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.     

乳腺癌和导管增生病变与1p染色体不稳定的关系

目的检测乳腺普通型导管增生(usual dutal hyperplasia,UDH)、乳腺不典型增生(atypical dutal hyperplasia,ADH)、乳腺导管原位癌(dutal carcinoma in situ,DCIS)及乳腺浸润性导管癌(invasive dutal carcinoma,IDC)中染色体1p微卫星杂合性缺失(loss of heterozygosity,LOH)/微卫星不稳定(microsatellite instability,MSI)的改变规律,探讨乳腺导管增生病变进展为IDC的遗传学改变特征。方法应用PCR法检测20例UDH、7例ADH、25例DCIS、25例IDC石蜡包埋组织,8%尿素变性聚丙烯酰胺凝胶电泳后银染检测染色体1p区间微卫星位点D1S193、D1S463、D1S2881、D1S2885、D1S234、D1S252的LOH/MSI。结果染色体1p的每个位点均发生LOH/MSI,其发生的频率UDH为55.0%、ADH为42.9%、DCIS为64.0%、IDC为72.0%,各组间差异无显著性(P0.05);微卫星位点D1S193、D1S2881、D1S2885、D1S252、D1S234在各组中LOH/MSI的频率差异均无显著性(P0.05)。D1S463的频率在UDH组为5.0%、ADH组为0、DCIS组为45.0%、IDC组为40.0%,其中UDH组与ADH组明显低于DCIS组、IDC组(P0.008)。结论 1p染色体遗传学不稳定是乳腺癌发生的早期事件,D1S463位点的改变可能在乳腺导管增生性病变进展为乳腺癌中起关键作用。     

头颈部鳞癌的微卫星不稳定性和杂合性丢失的初步研究

目的:探讨头颈部鳞癌的微卫星不稳定性(MSI)及杂合性丢失(LOH)。方法:选择来自3、5、6、8、9、13、17和18号染色体的15个微卫星标志对36例头颈部鳞癌标本和相应的外周血进行微卫星分析。结果:36例头颈部鳞癌中,27.8%(10/36)分别有1-8个位点存在MSI,MSI发生率较高的位点为:D17S520(22.9%)、D6S105(16.7%)和D8S264(13.9%)。在9p21-p22和3p14等处存在一定的LOH。微卫星异常的检出率与肿瘤分期、分级无相关性。结论:提示MSI是头颈部鳞癌中较为常见的遗传学变化,染色体9p21-p22和3p14区域可能存在与头颈部鳞癌有关的抑癌基因。     

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     

人肝癌P57~(kip2)基因失表达的遗传不稳定性

目的探讨肝细胞癌(HCC)P57kip2mRNA表达与遗传不稳定性之间的相互关系,以探索P57kip2基因失表达的机制。方法用原位分子杂交技术检测肝癌组织中P57kip2mRNA表达,用PCR-聚丙烯酰胺凝胶电泳-银染法检测LOH和MSI。结果在正常肝组织未见P57kip2mRNA表达,癌周肝硬化组与肝癌组阳性表达率均为26.7%(8/30)。3个位点均未发生LOH;在2个微卫星位点发生MSI,MSI总阳性率为16.7%。D11S1760位点发生MSI与P57kip2mRNA表达有关联性(P<0.05)。结论P57kip2mRNA表达异常提示其可能在HCC发生过程中起重要作用。MSI可能是肝癌发生过程中P57kip2mRNA表达异常的原因之一。     

