胃癌7号染色体长臂的杂合性缺失分析

目的:检测胃癌患者7号染色体长臂微卫星位点的杂合性缺失(loss of heterozygosity,LOH),以初步确定7号染色体长臂上与胃癌相关基因连锁最密切的微卫星多态位点及LOH的临床意义.方法:在70例原发性胃癌中应用多重PCR技术扩增覆盖整个7号染色体长臂的9个微卫星位点(平均遗传距离为10cm),聚丙烯酰胺凝胶电泳分离PCR产物,用GeneScan、Genotyper软件进行分析.结果:9个微卫星位点的LOH均可发生于原发性胃癌,总的LOH频率为34.3%(24/70),其中D7S486和D7S798位点的LOH频率较高,分别为24.0%(12/50)和19.2%(5/26);总的LOH频率随临床分期而显著增高(P=0.046),D7S486位点的LOH频率在淋巴结转移者显著高于无淋巴结转移者(P=0.015).结论:在7号染色体长臂D7S486和D7S798位点附近,可能存在与胃癌发展相关的抑癌基因.     

Allelic loss on chromosome 17 in human ovarian cancer

In order to identify a common region of deletion on chromosome 17 potentially containing a tumor-suppressor gene, 27 ovarian carcinomas and 3 ovarian tumors of low malignant potential (LMP) were examined for loss of heterozygosity (LOH) at 6 p arm and 10 q arm loci. Ninety percent of all tumors had deletions at one or more loci. On the p arm, there was a single near-common region of deletion on 17p 13.3 (D/7S30/ pYNZ22.1; 86% LOH), an intervening locus with a low LOH rate, and a more proximal locus on 17p11.2 (D/7S58/pEW301; 82% LOH) with a high LOH rate. In less aggressive tumors, LOH at Df 7S30 was not accompanied by LOH at p53. The q arm had a common region of deletion for high-stage carcinoma at D/7S579 (Mfd 188; 74% LOH) on q21, a locus tightly linked to the familial breast-ovarian-cancer syndrome (BRCAI) locus. D/7S579 was lost in all informative high-stage carcinomas and retained in all low-stage carcinomas and tumors of LMP. There may be at least 2 tumor-suppressor genes, an early-acting gene on the p arm and a gene on the q arm involved in tumor progression and metastasis.     

原发性胃癌染色体7q31的最小共同缺失区域及其意义

背景与目的:先前的研究显示7号染色体长臂(7q)在原发性胃癌有高频缺失;位于7q31的D7S486是7q上最高频的杂合性缺失(lossofheterozygosity,LOH)位点,且该位点的LOH频率与肿瘤的淋巴结转移显著相关;推测D7S486位点附近可能存在胃癌相关的抑癌基因(tumorsuppressorgene,TSG)。为能在更小的区域内找寻胃癌相关的TSGs,本研究通过检测原发性胃癌在7q31区域内微卫星标记位点的LOH情况,确定胃癌的最小共同缺失区域,并分析它们在胃癌发病中的可能作用。方法:以D7S486位点为中心,在位于其上下的7q31区域内选取平均遗传距离约0.5厘摩(centimorgan,cM)的12个微卫星标记。显微切割78例原发性胃癌和相应的正常胃粘膜组织,分别提取DNA;进行多重PCR扩增,聚丙烯酰胺凝胶电泳分离PCR产物,以GeneScan、Genotyper软件分析各微卫星位点的LOH情况。根据LOH结果作图,确定胃癌在7q31内的最小共同缺失区域,并与临床病理指标联系,分析区域缺失在胃癌发病中的可能作用。结果:12个微卫星标记位点均可在原发性胃癌中出现LOH,总的LOH频率为41.7%(40/72)。LOH的最高频位点是D7S486(位于7q31.2),为30.4.%(17/56);次高频位点是D7S650(位于7q31.3),为21.1%(8/38)。原发性胃癌在7q31内有两个最小共同缺失区域,分别为D7S2543～D7S486和D7S480～D7S650(长度均约为90kb)。D7S2543～D7S486区域缺失的频率与胃癌患者的临床分期和淋巴结转移显著相关(P=0.01和P=0.03);D7S480～D7S650区域缺失的频率与胃癌患者的临床分期显著相关(P=0.03),且该区域缺失仅出现于临床Ⅲ/Ⅳ期、T3/T4期或淋巴结转移的患者。结论:原发性胃癌在染色体7q31上存在两个最小共同缺失区域,分别为D7S2543～D7S486和D7S480～D7S650;在这两个区域内可能存在胃癌发展密切相关的抑癌基因。     

