BRAF基因V600E突变在甲状腺病变中的表达

目的 分析BRAF基因V600E突变在中国汉族人群甲状腺病变中的表达情况.方法 240例甲状腺病变中,乳头状癌(PTC)129例(其中经典型PTC 121例,滤泡型PTC 8例).滤泡癌12例,髓样癌4例,甲状腺隙瘤30例,结节性甲状腺肿30例,Lp状腺乳头状增生35例.采用常规酚.氯仿法抽提87例新鲜组织样本的总DNA,改良试剂盒法抽提153例石蜡组织样本的总DNA,经过PCR、测序,检测BRAF V600E突变.结果 在240例甲状腺病变中,BRAF V6OOE突变61例,发生率为25.4%.61例BRAF V600E突,变患者均为PTC,占PTC的47.3%.BRAF V600E突变在经典型PTC中的表达率(49.6%)与在滤泡型PTC中的表达率(12.5%)比较,差异有统计学意义(P<0.05).BRAF V600E突变与PTC患者临床病理参数间的差异无统计学意义(P>0.05).结论 BRAFV600E突变与PTC的发生、发展可能有着重要联系,BRAF V600E突变可以作为PTC诊断的特异性标记;改良的试剂盒法抽提石蜡组织DNA具有高效、简便、价格栩对低廉的优点.     

BRAF基因V600E突变在甲状腺病变中的表达

目的 分析BRAF基因V600E突变在中国汉族人群甲状腺病变中的表达情况.方法 240例甲状腺病变中,乳头状癌(PTC)129例(其中经典型PTC 121例,滤泡型PTC 8例).滤泡癌12例,髓样癌4例,甲状腺隙瘤30例,结节性甲状腺肿30例,Lp状腺乳头状增生35例.采用常规酚.氯仿法抽提87例新鲜组织样本的总DNA,改良试剂盒法抽提153例石蜡组织样本的总DNA,经过PCR、测序,检测BRAF V600E突变.结果 在240例甲状腺病变中,BRAF V6OOE突变61例,发生率为25.4%.61例BRAF V600E突,变患者均为PTC,占PTC的47.3%.BRAF V600E突变在经典型PTC中的表达率(49.6%)与在滤泡型PTC中的表达率(12.5%)比较,差异有统计学意义(P<0.05).BRAF V600E突变与PTC患者临床病理参数间的差异无统计学意义(P>0.05).结论 BRAFV600E突变与PTC的发生、发展可能有着重要联系,BRAF V600E突变可以作为PTC诊断的特异性标记;改良的试剂盒法抽提石蜡组织DNA具有高效、简便、价格栩对低廉的优点.

Abstract：

Objective To evaluate the expression of BRAF V600E mutation in 240 Chinese patients with thyroid lesions. Methods Two hundred and forty Chinese patients with thyroid lesions, including 129 papillary thyroid carcinomas (PTC) , 12 follicular carcinomas, 4 medullary carcinomas, 30 adenomas, 30 nodular goiters, and 35 papillary hyperplasia. DNA was extracted from thyroid biopsy and paraffin embedded thyroid tissues, and the expression of BRAF V600E mutation was detected by polymerase chain reaction and DNA sequencing assays. Results The presence of BRAF V600E mutation was found in 61 of the total group of 240 cases (25. 4%). It was only detected in PTC (47. 3%), and not detected in other types of malignant and benign thyroid lesions. There was a statistically significant difference between the expression of BRAF V600E mutation in classic type PTC (49. 6% ) and in follicular type PTC (12. 5% ,P <0. 05) , but statistical data did not show any correlation between BRAF V600E mutation and clinicopathologic parameters in PTC (P > 0. 05). Conclusions BRAF V600E mutation has a significant correlation with PTC and the detection of BRAF V600E mutation may be used as an important prognostic marker of PTC. Our new method of DNA extraction from paraffin embedded tissues is efficient and inexpensive.     

