口腔黏膜癌前病变及鳞癌中D9S1752微卫星位点变化的研究

目的探讨口腔黏膜癌前病变(口腔白斑、红斑)及口腔鳞癌组织中染色体D9S1752微卫星位点改变的情况。方法使用PCR结合变性高效液相色谱分析方法(DHPLC)对35例口腔黏膜癌前病变及15例口腔鳞癌D9S1752位点上的杂合性缺失(LOH)和微卫星不稳定(MSI)进行分析。结果50例口腔黏膜癌前病变及口腔鳞癌组织中10例标本出现LOH,9例标本出现MSI。经统计学处理,不同临床诊断及病理学分型之间的LOH或MSI检出率差异无显著性(P>0.05)。结论在口腔黏膜癌前病变和鳞癌发生时D9S1752微卫星位点已经出现了改变,可能在部分口腔癌的发生过程中发挥了一定的作用。     

白斑及鳞癌9p微卫星位点改变及与病理的关系

目的:探讨口腔白斑及鳞癌染色体9p上4个微卫星位点改变的状况及其与临床病理诊断的关系。方法:选择微卫星位点D9S171、D9S175 2、D9S1748和IFNA ,应用聚合酶链式反应-变性聚丙烯酰胺凝胶电泳-银染方法,检测3 9例口腔白斑及12例鳞癌,分析其微卫星不稳定(MSI)及杂合性缺失(LOH)状况。结果:不同病理组别间4个位点MSI及LOH总的检出率有显著性差异(P <0 .0 1)。其中LOH检出率在不同临床病理组别之间有显著性差异(P <0 .0 5 ) ;而MSI的检出率在不同临床病理组别之间无显著性差异(P >0 .0 5 )。不同病理组别间单个位点的MSI及/或LOH无显著性差异(P >0 .0 5 )。结论:口腔癌的发生是多阶段多基因共同作用的结果。4个微卫星位点附近可能存在与口腔鳞癌发生发展相关的抑癌基因。MSI在口腔癌前病变癌变早期即已发生,而LOH发生频率则随口腔癌前病变癌变的发生发展逐渐增高     

口腔癌前病变、口腔鳞癌3p、9p染色体微卫星位点杂合性缺失研究

目的：探讨3p,9p微卫星分析在口腔粘膜癌前病变中的改变及应用价值。方法：应用PCR为基础的微卫星分析技术，选取人正常口腔粘膜（8例），口腔白斑（31例）及口腔鳞状细胞癌（22例）标本，分析分别位于3p(D3S659),9p(D9S161,D9S157,D9S171)上四位点微卫星的改变。结果：在正常口腔粘膜（NOM）及单纯上皮增生组均未见等位基因的改变，在口腔鳞癌（OSCC）组中12／22（54．5％）存在至少一个位点的杂合性缺失（LOH），异常增生组中6／23（25．1％）存在至少一个位点的LOH。还观察到这几个位点均存在一定频率的微卫星不稳定性（MI），OSCC组9／22观察到MI，达40．9％，异常增生组中达21．9％，结论：3p,9p上可能存在与口腔鳞癌相关的抑癌基因，3p,9p上这四位点的改变可能系口腔鳞癌发生的早期分子事件，本研究提示该四位点微卫星不稳定性在一部分口腔癌发生中起一定作用。     