内源性端粒酶抑制基因遗传不稳定性与胃癌进展的关系

目的 研究8号染色体上D8S277位点的杂合性缺失(LOH)和微卫星不稳定性(MSI)对内源性端粒酶抑制基因(PINX1)蛋白表达的影响,阐明PINX1基因遗传不稳定性与胃癌进展的关系. 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D8S277位点的LOH和MSI,采用Envision免疫组织化学染色和Leica-Qwin计算机图像分析等方法. 结果 D8S277位点的LOH发生率在淋巴结转移组(21.15%)明显高于无淋巴结转移组(0,P<0.05);在胃癌TNM Ⅲ Ⅳ期(24.39%)显著高于Ⅰ Ⅱ期(3.33%,P<0.05).PINX1蛋白阳性率在无淋巴结转移组(78.95%)明显高于有淋巴结转移组(48.08%,P<0.05);TNM分期Ⅰ Ⅱ期(73.33%)明显高于Ⅲ Ⅳ期(43.90%,P<0.05);蛋白表达阴性组LOH阳性率为32.28%,明显高于蛋白阳性组的2.50%(P<0.01). 结论 PINX1基因的遗传不稳定性可能导致该抑癌基因突变,是肿瘤发生发展的一个因素,LOH和MSI通过不同的途径调控胃癌的发生和发展.     

HLA-Ⅰ基因微卫星变异与宫颈癌相关性的分析

目的　对宫颈癌组织进行HLA Ⅰ基因微卫星变异的分析与作图。方法　采用 8个位于HLA Ⅰ区域的微卫星多态性标记分析 30例宫颈癌活检标本微卫星变异的情况。结果　微卫星变异总的发生率为86 7% (2 6 / 30 ) ,微卫星位点C32 11的杂合性缺失频率最高 ,可达 5 0 0 % (15 / 30 ) ,并发现了一个微小缺失区域 ;位点D6S2 5 8的MSI频率最高 ,达 4 0 0 % (12 / 30 )。结论　HLA Ⅰ基因微卫星变异在宫颈癌的发生和发展过程中起着重要作用 ,位点C12 5～C32 11之间的微小缺失区域可能存在有宫颈癌相关抑癌基因。     

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。     

原发性胃癌中19p部分微卫星多态位点杂合性缺失分析

目的 筛选胃癌19p部分微卫星多态位点的杂合性缺失（loss of heterozygosite,LOH)频率,以初步确定19p上与胃癌相关基因连锁最密切的微卫星多态位点。方法 采用聚合酶链反应-单链长度多态（polymerase chain reaction-single strand length polymorophism,PCR-SSLP)-银染法选取19p上9对微卫星多态标记（D19S424,D19S216,D19S406,D19S413。D19S221,D19S226,D19S411,D19S883,D19S886）,对43例原发性胃癌的杂合性缺失情况进行了分析。结果 43例中22例至少在1个位点发生LOH,总缺失率为48.88%,这9个位点的LOH频率分别为29.63%,11.53%,33.33%,8.57%,13.15%,8.00%,6.45%,6.89%,10.71%,在D19S886也同时出现微卫星不稳定性（microsatellite instability,MSI)17.85%。结论 提示19p上的LOH缺失频发区域可能涉及与人类原发性胃癌发生发展相关基因的存在。     

鼻咽癌染色体1pter-p36.11杂合性缺失

目的构建鼻咽癌染色体1pter-p36.11(63.4cM)区域内的杂合性缺失精细图谱,为进一步寻找鼻咽癌相关基因提供依据。方法在比较基因组杂交和间期荧光原位杂交研究基础上,应用淋巴分离液将肿瘤组织的肿瘤细胞与淋巴细胞分离并以淋巴细胞DNA作相应正常对照,利用1pter-p36.11区域内的20个微卫星多态位点(平均间距3cM)对47例鼻咽癌活检组织用杂合性缺失(LOH)分析法进行LOH分析并绘制其精细缺失图谱。结果在47例鼻咽癌患者中,至少有一个位点存在LOH的有37例(82.2%),其中LOH频率最高的为D1S234(50.0%),位于1p36.13,其次为D1S2644(37.5%),位于1p36.22;微卫星不稳定频率最高的为D1S243(37.5%),位于1p36.33,其次为D1S199(30.2%),位于1p36.21;D1S234的LOH及D1S199的MI与临床分期相关无显著性意义（P>0.05）。结论鼻咽癌染色体1pter-p36.11之间63.4cM区域内有两个共同的缺失区,分别位于1p36.13(D1S234,2.0cM)与1p36.22(D1S436-D1S2644,6.3cM),其间有一个微卫星不稳定位点D1S199,D1S436-D1S199-D1S234区域内可能有一个或几个在早期与鼻咽癌形成相关的肿瘤抑制基因。     

