肝癌细胞株中肿瘤特异性抗原MAGE、GAGE、BAGE基因表达的研究

目的 研究MAGE、GAGE、BAGE基因在肝癌细胞株中的表达情况,评价这些肿瘤特异性抗原作为肿瘤分子标记以及肿瘤免疫治疗特异性靶位的可能性。 方法 用RT-PCR检测国内建株的肝癌细胞株SMMC-7721、QQY-7701、BEL-7402中MAGE-1、MAGE-3、GAGE1-8、GAGE1-2和BAGE基因mRNA的表达,以GAPDH基因作为检测内对照,并与非肿瘤肝穿组织比较。 结果 肝癌细胞株SMMC-7721表达MAGE-1和BAGE基因;QQY-7701表达MAGE-3和BAGE基因;BEL-7402表达MAGE-1和GAGE1-2基因;3株肝癌细胞至少表达其中一个基因。肝硬化病人肝穿刺组织中MAGE、GAGE、BAGE基因表达均为阴性。 结论 MAGE、GAGE、B A G E肿瘤特异性抗原可以作为肝癌早期诊断的分子标记,并具有作为肝癌免疫治疗特异性靶位的潜在价值。     

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma

BACKGROUND/AIMS: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.     

Melanoma Antigen Genes 1 and 2 Are Differentially Expressed in Human Gastric and Cardial Carcinomas

Background: MAGE genes encode for tumor-rejection antigens and are expressed in tumors of different histologic types but not in normal tissues, with the exception of testis and placenta. The aim of this study was to evaluate the frequency of MAGE-1 and -2 expression in gastric and in cardial carcinomas; these conditions have been described as two distinct diseases, having different etiologies, epidemiologic patterns, and gene mutations. Methods: Two groups of patients were studied: patients with distal gastric carcinoma and patients with carcinoma of the cardia. A group of patients with intestinal metaplasia in the gastric mucosa and controls were also included. All of them underwent upper GI endoscopy. Paired biopsy specimens were taken for routine histology and for RNA extraction, to study the expression of MAGE-1 and -2 genes. Results: None of the intestinal metaplastic samples or controls expressed MAGE-1 and -2 at detectable levels. Whereas 40% of the gastric cancer patients expressed either MAGE-1 or -2, 26.6% transcribed both. In the cardial cancer group, 20% of the cases expressed at least one MAGE, and only 6.6% expressed both genes. These results might reinforce the concept that cancer of the cardia is a distinct neoplastic disease with regard to esophageal and gastric (distal) carcinomas. Conclusions: Here we show that MAGE gene expression occurs in advanced stages of gastric and cardial cancer and therefore appears to be a late event. This might point to a reconsideration of their potential role in cancer immunotherapy.     

Melanoma antigen genes 1 and 2 are differentially expressed in human gastric and cardial carcinomas

BACKGROUND: MAGE genes encode for tumor-rejection antigens and are expressed in tumors of different histologic types but not in normal tissues, with the exception of testis and placenta. The aim of this study was to evaluate the frequency of MAGE-1 and -2 expression in gastric and in cardial carcinomas; these conditions have been described as two distinct diseases, having different etiologies, epidemiologic patterns, and gene mutations. METHODS: Two groups of patients were studied: patients with distal gastric carcinoma and patients with carcinoma of the cardia. A group of patients with intestinal metaplasia in the gastric mucosa and controls were also included. All of them underwent upper GI endoscopy. Paired biopsy specimens were taken for routine histology and for RNA extraction, to study the expression of MAGE-1 and -2 genes. RESULTS: None of the intestinal metaplastic samples or controls expressed MAGE-1 and -2 at detectable levels. Whereas 40% of the gastric cancer patients expressed either MAGE-1 or -2, 26.6% transcribed both. In the cardial cancer group, 20% of the cases expressed at least one MAGE, and only 6.6% expressed both genes. These results might reinforce the concept that cancer of the cardia is a distinct neoplastic disease with regard to esophageal and gastric (distal) carcinomas. CONCLUSIONS: Here we show that MAGE gene expression occurs in advanced stages of gastric and cardial cancer and therefore appears to be a late event. This might point to a reconsideration of their potential role in cancer immunotherapy.     