口腔白斑、赤斑及鳞癌9p、3p的LOH/MSI及其与细胞增殖关系的研究

目的:探讨口腔黏膜癌前病变口腔白斑(OLK)、赤斑(EK)及鳞癌(OSCC)组织中染色体9p上D9S171、D9S1752、D9S1748、IFNA及3p上D3S1266、D3S643、D3S966的LOH、MSI及其与细胞增殖关系的研究.方法:应用聚合酶链反应一变性聚丙烯酰胺凝胶电泳-银染方法,检测OLK、EK及OSCC染色体9p上D9S171、D9S1752、D9S1748、IFNA及3P上D3S1266、D3S643、D3S966的微卫星位点的LOH及MSI,将检测结果与细胞增殖水平进行相关分析.结果:口腔癌前病变及鳞癌组织中9p、3p上7个位点均出现LOH和/或MSI.不同病理组别单个位点的LOH/MSI检出率无显著性差异(p＞0.05).但综合9p上4个位点或3p上3个位点或9p 3p上7个位点微卫星改变发生率,发现:9p,3p,9p 3p上LOH及LOH MSI在不同病理组间差异显著.LOH,MSI改变状况与增殖水平关系密切.上皮异常增生程度加重,细胞增殖水平增高,LOH/MSI检出率增加.具体表现为9p、3p的LOH检出率与AgNOR计数呈正相关,MSI检出率与PCNA表达水平呈正相关.结论:9p和3p区域的基因异常是OSCC发生和发展过程高频分子事件,该区域可能存在抑癌基因.9p和3p上的微卫星改变状况在口腔癌的发生、发展中扮演重要角色,作用机制可能与其促进细胞增殖活性,使细胞无限增殖有关.     

OLK、EK和OSCC 3p微卫星改变及其与病理的关系

目的探讨口腔白斑、红斑及鳞状细胞癌染色体3p上3个微卫星位点杂合性缺失(LOH)、微卫星不稳定(MSI)改变状况及其与病理的关系。方法选择3p上3个微卫星位点,应用聚合酶链式反应-变性聚丙烯酰胺凝胶电泳-银染方法,研究39例口腔白斑,12例红斑,32例口腔鳞状细胞癌染色体3p上D3S1266、D3S643、D3S966的LOH、MSI的状况及其与病理的关系。结果口腔癌前病变及鳞状细胞癌组织中3p上3个位点均出现LOH和/或MSI。不同病理分级组间单个位点LOH或/和MSI发生率无显着性差异(P>0.05)。当综合3p上3个位点的LOH及MSI总的检出率进行统计学分析,LOH检出率在不同临床病理组别之间差异显著(P<0.01)。MSI检出率在不同临床病理组别之间差异无显著性(P>0.05)。MSI在口腔癌前病变癌变早期即已发生,LOH发生频率则随口腔癌前病变癌变的发生发展逐渐增高。结论3p区域的基因异常是口腔鳞状细胞癌发生和发展过程高频分子事件,该区域可能存在抑癌基因。3p上的微卫星改变状况在口腔癌的发生、发展中扮演重要的角色。     

口腔黏膜癌前病变癌变过程中染色体17p13.3区杂合性丢失的研究

目的　了解口腔黏膜良性增生、异常增生、鳞形细胞癌D17S6 95 (17p13.3)位点杂合性丢失 (lossofheterozygosity ,LOH)情况 ,为新的肿瘤抑制基因定位提供依据。方法　应用聚合酶链反应 (PCR) -聚丙烯酰胺尿素凝胶电泳 (PAUGE) -DNA银染方法 ,以D17S6 95位点微卫星多态标记为遗传标记 ,检测 2 9例口腔癌前病变及鳞癌组织的LOH。结果　 19例提供信息的病例中 ,1例良性增生、2例异常增生、1例鳞癌出现LOH。结论　 17p13.3区D17S6 95位点可能是口腔鳞癌发生的早期事件 ,提示该区域可能存在与口腔鳞癌发生发展密切相关的肿瘤抑制基因。     