Allelic alterations in nontumorous liver tissues and corresponding hepatocellular carcinomas from chinese patients

Allelic imbalance may play an important in tumor progression in hepatocarcinogenesis, but the genetic background of the corresponding nontumorous liver in hepatocellular carcinoma (HCC) is not well defined. We studied the incidence of loss of heterozygosity (LOH) and microsatellite instability (MSI) by microsatellite analysis in both nontumorous livers and the corresponding tumors, by comparing them with the normal DNA from Chinese patients with resected primary HCCs. We also evaluated the pathologic significance of the alterations. We used 18 highly polymorphic microsatellite markers on chromosomes 1, 3, 4, 7, 8, 9, 13, 16, 17, and 18. Our results showed that 70.6% (24 of 34) of the HCCs exhibited LOH at 1 or more loci, and that the overall fractional allelic loss (FAL) was 0.169. MSI was observed in only 1 tumor. In contrast, the nontumorous livers of the HCCs showed a very low incidence of LOH, with only a single LOH detected in 1 of 34 (2.9%) of the nontumorous livers, with an overall FAL index of 0.005. Tumors with LOH at 1 or more loci had significantly more frequent venous invasion (P = 0.019). Allelic loss at locus D9S199 (9p23) was seen more frequently in larger tumors (P = 0.031), and, less significantly, allelic loss at locus D16S516 (16q24.1) was seen more frequently in larger tumors (P = 0.059). LOH was common in predominantly hepatitis B virus-associated HCCs from Chinese patients. However, LOH or MSI in the corresponding cirrhotic or noncirrhotic livers was uncommon.     

肝细胞癌nm23H1基因遗传不稳定性与临床病理特征的相关性研究

目的： 研究人类17号染色体D17S396位点微卫星不稳定性和杂合性缺失，对肝细胞癌nm23H1蛋白表达的影响，阐明nm23H1基因遗传不稳定性与肝细胞癌及临床病理特征的关系，为揭示nm23H1基因作用机制和肿瘤发生、转移机理提供实验依据。方法： 采用石蜡包埋组织抽提DNA，PCR-单链构象多态性（PCR-SSCP），常规银染，Envision免疫组织化学和Leica-Qwin计算机图像分析等方法进行nm23H1基因遗传不稳定性研究。 结果： ① 48例肝细胞癌D17S396位点遗传不稳定性的发生率为35.42%。LOH的发生率在有无淋巴结或远处转移和有无肝内转移或门静脉栓的癌组织中有显著差异（P<0.01），临床TNM分期Ⅲ期LOH的发生率显著高于Ⅰ、Ⅱ期（P<0.01）。此外，在侵袭转移高危组LOH的发生率显著高于侵袭转移低危组 (P<0.01)。MSI检出率与肝细胞癌临床病理参数均无关。② nm23H1蛋白阳性率为56.25%，nm23H1蛋白阳性率在Edmondson分级Ⅲ +Ⅳ组低于Ⅰ+ Ⅱ组（P<0.01） ，在有淋巴或远处转移组显著低于无淋巴或远处转移组（P<0.01）,TNM分期Ⅲ期显著低于Ⅰ+Ⅱ期（P<0.01）；在侵袭转移高危组中nm23H1蛋白阳性率显著低于侵袭转移低危组 (P<0.01)。此外，计算机图像定量分析显示，在各临床病理参数影响下，nm23H1蛋白的表达强度没有差异。③ LOH阳性组中nm23H1蛋白阳性率为27.27％，显著低于LOH阴性组64.86％，两者差异显著（P<0.05）。结论： nm23H1基因的MSI和LOH通过相互独立的途径调控肝细胞癌的发生和转移，后者可抑制肝细胞癌局部nm23H1蛋白的表达，并赋予肝细胞癌高转移、预后差的特性。提高肝细胞癌局部nm23H1蛋白的表达，可减缓肿瘤的侵袭转移倾向，并改善预后。     

