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.     

Clonality analysis of multifocal papillary thyroid carcinoma by using genetic profiles

Papillary thyroid carcinoma (PTC) is the most common adult thyroid malignancy and often presents with multiple anatomically distinct foci within the thyroid, known as multifocal papillary thyroid carcinoma (MPTC). The widespread application of the next‐generation sequencing technologies in cancer genomics research provides novel insights into determining the clonal relationship between multiple tumours within the same thyroid gland. For eight MPTC patients, we performed whole‐exome sequencing and targeted region sequencing to identify the non‐synonymous point mutations and gene rearrangements of distinct and spatially separated tumour foci. Among these eight MPTCs, completely discordant mutational spectra were observed in the distinct cancerous nodules of patients MPTC1 and 5, suggesting that these nodules originated from independent precursors. In another three cases (MPTC2, 6, and 8), the distinct MPTC foci of these patients had no other shared mutations except BRAF V600E, also indicating likely independent origins. Two patients (MPTC3 and 4) shared almost identical mutational spectra amongst their separate tumour nodules, suggesting a common clonal origin. MPTC patient 7 had seven cancer foci, of which two foci shared 66.7% of mutations, while the remaining cancer foci displayed no common non‐synonymous mutations, indicating that MPTC7 has multiple independent origins accompanied by intraglandular disease dissemination. In this study, we found that 75% of MPTC cases arose as independent tumours, which supports the field cancerization hypothesis describing multiple malignant lesions. MPTC may also arise from intrathyroidal metastases from a single malignant clone, as well as multiple independent origins accompanied by intrathyroidal metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.

BRAF mutations are associated with some histological types of papillary thyroid carcinoma

Mutations in the BRAF gene have recently been detected in a wide range of neoplastic lesions with a particularly high prevalence in melanoma and papillary thyroid carcinoma (PTC). The hot-spot mutation BRAF(V599E) is frequently detected in PTC (36-69%), in contrast to its absence in other benign or malignant thyroid lesions. In order to unravel whether there is any association between the occurrence of the BRAF mutation and the histological pattern of PTC, in this study a previous series of 50 PTCs was extended to 134 cases, including ten cases of PTC-related entities-hyalinizing trabecular tumour (HTT) and mucoepidermoid carcinoma (MEC). Using PCR/SSCP and sequencing, the BRAF(V599E) mutation was detected in 45 of the 124 PTCs (36%). No mutations were detected in any case of HTT and MEC. BRAF(V599E) was present in 75% of Warthin-like PTCs and 53% of conventional PTCs, whereas no BRAF(V599E) mutations were detected in any of the 32 cases of the follicular variant of PTC. BRAF(V599E) was also detected in 6 of 11 cases of the oncocytic variant of PTC that displayed a papillary or mixed follicular-papillary growth pattern and in none of the four oncocytic PTCs with a follicular growth pattern. A distinct mutation in BRAF (codon K600E) was detected in three cases of the follicular variant of PTC. This study has confirmed the high prevalence of BRAF(V599E) in PTC and has shown that the mutation is almost exclusively seen in PTC with a papillary or mixed follicular-papillary growth pattern, regardless of the cytological features of the neoplastic cells. The results support the existence of an oncocytic variant of PTC that should be separated from the oncocytic variant of follicular carcinoma and suggest that the follicular variant of PTC may be genetically different from conventional PTC.     

