肾透明细胞癌VHL基因突变与Cyclin D1过度表达的相关性

背景与目的:VHL基因突变与肾透明细胞癌发生关系密切,而CyclinD1基因通过促进细胞过度增殖,对肾透明细胞癌发生和发展起重要作用。本研究拟初步探讨肾透明细胞癌中VHL基因突变的状况及其与CyclinD1表达上升的相关性。方法:取50例肾透明细胞癌手术标本,抽提病变组织和癌周正常组织中的基因组DNA和总RNA。通过PCR扩增VHL基因各外显子序列并测序以及内切酶鉴定的方法,确定肿瘤组织中VHL基因突变的比率和具体位点。通过RT-PCR和Westernblot方法,检测相应肿瘤组织中CyclinD1的表达水平是否发生改变。结果:VHL基因序列检测结果表明,有42例(84.0%)肾透明细胞癌标本的VHL基因发生各种形式的突变,有12(24.0%)例甚至发生2种以上的基因突变。在57例VHL基因外显子突变中,1号外显子突变有17例(29.8%);2号外显子突变有26例(45.6%);3号外显子突变有14例(24.6%)。CyclinD1的表达水平在42例VHL基因发生突变的组织中均有不同程度的上升,达到正常对照组的2～10倍(3.91±1.54倍)(P<0.01),另8例表现为正常。结论:在肾透明细胞癌中存在各种VHL基因突变,导致CyclinD1过度表达。     

53例国人肾透明细胞癌中PBRM 1基因的突变分析

目的 分析Polybromo 1（PBRM 1）基因在国人肾透明细胞癌中的突变特点,探讨基因突变与临床病理特征和预后的关系。方法 选取53例肾透明细胞癌患者,利用PCR结合直接测序的方法检测PBRM 1基因2～5号外显子、15～17号外显子的突变情况。分析PBRM 1基因突变与临床病理特征和预后的相关性。结果 53例样本中检测出PBRM 1基因突变14例,突变率为26％。突变较集中分布在第15、17外显子,第15外显子5例,第17外显子6例。PBRM 1基因突变组和无突变组的中位无进展生存期分别为5个月（95％CI：1.6～8.4个月）和14个月（95％CI：9.7～18.3个月）,中位生存期分别为7个月（95％CI：2.6～11.4个月）和18个月（95％CI：5.6～30.4个月）,差异均有统计学意义（P＜0.05）。 PBRM 1基因突变与性别、年龄、发病部位、分期及疾病控制率等无关（P＞0.05）。结论 中国人肾透明细胞癌中PBRM 1基因突变率较高,突变主要位于第15、17外显子。PBRM 1基因突变可能是肾透明细胞癌患者预后不良的危险因素。     

肾细胞癌VHL基因突变及异常甲基化的临床评价

 目的 评价ＶＮＬ基因异常在肾癌发生发展中的作用及其临床意义。方法 采用聚合酶链反应（ＰＣＲ）加单链构象多态性分析（ＳＳＣＰ）及多重对照聚合酶链反应（Ｍｕｌｔｉｐｌｘ－ＰＣＲ）检测 ４２例肾细胞癌组织、１８例远离肿瘤的正常肾脏及１０例正常肾脏组织中ＶＨＬ基因的突变、异常甲基化。结果１６／２８例（５７．１％）肾透明细胞癌、ＶＳ例（５０％）混合细胞癌中存在 ＶＨＬ基因突变，６／２８例（２１．４％）肾透明细胞癌、１／８例（１２．５％）混合细胞癌中存在异常甲基化，１例远离肿瘤的正常肾脏组织发现ＶＨＬ基因异常甲基化和１例肾透明细胞癌同时存在ＶＨＬ基因突变及异常甲基化；ＶＨＬ基因失活与肾癌组织类型及临床分期相关，与病理分级无关。结论 ＶＨＬ基因的检测可作为肾透明细胞癌的诊断指标，ＶＨＬ基困可望成为肾透明细胞癌基因治疗的重要目的基因。     

