非小细胞肺癌中hMLH1基因完全失活机制

目的 研究hMLH1基因完全失活机制及蛋白表达与非小细胞肺癌的关系.方法 应用PCR-变性聚丙烯酰胺凝胶电泳-银染方法 检测hMLH1基因杂合性缺失(LOH),利用甲基化敏感性限制性核酸内切酶HpaⅡ/MspⅠ酶切-PCR方法 检测异常甲基化;通过免疫组化染色方法 分析hMLH1基因蛋白表达情况.结果 40例NSCLC中,hMLH1基因LOH发生率为65%,甲基化发生率为67.5%,72.5% hMLH1蛋白表达阴性,均与对照组之间存在显著性差异.29例hMLH1蛋白表达缺失中有21例(72.4%)同时发生LOH和异常甲基化.结论 hMLH1基因异常在NSCLC发生中起着重要作用,其完全失活机制可能主要为:LOH 异常甲基化.     

子宫颈癌FHIT基因表达与杂合性丢失

目的　探讨抑癌基因FHIT基因在子宫颈癌发生中的作用及其失活的可能机制。方法　选取FHIT基因内 2个微卫星位点D3S130 0和D3S12 34,对 5 6例经显微切割分离肿瘤组织的原发性子宫颈癌进行杂合性丢失 (LOH)分析 ,同时采用免疫组化方法 (S P法 )检测FHIT的表达情况。结果　D3S130 0和D3S12 34的LOH发生率分别为 36 2 % (17/ 4 7)、32 7% (16 /4 9) ,5 2 % (2 9/ 5 6 )的子宫颈癌组织至少在一个位点存在LOH。 5 7 1%的子宫颈癌FHIT表达减弱或缺失 ,其中 71 9%存在FHIT基因LOH ,FHIT低表达与FHIT基因LOH之间有相关性 (P <0 0 1)。子宫颈鳞癌组织的LOH发生率及FHIT低表达率均高于腺癌 (6 4 3%vs 14 3% ,6 9 0 %vs 2 1 4 % ,P <0 0 5 )。子宫颈鳞癌LOH发生率及FHIT低表达与组织学分级、FI GO分期均不相关 (P >0 0 5 )。结论　FHIT基因可能在子宫颈鳞癌发生中起重要作用 ,杂合性丢失可能是FHIT基因失活的重要机制     

少突胶质细胞瘤中PTEN基因突变和10q 染色体的杂合性缺失分析

目的 探讨PTEN基因突变和10q染色体的杂合性缺失(LOH)在少突胶质细胞瘤发生和发展中的意义。方法 以55例少突胶质细胞瘤和混合性胶质细胞瘤为研究对象，PCR扩增PTEN基因所有9个外显子，其产物经变性梯度凝胶电泳(DGGE)后，DNA序列分析检测PTEN基因突变。应用不同颜色荧光标记10q微卫星标志物引物，PCR扩增后，其产物在自动测序仪上进行聚丙烯酰胺凝胶电泳，用Gene Scan软件分析LOH。结果 2／55例有PTEN基因突变，1例是PTEN的第9外显子的20号T碱基缺失导致终止密码351易位至347，另1例在139位发生C到A的转换，使得Phe变成Leu。23／55(42％)例出现染色体10q的LOH，其中15／55例位于10q^23.3(D10S541)，其为PTEN基因位点，12／55例10q微卫星标志物位点完全缺失。结论 在少突胶质细胞瘤中PTEN突变较少见，而10q LOH较频繁，尤其在恶性度高的少突胶质细胞瘤中表现明显，提示10q LOH与少突胶质细胞瘤恶性进展有关，且10q上可能存在其他抑癌基因。     