甲状腺乳头状癌BRAF V600E基因突变 及相关蛋白的表达

背景与目的:BRAF V600E基因突变可作为甲状腺乳头状癌靶向治疗的靶点,因此检测患者BRAF基因状态对于能否应用靶向药物治疗具有重要意义.观察BRAF V600E基因突变及突变蛋白VE1在甲状腺乳头状癌中的表达情况,并分析其与甲状腺乳头状癌的临床病理特征及其预后的关系.方法:采用DNA测序法及免疫组织化学法分别检测108例甲状腺乳头状癌、54例甲状腺腺瘤和54例结节性甲状腺肿标本中BRAF基因突变及其相关蛋白VE1的表达.结果:108例甲状腺乳头状癌基因突变率为67.6%,VE1表达率为64.8%,与甲状腺良性病变相比差异有统计学意义(P<0.05),与临床病理参数间无相关性.结论:甲状腺乳头状癌BRAF V600E基因突变率和BRAF V600E蛋白表达水平增高,可以作为鉴别甲状腺良、恶性肿瘤的有效指标.免疫组织化学法检测甲状腺乳头状癌BRAF V600E蛋白的表达与其基因突变的一致性高,可间接有效地反映BRAF V600E基因突变的状态.BRAF V600E基因突变及突变蛋白的表达与甲状腺乳头状癌患者预后无关.     

甲状腺乳头状癌BRAF基因突变及表达的临床意义研究

目的探讨BRAF基因点突变及B-raf蛋白表达在甲状腺乳头状癌发生中的临床意义。方法应用聚合酶链式反应(PCR)技术检测65例甲状腺病变石蜡组织中BRAF点突变,应用免疫组化方法检测112例甲状腺病变组织中B-raf蛋白的表达情况,并比较BRAF基因突变和B-raf蛋白表达的相关性。结果在46例甲状腺乳头状癌中有21例发生BRAF的点突变,突变率为45.7。BRAF基因突变位于第15外显子的1799位点,胸腺嘧啶突变为腺嘌呤(T1799A)。在结节性甲状腺肿和滤泡状癌中未检测到BRAF的突变。乳头状癌BRAF基因突变率与结节性甲状腺肿比较,差异具有统计学意义(P<0.05)。但是与患者的性别、年龄、组织学类型、淋巴结转移和肿瘤分期无相关性(P>0.05)。在乳头状癌、滤泡状癌和结节性甲状腺肿中B-raf蛋白表达阳性率分别为65.1、47.6和15.4。结果显示,乳头状癌B-raf蛋白阳性表达率与良性病变比较,差异具有统计学意义(P<0.05)。在乳头状癌中BRAF基因突变与B-raf蛋白表达水平呈正相关(P<0.05)。乳头状癌与滤泡状癌比较,B-raf蛋白表达水平两组间无统计学意义(P>0.05)。结论甲状腺乳头状癌BRAF基因突变率和蛋白表达水平的增高,提示BRAF基因在乳头状癌发病中可能发挥重要的作用。对甲状腺肿瘤的病理诊断也具有辅助价值。     

