人脑胶质瘤癌基因、抑癌基因最新研究进展

脑胶质瘤的形成与发展是癌基因的激活、过表达或扩增以及抑癌基因的缺失或突变失活的一个复杂的过程。目前在脑胶质瘤中已发现几十种癌基因异常和十余种抑癌基因的异常。其中较重要的癌基因有C-erbBl、C-sis、CDK4、c-myc、Bcl-2、bFGF、VEGF及MDM2等,抑癌基因有Rb、PTEN、P53、P16、NF2等。了解和掌握这些基因在脑胶质瘤中的不同作用,对临床治疗及预防胶质瘤具有重要意义。     

人脑胶质瘤细胞X线照射后癌基因表达的变化

目的:探讨癌基因表达的变化与辐射性细胞放射损伤的关系.方法:采用免疫组化技术检测人脑星形细胞瘤细胞系经X射线处理前后p53、c-myc、bcl-2基因表达的变化;采用原位杂交方法检测p53基因在mPNA水平表达的变化.结果:①处理组内p53、c-myc基因表达率明显高于对照组(P<0.001),bcl-2表达显著减弱(P<0.05),p53或c-myc与bcl-2基因表达呈显著性负相关(P<0.05).②处理组内p53在mRNA水平表达量明显高于对照组(P<0.05),p53基因在蛋白水平与mRNA水平表达呈显著性正相关(P<0.05),p53基因在蛋白水平与mRNA水平表达呈显著性正相关(P<0.05).结论:p53、c-myc和bcl-2基因在X线诱导的细胞损伤中起着重要的作用.本研究结果为脑胶质瘤的放射治疗和基因治疗提供了一条新线索.     

100例脑肿瘤中p53基因突变与P53蛋白积聚的研究

目的:研究不同类型脑肿瘤中的p53基因突变与P53蛋白积聚及其相关性。方法:采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析及免疫组化法检测100例脑肿瘤p53基因突变及蛋白表达。结果:p53基因突变率为11%(11/100),其中高恶度胶质瘤为37.5%(6/16),低恶度胶质瘤4.3%(1/23),脑膜瘤6.9%(2/29),转移瘤40.0%(2/5)。P53蛋白表达阳性率为22%(22/100),其中高恶度胶质瘤为62.5%(10/16),低恶度胶质瘤为26.1%(6/23),脑膜瘤10.3%(3/29),转移瘤60%(3/5);其他肿瘤均未发现p53基因突变或蛋白表达。P53蛋白表达阳性的22例中伴有p53基因突变者11例,多见于高恶度肿瘤。结论:p53基因失活在脑肿瘤恶性进展过程中起重要作用。p53基因突变与P53蛋白积聚相关,但并非唯一因素。     

脑胶质瘤中c-myc基因扩增、H-ras及P53基因突变的检测

胶质瘤的发生是由于染色体及基因异常所致，对癌基因ｃ－ｍｙｃ、Ｈｒａｓ及抑癌基因Ｐ５３的检测，发现Ｈｒａｓ与胶质瘤的发生有关，而胶质瘤的恶性程度与ｃ－ｍｙｃ癌基因的扩增及抑癌基因Ｐ５３的突变有关。材料和方法２２例肿瘤标本均为手术切除的新鲜瘤组织，经...     

EGFR、p53与Ki-67在国人胶质瘤中表达研究

表皮生长因子受体（epidermal grotwth factor receptor，EGFR）的过表达或扩增、激活以及抑癌基因p53的缺失、突变失活均是导致胶质瘤的主要分子事件。本文应用免疫组化方法对50例国人脑胶质瘤中表皮生长因子受体EGFR、p53蛋白及胶质瘤恶性程度的标志Ki-67标记指数（Ki-67 LI）联合检测，对胶质瘤分子生物学行为进行探讨，为基因治疗在胶质瘤中的应用提供理论依据。     