KiSS-1和MMP-9在肾透明细胞癌中的表达及其临床意义

目的:探讨KiSS-1基因和基质金属蛋白酶-9(MMP-9)与肾脏透明细胞癌侵袭、转移的关系,为研究肾透明细胞癌的转移机制及治疗提供理论基础。方法:利用免疫组化方法检测45例肾透明细胞癌和15例正常肾组织中KiSS-1、MMP-9的表达水平。结果:KiSS-1基因产物metastin在正常肾组织和肾透明细胞癌中的阳性表达率分别为40.0%和31.1%,差异无显著性(P>0.05);metastin表达与肾透明细胞癌的临床分期和淋巴结转移有关(P<0.05),与患者性别、年龄、肿瘤大小、肿瘤组织学分级等临床病理因素无关(P>0.05)。MMP-9在正常肾组织和肾透明细胞癌中的表达率分别为20.0%和64.4%,差异有显著性(P<0.05);MMP-9表达与肾透明细胞癌组织学分级、肿瘤临床分期及淋巴结转移有关(P<0.05),与患者性别、年龄、肿瘤大小等临床因素无关(P>0.05)。结论:metastin表达与肾透明细胞癌的临床分期和淋巴结转移有关,MMP-9可能促进肾透明细胞癌的侵袭和转移过程。两者有望成为判定肾透明细胞癌侵袭和转移能力的指标。     

VHL基因在肾透明细胞癌中的表达及其预后价值

目的明确Von Hippel-1indau(VHL)基因表达与肾透明细胞癌(CCRCC)分级、分期和预后的关系.方法采用免疫组化技术检测58例CCRCC标本中VHL的表达,比较其与Fuhrman分级、病理分期和预后的关系.结果VHL表达阳性率为56.9%(33/58).与分级和患者生存率均显著相关(P＜0.001),而与病理分期无关(P＞0.05).结论VHL的表达情况为判断预后提供了有用的信息.     

肾透明细胞癌组织IRS-5和IRS-6表达及其临床意义的探讨

目的：探讨肾透明细胞癌组织和对应癌旁正常组织中胰岛素受体底物IRS-5和IRS-6基因表达差异及其临床意义。方法：收集经病理确诊的52例肾透明细胞癌患者标本,取肾癌组织和对应的癌旁正常组织,采用实时荧光定量PCR法检测IRS-5和IRS-6基因的表达,并分析两者与临床病理参数之间的关系。结果：IRS-5在肾透明细胞癌组织中的表达水平显著高于对应癌旁正常组织（0．864±0．112VS0．762±0．104）,P=0．021。IRS-5mRNA的表达与肿瘤大小、临床分期及病理分级相关,P〈0．05;而与患者年龄、性别无关,P〉0．05。IRS-6在肾透明细胞癌组织和对应癌旁正常组织中均有表达,两者比较差异无统计学意义（01647±0．116vs0．6124±0．122）,P＝0．32。IRS-6mRNA的表达与患者年龄、性别、肿瘤大小、病理分级及临床分期亦无相关性,P〉0．05。结论：IRS－5基因在肾透明细胞癌中过表达,可能与肿瘤发生发展有关,对临床评价肿瘤恶性度有一定价值;IRS－6基因在肾透明细胞癌和癌旁组织中表达无差异,提示IRS－6基因在肾透明细胞癌中可能不起作用。     

嗜铬细胞瘤患者VHL基因突变的分析

目的 探讨散发性嗜铬细胞瘤患者VHL基因突变检测及其意义.方法 从41例散发性嗜铬细胞瘤患者的外周血及嗜铬细胞瘤组织中提取基因组DNA,进行VHL基因测序分析,存在基因突变者再进行家系成员筛查.以50例健康志愿者外周血提取的DNA样本作为对照.结果 3例散发性嗜铬细胞瘤患者的VHL基因第2外显子存在基因突变,其中1例为572位核苷酸由G突变为C,该突变导致120位编码氨基酸由精氨酸转变为苏氨酸;2例为623位核苷酸处插入一重复核苷酸T(TITGTtG),导致下游读码框移位.其余38例患者和50例健康志愿者未发现VHL基因突变.进一步对3例发生基因突变患者的家系成员进行筛查,分别筛查出3例G572C携带者、3例623(TTTGTtG)携带者.结论 散发性嗜铬细胞瘤患者中存在可能致病的VHL基阏突变,建议对散发性嗜铬细胞瘤患者进行VHL基因检测分析.VHL基因检测有望作为临床遗传性嗜铬细胞瘤基因诊断的指标之一.     

RhoB在肾透明细胞癌中的表达及临床意义

目的:探讨RhoB在肾透明细胞癌中的表达及临床意义.方法:采用实时定量PCR、Western blot和免疫组化方法检测RhoB在肾透明细胞癌组织中的表达情况.选取60例肾透明细胞癌组织标本,详细收集病人临床病理资料,通过免疫组化方法检测RhoB在肾透明细胞癌组织中的蛋白表达水平,并分析RhoB的蛋白水平与临床病理资料的关系.结果:与对应瘤旁肾组织相比,RhoB的mRNA及蛋白表达水平在肾透明细胞癌组织标本中明显降低;RhoB的蛋白表达水平在不同年龄、性别组间差异无统计学意义(P>0.05),而在肿瘤直径大小、T分期、临床分期和组织分级间差异有显著统计学差异(P<0.05).结论:RhoB的表达降低可能在肾透明细胞癌的肿瘤发生中发挥作用,且其表达下降可能与肾透明细胞癌的恶性进展有关.     