口腔鳞癌中染色体9p杂合性丢失的研究

目的:探讨口腔鳞状细胞癌中9号染色体短臂等位基因的杂合性丢失和微卫星不稳定与口腔鳞状细胞癌发生、发展之间的关系。方法:采用PCR法检测24例口腔鳞状细胞癌中染色体9p上8个微卫星多态位点。结果:在 24例口腔鳞状细胞癌中,10例(41167%)鳞癌组织至少有一个微卫星位点发生杂合性丢失。其主要发生在染色体 9p21的D9S171(21105%)和D9S304(10.00%),以及9p22-23的D9S168(22122%)和D9S162(15138%)。然而,这些基因位点的杂合性丢失与肿瘤病理学类型、肿瘤的大小及转移性在统计学上无显著相关性(P>0105)。此外,微卫星不稳定仅在2例患者中出现,没有1例患者符合微卫星不稳定的判定标准,即两个或两个以上的微卫星多态位点的异常。结论:本研究中发现的口腔鳞状细胞癌在染色体9p21-23区域发生高频率的杂合性丢失,提示在9p21-23区域可能存在多个与部分口腔鳞状细胞癌相关的肿瘤抑制基因,而微卫星不稳定与口腔鳞状细胞癌发生的关系不大。     

口腔颌面部肿瘤

口腔颌面部鳞癌化疗的临床研究；口腔鳞癌COX-2表达和肿瘤血管生成的研究；颊黏膜中分化鳞状细胞癌角蛋白谱的分析；口腔鳞状细胞癌中COX-2、VEGF和EGFR的表达及意义；口腔癌前病变及口腔癌中D9S171微卫星位点变化的研究；促凋亡分子Bad真核表达载体构建及其在人基底细胞癌细胞中的表达；雌激素受体在人舌癌细胞系中的表达。[编者按]     

口腔黏膜癌前病变及口腔癌杂合性缺失的研究进展

口腔黏膜癌前病变癌变监测是降低口腔癌发病率的关键 ,目前杂合性缺失 (LOH)及微卫星不稳定性(MI)的研究为口腔黏膜癌前病变癌变监测提供了分子生物学依据。本文结合近年来口腔黏膜癌前病变及口腔癌LOH及MI研究的有关文献 ,阐述LOH及MI在口腔癌发生中的作用 ,及其在口腔黏膜癌前病变癌变监测及防治中的临床意义     

端粒酶hTR基因在口腔癌前病变及鳞癌中的表达

目的探讨端粒酶ｈＴＲ基因在口腔癌前病变及鳞癌中的表达。方法用原位杂交技术检测８２例标本,其中正常口腔粘膜７例,上皮单纯性增生７例,上皮异常增生３０例,原位癌、鳞癌３８例。结果正常口腔粘膜及上皮单纯性增生组织中ｈＴＲ表达较弱,阳性细胞主要位于基底层,检出率２８．５６％（４／１４）。癌前病变中随着异常增生程度的增加,ｈＴＲ表达逐渐增强,阳性细胞逐步由基底层向棘层波及,检出率６０％（１８／３０）。原位癌及鳞癌均有较强的ｈＴＲ表达,检出率为８１．５８％（３１／３８）。结论端粒酶的激活可能出现在口腔癌前病变的晚期阶段,在口腔鳞癌的形成过程中起了关键性作用。     

Loss of heterozygosity and microsatellite instability on chromosome 2q in human oral squamous cell carcinoma

Allelic imbalance or loss of heterozygosity (LOH) and microsatellite instability (MSI) have been used to identify regions on chromosomes that may contain putative tumor suppressor genes. To obtain a detailed understanding of genetic alterations in oral cancer, 10 highly polymorphic markers mapped on chromosome 2 were used to examine 25 cases of oral squamous cell carcinoma (SCC). With these, we analyzed chromosome 2q for LOH in 25 primary oral SCCs and constructed a deletion map for this arm of the chromosome. LOH was detected in 16 (64%) of the 25 informative samples at one or more of the loci examined. MSI was observed in 5 (20%) of the 25 cases. Among the loci examined, LOHs were restricted to D2S1328 and D2S206 on chromosomes 2q14-21 and 2q36, respectively, with the former locus showing a rate of 5 (20.8%) and the latter a rate of 6 (25%) of the 24 informative cases. These observations taken in conjunction with data from 40 former cases analyzed at our laboratory suggest that the high incidence of LOH at chromosome 2q is associated with carcinogenesis of oral SCC. The regions that comprise the D2S1328 and D2S206 loci may play an important role in the development of oral SCC, perhaps containing sites that harbor a putative tumor suppressor gene.     