CaBP4基因突变与小儿癫痫的关系

目的 探讨CaBP4基因突变与小儿癫痫的关系。方法 选取2016年3月-2018年3月在本院就诊的儿童癫痫60例,检测CaBP4基因突变分布,分析所有患儿相关的临床资料。结果 60例癫痫患儿中CaBP4基因p.G155D突变阳性患儿为31例,其突变率为51.67%; CaBP4基因未突变患儿29例,未突变率为49.33%。CaBP4基因突变与小儿癫痫的性别无明显关系(P>0.05)。CaBP4基因突变与癫痫患儿的首次发病年龄和月发作频率有关,即CaBP4基因p.G155D突变患儿的首次发病年龄低于CaBP4基因未突变患儿(P<0.05),CaBP4基因p.G155D突变患儿的月发作频率高于CaBP4基因未突变患儿(P<0.05)。CaBP4基因突变与小儿癫痫患儿的发作程度有关,即CaBP4基因p.G155D突变患儿重度癫痫发作的比例高于中度癫痫发作和轻度癫痫发作的比例(P<0.05)。CaBP4基因突变与小儿癫痫发作类型有关,即CaBP4基因p.G155D突变患儿局灶性发作的比例显著高于全面性发作和起源不明的比例(P<0.05)。CaBP4基因突变与癫痫患儿的首次发病年龄、月发作频率、发作程度、发作类型有关(P<0.05),而与癫痫患儿的性别无关(P>0.05)。结论 CaBP4基因p.G155D突变与癫痫患儿的首次发病年龄、月发作频率频率、发作程度、癫痫发作类型有关; 癫痫患儿CaBP4基因p.G155D突变率存在差异。     

人脑胶质瘤PTEN基因变异的研究

目的进一步了解PTEN基因突变和缺失在人脑胶质瘤发生和恶性进展中的作用。方法应用聚合酶链反应-单链构象多态性(PCR-SSCP)结合银染技术和双重PCR分别检测10例正常脑组织、10例脑膜瘤、80例胶质瘤PTEN基因第5和第8外显子区域上的突变与缺失情况。结果发现10例正常脑组织和10例良性脑膜瘤均无PTEN基因点突变发生,80例胶质瘤分别有11例(13.75%)和27例(33.75%)发生基因点突变和基因缺失,且PTEN基因失活与星形细胞瘤病理分级明显相关(P<0.05),其中高恶性度胶质瘤(III、IV级)突变率(24.44%)和缺失率(60%)明显高于低恶性度(I、II级)胶质瘤(P<0.05)。结论PTEN基因突变或缺失与胶质瘤病理分级关系密切,可能属于胶质瘤恶性进展的后期事件。     

脑星形细胞瘤癌基因表达

52例星形细胞瘤应用免疫组化方法,检测癌基因p~(53)、C-erbB-2及增殖细胞核抗原(PCNA)的表达,结果发现①p53异常表达率为41.2%(24/52),C-erbB-2过度表达率为39%(20/52),PCNA(PI>0.05)增殖指数为77%(40/52),与对照组正常脑组织对比有显著差异(P<0.001).②p53,C-erbB-2,PCNA异常表达与病理级别有明显相关性.病理Ⅲ,Ⅳ级的阳性率分别为80%(16/20),40%(8/20),100%(20/20),(P0.05);C-erbB-2阳性组,PCNA指数;0.361±0.27,阴性组PCNA指数;0.399±0.39,两组间亦无差异(P>0.05).④11例胶质增生组织有1例(9%)显示p53表达,C-erbB-2,PCNA无表达.随访3年,病变复发,病理证实为星形细胞瘤Ⅰ～Ⅱ级,C-erbB-2,PCNA表达.结果提示:①星形细胞瘤p53、C-erbB-2及PCNA的异常表达可作为星形细胞瘤恶性程度及病人预后的指标,以p53过度表达尤为重要;②3种抗体的联合应用对星形细胞瘤发病机理研究及预测早癌发生有一定价值,p53异常表达主要是影响星形细胞瘤的分化,而C-erbB-2对肿瘤进展早期起一定作用.③胶质增生的胶质细胞具有恶性表型.     

