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.     

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。     

原癌基因RET与甲状腺乳头状癌关系的研究进展

许多肿瘤有其特异性的基因改变。RET是与甲状腺乳头状癌(papillary thyroid carcinoma)有着特殊关系的原癌基因。RET／PTC重排和甲状腺滤泡性癌中PAX8．PPAR7的重排,以及发生于乳头状肾细胞癌的基因重排,是迄今在人类上皮性癌中所确定的为数不多的融合基因。现已证实在甲状腺乳头状癌中存在着RET原癌基因的多种重排形式。     

甲状腺乳头状癌RET、CK19、TG、Ki-67的表达

目的 研究甲状腺乳头状癌RET、CK19、TG、Ki-67蛋白表达特点及其临床意义。方法 应用免疫组织化学SP法检测RET、CK19、TG、Ki-67蛋白在30例甲状腺乳头状癌、10例结节性甲状腺肿和18例癌旁正常甲状腺中的表达。结果 RET、CK19在乳头状癌的阳性率（66．7％、83．3％）明显高于结节性甲状腺肿和正常甲状腺阳性率（7．1％、25．0％）,两者差异有显著性（P〈0．01）。乳头状癌组及良性病例组TG表达阳性率差异无显著性（P〉0．05）。96．7％的乳头状癌Ki-67阳性细胞数小于10％。结论 RET及CK19在甲状腺乳头状癌表达增加,具有一定的病理诊断价值。     

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(...     

RET原癌基因与遗传性甲状腺样癌关系的研究进展

RET原癌基因的突变会引起MEN2A，MEN2B和FMTC。甲状腺髓样癌（MTC）是这三种遗传性综合征共同的临床特征。在MEN2A，MEN2GB和FMTC中，遗传性的RET突变仅通过点突变即可导致甲状腺髓样的发生。目前，从基因的角度来研究MTC的最新进展已使我们提高了诊断MTC的能力，促进了家族检测计划的发展。本文将主要就RET基因突变在遗传性和散发性MTC中所起的作用以及在患有遗传性MTC家族中确认RET突变的方法及临床意义作一综述。     

RET基因融合阳性非小细胞肺癌患者的临床病理学特征

目的探讨RET基因融合阳性非小细胞肺癌患者的临床病理学特征及治疗与预后。方法选取2018年8月至2020年4月江南大学附属医院存档的非小细胞肺癌标本1 089例, 采用多种基因融合检测试剂盒(荧光PCR法)检测1 089例石蜡包埋组织标本中RET、表皮生长因子受体、间变性淋巴瘤激酶、ROS1、KRAS、BRAF和HER2等基因状态。采用免疫组织化学法检测患者组织标本中PD-L1和错配修复相关蛋白的表达。分析患者RET基因融合与年龄、性别、吸烟史、分期、分化程度、病理学类型以及PD-L1、错配修复蛋白等表达的相关性。结果 1 089例非小细胞肺癌患者中共发现RET基因融合阳性患者22例(2.02%), 男性和女性各11例, 中位年龄63.5岁。22例RET基因融合阳性患者有20例腺癌, 其中腺泡亚型11例, 实体亚型5例, 贴壁生长型4例;另有鳞状细胞癌(非角化型)和肉瘤样癌(多形性癌)各1例。临床分期Ⅰ～Ⅱ期6例, Ⅲ～Ⅳ期16例。伴有淋巴结转移16例, 远处转移11例。RET基因融合阳性患者中有1例伴HER2突变。RET基因融合阳性肺癌患者中PD-L1的阳性比例分数≥1%的占54.5...     