散发性肾透明细胞癌的遗传学研究现状

 散发性肾透明细胞癌的发生机制十分复杂，不同于遗传性肾癌的单一遗传因素起着决定性作用，但3p缺失、VHL的基因突变等遗传学改变，可能会帮助临床进行肾癌病理亚型的鉴别诊断，基因表达谱中分析的一些标志物可能帮助预测预后     

散发性乳腺癌患者BRCA1基因突变分析

目的：探讨BRCA1基因突变在散发性乳腺癌发生和发展中的作用及在乳腺癌临床诊断和治疗中的应用前景。方法：应用PCR-SSCP和直接测序法检测30例散发性乳腺癌和15例正常乳腺组织中RRCA1基因外显子2、11和20的突变情况。结果：15例正常乳腺组织在3个外显子上郜未显示电泳异常．30例乳腺癌中有6例在外显子2上显示电泳条带异常．其中4例经测序证实有突变，1例在外显子2上，3例在内含子拼接区。BRCA1基因突变率在初诊年龄、临床分期和肿瘤体积上差异无统计学意义．但与肿瘤转移密切相关。结论：BRCA1基因突变与散发性乳腺癌的发生和发展密切相关，该基因突变筛查可作为一种预后指标。     

In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause tissue-specific tumors, with a striking genotype-phenotype correlation. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to pheochromocytoma, polycythemia, or combinations of hemangioblastoma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein (pVHL) has been implicated in many cellular activities including the hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix remodeling. We have expressed missense pVHL mutations in Vhl(-/-) murine embryonic stem cells to test genotype-phenotype correlations in euploid cells. We first examined the ability of mutant pVHL to direct degradation of the hypoxia inducible factor (HIF) subunits HIF1alpha and HIF2alpha. All mutant pVHL proteins restored proper hypoxic regulation of HIF1alpha, although one VHL mutation (VHL(R167Q)) displayed impaired binding to Elongin C. This mutation also failed to restore HIF2alpha regulation. In separate assays, these embryonic stem cells were used to generate teratomas in immunocompromised mice, allowing independent assessment of the effects of specific VHL mutations on tumor growth. Surprisingly, teratomas expressing the VHL(Y112H) mutant protein displayed a growth disadvantage, despite restoring HIFalpha regulation. Finally, we observed increased microvessel density in teratomas derived from Vhl(-/-) as well as VHL(Y112H), VHL(R167Q), and VHL(R200W) embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the cell, and that these activities can be separated via discrete VHL point mutations. The ability to dissect specific VHL functions with missense mutations in a euploid model offers a novel opportunity to elucidate the activities of VHL as a tumor suppressor.     

Low CAIX expression and absence of VHL gene mutation are associated with tumor aggressiveness and poor survival of clear cell renal cell carcinoma

We attempted to describe, in a series of clear cell renal cell carcinoma (RCC), the relationship between CAIX expression, VHL gene mutations, tumor characteristics and outcome. Radical nephrectomy was performed in 100 patients. Genomic DNA was extracted from frozen tumor samples. Four amplimers covering the whole coding sequence of the VHL gene were synthesized by PCR and sequenced. The monoclonal antibody M75 was used to evaluate CAIX protein expression immunohistochemically. VHL mutations were identified in 58 patients (58%) and high CAIX expression (>85%) was observed in 78 (78%). Tumors with VHL mutation showed higher CAIX expression than those without (p = 0.02). Low CAIX expression and absence of VHL mutation were associated with a more advanced tumors e.g., higher T stages and presence of metastases. VHL mutation and high CAIX expression predicted longer progression-free survival (p = 0.037) and disease-specific survival (p = 0.001), respectively. In combination, they defined three prognostic groups (p = 0.002): (i) good prognosis, defined as VHL mutation and high CAIX (2-year survival: 86%), (ii) intermediate prognosis with either VHL mutation or high CAIX (69%), and (iii) poor prognosis with no VHL mutation and low CAIX (45%, median survival 18 months). CAIX expression, but not VHL mutational status, was an independent prognostic factor in multivariate analysis. Taken together, CAIX expression and VHL mutational status are able to stratify patients with clear cell RCC into distinct groups with regards to clinicopathological variables and prognosis, with low CAIX expression and absence of VHL mutation being associated with a poor clinicopathological phenotype and diminished survival.     