实时剪切波弹性成像杨氏模量最大值诊断甲状腺乳头状癌BRAF V600E基因突变的价值

目的:探究实时剪切波弹性成像（SWE）杨氏模量最大值诊断甲状腺乳头状癌（PTC）鼠类肉瘤滤过性毒菌致癌同源体B1（BRAF）V600E基因突变的价值。方法:回顾性分析2018年1月~2019年6月宿迁市第一人民医院收治的110例PTC患者共124个结节的临床及超声影像学资料。患者经病理学检查均确诊为PTC,并完成了BRAF V600E基因检测以及SWE检查,临床资料完整。通过比较V600E基因突变型与野生型PTC的SWE检查结果,并绘制患者Emax值的ROC曲线,分析Emax值对PTC患者BRAF V600E基因突变的诊断效能。结果:BRAF基因检测结果显示:124个PTC结节BRAF基因检测V600E野生型共37个,V600E突变型共87个。野生型PTC结节的SWE图像多以蓝色为主,而突变型主要表现为红橙相间彩色混杂的图像。突变组PTC结节Emax、Emean、Eratio值均明显大于野生组,比较差异具有统计学意义（P<0.05）,ROC曲线分析显示:Emax值诊断V600E突变的AUC=0.846>0.5（95%CI:0.808~0.885, P<0.01）;Cut-off值为43.65 kPa,此时敏感度为91.45%,特异性为68.23%。以Emax≥43.65 kPa为临界值对124个结节进行诊断,结果显示:SWE诊断PTC结节为V600E基因突变型90个,野生型34个,正确率为86.29%;SWE诊断结果与基因检测结果一致性显著（Kappa值=0.665, P<0.01）。结论:PTC BRAF V600E基因突变型与野生型的SWE影像学特征具有明显差异,应用SWE的Emax值诊断PTC结节V600E基因突变的正确率和敏感性均较高,诊断价值显著,值得在临床上推广应用。     

A new BRAF gene mutation detected in a case of a solid variant of papillary thyroid carcinoma

BRAF gene mutations have been frequently detected in papillary thyroid carcinoma (PTC). Moreover, there is a close association between the type of mutation and the PTC histotype: BRAF(V600E) is associated with conventional PTC and with histological variants of PTC displaying a prominent papillary growth pattern, whereas BRAF(K601E) is associated with the follicular variant of PTC. We report the detection of a novel BRAF triplet deletion in a case of PTC displaying a predominantly solid growth pattern. The deletion leads to the replacement of a valine and a lysine by a glutamate in the BRAF activation segment (BRAF(VK600-1E)), thus mimicking partially the 2 BRAF mutations previously described. Our study reinforces the existence of a close relationship between the occurrence of some types of BRAF mutation and some PTC histotypes. The genetic study of more cases of the solid variant of PTC is necessary to find whether there exists a significant association between the occurrence of BRAF(VK600-1E) and such PTC histotype.     

The Relationship of the BRAFV600E Mutation and the Established Prognostic Factors in Papillary Thyroid Carcinomas

It has been shown that BRAF(V600E) mutation in papillary thyroid carcinomas (PTC) is associated both with pathogenesis and poor prognosis. In this study, we aimed to investigate the relationship of the BRAF(V600E) mutation and the established prognostic factors in a cohort of Turkish patients with PTC. Forty-six cases of papillary thyroid carcinoma have been evaluated for the presence of BRAF(V600E) mutation. BRAF(V600E) has been examined by restriction fragment length polymorphism. BRAF(V600E) mutation status has been compared with well-known histopathological and clinical prognostic parameters such as invasion of thyroid capsule, extrathyroidal extension, and the presence of lymph node and/or distant metastasis. We have found that BRAF(V600E) mutation was present in the majority of our cases (40/46). Considering the stage of the disease, five of the negative cases were in stage 1 while the remaining one was in stage 2. Only one BRAF(V600E) negative case has shown extrathyroidal extension and lymph node metastasis. All four patients with distant metastasis had BRAF(V600E) mutation. Statistical analyses revealed that there are no significant relationship between the BRAF(V600E) mutation and the established prognostic factors. We found a relatively higher BRAF(V600E) mutation rate in classical type PTC than in other similar studies. We think that the limited number of our cases may either weaken or mask some potentially important relationship between BRAF(V600E) mutation and the established prognostic factors.     