宫颈腺癌和腺上皮内肿瘤组织中PTEN基因的突变和蛋白表达

目的 探讨PTEN基因的突变和表达在宫颈腺癌发生中的作用.方法 应用免疫组织化学SP法检测42例宫颈腺癌、20例宫颈腺上皮内肿瘤和28例正常宫颈组织中PTEN蛋白表达,并采用聚合酶链反应-单链构象多态性(PCR-SSCP)法检测上述组织中PTEN基因第5、8外显子突变情况.结果 宫颈腺癌、宫颈腺上皮内肿瘤和正常宫颈腺上皮组织中PTEN蛋白的阳性表达率分别为54.8%(23/42)、25.0%(5/20)和100%(28/28),其差异均有统计学意义(P<0.05);黏液型和子宫内膜型宫颈腺癌组织中PTEN蛋白阳性表达率分别为9/19和2/10,其他组织学变型PTEN蛋白阳性表达率92.3%(12/13).宫颈腺癌、宫颈腺上皮内肿瘤和正常宫颈组织中PTEN基因第5、8外显子的突变率分别为19.0%(8/42)、45.0%(9/20)和0,其差异均有统计学意义(x<,2>=4.29,x2=12.70;均P<0.05);黏液型和子宫内膜型宫颈腺癌PTEN基因第5、8外显子的突变率分别为4/19和4/10,其他组织学变型未检测到突变.结论 宫颈腺癌的发生与PTEN基因的突变和蛋白表达产物缺失有关,PTEN基因的突变和蛋白表达产物缺失可能是宫颈腺癌发生中的早期事件.     

软组织平滑肌肉瘤染色体3p位点特异性的杂合性缺失

目的 对软组织平滑肌肉瘤(LMS)染色体3p上的5个特异性位点进行微卫星多态性分析,以明确这些位点染色体等位基因杂合性缺失(LOH)情况。方法 采用微卫星DNA-聚合酶链反应—银染法分析22例LMS 3p14．2-pter范围内的5个特异性位点LOH状态。结果 45．4％LMS至少在一个位点出现LOH,以D3s1295最高(36．8％),其次为D3s1289(10．5％),而D3s1293末检测到LOH。不同组织学分级、大小及不同部位的LMS其LOH发生差异无显著性。结论 LMS3p14．2—23范围发生杂合性缺失率较高,D3s1295及D3s1289位点所在的染色体区域及附近可能隐含与平滑肌肉瘤有关的抑癌基因。     

中国人肝癌分泌型卷曲相关蛋白1基因遗传的不稳定性

目的 研究8号染色体上D8S532位点的杂合性缺失(LOH)和微卫星不稳定性(MSI)对分泌型卷曲相关蛋白1基因(sFRP1)蛋白表达的影响,阐明 sFRP1 基因遗传不稳定性与肝癌进展的关系,为揭示sFRP1 基因作用机制和肿瘤发生发展机制提供实验依据. 方法 采用石蜡包埋组织抽提DNA,聚合酶链-单链构象多态性(PCR-SSCP)分析,常规银染检测D8S532位点的LOH和MSI,采用Envision免疫组织化学染色,Leica-Qwin计算机图像分析系统采图和Image-Pro P1uS(IPP)Version5.0专业图像分析软件分析蛋白表达. 结果 在36例肝癌中,D8S532位点LOH和MSI检出率分别为11.11%(4/36)和8.33%(3/36),D8S532位点MSI发生率在60岁以上年龄组(≥60) 26.67%(4/15),高于60岁以下年龄组(<60)0.00%(0/21, P <0.05).相比较于正常组织,86.11%(31/36)的sFRP1蛋白有不同程度表达下降,在肝癌组织中,sFRP1表达阳性率为52.78%(19/36),蛋白表达阴性组LOH阳性率为23.53%(4/17),明显高于蛋白阳性组的0.00%(0/19)( P <0.05). 结论 在中国人肝癌发生进展中,sFRP1蛋白表达下降甚至缺失是普遍现象, sFRP1 基因的遗传不稳定性可能是导致该抑癌基因突变、肿瘤发生的一个机制,LOH在sFRP1表达缺失的过程中起了重要作用.     