VEGF、bFGF、PTEN表达与胶质瘤恶性程度及预后因素的研究

目的研究血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、抑癌基因(PTEN)在胶质瘤的表达,并结合多种临床因素探讨其与肿瘤恶性程度及预后的关系。方法采用SABC免疫组化技术,检测50例胶质瘤VEGF、bFGF、PTEN的表达,并对可能影响预后的因素进行统计学分析。结果胶质瘤VEGF、bFGF、PTEN的表达在低级别组与高级别组之间存在显著差异(P<0.05);Logistic逻辑回归单因素分析显示,患者年龄、肿瘤切除范围、恶性程度、KPS、术后化疗、术后放疗、肿瘤bFGF和PTEN表达对3年存活率有意义;多因素分析表明,肿瘤的恶性程度、术前患者KPS及PTEN基因的突变或缺失情况是影响胶质瘤患者预后的主要因素。结论VEGF、bFGF、PTEN在胶质瘤中的表达与其恶性程度相关,肿瘤的恶性程度、术前患者KPS及PTEN基因的突变或缺失情况可作为考察胶质瘤患者预后的独立指标。     

人脑胶质瘤IDH1突变状态与MGMT启动子甲基化、P53和TERT突变相关性

目的 探讨脑胶质瘤异柠檬脱氢酶1（IDH1）突变与O6-甲基鸟嘌呤-DNA甲基转移酶 （MGMT）启动子甲基化状态、P53和端粒酶逆转录酶（TERT）突变之间的相关性。方法 收集2016年6月至2017年9月手术切除并经病理诊断为胶质瘤标本72例（WHO Ⅱ级14例,Ⅲ级19例,Ⅳ级39例）。采用PCR荧光探针法检测MGMT基因启动子甲基化状态,毛细管电泳法检测基因IDH1、P53、TERT突变情况;采用列联系数分析IDH1突变与MGMT基因启动子甲基化、P53、TERT突变状态的相关性。采用多因素Logistic回归分析检验IDH1突变的相关因素。结果 72例中,IDH1突变率为29.2%,MGMT启动子甲基化率为47.2%,P53突变率为41.4%,TERT突变率为50%。相关性分析发现IDH1突变与MGMT启动子甲基化（列联系数=0.44;P<0.001）、P53突变（列联系数=0.32;P<0.05）均有显著相关性,但与TERT启动子突变无明显相关性（P>0.05）。IDH1野生型胶质瘤中MGMT启动子甲基化与TERT启动子基因突变具有相关性（列联系数=0.28,P<0.05）。IDH1突变型胶质瘤中MGMT启动子甲基化与P53基因突变具有相关性（列联系数=0.27,P<0.05）。多因素Logistic回归分析,结果显示病人年龄、MGMT启动子甲基化是IDH1突变独立相关因素（P<0.05）。结论 胶质瘤IDH1突变可能与MGMT启动子甲基化、P53和TERT突变之间存在复杂的相互调节作用。     

Molecular-genetic characterisation of gliomas that recur as same grade or higher grade tumours

BACKGROUND: Due to their invasive growth, gliomas usually cannot be removed completely and almost always recur as same grade or higher grade malignancies. OBJECTIVE: To determine whether there were differences in the accumulation of genetic changes between the two types of glioma recurrence. METHODS: We genetically characterised 14 cases of lower grade glioma with a same grade recurrence, 12 cases of glioblastoma recurrence, and 14 cases of lower grade glioma with a higher grade recurrence. We investigated LOH (loss of heterozygosity) at 1p36, 10p15, the PTEN region in 10q23, the DMBT1 region in 10q25, 19q13, 22q13, LOH and mutation of TP53, and EGFR amplification. RESULTS: Genetic heterogeneity in the primary tumour was inferred in 3 cases of lower grade glioma with a higher grade recurrence. The cases of lower grade glioma with a higher grade recurrence displayed increased genetic instability in the recurrence (mean of 2.0 additional genetic changes per case) compared to cases with a same lower grade recurrence or those with a glioblastoma recurrence (mean of 0.6 and 0.8 additional changes per case, respectively). Compared to unselected primary glioblastomas, the glioblastomas that recurred as an operable tumour had infrequent EGFR amplification (8% v 30-40% of cases). CONCLUSIONS: Gliomas recurring as higher grade lesions might be genetically heterogeneous and accumulate more genetic changes than gliomas recurring as same grade lesions (whether originally low or high grade). Primary glioblastomas from patients for which the recurrence is operated because of prognostically more favourable clinical indices have infrequent EGFR amplification.     