Frequent loss of heterozygosity on chromosomes 1q, 5q, and 17p in human gastric carcinomas

Recently, loss or inactivation of genes at specific chromosomal loci has been considered to be one of the important mechanisms during the development of human tumors. In order to identify tumor suppressor genes for gastric carcinoma, we performed restriction fragment length polymorphism analysis on 48 human gastric carcinomas. Allele losses were investigated for 14 specific loci on chromosomes 1, 5, 6, 7, 10, 11, 12, and 17. Loss of heterozygosity on chromosome 17p13.1 (p53 locus) was detected in 13 (68%) of 19 informative cases. Well-differentiated adenocarcinoma showed high frequencies of allele losses on chromosomes 5q (60%) and 17p (67%) in early cancers and on chromosomes 1q (67%), 5q (36%), 7p (33%), 7q (39%), and 17p (73%) in advanced cancers. In poorly differentiated adenocarcinomas, loss of heterozygosity was detected on chromosomes 1p (38%), 12q (31%), and 17p (60%). Allele losses on chromosomes 1q, 5q, and 7p were not detected in poorly differentiated adenocarcinoma, their frequencies being significantly different between the two histological types. These results suggest that allele loss on chromosome 17p is a common event in gastric carcinoma, regardless of histological type, and that allele loss on chromosome 5q may play a role in the carcinogenesis of well-differentiated adenocarcinoma. Additionally, allele losses on chromosomes 1q and 7p may be involved in the progression of well-differentiated adenocarcinoma.     

Loss of heterozygosity analysis on the long arm of chromosome 7 in gastric carcinoma

Objective In order to defne the common deleted region related to primary gastric carcinomas in Chinese, the frequency of loss of heterozygosit (LOH) on human chromosome 7q and its clinicai significance were investigated. Methods A set of 9 microsatellite markers on 7q with an average genetic distance of l0cM were used to identify LOH by muiti -PCR amplification of matched tumor and non-tumor DNAs from 70 patients with primary gastric carcinoma. The PCR products were separated by electrophoresis in poiyacrylamide gels and analysed for LOH by using Genescan and Genotyper software. Results The total frequency of LOH at any iocus on 7q was 34.3% (24/ 70) in the tumors. Compared fo non -tumor DNA, LOH at D7S486 and D7S798 loci were higher, 24.0% (12/50) and 19.2% (5/26), respectively The total frequency of LOH on 7q was markedly higher with an increase in the clinical stage (P<0.05). The frequency of LOH at D7S486 in cases with lymph node metastasis was significanty higher than in cases without lymph node metastasis,P=0.015. Conclusion The higher incidence of LOH at O7S486 and D7S798 in primary gastric carcinoma compared to normal tissue suggests that the potential tumor suppressor genes (TSGs) involved in the progression of gastric carcinoma might be nearby these 2 locl. This work was supported by the National Natureal Science Foundation of China. (No.30070840)     

