胃癌组织p53基因缺失和异常表达

检测２０例行胃癌根治手术患者的癌组织和对应的正常粘膜组织中ｐ５３基因ＤＮＡ结构和蛋白表达。Ｓｏｕｔｈｅｒｎ印迹显示３０％（６／２０）的胃癌组织中存在ｐ５３基因部分缺失。应用抗ｐ５３单克隆抗体－ＰＡｂ１８０１和ＡＢＣ免疫组织化学技术检测冰冻切片组织中ｐ５３蛋白的表达，结果表明，５５％（１１／２０）胃癌组织中存在突变ｐ５３蛋白高表达，阳性染色部位位于细胞核和／或细胞浆中，所有正常组织标本中均未见阳性染色，低分化腺癌ｐ５３阳性表达率显著高于分化好的胃癌（Ｐ＝０．０１１６），ｐ５３蛋白高表达和等位基因缺失密切相关（ｒ＝０．５９）。结果提示，在人类胃癌形成和肿瘤演进过程中，ｐ５３基因失活是一个重要的事件，ｐ５３基因结构和产物的变化可以作为新的肿瘤相关标志，这对判断胃癌生物学行为，识别具有高度恶化倾向的肿瘤，估价胃癌病员的预后和指导临床治疗有一定的作用。     

DCC基因杂合性缺失与人结直肠癌转移关系的研究

ＤＣＣ基因位于染色体１８ｑ２１．３，是结直肠肿瘤发生发展中具有重要意义的抑癌基因。应用Ｓｏｕｔｈｅｒｎ印迹技术和限制性片断长度多态性（ＲＦＬＰ）研究了３３例结直肠癌病人癌组织及其正常粘膜组织基因组ＤＮＡ，其中１６例，占４９％具有杂合性，并检出６例，占３８％存在１８ｑ等位基因杂合性缺失。１８ｑ等位基因杂合性缺失与肝癌和淋巴结转移有明显相关性。提示分子遗传学研究在结直肠肿瘤临床上如估计肿瘤转移能力有一定意义。     

鼻咽癌染色体3p21-26的等位基因杂合性丢失研究

目的细胞遗传学研究表明,３号染色体缺失是鼻咽癌常见的染色体异常之一。分子生物学研究表明,染色体３ｐ在鼻咽癌中出现高频率的等位基因杂合性丢失（ＬＯＨ）。本研究将进一步确定鼻咽癌３ｐ等位基因杂合性丢失的频率及范围。方法应用位于３ｐ２１２６区域１６个微卫星多态性位点,对２４例低分化鼻咽癌患者进行了ＬＯＨ分析。结果２４例患者中有１６例存在杂合性丢失（６６．７％）。丢失频率最高的两个位点是Ｄ３Ｓ１５６０（５０％,１１／２１）和Ｄ３Ｓ１６２０（５０％,９／１８）。在具有丢失的１６例患者中,８例显示为１个连续的多个相邻位点的杂合性丢失区域,５例患者存在２个或２个以上的杂合性丢失区。病例１,３,４,７,８,１０,１６,１７,１８,１９和２２在Ｄ３Ｓ１５９７和Ｄ３Ｓ１２９７之间,表现为一个不同大小的杂合性丢失区。结论最小共同丢失区位于Ｄ３Ｓ１５６０Ｄ３Ｓ１６２０（３ｐ２５．３２６．２）之间,提示该区域有一个尚未克隆的、与晚期鼻咽癌明显相关的抑癌基因。     

PTEN基因甲基化与胃癌临床病理特征的关系

目的研究胃癌组织中抑癌基因第10号染色体缺失性磷酸酶张力蛋白基因（PTEN）表达异常与胃癌临床病理特征的关系。方法应用甲基化特异性PCR方法（MSP）检测45例胃癌及癌旁正常组织PTEN甲基化的表达情况。结果40．0％（18／45）的胃癌组织和2．2％（1／45）的癌旁正常组织PTEN基因发生甲基化,胃癌组织甲基化率显著增高（P〈0．05）;低分化腺癌甲基化率为60．0％（15／25）,高中分化腺癌甲基化率为15．0％（3／20）,二者差异具有统计学意义（P〈0．05）;发生淋巴结转移的24例胃癌组织中,13例PTEN基因发生甲基化,发生淋巴结转移的胃癌组织PTEN甲基化率明显高于无淋巴结转移胃癌组织（P〈0．05）。结论PTEN基因甲基化与胃癌的发生密切相关,PTEN基因甲基化在胃癌的发病过程中起重要作用。     

