/20 [65 %] mutation-positive) increased to 100 % when all three features were present (n?=?8/8 mutation-positive). To assess interobserver reproducibility, two pathologists blinded to the mutational status evaluated 30 PTCs (15 mutation-positive and 15 mutation-negative) after self-training on 10 PTCs with known BRAF ^V600E mutational status (five mutation-positive and five mutation-negative). The prediction of the mutation was achieved with substantial agreement (κ value, 0.79) and accuracy (25/30, 83 %). This study demonstrates that BRAF ^V600E mutation in papillary thyroid carcinoma can be predicted on morphology with accuracy and with substantial interobserver agreement.     

Association between BRAF V600E mutation and regional lymph node metastasis in papillary thyroid carcinoma

Background: BRAF V600E is the most frequent genetic alteration in papillary thyroid carcinoma (PTC); there are ongoing conflicts on its association with regional lymph node metastasis. And we aimed to test this association in a referred sample in a single institute in China. Methods: We analyzed BRAF V600E mutational status in the primary lesion of 150 PTC cases in Peking Union Medical College Hospital (PUMCH) and their corresponding lymph node metastasis (if present and available) using a validated Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) method. Results: Among 150 PTC cases, 121 (80.6%) primary tumors harbored BRAF V600E mutation, 66.9% (81/121) and 79.3% (23/29) had regional lymph node metastasis (LNM) in cases detected with and without BRAF V600E mutation, respectively (P = 0.195). The BRAF V600E mutational status of most of the metastatic lesions was not different to that of their primary foci (73 out of 76 cases, 96.1%, Kappa value = 0.893). The 3 inconsistent cases were all mutation positive for primary tumors and mutation negative for LNM. Conclusion: No association was established between BRAF V600E mutation and regional lymph node metastasis in PTC in Chinese patients.     

BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid.

BRAF mutations have been detected in 30% to 80% of papillary thyroid carcinomas (PTC). Several detection methods for BRAF mutation have been reported, but a direct comparison between different assay methods has not been previously reported. In this study, we examined the diagnostic utility of BRAF (T1799A) mutation in 71 cases of thyroid fine needle aspiration specimens using 4 different methods, including direct sequencing, Colorimetric Mutector Assay, real-time LightCycler polymerase chain reaction (LC PCR) with fluorescence resonance energy transfer probes, and an allele-specific LC PCR with CYBR green 1. BRAF mutation was detected in 31 of 58 cases of PTC, but not in 13 cases of non-PTC lesions. The 4 assay methods used in this study were sensitive, reliable, and comparable with each other (100% of specificity and 53.5% of sensitivity). PTC harboring BRAF mutation had higher extrathyroidal invasion and/or lymph node metastasis than PTC with wild-type BRAF. BRAF mutation analysis should be useful for the clinical diagnosis of PTC in cases of indeterminate fine needle aspiration specimen, because of the high degree of specificity. Our results indicate that there is similar sensitivity for the four detection methods. However, the allele-specific LC PCR with CYBR green 1 method is most rapid, easier to perform, and least expensive technique, and it can be readily performed in most molecular diagnostic laboratories.     

Investigation of BRAF V600E detection approaches in papillary thyroid carcinoma

Objective

The detection of BRAF V600E mutation in papillary thyroid carcinoma (PTC) may be helpful to offer diagnostic confirmation. Additionally, such detection may provide a targeted therapeutic approach for the radioactive iodine resistant patients to predict adverse outcomes. To compare the results of immunohistochemistry (IHC) method using the anti-BRAF V600E (VE1) antibody with the Quantitative real-time polymerase chain reaction (qPCR) approach in examining BRAF V600E mutation in PTC, we investigated the sensitivity and specificity of BRAF V600E (clone VE1) mouse monoclonal antibody in detecting the BRAF V600E mutation and correlated BRAF V600E mutation with clinicopathologic features in PTC.