人脑胶质瘤中Livin、p53的表达及其与肿瘤恶性程度的关系

目的 探讨Livin、p53在不同级别人脑胶质瘤中的表达及其与肿瘤恶性程度之间的关系.分析二者在胶质瘤细胞凋亡过程中的信号转导机制.方法 应用免疫组织化学技术检测Livin、p53蛋白在41例人脑胶质瘤和10例正常脑组织中的表达.结果 Livin、p53蛋白在10例正常脑组织中均不表达,在41例胶质瘤组织中表达率分别为87.8%和41.5%.Livin和p53蛋白在低级别胶质瘤中表达较低,在高级别胶质瘤中表达较高,两组比较差异有统计学意义(P＜0.05).Spearman等级相关分析显示,Livin蛋白与p53蛋白表达呈正相关(r=1.000,P＜0.01).结论 Livin和p53在人脑胶质瘤组织中表达上调,细胞凋亡受到抑制,引起肿瘤恶性增殖,与胶质瘤病程发展、恶性程度明显相关,这可能是胶质瘤恶性增殖的一个机制.     

Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry

Using polymerase chain reaction-assisted single-strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, mutations in the p53 tumor suppressor gene were examined in 19 low-and high-grade gliomas. By PCR-SSCP and nucleotide analyses, p53 gene mutation was seen in 7 gliomas. Out of the 7 mutations, 3 were located at the CpG site of the previously proposed hot-spot codons 248 and 273, 2 were at codons 171 and 214 and the other 2 were in intron 5, 1 at the splice acceptor site and the other in the vicinity of the splice donor site. The latter 4 mutations have not, or only rarely, been observed in gliomas or in other tumors. However, their effect on the structural and functional alteration of the p53 protein was suggested by positive intranuclear p53 immunostaining in neoplastic cells in 3 mutations including the 1 at the splice acceptor site. In connection with glioma grading, the p53 gene mutation was shown to have occurred in both low- and high-grade gliomas, often in most of the neoplastic cells, as suggested by lack of distinct normal bands and ladders in SSCP and direct sequencing, respectively. The absence of recurrence and malignant transformation over a considerably long postoperative time in our low-grade glioma cases suggested that the p53 gene mutation might not be sufficient for the progression from low- to high-grade gliomas. The frequency of detection of mutation was 7/19(37%) by PCR-SSCP, 8/19(42%) by immunohistochemistry and 10/19(53%) by both methods. The results of PCR-SSCP and immunohistochemistry were consistent in 14 cases(73.7%), but not in 5 cases(26.3%). Thus, the use of both methods was recommended to survey the occurrence of p53 gene mutation more accurately.Supported in part by the Fujisawa Foundation     

胶质瘤中EGFR扩增、过表达及其与肿瘤增殖关系的研究

目的探讨表皮生长因子受体（EGFR）在胶质瘤中扩增、过表达及其在肿瘤增殖中的作用机制。方法应用SP免疫组化法、RT—PCR、Southern印迹法检测6例正常脑组织、50例不同级别胶质瘤及2个体外胶质瘤细胞系中在EGFR蛋白水平、mRNA水平、DNA水平的表达及扩增情况，以及与肿瘤增殖活性的相关性。结果不同级别的胶质瘤中，表达与扩增程度不同，Ⅱ级与Ⅲ、Ⅳ级肿瘤之间比较具有显著性差异：EGFR过表达与Ki-67在胶质瘤中的表达呈正相关．而EGFR过表达与扩增并不一致。结论EGFR过表达与胶质瘤的增殖活性增高有密切相关性，推测EGFR过表达是肿瘤进展中的早期事件而非进展期事件。     