乳腺癌nm23—H1等位基因缺失与肿瘤淋巴结转移相关性的研究

应用核酸分子杂交技术检测了34例乳腺癌以及相应癌旁正常乳腺组织中肿瘤转移抑制基因nm23-H1等位基因缺失情况，分析该基因的等位基因缺失与肿瘤大小，发病年龄，雌激素受体状况以及腋下淋巴结转移之间的关系。结果发现nm23-H1等位基因缺失与肿瘤大小．发病年龄以及雌激素受体状况无关．而与腋下淋巴结转移关系十分密切．有转移病例nm23-H1等位基因缺失的百分率为333％（7／21）．无转移病例等位基因缺失的百分率为77％（1／13），两组病例对比差异有显著性（P＜0．05）。研究结果提示nm23-H1基因在抑制肿瘤转移方面具有重要作用。     

显微切割鼻咽癌组织16号染色体杂合性缺失的研究

目的：研究鼻咽癌中１６号染色体的缺失情况。方法：采用显微切割的方法获取肿瘤组织,然后用ＰＣＲ的方法以１６号染色体上的８对引物对３８例鼻咽癌进行分析。结果：３８例鼻咽癌组织中所有标本至少有一个位点出现有杂合笥缺失,其中Ｄ１６Ｓ５３３出现杂合性缺失的频率最高,占８６．１％（３１／３３）,此外,杂合性缺失频率超过５０％的有引物Ｄ１６Ｓ３９８、Ｄ１６Ｓ３００和Ｄ１６Ｓ４２０。分别占７８．８％（２６／３３）     

nm23－H1等位基因缺失与大肠癌转移相关性研究

ｎｍ２３基因是近年来发现的与肿瘤转移表型抑制相关的基因，称之为转移抑制基因。ｎｍ２３基因的等位基因缺失、突变和低表达与多种人类肿瘤转移相关。ｎｍ２３基因包括ｎｍ２３－Ｈ１及ｎｍ２３－Ｈ２两种亚型。我们利用Ｓｏｕｔｈｅｒｎ杂交技术检测了２３例大肠癌及其相应正常粘膜基因组ＤＮＡｎｍ２３－Ｈ１等位基因缺失情况，结果发现５例存在ｎｍ２３－Ｈ１等位基因缺失。有淋巴结、肝或其它脏器转移者，ｎｍ２３－Ｈ１等位基因缺失率为５７．１％（４／７），而无转移者为６．２％（１／１６）。两组比较差异有显著性意义（Ｐ＜０．００５）。ｎｍ２３－Ｈ１等位基因缺失与肿瘤大小、部位及分化程度无显著相关（Ｐ＞０．０５）。结果表明，ｎｍ２３－Ｈ１基因在抑制大肠癌转移方面起重要作用。     

子宫内膜癌中MTS1／p16基因缺失的研究

目的探讨ＭＴＳ１／ｐ１６基因缺失与子宫内膜癌发生发展的关系。方法采用聚合酶链式反应（ＰＣＲ）技术,扩增３２例子宫内膜癌组织的ＭＴＳ１／ｐ１６基因。结果３２例子宫内膜癌组织中,有６例出现ＭＴＳ１／ｐ１６基因缺失,缺失率为１８．８％;癌旁组织、正常子宫内膜组织和组织分级Ｇ１的癌组织未见ＭＴＳ１／ｐ１６基因缺失。结论ＭＴＳ１／ｐ１６基因缺失可能与子宫内膜癌病程进展有关,应对其进一步研究。     

Rb1基因mRNA在人胃癌中表达的研究

利用Rb1基因特异性cDNA探针,采用Northern杂交分析方法,检测16例经临床及病理确诊为胃癌的肿瘤组织及其癌旁组织标本中Rb1mRNNA的表达状态。2例(2/16)出现Rb1基因mRNA表达的缺失。提示:胃癌的发生可能与13号染色体的杂合性缺失及Rb1基因缺失有关。     

胃癌组织大肠癌丢失基因杂合性丢失及表达缺失的研究

为探讨大肠癌丢失基因（ＤＣＣ）改变在胃癌发生发展中的作用，应用ＰＣＲ－ＲＦＬＰ及ＲＴ－ＰＣＲ技术对手术切除胃癌组织ＤＣＣ基因的杂合性丢失及ｍＲＮＡ表达进行分析。５１例胃癌中发现杂合丢失１８例，占３５．３％。临床Ⅲ～Ⅳ期组杂合丢失率（５０．０％）显著高于Ⅰ～Ⅱ期组（１４．３％，Ｐ值＜０．０５）。杂合丢失与组织学类型、肿瘤大小、侵袭深度、淋巴转移无显著相关（Ｐ＞０．０５）。５１例胃癌中有２６例检测了ＤＣＣ基因ｍＲＮＡ表达，发现表达缺失８例，占３０．７％。表达缺失与杂合丢失及临床病理参数无关（Ｐ＞０．０５）。胃癌组织中存在着ＤＣＣ基因杂合丢失和ｍＲＮＡ表达缺失。杂合丢失是胃癌的晚期改变     

