甲状腺乳头状癌BRAF V600E、TERT和RET分子检测及与临床病理特征的关系

目的 探讨甲状腺乳头状癌(papillary thyroid carcinoma, PTC)中三种驱动基因BRAF V600E、TERT和RET与临床病理特征的关系。方法 收集4 678例PTC,2 637例患者行BRAF V600E和TERT启动子检测(C250T和C228T位点),另2 041例患者行BRAF V600E和RET检测，并分析以上3种基因与临床病理特征的关系。结果 PTC中BRAF V600E、TERT及RET基因阳性检出率分别为88.9%(4 161/4 678)、0.5%(12/2 637)、3.7%(75/2 041)。BRAF V600E突变仅与被膜侵犯及桥本甲状腺炎伴随相关(P<0.05);TERT启动子突变与患者年龄、肿瘤最大径、肿瘤数量、淋巴结转移、TNM分期均有关(P<0.05);RET基因融合与患者性别、肿瘤最大径、淋巴结转移、桥本甲状腺炎伴随有关(P<0.05)。双基因改变患者合计12例，包括1例BRAF V600E突变伴CCDC6-RET(Exon1-Exon12)基因重排变异，1例BRAF V600E突变伴NCOA4-RET(...     

甲状腺乳头状癌BRAFV600E基因突变对VEGF和MMP-9表达的影响

目的探讨甲状腺乳头状癌(PTC) BRAFV600E基因突变与多种分子标志物表达及肿瘤临床病理特征的关系。探讨该突变是否为预后不良指标。方法用实时荧光定量PCR检测标本BRAFV600EmRNA;用免疫组化PV法检测标本galectin-3、cyclin D1、VEGF和MMP-9蛋白表达。结果 104例PTC中的71例(68. 3%) BRAFV600E基因突变(+);突变组的肿瘤直径增大、肿瘤T分期较高、更易侵犯甲状腺被膜外及淋巴结转移;且VEGF、MMP-9蛋白表达阳性率升高(P0. 05)。二元Logist回归分析显示BRAFV600E基因突变(+)的PTC VEGF蛋白高表达,肿瘤直径增大、更易侵犯甲状腺被膜外组织且肿瘤T分期较高(P0. 05)。结论 BRAFV600E基因突变的PTC具有更高的侵袭性和转移潜能。     

新疆地区甲状腺乳头状癌中BRAF V600E基因的表达及意义

目的探讨新疆地区BRAF V600E基因点突变在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)中的表达,及其与临床病理特征的关系。方法收集原发性PTC 178例,石蜡包埋组织经QIAGEN试剂盒提取DNA,经Taq Man探针荧光定量PCR技术检测PTC中BRAF基因点突变。结果 178例原发性PTC中BRAF V600E基因点突变142例,突变率为79.8%(142/178)。患者年龄(≥45岁)、肿瘤直径(≤1 cm)、被膜外侵犯BRAF V600E基因点突变率高(P均0.05),而BRAF V600E基因点突变与患者性别、多灶性及淋巴结转移灶和肿瘤部位均无相关性(P均0.05)。结论新疆地区PTC中BRAF V600E基因点突变与临床病理特征密切相关,且具有一定特异性,可作为PTC诊断提供依据。     

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。     

中国人甲状腺乳头状癌中RET/PTC融合基因DNA序列特征的初步研究

目的应用长距离PER（long-distance PER，LD—PER），从甲状腺乳头状癌（papillary thyroid carcinoma，FTC）的基因组DNA中扩增RET／PTC1、3融合内含子，并分析融合点附近的DNA序列特征，探讨中国成人散发性PTC中RET／PTC融合基因的发生机理。方法20例新鲜FTC标本，用逆转录-PER方法检测RET／PTC基因的mRNA表达，确定阳性病例及融合基因类型后利用长距离PER扩增RET／PTC1和RET／PTC3的DNA片段。结果20例PTC新鲜组织中检测到RET／PTC1和RET／PTC3阳性病例各1例：（1）RET／PTC1融合基因的DNA片段大小为3091bp，融合点位于H4基因的第1内含子和RET基因的第11内含子。融合点呈端端吻合，无缺失、插入及重复序列等改变。（2）RET／PTC3（ELE1-RET）和交互性融合（reciprocal fusion）后形成的相应DNA片段（RET-ELE1），大小分别为2119bp和1568bp。RET／PTC3的融合点位于ELE1基因第5内含子的Alu序列内，在ELE1基因融合点上游有polyA序列。ELE1-RET的融合点的两个碱基（gg）无法判断来源于RET或ELE1基因。结合RET／PTC3与RET-ELE1，在融合点。ELE1基因有2个碱基（aa）缺失，RET基因有5个碱基（gttcc）缺失。结论Alu序列可能参与了RET／PTC3融合基因的形成。对于研究FTC中RET／PTC融合点附近的DNA序列特征及融合形成的机制，长距离PER是一种可行性比较高的研究方法。     