神经鞘瘤D22S268杂合缺失与组织学类型的相关性研究

目的　探讨神经鞘瘤组织学类型与NF2基因微卫星标记D2 2S2 6 8杂合性缺失 (LOH)的相关性。方法　选择与NF2基因密切相关的微卫星标记D2 2S2 6 8,应用PCR方法检测 35例神经鞘瘤患者的新鲜肿瘤组织标本。结果　病理分型为AntoniA型 2 0例 ,其中LOH 15例 ;AntoniB型 15例 ,LOH 0例。两种病理分型中D2 2S2 6 8LOH发生率存在着显著差异 (P <0 .0 1)。结论　NF2基因的LOH可能在AntoniA型的神经鞘瘤发生过程中起决定性作用。     

肺鳞状细胞癌的M6P/IGF2R突变

为了探测肺鳞状细胞癌的甘露糖 - 6-磷酸酶 /类胰岛素生长因子 ( M6P/IGF2 R)有否突变 ,作者通过组织显微镜解剖法收集样本 ,PCR扩增DNA,M6P/IGF2 R测序及免疫组化染色 ,对 2 2例新诊断尚未治疗的肺鳞状细胞癌样本进行了杂合性丢失 ( LOH)筛查。结果表明 ,肺鳞状细胞癌中常见 M6P/IGF2 R突变 ,其正常功能为肺癌抑制基因。 1 9/2 2例( 86% )有 LOH信息 ,其杂合信息为该受体 3′-非翻译区的多聚二核苷酸重复序列和四核苷酸插入或缺失性多态。在 1 1 /1 9例 ( 5 8% ) M6P/IGF2 R基因座有 LOH信息的患者中 6女 1 3男 ,平均年龄 …     

甲状腺肿瘤3号染色体短臂杂合性缺失的研究

目的 探讨甲状腺肿瘤中3号染色体短臂(3p)杂合性缺失(LOH)状态及其临床意义.方法 收集74例甲状腺肿瘤标本,包括20例甲状腺腺瘤(FA)、24例滤泡性甲状腺癌(FTC)和30例乳头状甲状腺癌(Prc).通过PCR扩增和银染分析其3p上11个微卫星位点的杂合性缺失状态.结果 FFC的LOH频率达到71%(17/24),PTC中30%(9/30),FA中10%(2/20).FFC的3p LOH频率显著高于FA和PTC(P<0.01).FTC中存在两个最小共同缺失区,分别位于3p26-pter和3p14.2-3p22.PTC上存在一个最小共同缺失区,位于3p 25.2-26.1.结论 FTC的3p LOH频率显著高于FA和PTC.3p的3个最小缺失区上可能存在着与FTC和PTC发生发展相关的肿瘤抑制基因.     

卵巢癌PTEN基因的等位基因不平衡与突变

在人类许多癌症的染色体 1 0 q2 3.3的纯合子区分离出一种候选抑癌基因即 PTEN基因 (也称MMAC1和 TEP1 ) ,多数癌的 PTEN基因都发生缺失和突变。据报道在子宫内膜样型卵巢癌和子宫内膜增生中 PTEN基因常发生突变 ,相反 ,也有报道卵巢癌很少发生 PTEN基因突变。为了证实卵巢癌 PTEN基因的突变总频率及组织学特异性 ,作者应用位于 1 0 q2 3.3的多态标记( D1 0 S2 1 5 ,D1 0 S5 4 1 ,D1 0 S1 75 3和 AFMA1 1 4XBI等 )进行微卫星分析 ,研究了 33例卵巢癌、3例卵巢交界瘤及 7种卵巢癌细胞系。结果表明 ,在 2 2 /36中杂合率为 61 …     

PTEN/MMAC1/TEP1在肺癌中的丢失和失活

目的 了解抑制癌基因ＰＴＥＮ／ＭＭＡＣ１／ＴＥＰ１（以下简称ＰＴＥＮ）在肺癌中的缺失和失活。方法 选用２４例具有正常对照的肺癌新鲜标本,１８例小细胞肺癌石蜡切片,用聚合酶链反应及杂合性丢失分析法检测ＰＴＥＮ的杂合笥丢失;并采用原位杂交法、免疫组化化学染色法、Ｗｅｓｔｅｍ ｂｌｏｔ法观察肺癌标本中ＰＴＥＮ ｍＲＮＡ和蛋白水平的表达。对１株肺腺癌和３株小细胞肺癌系进行ＰＴＥＮ的Ｓｏｕｔｈｅｒｎ、Ｎｏｒ     

Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis.     

Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer

The PTEN tumor suppressor gene on 10q23.3, responsible for the Cowden and Bannayan-Zonana syndromes, encodes a dual-specificity phosphatase able to dephosphorylate both tyrosine phosphate and serine/threonine phosphate residues. Mutational inactivation of PTEN has been reported in various malignancies, including endometrial cancers, ovarian cancers, and glioblastomas. In this study, we investigated PTEN gene mutations in 10 gastric cancer cell lines and 58 primary gastric cancers by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP). Hypermethylation of promoter region CpG islands, an alternative mechanism of gene inactivation to coding region mutations, was also evaluated by methylation specific PCR (MSP). Only one (1.7%) of the 58 primary tumors carried a somatic 5-bp deletion in intron 7 of PTEN, which did not alter the mRNA sequence, and no mutations were detected in any of the cell lines. Similar levels of PTEN mRNA expression were observed in all cell lines and primary tumors studied by RT-PCR, and PTEN promoter CpG islands remained unmethylated. Therefore, we conclude that PTEN does not participate in gastric carcinogenesis as a tumor suppressor gene.     

PTEN gene loss, but not mutation, in benign and malignant phaeochromocytomas

Mutations of the 'phosphatase and tensin homologue deleted on chromosome 10' (PTEN/MMAC1) gene have been associated with a variety of human cancers, including prostate cancer, glioblastoma, and melanoma. The gene is thought to be one of the most frequently mutated tumour suppressor genes and inactivation of PTEN is associated with disease progression and angiogenesis. High vascularization and resistance to chemo- and radio-therapy are two well-established features of phaeochromocytomas (PCCs). Furthermore, benign and malignant PCCs are found in several PTEN knockout mouse models. This study therefore evaluated whether inactivation of PTEN may be involved in the tumourigenesis of PCC in man and whether PTEN abnormalities may help to define the malignant potential of these tumours. Tumour and germline DNA was analysed from 31 patients with apparently sporadic PCC, including 14 clinically benign and 17 malignant tumours, for loss of the PTEN gene locus, mutations in the PTEN gene, and for PTEN protein expression by immunohistochemistry. Loss of heterozygosity (LOH) analysis showed loss of PTEN in four malignant tumours (40%) and in one benign tumour (14%). However, no mutations of PTEN were observed. Immunohistochemistry showed no correlation with clinical behaviour and/or LOH status. The results indicate that inactivation of the PTEN/MMAC1 gene may play a minor role in the development of malignant phaeochromocytomas.     

Biallelic inactivating mutations and an occult germline mutation of PTEN in primary cervical carcinomas

A tumor suppressor gene on chromosome sub-band 10q23.3, PTEN, is frequently mutated or deleted in a variety of human cancers. Germline mutations in PTEN, that encodes a dual-specificity phosphatase, have been implicated in two hamartoma-tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Zonana syndrome. Although cervical cancer is not a known component of these two syndromes, loss of heterozygosity (LOH) of markers on chromosome arm 10q is frequently observed in cervical cancers. To determine the potential role that PTEN mutation may play in cervical tumorigenesis, we screened 20 primary cervical cancers for LOH of polymorphic markers within and flanking the PTEN gene, and for intragenic mutations in the entire coding region and exon-intron boundaries of the PTEN gene. LOH was observed in 7 of 19 (36.8%) cases. Further, one sample may have homozygous deletion. Three (15%) intragenic mutations were found: two were somatic missense mutations in exon 5, that encodes the phosphatase motif, and an occult germline intronic sequence variant in intron 7, that we show to be associated with aberrant splicing. All three samples with the mutations also had LOH of the wild-type allele. These data indicate that disruption of PTEN by allelic loss or mutation may contribute to tumorigenesis in cervical cancers. In cervical cancer, unlike some other human primary carcinomas, e.g., those of the breast and thyroid, biallelic structural PTEN defects seem necessary for carcinogenesis. Further, one in 20 unselected cervical carcinomas was found to have a germline PTEN mutation; it is unclear whether the patient with this mutation had Cowden disease or a related syndrome.     