肿瘤-睾丸抗原MAGE-1、MAGE-3、SSX-2及NY-ESO-1在胃癌中的表达

目的　检测肿瘤 睾丸 (cancer testis ,CT)抗原MAGE 1、MAGE 3、NY ESO l及SSX 2基因mRNA在胃癌组织中的表达 ,探索其在胃癌免疫治疗中的应用价值。方法　用逆转录聚合酶链反应 (RT PCR)方法对胃癌患者癌组织、相应癌旁组织和对照组织(慢性胃炎和正常胃组织 )中的MAGE 1、MAGE 3、NY ESO 1及SSX 2基因mRNA进行检测 ,结合临床指标进行分析 ,随机选取RT PCR阳性扩增产物进行序列测定。结果　在在检测的 5 0例胃癌组织中 ,MAGE 1、MAGE 3、NY ESO 1及SSX 2基因mRNA阳性率分别是 48% (2 4/5 0 )、42 % % (2 1/5 0 )、11% (6/5 0 )和 8% (4 /5 0 ) ;多个CT抗原基因mRNA在胃癌中同时表达 ,至少表达一种CT抗原者达76% (3 8/5 0 ) ,表达两种或两种以上者为 40 % (2 0 /5 0 ) ,表达三种或三种以上者为 10 % (5 /5 0 ) ,同时表达四种者为 6% (3 /5 0 )。而相应的癌旁组织和对照组织中均未检测到四种目的基因的表达。DNA测序结果表明RT PCR产物确为这四种基因的目的片段。MAGE 1、MAGE 3、NY ESO 1及SSX 2基因的表达与年龄、性别、肿瘤大小、分化程度、浸润深度和淋巴结转移等无显著相关性 (P >0 .0 5 )。结论　CT抗原 (MAGE 1、MAGE 3、NY ESO 1及SSX 2 )基因在胃癌组织中呈高特异表达 ,这使得以这些基因编码的蛋     

肿瘤特异性肿瘤/睾丸抗原在肝癌组织中的表达

目的 研究7种主要肿瘤/睾丸(CT)抗原MAGE-1、MAGE-3、MAGE-4、MAGE-10、NY-ESO-1、SSX-2、SCP-1在原发性肝细胞癌(HCC)患者癌组织中的表达、编码基因的变异状况及与临床指标的关系。 方法 收集30例肝癌患者的癌和癌旁组织,采用特异性引物逆转录聚合酶链反应检测7种CT抗原的表达,并对PCR产物进行测序分析。结果 在30例HCC患者中,MAGE-1、MAGE-3、MAGE-4、MAGE-10、NY-ESO-1、SSX-2、SCP-1在癌组织中的表达率分别为66.7％、70.0％、20.0％、36.7％、40.0％、33.3％和33.3％,而癌旁没有表达。癌组织中至少表达1种、2种和3种CT抗原的阳性率分别为90.0％、70.0％和53.5％。我国肝癌表达的7种CT抗原编码基因与国外报道的相比,同源性非常高。MAGE-10和SCP-1的表达与甲胎蛋白的水平相关,MAGE-3和SSX-2表达与平均年龄相关。 结论 7种CT抗原在HCC患者癌组织中均有表达,阳性率为20.0％-70.0％,其编码基因序列高度保守。一些CT抗原的表达与临床指标存在相关性。     

Analysis of cancer/testis antigens in sporadic medullary thyroid carcinoma: expression and humoral response to NY-ESO-1

Cancer/testis antigens (CTA) are tumor-associated antigens expressed during ontogenesis, in a number of solid tumors but not in normal tissues except testis. Most of these CTA are highly immunogenic, eliciting a humoral and cellular response in the patients with advanced cancer, and are useful for tumor-specific immunotherapy. Medullary thyroid carcinoma (MTC) is a neoplasm derived from the parafollicular cells of the thyroid and occurs in either a sporadic or a familial form. In the present study, we examined by RT-PCR the expression of a number of genes encoding CTA in 23 surgical samples of sporadic MTC. Among the 11 cDNA antigens examined, RAGE, MAGE-4, and GAGE 1-2, were not expressed in any of the tissues. SSX 2 was present only in one tissue, whereas BAGE, GAGE 1-6, MAGE-1, MAGE-2, MAGE-3, and SSX 1-5 were detected in two to five samples. NY-ESO-1 cDNA was the most frequent, being detected in 15 of 23 examined samples (65.2%). Six (26.1%) tissues did not express any CTA-specific mRNA, whereas 10 tumors expressed only one gene (43.5%), 3 (21.4%) expressed 2 genes, and 4 displayed a broad CTA gene expression. NY-ESO-1 expression in primary MTC tissues significantly correlated with tumor recurrence. The presence of specific anti-NY-ESO-1 antibodies was searched in the sera of MTC-affected patients examined by ELISA using recombinant NY-ESO-1 protein. A humoral response against this CTA was detected in 6 of 11 NY-ESO-1 expressing patients (54.5%), and in 1 of 6 patients with NY-ESO-1-negative tumor. No anti-NY-ESO-1 antibodies were detected in healthy subjects (n = 17). The presence of anti-NY-ESO-1 antibodies was searched also in the sera of MTC affected patients whose tissues were not available for CTA analysis. Anti-NY-ESO-1 antibodies were present in 15 of 42 sera (35.7%), demonstrating that MTC is a neoplasm frequently associated with humoral immune response to NY-ESO-1. Serological survey may be useful as a way to identify patients with humoral immune response to NY-ESO-1 that provide a new attractive target for vaccine-based immunotherapy of MTC.     