RET原癌基因与遗传性甲状腺髓样癌关系的研究进展

RET原癌基因的突变会引起 MEN2 A、MEN2 B和 FMTC。甲状腺髓样癌 (MTC)是这三种遗传性综合征共同的临床特征。在 MEN2 A、MEN2 B和 FMTC中 ,遗传性的 RET突变仅通过点突变即可导致甲状腺髓样癌的发生。目前 ,从基因的角度来研究 MTC的最新进展已使我们提高了诊断 MTC的能力 ,促进了家族检测计划的发展。本文将主要就 RET基因突变在遗传性和散发性 MTC中所起的作用以及在患有遗传性 MTC家族中确认 RET突变的方法及临床意义作一综述     

Twist基因蛋白表达在甲状腺乳头状癌诊断与鉴别诊断中的意义

目的探讨Twist基因蛋白在甲状腺乳头状癌中的表达状况,寻找更特异的标记物,用于甲状腺乳头状癌（PTC）与滤泡状腺瘤（FA）和良性乳头病变（BPL）的鉴别诊断。方法以50例PTC为研究组,以48例FA和47例BPL作对照组。在自制组织芯片上行免疫组织化学（SP法）标记,检测Twist基因蛋白和HBME-1,并以CK19进行对照。结果3种指标（Twist、HBME-1、CK19）阳性表达率：在PTC组分别为100％（48／48）、94．0％（47／50）和78．0％（39／50）;在FA组分别为0、6．7％（3／45）和0;在BPL组分别为7．0％（3／43）、2．1％（1／47）和0。PTC组分别与FA和BPL组比较差异均有统计学意义（P=0．000）。3种指标在鉴别诊断甲状腺良、恶性病变的灵敏度分别为：100％、96．4％和97．7％,特异度分别为：94．0％、95．7％和95．1％,准确度分别为：78．0％、100％和91．9％。结论Twist可应用于辅助诊断PTC,并可应用在对FA或BPL的鉴别诊断。     

RET/RAS/BRAF基因驱动甲状腺乳头状癌共表达关键基因的生物信息学分析

目的应用生物信息学方法分析RET/RAS/BRAF基因驱动甲状腺乳头状癌(papillary thyroid carcinoma, PTC)的共表达关键基因。方法从GEO下载数据集GSE58545,利用在线分析GEO2R工具筛选差异表达基因,通过韦恩图交集寻找共表达基因,应用GO、KEGG、Matascape等数据库分析共表达基因的功能及通路,应用Cytoscape软件和STRING网络明确关键基因,应用Oncomine数据库分析关键基因的表达,应用Ualcan数据库验证TCGA数据库中关键基因在PTC中的表达趋势及临床病理联系,在HPA数据库确认关键基因编码蛋白在PTC中的表达。结果 KEGG、Canonical、Reactome通路分析筛选出来的34个RET/RAS/BRAF基因驱动PTC的共表达基因,分别与转录失调、p53蛋白调控免疫球蛋白死亡结构域、MET信号转导事件有关,从中挖掘出10个关键基因:MET、DPP4、ENTPD1、MUC1、SPINT1、BHLHE40、CLDN1、GALNT7、TGFA、TYMS,进一步由Oncomine和Ualcan数据库的临床病理资料证实关键基因mRNA的表达均显著高于正常组织(P0.01),除了ENTPD1、TGFA、TYMS基因,其他7个基因mRNA的表达均与PTC淋巴结转移相关(P0.01),目前尚未见ENTPD1、BHLHE40、GALNT7和TYMS这4个分子在PTC中的研究报道。结论该实验利用生物信息学方法筛出的关键基因,丰富了RET/RAS/BRAF基因驱动PTC中发挥作用的关联基因成员,为PTC的精准危险度分层拓宽有关基因变异及其分子机制探索,它们有望成为放射性碘难治性或手术不能切除PTC患者潜在的分子标志物及靶点。     

Overexpression of wild-type c-RET and zero prevalence of RET/PTC rearrangements are associated with papillary thyroid cancer (PTC) in Kuwait

Background

Papillary thyroid carcinoma (PTC) is common in Kuwait. The activation of the RET oncogene by DNA rearrangement (RET/PTC) is known to have an important role in PTC carcinogenesis. However, the real frequency of the RET/PTC expression in PTC is variable between different studies. This study seeks to determine the prevalence of RET/PTC and to analyze the RET oncogene expression associated with PTC in Kuwait.