野生型RET和RET/PTC融合基因在成人散发性甲状腺乳头状癌中的表达及其意义

目的探讨野生型BET（WT-RET）及RET／PTC1、3融合基因在成人散发性甲状腺乳头状癌（PTC）中的表达及其与临床病理学指标的关系和意义。方法用逆转录-聚合酶链反应（RT-PCR）检测102例石蜡与新鲜（43例）甲状腺病变组织（PTC66例,对照组各种良恶性肿瘤及良性病变共36例）中WT-RET和RET／PTC1、3融合基因的表达并结合临床资料进行分析。结果（1）62％（41／66）PTC患者≥40岁。38％（25／66）PTC伴淋巴细胞性甲状腺炎,59％（39／66）伴淋巴结转移,5例（7.6％）有远处转移。（2）RET原癌基因的酪氨酸激酶区（BET-TK）检出率为68．1％（45／66）。BET原癌基因断裂点（BP）与TK的同时检出率在PTC中28．8％（19／66）,腺瘤中12．5％（1／8）,表明存在WT-BET转录物。（3）RET／PTC检出率21．2％（14／66）,其中5例BET／PTC1阳性（7．6％）,9例RET／PTC3阳性（13．6％）。6例（9％）PTC同时表达BET／PTC和WT-BET。36例对照组病例中未检测到RET／PTC融合基因。（4）统计学分析,PTC病例中WT-BET与RET／PTC1融合基因的表达与性别、年龄、肿瘤大小、多灶性、伴淋巴细胞浸润及淋巴结转移等临床病理学指标无关（P〉0．05）。结论RET／PTC融合基因在散发性成人PTC中表达率低,其诊断和判断预后的价值不大。WT-BET在甲状腺肿瘤的滤泡形成过程中起一定作用。     

甲状腺癌中BARF基因突变和RET融合基因的分子病理检测分析

目的：探讨甲状腺癌中BRAF基因突变和RET融合基因各亚型情况。方法：应用Taqman-ARMS方法检测106例甲状腺癌中BRAF基因突变和RET融合基因情况。结果：甲状腺癌中BRAF基因总突变率为66.98%(71/106),均为V600E突变,BRAF基因突变在年龄(<49岁)(47/61,77.05%)、被膜无浸润或转移(40/56,71.43%)和临床分期Ⅰ或Ⅱ(37/52,71.15%)中较高(P<0.05);RET融合基因总阳性率为4.72%(5/106),RET融合基因与性别、年龄、肿瘤大小、被膜是否浸润及转移和临床分期无关(P>0.05)。结论：甲状腺癌患者中BRAF基因存在较高的突变率,低龄、被膜未浸润或转移及Ⅰ或Ⅱ期甲状腺癌多见,RET融合基因中融合伙伴CCDC6-或NCOA4-较为常见。     

甲状腺新鲜标本直接涂片法BRAFV600E基因检测的应用及临床意义

<正>甲状腺乳头状癌(thyroid papillary carcinoma,PTC)是最常见的甲状腺恶性肿瘤,占80%～85%[1]。近年来,其发病率逐年递增,甚至部分地区10年增长了近10倍,成为发病率增长最快的恶性肿瘤[2]。BRAFV600E基因突变是PTC中最常见的突变类型,也是最具特征性的肿瘤生物学标记[3]。研     

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。     

甲状腺乳头状癌中NTRK基因融合变异的检测比较

目的探讨甲状腺乳头状癌(papillary thyroid carcinoma, PTC)中NTRK基因变异频次, 分析免疫组织化学法检测TRK蛋白预测NTRK基因融合变异的可行性。方法收集2017年6月至2020年6月深圳市人民医院病理科存档的848例PTC组织样本, 其中男性242例, 女性606例, 患者年龄范围9～83岁。利用免疫组织化学检测848例PTC组织中TRK的表达情况;利用基于DNA的下一代测序检测150例PTC中NTRK融合变异。结果免疫组织化学检测TRK阳性病例120例, 下一代测序检测发现NTRK融合病例13例, PTC中NTRK融合频次为1.5%(13/848)。免疫组织化学检测PTC中NTRK融合的灵敏度和特异度分别为100%和21.9%, 免疫组织化学检测弱阳性、中等阳性和强阳性的特异度分别为23.8%、76.9%、93.8%;预测NTRK基因融合的特异度随免疫组织化学染色强度增加而增加;在BRAF V600E阴性的样本中, 免疫组织化学检测TRK弱阳性和中等阳性的特异度提升至62.5%和96.8%。下一代测序检测出EML4、ETV6、CDH1、GJD2、...     