The von-Hippel-Lindau (VHL) tumor suppressor gene is not mutated in sporadic ovarian carcinomas

The von Hippel-Lindau (VHL) tumor suppressor gene has been shown to be mutated frequently not only in neoplasms from von Hippel-Lindau disease, but also in sporadic clear cell renal carcinoma. In order to reveal the possible role of the VHL tumor suppressor gene in the development of ovarian carcinoma, a total of 71 primary sporadic ovarian carcinomas were analyzed for the presence of mutations in the exon 2 and 3 of the VHL tumor suppressor gene, using the polymerase chain reaction with single strand conformation polymorphism analysis. No mutations in the VHL gene were found in any of the tumors analyzed. This result shows that the VHL tumor suppressor gene does not play a major role in the tumorigenesis of sporadic ovarian carcinoma.     

Cloning of the Rat Homologue of the von Hippel-Lindau Tumor Suppressor Gene and Its Non-somatic Mutation in Rat Renal Cell Carcinomas

Recently, von Hippel-Lindau ( VHL ) gene mutations were detected in non-inherited, sporadic human renal cell carcinomas (RCs) at a high frequency. In order to determine whether or not the VHL gene is also a critical gene in rat RCs, we cloned and sequenced the rat homologue of human VHL gene and searched for mutations of the VHL gene in rat RCs. Mutations in the VHL gene were not detected in spontaneous RCs of the Eker rat model or in ferric nitrilotriacetate-induced rat RCs using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. These data indicate that mutation of the VHL tumor suppressor gene is not an event in rat renal carcinogenesis, at least in our present systems.     

Analysis of the TSC1 and TSC2 genes in sporadic renal cell carcinomas.

The genetic events involved in the aetiology of non-clear-cell renal cell carcinoma (RCC) and a proportion of clear cell RCC remain to be defined. Germline mutations of the TSC1 and TSC2 genes cause tuberous sclerosis (TSC), a multi-system hamartoma syndrome that is also associated with RCC. We assessed 17 sporadic clear cell RCCs with a previously identified VHL mutation, 15 clear-cell RCCs without an identified VHL mutation and 15 non-clear-cell RCCs for loss of heterozygosity (LOH) at chromosomes 9q34 and 16p13.3, the chromosomal locations of TSC1 and TSC2. LOH was detected in 4/9, 1/11 and 3/13 cases informative at both loci. SSCP analysis of the whole coding region of the retained allele did not reveal any cases with a detectable intragenic second somatic mutation. Furthermore, RT-PCR analysis of TSC1 and TSC2 on total RNA from 8 clear-cell RCC cell lines confirmed expression of both TSC genes. These data indicate that biallelic inactivation of TSC1 or TSC2 is not frequent in sporadic RCC and suggests that the molecular mechanisms of renal carcinogenesis in TSC are likely to be distinct.     

VHL gene alterations in renal cell carcinoma patients: novel hotspot or founder mutations and linkage disequilibrium

Mutations in the von Hippel-Lindau (VHL) gene are frequently detected in human sporadic renal cell carcinoma (RCC). We analysed 102 Swedish RCCs for VHL mutations by PCR-SSCP and sequencing. In 47 patients (46.1%), 70 different mutations were found, and most of them represented novel variations of the VHL gene. Mutations in the VHL gene were found in 54% of clear cell renal cell carcinomas (CCRCC) and in 18% of chromophilic cancers but in no chromophobe cancers or oncocytomas (P=0.016). Three novel hotspot or founder mutations were detected in our study: four CCRCCs carried a missense mutation (glutamic acid to lysine) at codon 160 which is critical in the stabilization of the H1 helix of the alpha domain and the alpha-beta domain interface in the VHL protein. Five CCRCCs and one chromophilic RCC harbored a 15-nucleotide in-frame deletion (codons 41-45) at a duplex tandem repeat sequence site. Moreover, this deletion was in linkage disequilibrium with a C-->T transition in the promoter region. The frequency of linkage was 17 times more common than chance. Five patients with this linked mutation resided in the same hospital district and at least three of them showed the two sequence variants in the tumor-adjacent tissue. In 5/6 patients the wild-type allele was lost in the tumor samples, suggesting a causal role for the mutations in RCC. These linked mutations might be novel polymorphisms maintained in a relative isolated population. Multiple mutations in VHL were found in 17 tumors out of 47 tumors with the VHL mutation. A higher multiple mutation detected rate (33%) was observed in grade 3 CCRCCs than those in grade 1 (22%) and grade 2 (9%) (P=0.04). This is evidence on the association between VHL mutation and extent of nuclear atypia.