Methods

RET expression and DNA rearrangements (RET/PTC 1, RET/PTC 2 and RET/PTC 3) were studied by RT–PCR in different thyroid diseases. Results were confirmed by the Southern blot and by immunohistochemistry. Quantitative real-time PCR was used to determine the level of RET mRNA expression in PTCs.

Results

Wild-type (nonrearranged) c-RET oncogene was overexpressed in 60% of PTC cases and absent in follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC), follicular adenomas (FA) or normal thyroid. No RET/PTC rearrangement was detected in any sample. The c-RET expression in Hashimoto's thyroiditis and multinodular goiter was limited to follicular cells with PTC-like nuclear changes.

Conclusions

The overexpression of wild-type c-RET is a characteristic molecular event of PTCs in Kuwait. The prevalence of RET/PTC is zero and among the lowest recorded in the world.     

Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma

RET/PTC1 and RET/PTC3 are the markers for papillary thyroid carcinoma. Their reported prevalence varies broadly. Nonrearranged c-RET has also been detected in a variable proportion of papillary carcinomas. The published data suggest that a wide range in expression levels may contribute to the different frequency of c-RET and, particularly, of RET/PTC detection. However, quantitative expression analysis has never been systematically carried out. We have analyzed by real-time RT-PCR 25 papillary carcinoma and 12 normal thyroid samples for RET/PTC1, RET/PTC3 and for RET exons 10-11 and 12-13, which are adjacent to the rearrangement site. The variability in mRNA levels was marked and four carcinoma groups were identified: one lacking RET/PTC rearrangement with balanced RET exon levels similar to those of the normal samples (7/25 cases, 28%), the second (6/25 cases, 24%) with balanced RET expression and very low levels of RET/PTC1, the third with unbalanced RET exons 10-11 and 12-13 expression, high RET/PTC1 levels but no RET/PTC3 (7/25 cases, 28%), and the fourth with unbalanced RET expression, high RET/PTC1 levels and low levels of RET/PTC3 (5/25 cases, 20%). Papillary carcinomas with high RET/PTC1 expression showed an association trend for large tumor size (P=0.063). Our results indicate that the variability in c-RET and RET/PTC mRNA levels contributes to the apparent inconsistencies in their reported detection rates and should be taken into account not only for diagnostic purposes but also to better understand the role of c-RET activation in thyroid tumorigenesis.     

RET/PTC and CK19 expression in papillary thyroid carcinoma and its clinicopathologic correlation

Recently, the rearrangement of RET proto-oncogene has been reported to be the most common genetic change in papillary thyroid carcinoma (PTC). However, its prevalence has been reported variably and its relation to clinical outcome has been controversial. The characteristic nuclear features of PTC usually render the diagnosis, but problem arises with equivocal cytologic features that are present focally. Although there remains some controversy, CK19 has been reported to be a useful ancillary tool for diagnosis of PTC. To evaluate the expression rate of RET/PTC rearrangement and CK19 in PTCs in a Korean population, we studied 115 papillary thyroid carcinomas in 3 mm-core tissue microarray based immunohistochemical analysis. The prevalence of Ret protein expression was 62.6% and the CK19 immunoreactivity was 80.9%. There was no statistically significant association between the Ret positivity and CK19 immunoreactivity, although the percent agreement of the two was relatively high. The clinicopathological variables did not correlate with the expression of Ret. In conclusion, the prevalence of Ret protein expression and its clinicopathological implications in a Korean population are not much different from those reported in previous studies. However, its detection via immunohistochemistry can be a useful diagnostic tool for diagnosing papillary thyroid carcinoma in conjunction with CK19.     

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.     

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.