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.     

Alterations of the BRAF gene in thyroid tumors

BRAF belongs to the RAF family of protein kinases that are important components of the MAPK signaling pathway mediating cell growth, differentiation and survival. Activating point mutation of the BRAF gene resulting in V600E (previously designated as V599E) is a common event in thyroid papillary carcinoma, being found in approx 40% of this tumor. It has strong association with classical papillary carcinoma and tall cell and possibly Warthin-like variants. This mutation also occurs in thyroid poorly differentiated and anaplastic carcinomas, usually those containing areas of papillary carcinoma. Alterations in the BRAF gene do not overlap with RAS mutations and RET/PTC rearrangement, indicating that activation of one of the effectors of the MAPK pathway is sufficient for papillary thyroid carcinogenesis. Recently, another mechanism of BRAF activation has been identified, which involves chromosome 7q inversion that results in the AKAP9-BRAF fusion. It is rare in sporadic papillary carcinomas and is more common in tumors associated with radiation exposure. Yet another mechanism of BRAF activation may involve copy number gain, which is seen in a significant portion of thyroid follicular tumors of both conventional and oncocytic (Hürthle cell) types.     

Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma.

The tall-cell variant (TCV) of papillary thyroid carcinoma (PTC), characterized by tall cells bearing an oxyphilic cytoplasm, is more clinically aggressive than conventional PTC. RET tyrosine kinase rearrangements, which represent the most frequent genetic alteration in PTC, lead to the recombination of RET with heterologous genes to generate chimeric RET/PTC oncogenes. RET/PTC1 and RET/PTC3 are the most prevalent variants. We have found RET rearrangements in 35.8% of TCV (14 of 39 cases). Whereas the prevalences of RET/PTC1 and RET/PTC3 were almost equal in classic and follicular PTC, all of the TCV-positive cases expressed the RET/PTC3 rearrangement. These findings prompted us to compare RET/PTC3 and RET/PTC1 in an in vitro thyroid model system. We have expressed the two oncogenes in PC Cl 3 rat thyroid epithelial cells and found that RET/PTC3 is endowed with a strikingly more potent mitogenic effect than RET/PTC1. Mechanistically, this difference correlated with an increased signaling activity of RET/PTC3. In conclusion, we postulate that the correlation between the RET/PTC rearrangement type and the aggressiveness of human PTC is related to the efficiency with which the oncogene subtype delivers mitogenic signals to thyroid cells.     

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.

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.     

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。     

Diffuse sclerosing variant of papillary thyroid carcinoma: lack of BRAF mutation but occurrence of RET/PTC rearrangements.

Diffuse sclerosing variant of papillary thyroid carcinoma (PTC) is a rare tumour with a characteristic morphology as well as a strong preponderance for younger female patients. The T1799A missense mutation in exon 15 of the BRAF gene and RET/PTC rearrangement have been identified as the dominant genetic tumour initiation events in the pathogenesis of PTC leading to a constitutive activation of the RAS-RAF-MAPK pathway. In order to elucidate the pathogenesis of diffuse sclerosing variant of PTC, the prevalence of BRAF mutation and RET/PTC were determined by RT-polymerase chain reaction and DNA-sequence analysis in tumour samples of seven patients with this variant (all female, age range 15-61 years, mean 33.3 years) without prior radiation exposure. None of these cases showed a BRAF mutation. RET/PTC1 (two out of seven) and RET/PTC3 (one out of seven), which have been shown in large PTC series to comprise together more than 90% of RET/PTC types, were found in <50% of the cases investigated. All seven samples expressed the RET tyrosine kinase domain but lacked its extracellular domain potentially suggesting the existence of rare types of RET/PTC rearrangement in the four remained cases of diffuse sclerosing variant of PTC. Regarding this subtype, our study confirmed the paradigm of a mutual exclusivity between RET/PTC and BRAF in PTC. Additionally, this rare variant of papillary thyroid carcinoma may represent a tumour type susceptible to RET-targeted therapies.