胃癌及癌前病变组织染色体7q31.1杂合性缺失的意义

目的 检测胃癌及不典型增生细胞染色体7q31.1区域的杂合性缺失（LOH）,绘制胃癌及癌前病变7q31.1区域等位基因缺失图谱,确定其常见最小缺失区域,探索胃黏膜上皮癌变过程不同阶段的分子遗传学改变。方法 在胃癌及胃黏膜组织石蜡切片上行显微切割,获得胃癌及胃黏膜上皮不典型增生细胞。用高密度微卫星标志结合PCR技术检测胃癌及癌前病变细胞染色体7q31.1杂合性缺失,绘制胃癌及癌前病变染色体7q31.1等位基因的缺失图谱。结果 发现胃癌染色体7q31.1至少有一个位点存在杂合性缺失的21例,占70.0%（21/30）;D7S2459、D7S523、D7S2502、D7S486、D7S480、D7S650、D7S2486各位点杂合性缺失频率分别为10.0%、6.7%、23.3%、43.3%、26.7%、26.7%、20.0%;缺失图谱分析显示胃癌常见最小缺失区域位于D7S2502~D7S480之间。在胃黏膜不典型增生组织中,至少一个位点等位基因缺失的11例,占36.7%（11/30）,其中缺失频率最高的微卫星位点是D7S480为23.3%(7/30);不同程度胃黏膜不典型增生患者中染色体7q31.1 LOH阳性率比较差别有统计学意义（P<0.01）。结论 胃癌染色体7q31.1常见最小缺失区域在D7S2502~D7S480之间,在D7S486附近可能存在与胃癌相关的抑癌基因。在胃黏膜癌前病变阶段（不典型增生）可检测出染色体7q31.1区域的杂合性缺失,7q31.1 LOH可能是胃癌发生极早期的分子事件之一。     

Primary Gastric Carcinoma Cells Frequently Lose Heterozygosity at the APC and MCC Genetic Loci

Loss of heterozygosity (LOH) at APC and MCC gene loci (both mapped to 5q21) was investigated in 24 surgical specimens of primary gastric carcinomas using the polymerase chain reaction after tumor cell enrichment by cell sorting based on differences in DNA content. LOH at APC and/or MCC was detected in 87% (13/15) of the cases; at the APC in 86% (12/14) and at the MCC locus in 100% (7/7). LOH at the APC locus was always accompanied by LOH at the MCC locus. LOH at the APC and/or MCC was found in both differentiated and undifferentiated types in both early and advanced stages of gastric carcinoma. Thus, LOH at APC and/or MCC is considered to be one of the most prevalent genetic alterations in human gastric carcinoma and occurs at an early stage of the carcinogenesis.     

中国人胃癌组织微卫星DNA的杂合性缺失研究

目的从多位点筛选中国人胃癌的杂合性缺失（ＬＯＨ）频率,以初步确定与胃癌的致病基因连锁最密切的ＭＳｍａｒｋｅｒ。方法选择了３０个多态微卫星标记,采用ＰＣＲ、聚丙烯酰胺凝胶电泳及银染技术,对４２例胃癌组织进行了ＬＯＨ分析。结果在２９个位点上检测到总的ＬＯＨ频率为２７．７％。高频率的ＬＯＨ主要集中在Ｄ３Ｓ１５７７、Ｄ３Ｓ１０６７、Ｄ６Ｓ４３０、Ｄ１５Ｓ２０５、Ｄ１７Ｓ９４５等位点。ＬＯＨ频率均在４０％以上。其次是Ｄ５Ｓ４０７、Ｄ２Ｓ１３６、Ｄ２Ｓ１４７、Ｄ１３Ｓ１７５、Ｄ１４Ｓ６８、Ｄ８Ｓ２７９,其ＬＯＨ频率均在３０％以上。不同病理分型的胃癌,ＬＯＨ频率存在明显差异。印戒细胞癌的ＬＯＨ明显高于低分化胃癌（Ｐ＜０．０１）;低分化胃癌的ＬＯＨ频率亦明显高于高分化胃癌（Ｐ＜０．０１）。４例高分化胃癌均未发现ＬＯＨ。结论高频率的ＬＯＨ位点附近,除了已克隆的癌基因、抑癌基因外,可能还含有未知的胃癌易感基因。这一结果表明,恶性程度高的胃癌涉及更多的等位基因丢失     

Loss of Heterozygosity Analysis on the Long Arm of Chromosome 7 in Gastric Carcinoma