EGFR过表达和基因扩增状态差异与胶质瘤临床病理和细胞凋亡关系研究

目的探讨人类表皮生长因子受体（EGFR）在胶质瘤中的表达和基因扩增状态,并分析其与肿瘤细胞凋亡的关系。方法采用Envision免疫组化二步法、westernblot和荧光原位杂交法（FISH）检测10例正常脑组织、60例不同级别原发性胶质瘤和25例继发性胶质母细胞瘤组织中EGFR蛋白水平和基因扩增状态,并分析其与肿瘤凋亡蛋白P53的相关性。结果在不同级别的原发性胶质瘤组织中,EGFR的阳性表达与扩增程度不同,I～Ⅱ级组与Ⅲ～Ⅳ级组间比较具有统计学差异（P〈0．05）;原发性胶质瘤中EGFR免疫组化阳性率和基因扩增率分别为85％和40％,显著高于继发性胶质母细胞瘤（28％和28％）,差异均具有统计学意义（P均〈0．05）;EGFR过表达与基因扩增状态并不一致,在有EGFR扩增的原发性胶质母细胞瘤中均有EGFR过表达,在EGFR过表达的肿瘤中仅有31．03％EGFR扩增;在胶质瘤中EGFR阳性表达与P53呈正相关（r=14．22,P：0．001）。结论EGFR在不同级别胶质瘤中差异性过表达和扩增,且与肿瘤细胞凋亡相关,其基因扩增状态在原发性和继发性胶质母细胞瘤中存在差异,EGFR可作为胶质母细胞瘤治疗的一个重要靶点。     

Concurrent EGFR amplification and TP-53 mutation in glioblastomas

Glioblastoma multiforme is the most common and most aggressive of the primary brain tumors. The mean survival of patients is 10-12 months. Conventional therapy of surgery, radiation and chemotherapy is largely palliative. Cytogenetically, karyotypes of glioblastomas are very complex with trisomy 7 and monosomy 10 as the most frequent abnormalities. A genetic alteration that is significantly more frequent in primary than in secondary glioblastomas, the latter arising from preceding low-grade gliomas, is epidermal growth factor receptor gene (EGFR) amplification, whereas TP-53 mutations are significantly more frequent in low-grade gliomas and secondary glioblastomas derived there- from. We report the histological and genetic study of two glioblastomas, one case arising de novo and the other case arising 3 years after a previously diagnosed anaplastic astrocytoma, with concurrent EGFR amplification and TP-53 mutation. These anomalies were initially deemed as mutually exclusive. However, a small percentage of cases have been found with both anomalies although at a significantly lower level than could be expected. We have analyzed these two cases cytogenetically and by molecular studies in order to detect additional alterations associated with this phenotype. Cytogenetically, both cases showed in common the monosomy of chromosomes 10 and 17. At the molecular level, a rare mutation of TP-53 was found in the secondary glioblastoma and hypermethylation of the promoter region of p16(INK4a) and p14(ARF) genes were observed in the primary and secondary glioblastoma, respectively.     

Evaluation of p53 protein expression and proliferative potential based on the MIB-1 positive index in astrocytic gliomas