Epigenetic PTEN silencing in malignant melanomas without PTEN mutation

A tumor suppressor gene at 10q 23.3, designated PTEN, encoding a dual specificity phosphatase with lipid and protein phosphatase activity, has been shown to play an important role in the pathogenesis of a variety of human cancers. Germline mutations in PTEN cause Cowden syndrome (CS), which is characterized by multiple hamartomas and a high risk of breast and thyroid cancers. Frequent loss of heterozygosity at 10q is found in both early and advanced-stage sporadic melanomas; however, mutations or deletions in PTEN are detected mainly in melanoma cell lines. In this study, we examined PTEN expression in 34 unselected sporadic melanomas (4 primary melanomas, 30 metastases) using immunohistochemistry and correlated this with the results of structural studies of this gene. Immunostaining of 34 melanoma samples revealed no PTEN expression in 5 (15%) and low PTEN expression in 17 (50%), whereas the rest of the tumors (35%) had high levels of expression. Hemizygous deletion was found in 32% of the tumors but neither intragenic PTEN mutation nor biallelic deletion was found in any of the samples. Of the 5 melanomas showing no PTEN expression, 4 had no mutation or deletion of PTEN. Of the 13 tumors having weak PTEN immunoreactivity and informative loss of heterozygosity results, 6 had evidence of hemizygous allelic loss of PTEN while the remaining 7 had intact PTEN. These results strongly support PTEN as a major tumor suppressor on 10q involved in melanoma tumorigenesis and suggest an epigenetic mechanism of biallelic functional inactivation not previously observed in other cancers where PTEN might be involved.     

10q23.3 loss of heterozygosity is higher in lymph node-positive (pT2-3,N+) versus lymph node-negative (pT2-3,N0) prostate cancer

Loss of heterozygosity (LOH) in the region of 10q23.3 has been associated with multiple tumors, including glioblastoma multiforme, melanoma, endometrial carcinoma, and prostate carcinoma. The tumor suppressor gene, PTEN/MMAC1, is also located in this region, and, in addition to other tumor types (eg, glioblastoma multiforme, endometrial, and melanoma), PTEN/MMAC1 mutations have been found in prostate cancer cell lines, xenografts, and hormone refractory prostate cancer tissue specimens. The aim of this study was to evaluate LOH at 10q23.3 as a marker of cancer progression in node-positive prostate cancer. Genetic alterations in the region of 10q23.3 were assessed in 23 node-positive (pT2-3, N+) and 44 node-negative prostate (pT2-3, N0) cancers with D10S532, D10S1687, D10S541, and D10S583 flanking polymorphic genetic markers; PTENCA, a genetic marker within PTEN/MMAC1, was also tested. Using DNA from paired normal and microdissected tumor samples, LOH at microsatellite loci was determined after polymerase chain reaction amplification. LOH in at least 1 marker was identified in 14% (6 of 44) of lymph node-negative and 43% (10 of 23) of lymph node-positive prostate cancers (chi-square test, P = .007). This increase in genetic alterations in node-positive prostate cancer suggests that 10q23.3 is a marker for metastatic progression.     

Promoter methylation of the PTEN gene is a common molecular change in breast cancer

About 25-50% of women with Cowden disease, a syndrome associated with germ-line mutations of the PTEN gene (at 10q23), develop breast cancer (BC), but PTEN mutations have been found in only 5% of sporadic BCs. However, 29-48% of BCs display loss of heterozygosity in 10q23, and about 40% of BCs show a decrease or absence of PTEN protein levels at the time of diagnosis. Promoter hypermethylation has been identified as an alternative mechanism of tumor-suppressor gene inactivation, but its importance in PTEN silencing in sporadic BC is unknown. We investigated PTEN promoter hypermethylation in 90 sporadic BCs and its correlations with 11 molecular and pathologic parameters, including mRNA levels of PTEN. The study, a methylation-specific PCR assay, was carried out with methylated specific primers designed in a region with scarce homology with the psiPTEN pseudogene. Expression was analyzed by real-time PCR. We found that the PTEN promoter was hypermethylated in 43 BCs (48%). PTEN hypermethylation was associated with ERBB2 overexpression, larger size, and higher histologic grade (P=0.012, 0.03, and 0.009, respectively). We concluded that PTEN promoter hypermethylation is a common event in sporadic BC, correlating with other well-established prognostic factors of this malignancy. Additionally, PTEN mRNA expression was lower in tumors with aberrant methylation.     