Objective  In order to defne the common deleted region related to primary gastric carcinomas in Chinese, the frequency of loss of heterozygosit (LOH) on human chromosome 7q and its clinicai significance were investigated. Methods  A set of 9 microsatellite markers on 7q with an average genetic distance of l0cM were used to identify LOH by muiti -PCR amplification of matched tumor and non-tumor DNAs from 70 patients with primary gastric carcinoma. The PCR products were separated by electrophoresis in poiyacrylamide gels and analysed for LOH by using Genescan and Genotyper software. Results  The total frequency of LOH at any iocus on 7q was 34.3% (24/ 70) in the tumors. Compared fo non -tumor DNA, LOH at D7S486 and D7S798 loci were higher, 24.0% (12/50) and 19.2% (5/26), respectively The total frequency of LOH on 7q was markedly higher with an increase in the clinical stage (P<0.05). The frequency of LOH at D7S486 in cases with lymph node metastasis was significanty higher than in cases without lymph node metastasis,P=0.015. Conclusion  The higher incidence of LOH at O7S486 and D7S798 in primary gastric carcinoma compared to normal tissue suggests that the potential tumor suppressor genes (TSGs) involved in the progression of gastric carcinoma might be nearby these 2 locl. This work was supported by the National Natureal Science Foundation of China. (No.30070840)     

Genetic heterogeneity of surgically resected prostate carcinomas and their biopsy specimens is related to their histologic differentiation

BACKGROUND: In human prostate carcinogenesis, many genetic analyses including conventional loss of heterozygosity (LOH) studies and microsatellite LOH analyses using the polymerase chain reaction method have revealed frequent LOH events at specific regions on chromosomes 3p, 7q, 8p, 10q, 16q, 17q, and 18q. METHODS: Using the laser-captured microdissection method, the authors extracted genomic DNA from 23 cases of prostate carcinomas including 59 different lesions and 8 biopsy specimens. Using (32)P-labeled primers, the authors analyzed six microsatellite loci (D3S647, D3S1228, D7S522, D8S137, NEFL, and D10S190) at which frequent LOH events have been reported. RESULTS: Of 10 cases in which the authors found LOH at any of the loci, 8 cases showed a heterogeneous LOH pattern. In four cases, the authors also found replication error (RER) at some of the loci examined. There was no significant relation between histologic differentiation and frequency of LOH or RER events. The overall LOH rate was found to be significantly lower in foci at classification pT2 (1 of 28 foci, 3%) compared with those at classification pT3 (13 of 44 foci, 30%). In pT3 samples, LOH events in extraglandular foci (9 of 23 foci, 39%) tended to be more frequent compared with those in intraglandular foci (8 of 41 foci, 20%). The patterns of LOH events in biopsy specimens correlated well with those in foci from surgical material showing the same histologic characteristics. CONCLUSIONS: Prostate carcinoma is a genetically multicentric carcinoma, and the genetic heterogeneity is well correlated with histologic differentiation. The frequency of LOH events increased according to the degree of tumor progression.     

Commonly deleted region on the long arm of chromosome 7 in differentiated adenocarcinoma of the stomach.

Loss of heterozygosity (LOH) at several chromosomal loci is a common event in human malignancies. Frequent LOH on the long arm of chromosome 7 has been reported in various human malignancies, and investigators have identified the most common site of LOH as 7q31.1. We have identified ten chromosomal loci, including chromosome 7q, that have been shown by previous allelotype study to be sites of frequent LOH in differentiated adenocarcinoma of the stomach. In the present study, we performed a polymerase chain reaction (PCR) microsatellite analysis to define the common deleted region on 7q, using 14 polymorphic microsatellite markers in matched tumour and non-tumour DNAs from 53 patients with primary gastric carcinoma of the differentiated type. LOH at any locus on 7q occurred in 34% (18 out of 53) of the tumours. Although many tumours exhibited total or large interstitial deletions, we determined the smallest common deleted region to be at D7S480 (7q31.1). This is identical to the region identified for other human malignancies. These observations indicate that a putative tumour suppressor gene at 7q31.1 may be involved in the pathogenesis of differentiated adenocarcinoma of the stomach.     