甲状腺乳头状癌中TROP-2表达的临床意义及其诊断价值

目的观察人类滋养层细胞表面抗原2(TROP-2)在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)中的表达,探讨TROP-2在PTC中表达的临床意义及其诊断价值。方法采用免疫组化EnVision法检测100例甲状腺恶性病变(PTC 75例、滤泡性癌10例、髓样癌10例、差分化癌5例)、45例良性病变(正常甲状腺10例、结节性甲状腺肿10例、桥本甲状腺炎15例、滤泡性腺瘤10例),5例具有乳头样核特征的非浸润性甲状腺滤泡性肿瘤(non-invasive folliculaRthyroid neoplasms with papillary-like nucleaRfeatures,NIFTP)中TROP-2的表达;ARMS法检测PTC中BRAF V600E基因突变。分析TROP-2对PTC诊断的敏感性和特异性,及其与PTC临床病理特征以及BRAF V600E基因突变的关系。结果TROP-2在PTC中的阳性率为81.3%(61/75),在其他甲状腺恶性肿瘤和良性病变中均阴性,TROP-2对PTC诊断的敏感性为81.3%,特异性为100%。TROP-2表达与PTC淋巴结转移有关(P<0.05),与患者年龄、性别、肿瘤部位及临床分期无关(P>0.05),经典型PTC中TROP-2表达高于滤泡亚型PTC(P<0.05)。PTC中TROP-2表达与BRAF V600E基因突变呈正相关(P<0.05)。结论TROP-2是一种具有高度特异性和敏感性的诊断PTC的标志物,TROP-2检测可预测PTC的临床生物学行为和BRAF V600E基因突变状态,并对于形态学变形的PTC、微小型PTC和PTC的甲状腺内扩散的诊断和识别具有重要价值。     

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 does not predict recurrence after long-term follow-up in TNM stage I or II papillary thyroid carcinoma patients

The BRAF V600E mutation may serve as a marker of disease recurrence in well-differentiated papillary thyroid cancer (PTC). Our aim was to study if TNM stage I or II PTC patients, with and without recurrence after long-term follow-up would differ in BRAF status. BRAF status was retrospectively determined in tumour tissue from a cohort of low-risk PTC patients (n = 461) with and without recurrence after 16 years of follow-up. Initial treatment was total thyroidectomy (TTE) and radioiodine remnant ablation (RRA). Forty-six patients (9.9%) experienced disease recurrence. BRAF mutation was positive in 66% (17/26) of patients with and 68% (17/25) without recurrence (p = NS). Fifty per cent of BRAF positive and 53% of BRAF negative patients experienced disease recurrence (p = NS). Time to recurrence was 52 (range 18-144) and 36 (range 16-71) months, respectively (p = NS). Primary tumour size, nodal metastasis and local infiltration at presentation did not differ between BRAF positive and negative patients (2.0 vs 2.2 cm, 21% vs 35% and 6% vs 12%, respectively, all p = NS). Taken together, BRAF V600E is common in Finnish patients with low-risk PTC but does not predict recurrence after long-term follow-up after initial treatment with TTE and RRA.     

甲状腺乳头状癌中BRAFV599E点突变与RET/PTC融合基因的检测

目的检测甲状腺乳头状癌(PTC)及其他类型甲状腺良恶性肿瘤中BRAFV599E的点突变及 RET/PTC1、3融合基因的表达状况,探讨二者与PTC临床病理学特征的关系.方法用聚合酶链反应(PCR)及逆转录(RT)-PCR分别检测95例石蜡与新鲜甲状腺病变组织中BRAFV599E点突变和RET/PTC1、3融合基因.结果 (1)仅在PTC中检测到BRAFV599E的突变,突变率56%(37/66),在经典型PTC和高细胞型PTC中突变率分别为70%(29/41)和2/3,在滤泡型PTC及其他类型甲状腺病变中未检测到BRAFV599E的突变.统计学分析BRAF突变与性别、年龄、伴慢性淋巴细胞浸润及淋巴结转移无明显关系(P>0.05).(2)PTC中RET/PTC检出率21.2%(14/66),其中5例RET/PTC1阳性(7.6%),9例RET/PTC3阳性(13.6%).RET/PTC融合基因阳性的14例PTC中未检测到BRAFV599E突变.其余29例良恶性病例中未检测到RET/PTC融合基因.RET/PTC融合基因的表达与PTC的临床病理学特征无明显关系(P>0.05).结论 (1)BRAFV599E突变和RET/PTC融合基因是PTC较特征性的遗传学改变,可作为PTC诊断和鉴别诊断提供分子学的依据,BRAFV599E突变可能是甲状腺乳头状癌表型的重要决定因素之一;(2)BRAFV599E突变与PTC的经典型和高细胞型两种主要亚型密切相关;(3)BRAFV599E突变与RET/PTC融合基因可能在PTC中是独立事件.     

mRNA BRAF expression helps to identify papillary thyroid carcinomas in thyroid nodules independently of the presence of BRAFV600E mutation

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.     