甲状腺结节穿刺标本中BRAF V600E突变检测的临床意义

背景与目的：在甲状腺乳头状癌（papillary thyroid carcinoma，PTC）中，BRAF V600E突变是迄今报道最多的基因突变。检测甲状腺穿刺细胞中的BRAF V600E突变有助于提高细针抽吸细胞学检查（fine-needle aspiration cytology，FNAC）诊断的准确性。本研究对甲状腺穿刺细胞液进行BRAF V600E突变检测，与术后组织病理学诊断结果进行比较，评估BRAF V600E突变的术前诊断价值。方法：回顾性分析2016年6月—2017年4月在复旦大学附属肿瘤医院就诊的563例甲状腺结节患者的B超引导下FNAC标本中的BRAF V600E突变结果，所有病例用QIAamp DNA Mini Kit提取DNA，并用突变特异性扩增系统（amplification refractory mutation system，ARMS）方法检测BRAF V600E突变。结果：563例患者的FNAC标本中，ARMS方法检测成功率为99.3%，男女比例为1.0∶3.7，平均年龄（45.0±0.9）岁。以组织病理学诊断作为金标准对209例接受手术治疗的患者进行诊断，细胞学诊断PTC的灵敏度为86.6%，特异度为100.0%；细胞学联合BRAF V600E诊断PTC的灵敏度为92.1%，特异度为100.0%。FNAC联合BRAF V600E检测的诊断准确率高于细胞学诊断。组织病理学诊断为PTC的患者中，有11例患者细胞学诊断未见肿瘤细胞，但BRAF V600E检测有突变，其中9例为微小PTC。结论：用ARMS方法检测FNAC标本中BRAF V600E基因突变，检测成功率高，是临床易于开展的术前辅助诊断方法。将细胞学诊断与BRAF V600E检测结果相结合，可提高PTC的检出率，提高术前诊断的准确率。     

甲状腺乳头状癌18F-FDG PET/CT显像葡萄糖代谢与BRAF突变的相关性研究

目的 评估甲状腺乳头状癌(Papillary thyroid carcinoma,PTC)患者¹⁸F-FDG PET/CT的最大标准摄取值(SUV_max)与BRAF突变的相关性。方法 回顾性分析我院在甲状腺切除术前接受¹⁸F-FDG PET/CT显像和活检的患者51例(平均年龄为49.3±12.9岁),病理诊断为PTC 48例,甲状腺滤泡状癌(Follicular thyroid carcinoma,FTC)3例。采用基因检测法检测BRAF V600E突变状态,半定量分析法测定甲状腺结节灶的SUV_max,分析患者临床资料包括性别、年龄、肿瘤大小和甲状腺球蛋白浓度与SUV_max之间的相关性;将患者分为BRAF V600E突变组和未突变组,分析比较两组间SUV_max值的差异。结果 在PTC患者中,33例患者肿瘤发生BRAF V600E突变,15例患者肿瘤未发生突变,BRAF V600E突变组的SUV_max显著高于未突变组(5.5±3.9 vs. 2.2±1.2,P=0.002);肿瘤直径≥1cm的患者的SUV_max明显高于肿瘤＜1cm(P＜0.05);甲状腺球蛋白浓度升高患者的SUV_max高于正常甲状腺球蛋白浓度的患者(P＜0.05);FTC组中未观察到BRAF V600E突变。结论 PTC患者携带BRAF V600E突变基因相对于未携带者具有更高的SUV_max值,不同肿瘤大小、血清甲状腺球蛋浓度的PTC患者SUV_max值存在统计学差异。     

BRAF V600E突变与甲状腺乳头状癌临床病理特征的相关性

目的:探讨BRAF V600E基因突变对甲状腺乳头状癌（PTC）临床病理特征的影响。方法:收集经甲状腺切除术且病理证实为PTC患者的临床资料,分析BRAF V600E基因突变与PTC临床病理特征、甲状腺功能的关系。结果:PTC患者882例,发生BRAF V600E基因突变者722例（81.86%）,单因素分析显示,突变组癌灶多发、双侧、包膜外侵犯比例、复发危险度分层中/高危及TNM分期Ⅲ/Ⅳ期明显高于未突变组,差异均有统计学意义（均P＜0.05）;而淋巴结转移率、转移数目和直径均无统计学差异;突变组TPOAb、TGAb、TSH水平低于未突变组,差异均有统计学意义（均P＜0.05）,FT4、FT3水平无统计学差异（均P＞0.05）。分别行多因素Logistic回归分析显示:BRAF V600E基因突变与癌灶多发、包膜外侵犯显著相关（OR值分别为1.722、1.436,均P＜0.05）;原发癌灶直径>1 cm、BRAF基因突变、TGAb异常升高与PTC患者包膜外侵犯呈独立正相关（OR值分别为2.862、1.619、1.532,均P＜0.05）。结论:BRAF V600E基因突变的PTC易发多灶和包膜外侵犯,提示可能有不良预后;癌灶直径>1 cm、BRAF V600E基因突变及TGAb异常升高是PTC包膜外侵犯的独立危险因素。     