Genetic alterations on chromosome 16 and 17 are important features of ductal carcinoma in situ of the breast and are associated with histologic type

We analysed the involvement of known and putative tumour suppressor- and oncogene loci in ductal carcinoma in situ (DCIS) by microsatellite analysis (LOH), Southern blotting and comparative genomic hybridization (CGH). A total of 78 pure DCIS cases, classified histologically as well, intermediately and poorly differentiated, were examined for LOH with 76 markers dispersed along all chromosome arms. LOH on chromosome 17 was more frequent in poorly differentiated DCIS (70%) Compared to well-differentiated DCIS (17%), whereas loss on chromosome 16 was associated with well- and intermediately differentiated DCIS (66%). For a subset we have done Southern blot-and CGH analysis. C-erbB2/neu was amplified in 30% of poorly differentiated DCIS. No amplification was found of c-myc, mdm2, bek, flg and the epidermal growth factor (EGF)-receptor. By CGH, most frequent alterations in poorly differentiated DCIS were gains on 8q and 17q22-24 and deletion on 17p, whereas in well-differentiated DCIS amplification on chromosome 1q and deletion on 16q were found. In conclusion, our data indicates that inactivation of a yet unknown tumour suppressor gene on chromosome 16q is implicated in the development of most well and intermediately differentiated DCIS whereas amplification and inactivation of various genes on chromosome 17 are implicated in the development of poorly differentiated DCIS. Furthermore these data show that there is a genetic basis for the classification of DCIS in a well and poorly differentiated type and support the evidence of different genetic routes to develop a specific type of carcinoma in situ of the breast.     

Analysis of Genetic Alterations Related to the Development and Progression of Breast Carcinoma

To study genetic alterations related to the development and/or progression of breast carcinoma, we examined amplification of the ERBB2, INT2, and MYC genes, as well as loss of heterozygosity (LOH) at loci on 11p, 16q, 17p ( D17S5 and TP53 ), 17q ( D17S74 and NME1 ), and 18q by restriction fragment length polymorphism analysis. The subjects were 26 patients with small breast carcinomas (≦2 cm) and 88 patients with larger breast carcinomas (2 to < 5 cm). All patients were free of distant metastasis. As tumor diameter increased, the frequency of oncogene amplification and LOH at all loci except D17S5 increased. However, there was no relationship between tumor diameter and amplification of specific oncogenes or allelic loss at specific loci. LOH at D17S5 was detected in 40% of small breast carcinomas (≦2 cm) and 43% of larger breast carcinomas (2 to < 5 cm). There was a significant correlation of LOH at D17S5 with INT2 amplification or with LOH on 11p, 16q, and 18q. These findings suggest that LOH at D17S5 may be involved in the early stage of breast carcinoma development, while INT2 amplification and LOH at 11p, 16q, and 18q appear to be genetic alterations that occur with tumor progression. In addition, as lymph node metastases were significantly related to amplification of the ERBB2 and MYC genes, and LOH of the NME1 gene, these genetic alterations may play a role in the mechanism of lymph node metastases.     

Loss of Heterozygosity at the bcl-2 Gene Locus and Expression of bcl-2 in Human Gastric and Colorectal Carcinomas

The loss of heterozygosity (LOH) at the bcl -2 gene locus and the expression of the bcl -2 gene were examined in gastric and colorectal carcinoma cell lines and carcinoma tissues. LOH at the bcl -2 locus was detected in 24% (4/17) of gastric and 60% (6/10) of colonic carcinomas, all of which were well differentiated adenocarcinomas, whereas LOH was not seen in poorly differentiated ones. On the other hand, 24% (5/21) of poorly differentiated stomach cancers overexpressed bcl -2 gene, whereas no overexpression was detected in well differentiated stomach cancer. Three gastric and three colorectal carcinoma cell lines, all of which were derived from poorly differentiated adenocarcinomas, expressed considerable levels of bcl -2 mRNA and protein. These results suggest that LOH at the bcl -2 locus is frequently associated with well differentiated adenocarcinomas of the stomach and colon, and bcl -2 overexpression has implications for the development of poorly differentiated adenocarcinomas of the gastrointestinal tract.     

Allelic imbalance on the long arm of chromosome 5 in oral squamous cell carcinoma

To obtain a more detailed estimate of chromosome 5 loci involvement in oral squamous cell carcinoma (SCC), thirty-two oral SCCs were examined for loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) by PCR-LOH assay using thirteen microsatellite markers. LOH was observed in 16 (51.6%) of 31 informative cases. The incidence or the number of regions showing LOH was significantly higher in moderately and poorly differentiated tumors than in the well differentiated ones. Among the loci tested, D5S178 exhibited LOH in 11 (39.3%) of 28 cases, suggesting this to be the site, at 5q, of a novel candidate tumor suppressor gene. These data indicate that the incidence of LOH at chromosome 5q is high and is associated with oral tumor differentiation.     