口腔鳞癌中染色体9q22-31的杂合性丢失

目的 检测染色体9q在口腔鳞癌发生发展中的作用。方法 应用聚合酶链反应检测22例经显微切割的口腔鳞癌组织中DNA微卫星多态标记D9S299（9q22-31)。结果 杂合性丢失检出率为43.8%(7/16)，其中高分化鳞癌为33.3%，而低分化鳞癌为75%。结论 染色体9q22-31区域在口腔鳞癌发生发展中起重要作用。提示此区域可能存在与口腔鳞癌发生发展密切相关的抑癌基因。     

Allelic imbalance on the long arm of chromosome 21 in human oral squamous cell carcinoma: relationship between allelic imbalances (LOH and MSI) and clinicopathologic features

Frequent allelic imbalances, including loss of heterozygosity (LOH) and microsatellite instability (MSI), have been found on the long arm of chromosome 21 (21q) in several types of human cancer. This study was designed to identify the tumor suppressor locus (or loci) associated with oral squamous cell carcinoma (SCC) on 21q. In order to understand the details of genetic alterations on chromosome 21, we performed polymerase chain reaction analysis of microsatellite polymorphisms corresponding to ten loci on this chromosome. We examined forty primary tumor tissues, forty corresponding normal tissues, and seven lymph node metastatic tissues. We identified novel tumor suppressor loci in this region in primary oral SCCs. To further determine the role of 21q deletions in oral cavity carcinogenesis, forty oral SCCs were examined for allelic imbalances (LOH or MSI) at 21q using ten microsatellite markers. Among these forty patients, twenty-six (65%) showed LOH at one or more loci. Deletion mapping of these tumors revealed four discrete, commonly deleted regions on the chromosome arm. Furthermore, we detected MSI in seventeen of those tested cases (42.5%). We compared our results with the clinicopathologic features. A number of sites displaying LOH at 21q could be detected in early stage lesions, and the frequencies of LOH tended to be higher in later clinical stages, but no statistical correlation was observed. Our results strongly suggest that allelic imbalances on 21q are involved in the development of oral SCC and that at least four different putative tumor suppressor genes contributing to the pathogenesis of this disease are present on 21q. Furthermore, allelic loss on 21q appears to be a useful indicator for evaluating the malignancy and prognosis of oral SCC, because the LOH of recurrent cases was more frequent than that of non-recurrent ones.     

Frequent allelic loss/imbalance on the short arm of chromosome 3 in tongue cancer.

Frequent allelic imbalances including loss of heterozygosity (LOH) and microsatellite instability (MSI) on the short arm of chromosome 3 (3p) have been found in several types of human cancer. This study was designed to identify the tumor suppressor locus (or loci) on 3p associated with tongue squamous cell carcinoma (SCC). Among 16 patients with tongue SCC tested, 7 (44%) of 16 informative cases showed LOH at one or more loci. Deletion mapping of these 16 tumors revealed two discrete, commonly deleted regions on the chromosome arm. Our data support the notion that tumor suppressor gene(s) contributing to the progression of tongue squamous cell carcinoma reside on 3p24 and 3p25.     

Frequent promoter hypermethylation of RASSF1A and p16INK4a and infrequent allelic loss other than 9p21 in betel-associated oral carcinoma in a Vietnamese non-smoking/non-drinking female population