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.     

LIMD2 Is Overexpressed in BRAF V600E-Positive Papillary Thyroid Carcinomas and Matched Lymph Node Metastases

We previously described that LIM domain containing 2 (LIMD2) overexpression was closely correlated with metastatic process in papillary thyroid carcinoma (PTC). We here evaluated the expression of LIMD2 in a series of non-metastatic and metastatic PTC and their matched lymph node metastases via immunohistochemistry. LIMD2 was expressed in 74 (81%) of primary PTC and 35 (95%) of lymph node metastases. Sub-analysis performed in 37 matched samples demonstrated that in four cases, LIMD2 is expressed in lymph node metastases, while it is not expressed in primary tumors. Moreover, in eight cases, the staining intensity of LIMD2 was stronger in the patient-matched lymph node metastases than in the primary tumors. Next, the expression of LIMD2 was correlated with clinical pathological parameters and BRAF V600E and RET/PTC mutational status. The expression of LIMD2 in primary tumors was correlated with the presence of BRAF V600E mutation (P =?0.0338). Western blot analysis in thyroid cell lines demonstrated that LIMD2 is expressed in two PTC cell lines, while it is not expressed in normal thyroid and follicular thyroid carcinoma cell lines. Importantly, its expression was higher in a PTC cell line that harbors BRAF V600E mutation than in a PTC cell line that harbors RET/PTC1. The available genomic profiling data generated by The Cancer Genome Atlas Research Network confirmed that LIMD2 expression is higher in BRAF-like PTC samples. Our data suggest that LIMD2 may play an important role in the metastatic process of PTC, predominantly in BRAF V600E-positive tumors.     

Immunohistochemical analysis using a BRAF V600E mutation specific antibody is highly sensitive and specific for the diagnosis of hairy cell leukemia

Hairy cell leukemia (HCL) is usually diagnosed by morphology and flow cytometry studies. However, it is challenging sometimes to distinguish HCL from its mimics. Recently, the BRAF V600E mutation has been described as a disease-defining molecular marker for HCL which is present in nearly all cases of HCL but virtually absent in mimics of HCL. In this study, we investigated the possibility of using immunohistochemical detection of the BRAF V600E mutant protein to differentiate HCL from its mimics. A total of twenty-eight FFPE tissue specimens were studied, including HCL (n=12), HCL variant (HCL-v, n=3), splenic marginal zone lymphoma (SMZL, n=6), and other marginal zone lymphomas (MZL, n=7). Immunohistochemical studies were performed using a mouse monoclonal antibody (clone VE1, Spring Bioscience, CA) specific for BRAF V600E mutation. Molecularly confirmed BRAF V600E mutation positive and negative cases were used as the positive and negative controls respectively. All 12 cases of HCL showed cytoplasmic BRAF V600E protein expression in leukemia cells by immunohistochemical study regardless of tumor burden, whereas all cases of HCL mimics including HCL-v, SMZL, and MZL were negative for BRAF V600E protein. Using this BRAF V600E mutation specific antibody, this immunohistochemical study has 100% sensitivity and 100% specificity for the diagnosis of HCL in our cohort. In conclusion, immunohistochemical detection of the BRAF V600E mutant protein is highly sensitive and specific for the diagnosis of HCL. Compared to PCR or sequencing-based methodologies, immunohistochemistry is a relatively rapid and inexpensive alternative for the differential diagnosis between HCL and its mimics.     

Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis

Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.     