乳腺癌与癌前病变的微卫星不稳定性与3p杂合性缺失

目的探讨微卫星不稳定性(MSI)与3号染色体短臂杂合性缺失(LOH)在乳腺癌发生机制中的作用及其临床病理学意义。方法采用PCR-SSLP及银染方法检测41例乳腺癌和13例癌前病变中MSI状态以及3p上11个微卫星位点LOH发生情况;应用免疫组化SP法检测错配修复(MMR)基因hMLH1、hMSH2和3p上相关基因的表达情况。结果(1)乳腺癌MSI发生率为36.6%,低分化乳腺癌MSI发生率明显高于高中分化组;癌前病变的发生率为15.4%;良性增生均表现微卫星稳定。(2)乳腺癌中微卫星异常主要分布于D3S1766、D2S2739和TP53 3个位点,癌前病变和乳腺癌具有相同的高频不稳定位点D3S1766和D2S2739。(3)97.0%乳腺癌患者发生3p LOH,检出率较高的位点分别是D3S1295、D3S1029和D3S1038。癌前病变患者3p LOH发生率为41.7%,缺失率较高的位点是D3S1295和D3S1029。(4)hMLH1和hMSH2蛋白在乳腺癌中阳性表达率分别为45.0%和40.0%,在癌前病变中分别为61.5%和76.9%,均明显低于良性增生组(P<0.05)。乳腺癌中两种蛋白阳性率与组织学分级相关(P<0.05),高分化组明显高于低中分化组。MSI 乳腺癌和癌前病变均表现MMR蛋白表达缺失。结论错配修复基因表达缺陷和MSI是乳腺癌多步骤发展过程的早期事件,在进展阶段MSI与乳腺癌低分化相关;D3S1766和D2S2739可能是检测乳腺癌与癌前病变MSI较敏感的位点。3p最小共同缺失区位于3p14-p25,提示该区域有与乳腺肿瘤发生发展相关并影响其生物学行为的候选抑癌基因。     

Loss of heterozygosity at chromosome 6q in preinvasive and early invasive breast carcinomas.

We have used polymerase chain reaction (PCR) analysis to study the incidence of allelic imbalance at four polymorphic microsatellite markers on chromosome 6q25.1-27, three dinucleotide repeats and one trinucleotide repeat, for microdissected tumour foci from a group of 75 ''early'' breast carcinomas. The tumours comprised 16 preinvasive cases of ductal carcinoma in situ (DCIS) and 59 mammographically detected early invasive carcinomas. Loss of heterozygosity (LOH) was detected at all four loci and in all types and grade of disease. The frequency of LOH ranged from 23% to 50% depending on the marker studied. The highest frequency of LOH was observed at the D6S186 locus for the cases of DCIS and at the oestrogen receptor locus for the invasive carcinomas. These data suggest that the inactivation of tumour-suppressor genes within this region on chromosome 6q is important for the development of these early lesions.     

Loss of heterozygosity analysis on chromosome 5p defines 5p13-12 as the critical region involved in tumor progression of bladder carcinomas

We have previously observed loss of heterozygosity (LOH) at a single locus (del-27) on human chromosome 5p13-12 to correlate with bladder tumor progression. In this study, we examined 33 bladder tumors for their pattern of allelic loss on chromosome 5p using 7 microsatellite markers. In 14 of 15 bladder tumors with LOH at locus del-27, allelic loss was confined to chromosomal region 5p13-12. This region included the microsatellite marker D5S2025 that showed LOH in 5 of 11 (45%) informative cases with LOH at del-27. This suggests that D5S2025 and del-27 are located within a single critical region of LOH on 5p13-12 harboring a tumor suppressor gene involved in bladder tumor progression. Recurrent LOH at other loci was observed at microsatellite markers located at 5p15. However, these losses appeared to be independent of LOH at 5p13-12 and occurred predominantly in poorly differentiated (G3) and advanced (T3-T4) tumors.