BACKGROUND: Betel-chewing, a risk factor for oral carcinoma, is a common habit of elderly Vietnamese females, but concomitant habits of tobacco and alcohol are uncommon. METHODS: In the present study, 36 paraffin-embedded betel-associated oral carcinoma samples including 27 squamous cell carcinoma (SCC) and nine verrucous carcinomas (VC) were analyzed for the hypermethylation of tumor suppressor genes (TSGs) and loss of heterozygosity (LOH) of important TSG loci. Methylation-specific polymerase chain reaction (MSP) was used to identify promoter hypermethylation of p16INK4a and RASSF1A. For LOH analysis, 39 microsatellite markers at 12 chromosomal arms were examined by p olymerase chain reaction (PCR)-based microsatellite assay. RESULTS: Hypermethylation of p16IKN4a was detected in 63% of SCC and 67% of VC. In addition, LOH at 9p21 (locus for p16INK4a) was 58% for SCC and 22% for VC, and hypermethylation of RASSF1A was 93% for SCC and 100% for VC. LOH at 3p21.3-3p22.1 (where RASSF1A is located) was detected in only 12% of SCC and 0% of VC. LOH of other chromosomal arms were infrequent. Conclusion: As LOH for chromosomes other than 9p was uncommon, epigenetic silencing of RASSF1A and p16INK4a gene expression by promoter hypermethylation may play a critical role in betel-associated oral carcinogenesis.     

微卫星位点TP53、D9S1747、D9S162及RPS6的杂合性缺失与口腔鳞状细胞癌的关系

目的 检测微卫星位点TP53(位于17p13区)、D9S1747、D9S162、RPS6(均位于9p21区)在口腔鳞状细胞癌中的杂合性缺失(1oss of heterozygosity,LOH)和微卫星不稳定(microsatellite instability,MI)与临床病理因素及预后的关系.方法 取71例符合纳入标准的口腔鳞状细胞癌病变组织,用基因组DNA快速提取纯化试剂盒提取肿瘤组织及对应正常淋巴结或外周血的DNA,聚合酶链反应(PCR)变性电泳检测上述位点的基因变化.结果 所有位点在癌组织中的LOH和MI总发生率为68%(48/71);TP53(17p13)为56%(35/63),9p21(RPS6+D9S1747+D9S162)为59%(40/68);9p21的LOH、MI与WHO组织学分级和淋巴结转移有关(P＜0.05);17p13的LOH、MI与WHO组织学分级(P＜0.01)和临床分期有关(P＜0.05);TP53(17p13)和9p21在肿瘤组织中的LOH和MI对口腔鳞状细胞癌的预后有显著影响(P＜0.05);TP53(17p13)LOH阳性组术后生存率显著低于阴性组(P＜0.01);9p21的LOH和MI阳性组术后生存率显著低于阴性组(P＜0.05).结论 所检测位点的LOH和MI与口腔鳞状细胞癌的病理分级、临床分期和淋巴结转移有关;癌组织中TP53的变化是口腔鳞状细胞癌预后较差的独立影响因素.

Abstract：

Objective To investigate the correlation between the frequency of molecular abnormalities of 4 loci at chromosomal 9p21 (D9S1747 ,D9S162, RPS6) and 17p13 (TP53) and the clinical characteristics and prognosis. Methods The oral squamous cell carcinoma(OSCC) lesions in 71 patients were manually microdessected. Genomic DNA from these lesions and normal lymphnode tissu or peripheral blood of the same patients were extracted using the Watson's tissue kit. The loss of heterozygosity(LOH) and microsatellite instability (MI) of 17p13 and 9p21 were analyzed by PCR-page electrophoresis after DNA extraction. Results LOH and MI were detected in the OSCC of 48 patients (68%). The LOH and MI frequency at chromosomes 17p13 and 9P21 were 56% (35/63) and 59% (40/68) respectively. The LOH and MI frequency at 9p21 was significantly associated with WHO grading ( P ＜ 0.01 ) and lymphonode metastasis (P ＜0.01 ). The LOH and MI frequency at 17p13 was significantly associated with clinical stage (P ＜ 0.05 ). TP53 genetic aberration and 9p21 genetic aberration were significant prognostic factors for OSCC. The prognosis was poor in the LOH and MI positive group of chromosome 17p13 and 9p21. The frequency of LOH and MI at TP53 was the only independent factor for overall survival ( P ＜ 0.05 ).Conclusions The LOH and MI of 17p13 and 9p21 were related to clinical stage and lymphonode metastasis. LOH of TP53 was an independent prognostic factor for OSCC.