Allelotype of gastric adenocarcinoma

Gastric adenocarcinoma is a leading cause of cancer mortality world-wide. Yet, the underlying molecular events important in the development of this cancer are largely undefined. Thus, we performed a comprehensive survey for allelic loss on our panel of xenografted human gastric carcinomas. Contaminating normal stromal cells of primary cancers often limit mutational analyses. Xenografted samples of our gastric carcinomas provided optimally enriched tumors for neoplasia that clearly and sensitively demonstrated genetic alterations. Additionally, total absence of allelic signals in these xenografted samples confirmed true loss of alleles rather than just allelic imbalance. Analysis of at least two highly polymorphic microsatellite markers per nonacrocentric chromosomal arm in our xenografted human gastric carcinomas demonstrated significant loss of heterozygosity well above background levels at 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q. Several of these loci represent novel findings of significant loss in gastric cancers. On chromosome 17p, p53 is known to be inactivated either by mutation or deletion in a majority of gastric carcinomas. The critical target(s) of inactivation in gastric cancers at these other loci remain to be characterized.     

Allele loss in human gastric carcinomas - relation to tumor progression and differentiation

The sequence of genetic changes associated with the development of gastric carcinoma remains unclarified despite the numerous genetic and chromosomal abnormalities that have been implicated so far in this process. We investigated the frequency and pattern of allele loss in 68 gastric carcinomas, with the aim of identifying genetic changes putatively involved in the histologic differentiation and/or progression of gastric cancer. Allele loss was investigated using 12 RFLP and 11 microsatellite markers localized at 22 different loci from 9 autosomal chromosomes. Allele loss in at least one chromosome arm was detected in 41 out of the 68 cases (60%). A high ratio of allele loss was significantly associated with the masculine gender and aneuploidy. The chromosome arms most commonly affected were 3p (57%), 17p (44%), and 6q (32%). Alterations at these chromosome arms were also frequently found (greater than or equal to 40%) in the six less advanced gastric carcinomas of the series, thus suggesting that genetic changes involving these chromosomes are early events in gastric tumorigenesis. Genetic changes at 5q and 17p loci were only observed in gastric carcinomas of the intestinal and atypical (unclassified) types, thereby indicating a possible role of genes located at these chromosome arms in the differentiation of gastric carcinoma.     

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.     

Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer.

Frequent loss of heterozygosity at chromosomal loci in a specific tumor type may indicate the presence of a tumor suppressor gene. We have examined loss of heterozygosity on chromosome 8p in paired tumor and constitutional DNA from 346 patients representing seven different types of human cancer. Frequent allelic losses were observed in hepatocellular carcinoma (22 of 46 cases, 47.8%), in colorectal cancer (12 of 26, 46.2%), and in non-small cell lung cancer (14 of 35, 40.0%), in contrast to low frequencies detected in breast cancer (5 of 56, 8.9%) and renal cell carcinoma (2 of 27, 7.4%). Ovarian cancer and gastric cancer showed intermediate frequencies of 33.3% and 22.2%. Subsequent analysis of 120 hepatocellular carcinomas and 94 colorectal cancers with five polymorphic markers along the short arm of chromosome 8 defined commonly deleted regions within the same chromosomal interval, 8p23. 1-8p21.3, suggesting that one or more tumor suppressor genes for both cancers may be present in that region.     

Definition and refinement of chromosome 8p regions of loss of heterozygosity in gastric cancer.

Loss of heterozygosity at several chromosomal loci is a common feature of the malignant progression of human tumors. These regions are thought to harbor one or more putative tumor suppressor gene(s) playing a role in tumor development. Allelic losses on the short arm of chromosome 8 (8p) have been reported as frequent events in several cancers, and three commonly deleted regions have been defined at 8p11.2-12, 8p21-22, and 8p23.1. To evaluate the possible involvement of these regions in gastric cancer, we used eight microsatellite markers to perform an extensive analysis of allele loss at 8p21-22 in 52 cases of primary gastric adenocarcinoma. We found that 44% of tumors showed allelic loss for at least one marker at 8p21-22. The critical region of loss was found to be between markers LPL and D8S258, which displayed loss of heterozygosity in 39% and 33% of cases, respectively. This region is centromeric to the LPL locus and centered on the D8S258 locus. We conclude that 8p22 deletion is a frequent event in gastric cancer and suggest the presence of a putative tumor suppressor gene near the D8S258 locus. Initial steps were taken toward the identification of this gene, which is likely to play an important role in the pathogenesis of gastric cancer and of other tumors as well.     