肿瘤-睾丸抗原MAGE-1和NY-ESO-1 mRNA在胃肠间质瘤中的表达

目的:探讨肿瘤-睾丸抗原(CTA)MAGE-1和NY-ESO-1作为胃肠间质瘤(GISTs)免疫治疗特异性靶点及MAGE-1和NY-ESO-1 mRNA作为辅助GISTs危险度分级指标的可能性.及其与GISTs生物学行为的关系.方法:采用逆转录-聚合酶链反应(RT-PCR)技术,检测30例GISTs中MAGE-1和NY-ESO-1mRNA的表达,并取正常胃肠道组织作为阴性对照组,同时分析MAGE-1和NY-ESO-1mRNA表达与病理特征的关系.结果:正常对照组中无阳性表达,30例GISTs中,18例至少表达一种CTA,MAGE-1和NY-ESO-1 mRNA在GISTs中的表达率分别为30%和47%.MAGE-1和NY-ESO-1 mRNA表达与患者年龄、性别和病理类型无关,而与肿瘤生长部位、肿瘤大小及危险度分级有关(P<0.05).MAGE-1和NY-ESO-1 mRNA在低危、中危、高危三个组中表达量随着危险度分级的升高而增高,三组间差异有统计学意义(P<0.05).MAGE-1和NY-ESO-1 mRNA在GISTs中的表达不存在相关性(r=0.018,P>0.05).结论:MAGE-1和NY-ESO-1 mRNA在GISTs中的高特异性表达,其抗原有望成为GISTs免疫治疗特异性的靶点:MAGE-1和NY-ESO-1 mRNA表达与GISTs危险度分级有关,MAGE-1和NY-ESO-1 mRNA有望成为辅助GISTs危险度分级的诊断指标.     

The Sialyl Lewis X expression in hepatocarcinogenesis: potential predictor for the emergence of hepatocellular carcinoma

BACKGROUND/AIMS: Sialyl Lewis X (sLeX), one of the cancer-associated glycoproteins, has been reported to be expressed in both liver tissue from various types of liver disease and hepatocellular carcinoma. In order to clarify sLeX expression during the early stage of hepatocarcinogenesis, we examined sLeX expressions in either liver tissue specimens without any nodular lesions, dysplastic nodules or hepatocellular carcinomas. METHODOLOGY: Immunohistochemical observations were performed using a monoclonal antibody for sLeX. As for the liver tissue specimens, 8 livers without any chronic liver disease and 42 diseased livers were examined, while for the nodular lesions, 5 dysplastic nodules (borderline lesions) and 47 hepatocellular carcinomas were examined in this study. RESULTS: sLeX was not expressed in all 8 normal livers. sLeX was expressed membraneously in 8 of 15 (53%) chronic hepatitic liver tissue specimens, in 8 of 9 (89%) precirrhotic liver tissue specimens and in 16 of 18 (89%) cirrhotic liver tissue specimens. The incidence of sLeX expression on hepatocytes in both pre-cirrhotic and cirrhotic liver tissue was higher than that in chronic hepatitic liver tissue (P < 0.05). The sLeX expression in liver tissue was positive in all 14 liver tissue specimens containing multiple hepatocellular carcinomas, in which at least one of the nodules was a well-differentiated hepatocellular carcinoma regarded as multicentric hepatocellular carcinomas. In 18 of 28 (64%) liver tissue specimens without multicentric hepatocellular carcinomas, sLeX was positive and the difference was statistically significant (P < 0.05). In nodular lesions, sLeX was negative in 5 dysplastic nodules (borderline lesions). In hepatocellular carcinoma, 14 of 47 (30%) hepatocellular carcinoma nodules showed a positive expression. Six of 14 (43%) well-differentiated hepatocellular carcinomas were positive on the cell membrane. Four of 23 (17%) moderately differentiated hepatocellular carcinomas were positive on the cell membrane, while one of 23 (4%) moderately differentiated hepatocellular carcinoma was positive in the cytoplasm. In addition, 3 of 10 (30%) poorly differentiated hepatocellular carcinomas were positive in the cytoplasm. CONCLUSIONS: These results suggested that the sLeX-positive liver tissue specimens possessed a high degree of carcinogenicity and therefore sLeX expression in the diseased liver might be a good predictor for hepatocellular carcinoma emergence. At the same time, the suppression of sLeX occurred at a very early stage of hepatocarcinogenesis. In addition, the phenotype of sLeX was also considered to change during the progression of hepatocarcinogenesis.     