Immunohistochemical detection of the BRAF V600E mutation in melanoma patients with monoclonal antibody VE1

A novel mutation‐specific monoclonal antibody VE1 was generated to detect BRAF V600E mutation with immunohistochemistry. This study aims to investigate the sensitivity and specificity of immunohistochemistry compared with conventional Sanger sequencing and to evaluate whether IHC would become the routine screening method of BRAF V600E mutation. A total of 84 cases of melanoma lesion specimens were selected to make the tissue microarray and to perform IHC with VE1 antibody. Simultaneously Sanger sequencing was applied to test and verify. VE1 has a high specificity (100%) and sensitivity (72.2%), and the concordance between the two techniques is excellent (93.8% cases coherent and kappa = 0.801). As a rapid, cost‐effective method, IHC may become the routine diagnostic means for the detection of BRAF V600E mutation of malignant melanomas in the near future, and the recommended detection process is initial immunohistochemical staining for positive cases, followed by molecular techniques for negative or ambiguous cases.     

Mutant BRAF induces DNA strand breaks, activates DNA damage response pathway, and up-regulates glucose transporter-1 in nontransformed epithelial cells

Although the oncogenic functions of activating BRAF mutations have been clearly demonstrated in human cancer, their roles in nontransformed epithelial cells remain largely unclear. Investigating the cellular response to the expression of mutant BRAF in nontransformed epithelial cells is fundamental to the understanding of the roles of BRAF in cancer pathogenesis. In this study, we used two nontransformed cyst108 and RK3E epithelial cell lines as models in which to compare the phenotypes of cells expressing BRAF(WT) and BRAF(V600E). We found that transfection of the BRAF(V600E), but not the BRAF(WT), expression vector suppressed cellular proliferation and induced apoptosis in both cell types. BRAF(V600E) generated reactive oxygen species, induced DNA double-strand breaks, and caused subsequent DNA damage response as evidenced by an increased number of pCHK2 and γH2AX nuclear foci as well as the up-regulation of pCHK2, p53, and p21. Because BRAF and KRAS (alias Ki-ras) mutations have been correlated with GLUT1 up-regulation, which encodes glucose transporter-1, we demonstrated here that expression of BRAF(V600E), but not BRAF(WT), was sufficient to up-regulate GLUT1. Taken together, our findings provide new insights into mutant BRAF-induced oncogenic stress that is manifested by DNA damage and growth arrest by activating the pCHK2-p53-p21 pathway in nontransformed cells, while it also confers tumor-promoting phenotypes such as the up-regulation of GLUT1 that contributes to enhanced glucose metabolism that characterizes tumor cells.     

Different clonal origin of bilateral papillary thyroid carcinoma, with a review of the literature

Papillary thyroid carcinoma (PTC) often presents with two or more anatomically separate foci. A long-standing argument is whether this multifocality is the result of multiple independent tumors (“multicentricity”) or of intrathyroidal spread originating from a single tumor mass, presumably through permeation of intrathyroidal lymph vessels. We reexamined this issue with a clonality assay and compared our results with those in the literature. A total of 27 nodules from 11 female patients with bilateral PTC treated with total thyroidectomy were investigated for clonality using the HUMARA assay. Eight of 11 cases were informative (72.7 %). All but one of tumor foci showed a monoclonal population. The outlier sample gave a value indicative of balanced X-inactivation in one nodule. The monoclonality was concordant in three patients, discordant in three, and mixed in two (with both concordant and discordant results). Interestingly, in both of the latter cases (composed of over two samples per case), the contralateral nodules were discordant. Moreover, all four ipsilateral nodules were concordant. The results of our study suggest that some cases of multifocal PTC are the result of true multicentricity, whereas others are the consequence of intrathyroid spread by an originally single tumor mass. These conclusions support those made in the past years on the basis of morphologic considerations. Specifically, the incidental finding of two or more microscopic foci of PTC widely separate from each other was felt to favor multicentricity, whereas the finding of multiple ipsilateral foci of PTC within vascular spaces, often accompanied by multiple lymph node metastases, suggested intrathyroid spread; the most striking manifestation of this phenomenon being seen in the diffuse sclerosing variant of PTC.