Fifty-seven patients with infiltrative astrocytic gliomas, comprising 29 cases of glioblastoma, 13 cases of anaplastic astrocytoma and 15 cases of low-grade astrocytoma, were examined. In 12 cases of astrocytic glioma, the p53 gene mutation was investigated by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). p53 overexpression was observed in 20 (35%) of the 57 patients with astrocytic gliomas. The occurrence of p53 overexpression was apparently correlated with the histological grading of the astrocytic gliomas. Of the 20 patients with glioblastoma who were less than 60 years old (mean age ± SD, 39.2 ± 15.0 years), 50% demonstrated p53 overexpression, whereas only 2 (22%) patients with glioblastoma who were more than 60 years old (mean age ± SD, 65.1 ± 5.8 years) had p53 overexpression. A substantial difference in survival associated with glioblastoma was noted between the p53-positive group (mean survival 28.9 months) and the p53-negative group (mean survival 11.3 months) based on Kaplan-Meier survival curves (P= 0.01). Even among patients with glioblastoma who were less than 60 years old, a better survival rate was recognized in those with p53 overexpression than in those without p53 (P= 0.03). The MIB-1 indices tended to increase with tumor malignancy, and a poor survival time was significantly associated with elevated values for the MIB-1 index. A substantial difference in survival between the two groups of patients who had MIB-1 indices of above 5% and below 5% was evident from the Kaplan-Meier survival curves (P= 0.04). The group that had no p53, and MIB-1 indices of above 5%, was found to show the malignant gliomas most frequently. Although most of the gliomas with p53 overexpression demonstrated MIB-1 indices of above 5%, there was no significant correlation between p53 protein expression and the MIB-1 index. Of the 12 patients with astrocytic glioma who were examined by PCR-SSCP, 3 (one glioblastoma and 2 anaplastic astrocytomas) revealed p53 gene mutation correlated with p53 (DO-7) overexpression.     

Loss of alleles in brain tumours: Distribution and correlations with clinical course

Gliomas (n = 44) and meningiomas (n = 24) of different grades of malignancy were analysed for allele losses at loci on chromosomes 10, 13, 17 and 22. Deletions of genetic material on these chromosomes occurred in gliomas without being restricted to any histological entity. The frequency of chromosome-l0-specific allele losses increased significantly with the age of the patients and with the grade of malignancy of the tumours. Deletions of chromosome 10 material were associated with a poor prognosis. The glioblastomas of patients aged over 70 years lacked the loss of the entire chromosome 10, even in tumours with EGFR gene amplification. Deletions at loci of chromosomes 13, 17 and 22 were observed in 18–32% of all gliomas, independent of grade of malignancy, patients' age, EGFR gene amplification and clinical course. Only chromosome-22-specific allele losses were found preferentially in gliomas of female patients. Loss of chromosome 22 alleles in 44% was the only mutation detected in meningiomas. This occurred independently of grade of malignancy and biological factors.Supported by a grant of the Deutsche Krebshilfe     

p53 protein alterations in adult astrocytic tumors and oligodendrogliomas

BACKGROUND: p53 is a tumor suppressor gene implicated in the genesis of a variety of malignancies including brain tumors. Overexpression of the p53 protein is often used as a surrogate indicator of alterations in the p53 gene. AIMS: In this study, data is presented on p53 protein expression in adult cases (>15 years of age) of astrocytic (n=152) and oligodendroglial (n=28) tumors of all grades. Of the astrocytic tumors, 86% were supratentorial in location while remaining 14% were located infratentorially - 8 in the the cerebellum and 13 in the brainstem. All the oligodendrogliomas were supratentorial. MATERIALS AND METHODS: p53 protein expression was evaluated on formalin-fixed paraffin-embedded sections using streptavidin biotin immunoperoxidase technique after high temperature antigen retrieval. RESULTS: Overall 52% of supratentorial astrocytic tumors showed p53 immunopositivity with no correlation to the histological grade. Thus, 58.8% of diffuse astrocytomas (WHO Grade II), 53.8% of anaplastic astrocytomas (WHO Grade III) and 50% of glioblastomas (WHO Grade IV) were p53 protein positive. In contrast, all the infratentorial tumors were p53 negative except for one brainstem glioblastoma. Similarly, pilocytic astrocytomas were uniformly p53 negative irrespective of the location. Among oligodendroglial tumors, the overall frequency of p53 immunopositivity was lower (only 28%), though a trend of positive correlation with the tumor grade was noted - 25% in Grade II and 31.5% in grade III (anaplastic oligodendroglioma). Interestingly, p53 labeling index (p53 LI) did not correlate with the histopathological grade in both astrocytic and oligodendroglial tumors. CONCLUSIONS: Thus, this study gives an insight into the genetic and hence biological heterogeneity of gliomas, not only between astrocytic tumors vs. oligodendrogliomas but also within astrocytic tumors with regard to their grade and location. With p53 gene therapy trials in progress, this will possibly have future therapeutic implications.