甲状腺乳头状癌中CyclinD1表达和BRAF（V600E）突变的相关性及意义

目的：探讨甲状腺乳头状癌( papillary thyroid carcinoma, PTC)中Cyclin D1表达上调和BRAF( V600E)突变的相关性及临床意义,分析二者在PTC发生、发展中的作用。方法采用免疫组化EnVision两步法检测52例PTC和52例甲状腺良性病变(结节性甲状腺肿25例、桥本甲状腺炎15例、滤泡性腺瘤12例)中Cyclin D1的表达;采用PCR法及DNA测序法检测上述标本中BRAF(V600E)突变状况,分析Cyclin D1表达上调和BRAF(V600E)突变的相关性及与PTC临床病理特征的关系。结果52例PTC中Cyclin D1阳性率为84．6%,免疫反应评分(4．6±2．4)与甲状腺良性病变(1．3±1．6)比较差异有统计学意义(t=8．525,P<0．01);Cyclin D1表达上调与肿瘤淋巴结转移、包膜侵犯、肿瘤分期(Ⅲ+Ⅳ期)及BRAF(V600E)突变相关(P<0．05),与患者年龄、性别、肿瘤结节数目及直径无关。52例PTC中BRAF(V600E)突变率63．5%,甲状腺良性病变中突变率为0,差异有统计学意义(P<0．01),BRAF(V600E)突变与肿瘤淋巴结转移、包膜侵犯及肿瘤分期(Ⅲ+Ⅳ期)密切相关(P<0．05),与患者年龄、性别、肿瘤结节数目及直径无关。 BRAF(V600E)突变组中Cyclin D1表达强度更强,阳性率显著高于野生组(突变组100%,野生组57．9%),免疫表达评分均值高于野生组[突变组(5．7±1．6),野生组(4．0±2．5)],两组比较差异有统计学意义(t=2．652,P<0．05)。结论 Cyclin D1表达上调与BRAF(V600E)突变呈正相关,二者与PTC的发生、发展密切相关,可作为评估PTC侵袭转移能力的指标。     

Immunohistochemistry is highly sensitive and specific for detecting the BRAF V600E mutation in papillary thyroid carcinoma

The V600E mutation in the B-type Raf kinase (BRAF) gene is a common genetic change in cases of papillary thyroid carcinoma (PTC) that appears to play a key role in the development and progression of this disease. We sought to assess the sensitivity and specificity of immunohistochemical detection of this mutation with a V600E mutated BRAF antibody in a Chinese PTC cohort. In this study, we used fully automated immunohistochemistry (IHC) assay with a BRAF V600E (VE1) mouse monoclonal primary antibody to screen for the BRAF V600E mutation in 556 cases of PTC. Moreover, to verify the IHC staining results, real-time PCR was applied to detect this mutation in the same patient cohort. Among the 556 cases in the examined primary PTC cohort, 414 (74.5%) cases and 419 (75.4%) cases were positive for the BRAF V600E mutation by IHC staining and by real-time PCR, respectively. The real-time PCR results indicated that the sensitivity and specificity of IHC staining for the BRAF V600E mutation were 98.8% and 100%, respectively. The BRAF V600E mutation was common among Chinese patients with primary PTC, and was strongly correlated with older patient age and the conventional subtype of PTC but was not associated with parameters of clinicopathological aggressiveness. The fully automated IHC is a reliable technique that can serve as an alternative to molecular biological approaches for the routine detection of the BRAF V600E mutation in PTC patients.     

Presence of the BRAF V600E point mutation in morphologically benign appearing thyroid inclusions of cervical lymph nodes

The biologic nature of morphologically bland-appearing thyroid inclusions in cervical lymph nodes continues to be a controversial topic. The diagnosis of benignity entails a much more conservative clinical approach than does malignancy. Arriving at the correct interpretation, however, can be difficult when only morphologic examination is performed. Incorporating the use of the recently identified BRAF V600E point mutation, a highly specific biomarker for papillary carcinoma of the thyroid, may provide a useful adjunct in assessing the biologic nature of morphologically bland-appearing thyroid inclusions in cervical lymph nodes. In this case report, bland thyroid inclusions were noted in addition to a primary papillary carcinoma of the thyroid and morphologically recognizable cervical lymph node metastasis. Cells from these separate entities were procured by lasercapture microdissection, and the DNA was isolated, amplified, and sequenced. Molecular analhsis provided integral data indicating these morphologically bland thyroid inclusions were malignant, findings not readily apparent by morphologic examination alone.