ddPCR技术和Sanger测序法检测甲状腺乳头状癌患者BRAF V600E基因突变的比较分析

目的 探讨微滴式数字PCR(droplet digital PCR,ddPCR)技术在甲状腺乳头状癌(papillary thyroid cancer,PTC)患者BRAF V600E基因突变检测中的应用.方法 应用ddPCR技术和Sanger测序法同时检测病例组90例PTC患者及对照组19例甲状腺良性病变患者术后福尔...     

BRAF~(V600E)突变与甲状腺乳头状癌

BRAF基因属于RAF基因家族,其编码的蛋白在RAS-RAF-MEK-ERK信号转导调节途径中起重要作用。最近国外研究发现,BRAFV600E突变与甲状腺乳头状癌(PTC)的发生、发展密切相关,为未来PTC的诊断和治疗提供了新的突破口。现综述BRAFV600E突变与PTC的研究进展。     

BRAF V600E突变对甲状腺乳头状癌发生及预后的影响

甲状腺乳头状癌(papillary thyroid cancer,PTC)是甲状腺癌最常见的病理类型.PTC通常预后良好,但近年来甲状腺癌的发病率逐年攀升.随着患病人数的不断增多,中晚期难治性甲状腺癌患者不再少见.因此,越发庞大的甲状腺癌患者群体的管理与诊治已成为巨大的考验.BRAF V600E基因突变是乳头状癌经典DNA相关标志物,目前已被广泛应用于甲状腺癌的术前诊断和预后评估,并且作为潜在的治疗靶点受到越来越多的关注.因此,正确全面的了解BRAF V600E基因突变可以帮助我们对PTC的发生、发展及生物学行为有更进一步的了解,并为PTC患者管理方式与治疗策略提供新的方向.     

BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC

Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAF(V599E) mutation in sporadic PTC and in PTC-derived cell lines. The BRAF(V599E) mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAF(V599E) mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAF(V599E) mutation. BRAF(V599E) mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAF(V599E) mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAF(V599E) mutation is frequent in the etiopathogenesis of PTC. The BRAF(V599E) mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAF(V599E) may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.     

BRAF V600E mutation in anaplastic thyroid carcinomas and their accompanying differentiated carcinomas

Frequency of a BRAF V600E mutation in anaplastic thyroid carcinoma, which is thought to be derived mainly from papillary carcinoma by multi-step carcinogenesis, is much lower than that in papillary carcinomas. To clarify this phenomenon, we analysed BRAF V600E mutation in 20 cases of anaplastic carcinoma and 13 accompanying differentiated carcinomas. Among twenty cases of anaplastic carcinomas, nine and four accompanied papillary and follicular carcinomas, respectively. BRAF V600E mutation was found in four (20%) cases. BRAF V600E mutation was found in three of nine (33.3%), none of four and one of seven (14.3%) anaplastic carcinomas with papillary carcinoma, follicular carcinoma and without differentiated components, respectively. All three papillary carcinomas accompanied by anaplastic carcinoma with a BRAF V600E mutation were also shown to have a BRAF V600E mutation. In summary, BRAF V600E mutation was occasionally observed in anaplastic carcinomas with papillary carcinoma, and the low frequency of BRAF V600E mutation in anaplastic carcinoma was thought to be due to the low frequency of anaplastic carcinomas with papillary carcinoma. These findings raise a question about the classical model of anaplastic transformation and suggest some roles of thyroid cancer stem cells in the generation of anaplastic carcinoma.     

Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells

The activating mutation BRAF(T1796A) is the most prevalent genetic alteration in papillary thyroid carcinomas (PTC). It is associated with advanced PTCs, suggesting that this oncoprotein confers thyroid cancers with more aggressive properties. BRAF(T1796A) is also observed in thyroid micropapillary carcinomas and may thus be an early event in tumor development. To explore its biological consequences, we established doxycycline-inducible BRAF(V600E)-expressing clonal lines derived from well-differentiated rat thyroid PCCL3 cells. Expression of BRAF(V600E) did not induce growth in the absence of thyrotropin despite increasing DNA synthesis, which is likely explained because of a concomitant increase in apoptosis. Thyrotropin-dependent cell growth and DNA synthesis were reduced by BRAF(V600E) because of decreased thyrotropin responsiveness associated with inhibition of thyrotropin receptor gene expression. These results are similar to those obtained following conditional expression of RET/PTC. However, in contrast to RET/PTC, BRAF activation did not impair key activation steps distal to the thyrotropin receptor, such as forskolin-induced adenylyl cyclase activity or cyclic AMP-induced DNA synthesis. We reported previously that acute RET/PTC expression in PCCL3 cells did not induce genomic instability. By contrast, induction of BRAF(V600E) expression increased the frequency of micronuclei by both clastogenic and aneugenic events. These data indicate that BRAF(V600E) expression confers thyroid cells with little growth advantage because of concomitant activation of DNA synthesis and apoptosis. However, in contrast to RET/PTC, BRAF(V600E) may facilitate the acquisition of secondary genetic events through induction of genomic instability, which may account for its aggressive properties.     

Mutationally activated BRAF(V600E) elicits papillary thyroid cancer in the adult mouse

Mutated BRAF is detected in approximately 45% of papillary thyroid carcinomas (PTC). To model PTC, we bred mice with adult-onset, thyrocyte-specific expression of BRAF(V600E). One month following BRAF(V600E) expression, mice displayed increased thyroid size, widespread alterations in thyroid architecture, and dramatic hypothyroidism. Over 1 year, without any deliberate manipulation of tumor suppressor genes, all mice developed PTC displaying nuclear atypia and marker expression characteristic of the human disease. Pharmacologic inhibition of MEK1/2 led to decreased thyroid size, restoration of thyroid form and function, and inhibition of tumorigenesis. Mice with BRAF(V600E)-induced PTC will provide an excellent system to study thyroid tumor initiation and progression and the evaluation of inhibitors of oncogenic BRAF signaling.     

BRAF Mutations in Iranian Patients with Papillary Thyroid Carcinoma

Background: Papillary thyroid cancer or papillary thyroid carcinoma (PTC) is the most common thyroidcancer. The fact that it occasionally occurs in women aged 30-40 years old suggests that genetic alterations areinvolved its genesis. Recently, activator mutations in BRAF gene have been relatively frequently discovered.Materials and Methods: In this study, we tested 63 DNA samples from PTC patients to identify the V600Emutation frequency in the Ahvaz population. DNA was isolated from formalin fixed paraffin-embedded (FFPE)PTC tumor tissues. Genotyping was performed by PCR-RFLP and confirmed by direct DNA sequencing of asubset of PCR products. PCR-RFLP data were reported as genotype frequencies and percentages. Results: Fortynine out of 63 patients (77.8%) had a mutated heterozygote form while 14 (22.2%) showed normal genotype butnone demonstrated a mutant homozygote genotype. The frequency of V600E mutation was significantly high inPTC patients. Conclusions: These findings support involvement of V600E mutations in PTC occurrence in Iran.Assessment of correlations between BRAF V600E mutations and papillary thyroid cancer progression needs tobe performed.     

BRAF V600E mutation and p27 kip1 expression in papillary carcinomas of the thyroid <or=1 cm and their paired lymph node metastases

BACKGROUND: BRAF(V600E) mutation and p27(kip1) expression have been introduced as novel indicators that may predict prognosis in different tumors, as well as in papillary thyroid carcinomas. METHODS: Tissue samples from 214 consecutive patients who underwent total or near-total thyroidectomy with histological diagnosis of papillary thyroid carcinoma (PTC) 相似文献