肝细胞癌肿瘤抑制基因的杂合性缺失和微卫星不稳定性研究

目的　了解肿瘤抑制基因(TSG)杂合性缺失(LOH)与微卫星不稳定性(MSI)在肝细胞癌发生机制中的作用,并探讨其临床病理学意义。方法　采用显微组织切割基础上的DNA直接测序法,从92例手术切除肝细胞癌中筛选出36例信息性肝细胞癌进行6种TSG(APC、DCC、MCC、OGG1、p53和RB1)的LOH检测,对其中15例肝细胞癌进行13个多态性微卫星位点的LOH和MSI检测,并与临床病理学参数的相关性进行统计学分析。结果　TSG的LOH总发生率为41 .7% (15 /36),仅MCC基因未出现LOH。15例肝细胞癌中有9例(60% )发生微卫星LOH,占检测微卫星的46 .2% (6 /13),但无1例肝细胞癌出现MSI。若将APC、OGG1和DCC基因LOH作为Ⅰ型(n=7 ),将p53和RB1基因LOH作为Ⅱ型(n=8)进行统计学处理,则两组肝细胞癌的平均瘤体直径分别为( 2. 9 ±1 .7)cm和(7 .2 ±3 .4)cm (P<0 .01),两组患者术后平均生存期分别为( 72. 0 ±38 6 )个月和(51 .0±30. 4)个月(P<0 .05 )。肝细胞癌基因变异型与患者的年龄、性别、血清甲胎蛋白水平、HBsAg阳性率、合并肝炎/肝硬化、肝细胞癌分化程度和组织学类型之间无明显相关性。结论　在肝细胞癌发生的多阶段演进与多基因变异过程中,LOH路径所起的作用要比MSI路径更大。Ⅰ型基因变异(APC、OGG1和DCC)主要在肝细胞癌早期阶段起作     

Molecular characterization of undifferentiated-type gastric carcinoma

As the great majority of gastric cancers develop histologically differentiated, and a significant proportion of differentiated-type carcinomas progress to become undifferentiated, both histological types are likely to share several common genetic abnormalities, such as p53 mutations at advanced stages. However, a subset of gastric cancers develop as undifferentiated carcinomas, including signet-ring cell carcinoma and poorly differentiated adenocarcinoma, and the molecular pathogenesis of this tumor type remains largely unknown. To characterize the molecular features of undifferentiated-type gastric carcinomas that developed as undifferentiated-type, we examined for p53, APC, and epithelial (E)-cadherin gene mutations, microsatellite alterations including loss of heterozygosity (LOH) and microsatellite instability (MSI), and hypermethylation of the E-cadherin gene promoter in 26 early undifferentiated gastric carcinomas, consisting of 14 signet-ring cell carcinomas and 12 poorly differentiated adenocarcinomas. E-cadherin expression was evaluated immunohistochemically. p53 mutations were detected in only one poorly differentiated adenocarcinoma sample (3.8%; 1/26), whereas no APC or E-cadherin mutations were found. LOH was present only at D8S261 on the short arm of chromosome 8 in 2 of 14 (14%) informative tumors, both of which were poorly differentiated adenocarcinomas, and MSI was not observed in any of the tumors. No signet-ring cell carcinomas have been found to carry gene mutations or microsatellite alterations. In contrast, hypermethylation of the E-cadherin promoter occurred in 69% (18/26) of the tumors; 57% (8/14) of signet-ring cell carcinomas, and 83% (10/12) of poorly differentiated adenocarcinomas, and was significantly associated with loss or reduced expression of E-cadherin. Thus, whereas tumor suppressor gene mutation, LOH, and MSI were less common in undifferentiated-type early gastric carcinomas, epigenetic inactivation of E-cadherin via promoter hypermethylation may be an early critical event in the development of undifferentiated tumors.     