MAGE-1基因在肝细胞性肝癌中的表达

目的探讨ＭＡＧＥ－１基因编码的肿瘤相关抗原作为肝细胞性肝癌（ＨＣＣ）主动免疫治疗攻击靶点的可能性。方法用ＲＴ－ＰＣＲ的方法检测了３９例ＨＣＣ及癌旁组织、３种人肝癌细胞株和５例非ＨＣＣ病例肝组织中ＭＡＧＥ－１基因的表达。结果ＨＣＣ癌组织中ＭＡＧＥ－１基因表达率明显高于癌分组织中的表达率,５８．９％（２／３９）对１０．３％（４／３９）,Ｐ＜００１。３种细胞株均为阳性表达。结论ＭＡＧＥ－１基因可作为主动免疫治疗的攻击靶点,以进一步研究设计新的ＨＣＣ免疫治疗策略。     

肝细胞癌相关肿瘤抗原基因的筛选与分析

目的 筛选和分析肝细胞癌相关的肿瘤抗原。方法 用肝细胞癌组织构建cDNA表达文库，通过重组cDNA表达文库血清学分析法，用自体患者和异体患者的血清对文库进行筛选。将所得阳性噬菌体克隆体内剪切获得其pBK-CMV噬菌粒。通过限制性核酸内切酶鉴定插入cDNA片段，并作测序和生物信息学分析。同时将阳性克隆中的LIMS1插入片段进行原核表达。结果 自体血清筛选得到14种基因，异体筛选获得11种基因，两组中均筛选到kinectin，共有24种肝细胞癌相关的肿瘤抗原基因。其中有8种基因目前还不清楚其功能，其余16种基因根据确定的或者推断的功能可以分成8组。LIMS1原核重组蛋白表达成功。结论 本研究为肝细胞癌的免疫治疗提供了候选基因，并为理解肝细胞癌发生和发展的机制提供了线索。     

Endothelial cell marker expression in dysplastic lesions of the liver: an immunohistochemical study

BACKGROUNDS/AIMS: Hepatocellular carcinoma usually contains continuous capillary vessels lacking the differentiation markers specific for normal sinusoidal endothelial cells. We therefore aimed to search for alterations in endothelial cell marker expression in precancerous liver lesions. METHODS: Expression of the endothelial cell markers CD31, CD34 and BNH9 was analyzed in 138 dysplastic lesions from 40 cirrhotic patients (20 with and 20 without hepatocellular carcinoma). RESULTS: No expression of the three endothelial cell markers was detected in cirrhotic nodules and in non dysplastic regenerative macronodules. The three markers were detected in 29.8% of dysplastic lesions and 47% of hepatocellular carcinomas. At least one marker was detected in 75% of dysplastic lesions and 100% of hepatocellular carcinomas. The three markers were more frequently expressed in areas of small cell than of large cell change (34 vs 10%). No correlation was found with the grade of dysplasia, the occurrence of arterialization and the association with hepatocellular carcinoma. CONCLUSIONS: Alterations in the hepatic microcirculation comparable to those observed in hepatocellular carcinoma are present in a significant proportion of dysplastic lesions of the liver and may be indirect markers of the process of liver carcinogenesis.