PTEN mutational spectra,expression levels,and subcellular localization in microsatellite stable and unstable colorectal cancers

PTEN on 10q23.3 encodes a dual-specificity phosphatase that negatively regulates the phosphoinositol-3-kinase/Akt pathway and mediates cell-cycle arrest and apoptosis. Germline PTEN mutations cause Cowden syndrome and a range of several different hamartoma-tumor syndromes. Hereditary nonpolyposis colon cancer (HNPCC) syndrome is characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. Although both colorectal carcinoma and endometrial carcinoma are the most frequent component cancers in HNPCC, only endometrial cancer has been shown to be a minor component of Cowden syndrome. We have demonstrated that somatic inactivation of PTEN is involved in both sporadic endometrial cancers and HNPCC-related endometrial cancers but with different mutational spectra and different relationships to MSI. In the current study, we sought to determine the relationship of PTEN mutation, 10q23 loss of heterozygosity, PTEN expression, and MSI status in colorectal cancers (CRCs). Among 11 HNPCC CRCs, 32 MSI+ sporadic cancers, and 39 MSI- tumors, loss of heterozygosity at 10q23.3 was found in 0%, 8%, and 19%, respectively. Somatic mutations were found in 18% (2 of 11) of the HNPCC CRCs and 13% (4 of 32) of the MSI+ sporadic tumors, but not in MSI- cancers (P = 0.015). All somatic mutations occurred in the two 6(A) coding mononucleotide tracts in PTEN, suggestive of the etiological role of the deficient MMR. Immunohistochemical analysis revealed 31% (14 of 45) of the HNPCC CRCs and 41% (9 of 22) of the MSI+ sporadic tumors with absent or depressed PTEN expression. Approximately 17% (4 of 23) of the MSI- CRCs had decreased PTEN expression, and no MSI- tumor had complete loss of PTEN expression. Among the five HNPCC or MSI+ sporadic CRCs carrying frameshift somatic mutations with immunohistochemistry data, three had lost all PTEN expression, one showed weak PTEN expression levels, and one had mixed tumor cell populations with weak and moderate expression levels. These results suggest that PTEN frameshift mutations in HNPCC and sporadic MSI+ tumors are a consequence of mismatch repair deficiency. Further, hemizygous deletions in MSI- CRCs lead to loss or reduction of PTEN protein levels and contribute to tumor progression. Finally, our data also suggest that epigenetic inactivation of PTEN, including differential subcellular compartmentalization, occurs in CRCs.     

Genetic alterations in pediatric high-grade astrocytomas

High-grade astrocytomas are tumors that are uncommon in children. Relatively few studies have been performed on their molecular properties and so it is not certain whether they follow different genetic pathways from those described in adult diffuse astrocytomas. In this study, we evaluated 24 pediatric high-grade astrocytomas (11 anaplastic astrocytomas and 13 glioblastomas) all of which were sporadic and primary. We studied mutations of p53, phosphatase and tensin homolog (PTEN), loss of heterozygosity (LOH) of chromosomes 17p13, 9p21 and 10q23-25, amplification of epidermal growth factor receptor (EGFR), and overexpression of EGFR and p53 protein. In addition, we searched for microsatellite instability (MSI) by using MSI sensitive and specific microsatellite markers. p53 mutations were found in 38% (9/24) of the high-grade astrocytomas and all brain stem tumors except 2 (71%, 5/7) had p53 mutations. PTEN mutations were found in 8% (2/24) of high-grade astrocytomas. However, no EGFR amplification was found in any of them. LOH was found at 17p13.1 in 50% (3/6 informative tumors), 9p21 in 83% (5/6 informative tumors), and 10q23-25 in 78% (7/9 informative tumors). Four tumors showed MSI, and 2 of them that showed widespread MSI were regarded as tumors with replication error (RER+) phenotype. All 4 tumors with MSI showed concurrent LOH of 9p21 and 10q23-25. Combining gene alterations, LOH, MSI, and gene mutations, inactivation of both alleles of PTEN and p53 was found in 57% (4/7 informative tumors) and 50% (3/6 informative tumors) of the cases respectively. We conclude that development of pediatric high-grade astrocytomas may follow pathways different from the primary or secondary paradigm of adult glioblastomas. In a subset of these tumors, genomic instability was also implicated.