Involvement of genetic instability in the downregulation of sFRP1 in Chinese patients with hepatocellular carcinoma

Secreted frizzled‐related protein 1 (sFRP1) is a new tumor suppressor based on recent researches, but the correlation of the genetic instability of sFRP1 gene with the clinicopathologic features of the hepatocellular carcinoma (HCC) has not been studied in Chinese people. In this study, 42 pairs of paraffin‐embedded HCC and adjacent non‐carcinoma tissues were examined for the loss of heterozygosity (LOH) and microsatellite instability (MSI) of two microsatellite markers D8S532 and D8S1722 located in the vicinity of the sFRP1 gene. Envision immunohistochemistry was used to assess the expression of sFRP1. We found that the reduced expression of the sFRP1 protein was frequently observed in Chinese patients with HCC, which may at least partially result from the genetic instability, especially LOH. The LOH‐associated sFRP1 downregulation may play an important role in the development of HCC. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Microsatellite instability is uncommon in lymphoepithelioma-like carcinoma of the lung

Primary lymphoepithelioma-like carcinoma of the lung (LELC) shares some morphologic and clinical characteristics with malignancies associated with microsatellite instability (MSI). The aims of our study were to determine the MSI status in LELC and compare these findings with stage I non-small cell lung carcinoma (NSCLC) with marked lymphocytic host response (MLHR). We assessed MSI by a DNA-based polymerase chain reaction assay using mononucleotide (BAT25 and BAT26) and dinucleotide (D2S123, D5S346, and D17S250) repeats. MSI was detected in 2 (29%) of 7 LELC cases with only 1 marker (D17S250), and in 3 (19%) of 16 NSCLC cases with MLHR with only 2 markers (1D2S123 and 2 D17S250). Loss of heterozygosity (LOH) was detected at 1 or 2 of 3 dinucleotide repeats in 11 NSCLC cases (69%) with MLHR and 3 LELC cases (43%) (P = .36). The overall frequencies of LOH in NSCLC with MLHR were 29% and 19% in LELC (P = .55). MSI is very uncommon in LELC, indicating that MSI is not an important event in carcinogenesis for this tumor subtype. The presence of LOH suggests a probable role of tumor suppressor genes in LELC carcinogenesis.     

Detection of microsatellite instability by real time PCR and hybridization probe melting point analysis

Microsatellite alterations can be found in a number of tumors. There are two types of alterations: loss of heterozygosity (LOH), which can be detected in the majority of colorectal cancers (CRC), and microsatellite instability (MSI). MSI occurs in about 15% of CRC with a mutator phenotype and are the hallmark of hereditary nonpolyposis colorectal cancers (HNPCC). Furthermore, MSI can also be detected in other tumors which are part of the HNPCC tumor spectrum (eg, gastric, ovarian, and endometrial carcinomas). Usually, a set of microsatellite markers is amplified by PCR followed by gel or capillary electrophoresis to separate PCR amplicons and by detection of the markers using autoradiography (Thibodeau et al, 1993), silver staining (Schlegel et al, 1996), or fluorescence techniques (Gyapay et al, 1996; Mansfield et al, 1994). We have established a technique to detect MSI by LightCycler PCR and melting point analysis using sequence-specific hybridization probes (HyProbes) labeled with LightCycler dyes, LCRed640 and LCRed705. Amplification of microsatellites by real-time PCR is followed by melting point analysis to display alterations in the length of repetitive sequences, thereby avoiding any electrophoretical separation of amplified DNA. Two mononucleotide markers (BAT25 and BAT26) were tested in 81 formalin-fixed and paraffin-embedded colorectal cancer samples with matched normal tissues from 21 MSI tumors and 60 tumors with microsatellite stability. Amplification and melting point determination of BAT26 and BAT25 was possible in 129/162 (80%) and 123/162 (76%) formalin-fixed and paraffin-embedded tissue samples, respectively. MSI could be detected specifically with both BAT25 and BAT26 markers only in MSI-high tumors (> or =40% MSI rate, determined with microsatellite reference panel, BAT25, BAT26, D5S346, D2S123, D17S250; Boland et al, 1998; Dietmaier et al, 1997). This new technique allows MSI detection within less than a hour and provides a basis for fast, high-throughput MSI analysis.