Genetic alterations of the KLF6 gene in gastric cancer

The KLF6 is a zinc-finger tumor suppressor that is frequently mutated in several human cancers and broadly involved in differentiation and development, growth-related signal transduction, cell proliferation, apoptosis, and angiogenesis. To determine whether genetic alterations of KLF6 gene are involved in the development and/or progression of gastric cancer, we have screened a set of 80 sporadic gastric cancers for mutations and allele loss of the KLF6 gene. Four missense mutations, S155R, P172 T, S180L, and R198 K, were detected in transactivation domain of the KLF6 gene and one of them had biallelic mutations with somatic mutation of one allele and loss of the remaining allele. All of the cases with mutation were of advanced intestinal-type gastric cancer with lymph node metastasis. In addition, 16 (43.2%) of 37 informative cases showed allelic loss at KLF6 locus. Interestingly, allelic loss was also frequent in intestinal-type gastric cancer. Therefore, our data suggest that genetic alterations of KLF6 gene might play an important role in the development or progression of sporadic gastric cancers.     

Loss of heterozygosity on the long arm of chromosome 11 in colorectal tumours.

We have examined a series of human colorectal adenomas, carcinomas and cell lines derived from human colorectal cancer for loss of heterozygosity (LOH) on chromosome 11q22-23 by polymerase chain reaction (PCR) amplification of a microsatellite polymorphism of the dopamine D2 receptor (DRD2) locus. LOH was demonstrated in 5/30 (16.7%) adenomas and 23/68 (33.8%) carcinomas. Only 2/20 (10%) cell lines showed homozygosity which could potentially be as a consequence of LOH. This moderate level of loss in the tumour samples was probably not an underestimation as a result of excessive stromal contamination because high rates (68-77%) have been detected in the same samples on chromosomes 17 and 18. In contrast to a previous report, LOH in carcinomas at 11q22-23 occurred at a lower frequency and was not associated with Dukes'' stage, degree of differentiation, mucin production or the location of the cancer. However, a significant association was found between LOH on chromosome 11 and chromosome 14. Thus, inactivation of any putative tumour-suppressor gene at 11q22-23 by LOH is not a very common event in the development of colorectal tumours, but may be biologically significant if accompanied by chromosome 14 deletions.     

Ala228 variant of trail receptor 1 affecting the ligand binding site is associated with chronic lymphocytic leukemia, mantle cell lymphoma, prostate cancer, head and neck squamous cell carcinoma and bladder cancer

Allelic loss of chromosome 8p21-22 is a frequent event in various human cancers including mantle cell lymphoma (MCL), prostate cancer, head and neck squamous cell carcinoma (HNSCC) and bladder cancer. The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, including TNFRSF10A and TNFRSF10B, are located within this chromosomal region. Since recent studies demonstrate that chronic lymphocytic leukemia (CLL) and prostate cells are TRAIL induced apoptosis, TRAIL-receptors are strong tumor suppressor candidate genes in human cancers exhibiting loss of chromosomal material in 8p21.3. However, no mutation of the TRAIL receptor genes has been reported in CLL, MCL, prostate cancer, HNSCC so far. In this study we analyzed the complete coding region of TNFRSF10A and TNFRSF10B in a series of 32 MCL and 101 CLL samples and detected a single nucleotide polymorphism (SNP) in TNFRSF10A (A683C) with tumor specific allele distribution. We examined allele distribution in 395 samples of different tumor entities (prostate cancer, n = 43; HNSCC, n = 40; bladder cancer, n = 179) and compared them to 137 samples from healthy probands. We found the rare allele of TNFRSF10A is more frequent in CLL, MCL, prostate cancer, bladder cancer and HNSCC. The A683C polymorphism did not cosegregate with other TNFRSF10A polymorphisms previously described. Thus screening for 683A-->C nucleotide exchanges may become important in diagnosis and/or treatment of these malignancies.     

Clonal allele loss in gastrointestinal cancers

Using a panel of DNA probes for hypervariable DNA regions we screened 52 gastrointestinal carcinomas for clonal allele losses on chromosomes 1, 5, 7, 12, 16 and 17. A total of 24/35 informative cases of colorectal cancers showed loss of constitutional heterozygosity at a locus on chromosome 17p, while 9/31 cases informative for a locus on 5q showed allele loss. Loss of sequences at 5q was linked to allele loss at 17p with a single exception. In gastric cancers loss of heterozygosity most frequently occurred at 1q (5/10 tumours) and at 12q (6/11 tumours). Gastrointestinal tumours show consistent chromosomal losses and the loci involved are different in gastric and colorectal cancers.