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。     

口腔鳞癌浸润前沿、中心和癌间质TP53、RPS6基因变化的研究

目的检测微卫星位点TP53、RPS6在口腔鳞癌浸润前沿、中心及癌间质细胞中的基因变化，揭示口腔鳞癌的生物学行为。方法运用激光捕获显微切割分别获取同一肿瘤浸润前沿、中心及癌间质足量的细胞，提取DNA，PCR-变性PAGE电泳检测TP53和RPS6位点的基因变化。结果浸润前沿、中心和癌间质中TP53和RPS6存在杂合性缺失（10ssofheterozygosity，LOH）和微卫星不稳定（microsatelliteinstability，MI），发生率从23．5％（4／17）到64．7％（11／17），且LOH和（或）MI的模式存在不同。上皮TP53和RPS6的LOH和MI发生率分别为70．6％（12／17）和64．7％（11／17），间质为43．8％（7／16）和23．5％（4／17），癌实质较间质发生率高，差异有统计学意义（P〈0．05）。浸润前沿TP53的LOH发生率为64．7％（11／17），中心为60．0％（9／15）。RPS6在浸润前沿的LOH和MI为58．8％（10／17），中心为29．4％（5／17）。两位点总发生率分别为62．5％（20／32）和44．1％（15／34），差异有统计学意义（P〈0．05）。结论部分口腔鳞癌样本浸润前沿、中心和肿瘤间质的基因变化不同，癌实质基因变化率与肿瘤分化程度有关。     

p53-p21waf1通路在口腔鳞状细胞癌中心体扩增中的作用及意义

目的 了解口腔鳞状细胞癌(oral squamous cell cancer,OSCC)p21waf1蛋白表达与中心体扩增的相关性,探讨p53-p21waf1通路在OSCC中心体扩增中的作用及意义.方法 8例正常口腔黏膜及27例OSCC石蜡包埋组织,采用间接免疫荧光双重染色法了解OSCC组织中心体扩增情况;采用流式细胞术及免疫组织化学方法对相应组织p21waf1蛋白表达量及突变型p53蛋白进行检测,分析三者间的相关性.结果 中心体数目扩增(＞2个/细胞)可见于78%(21/27)的OSCC组织,p21waf1蛋白表达量在有中心体扩增的OSCC组织中[(0.878±0.081)]低于无中心体扩增的OSCC组织[(0.952±0.018),t=3.838,P=0.001],OSCC组织中心体扩增程度与p21waf1蛋白表达量间存在负相关关系(r=0.472,P＜0.05);p21waf1蛋白表达量在OSCC组织p53阳性组[(0.823±0.071)]低于p53阴性组[(0.909±0.075),t=3.905,P＜0.01],两者间存在负相关关系(r=-0.491,P＜0.05).结论 p53-p21waf1通路可能参与了OSCC中心体循环调控,p53突变导致的p21waf1蛋白表达下调在OSCC中心体扩增中可能起一定作用.     

口腔鳞癌HPV16、18感染与多基因表达的相关性研究

目的:检测口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)p53、Ki-67、Rb、p16和cyclin D1蛋白表达与高危型人类乳头状瘤病毒(human papilloma virus,HPV)感染的相互关系.方法:对73例OSCC组织标本,应用免疫组织化学ABC法检测病变组织中p...     

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。     

口腔癌前病变及口腔癌中D9S171微卫星位点变化的研究

目的 探讨口腔粘膜癌前病变及口腔鳞癌组织中染色体D9S171微卫星位点变化的情况。方法 使用PCR结合琼脂糖凝胶电泳条带Genetoolsanalysis software分析对35例口腔粘膜癌前病变及15例口腔鳞癌的D9S171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例口腔粘膜癌前病变度口腔癌组织中，D9S171位点上21例出现微卫星改变（LOH＋MSI），其中8例标本出现LOH，13倒标本出现MSI。不同临床诊断及病理学分型之间的LOH或MSI检出率经统计学检验无显著性差异（P〉0．05）。结论 D9S171可能为口腔癌前病变癌变过程中的异常基因之一。在口腔癌前病变癌变的发展过程中扮演一定角色。     

p53和增殖细胞核抗原在口腔白斑和口腔鳞状细胞癌中的表达及意义

目的 检测正常口腔黏膜、口腔黏膜白斑和口腔鳞状细胞癌(OSCC)中p53和增殖细胞核抗原(PCNA)表达,探讨p53和PCNA在OSCC癌变过程中的规律及相关性.方法 口腔黏膜白斑标本20例、OSCC标本31例及正常口腔黏膜10例,采用免疫组化SP法检测其p53和PCNA的表达,应用SPSS 11.0软件包对结果进行χ2检验、单因素方差分析和线性回归分析.结果 p53在正常黏膜组、白斑组与鳞状细胞癌组的表达率分别为0%、35%和77.4%,PCNA在上述三组中表达率分别为30%、70%和96.8%,两种表达的组间比较差异有统计学意义(P<0.05);p53蛋白与PCNA表达呈正相关(P<0.01).结论 p53基因和PCNA表达水平均与OSCC发展进程密切相关.     

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.     

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。     

9p21耗竭与头颈癌复发的关系

遗传物质的部分丢失为发现肿瘤抑制基因(tumor suppressor gene,TSG)提供了有力证据,遗传物质的耗竭可通过某些特殊等位基因的杂合性丢失(loss of heterozygosity,LOH)而予检测。杂合性丢失见于许多肿瘤系统的多个位点,在头颈部鳞癌(head and neck squamous cell carcinoma,HNSCC),已经证明LOH发生于3p,8p,9p,10q,11p,13q,17p和18q。其中以染色体9的短臂最为常见,发生率为45％～72％不等。在癌前病变中,LOH的发生率也相对较高,9p常见的耗竭区位于9p21～22的D9 S156和D9 S165。     

Lack of association between denture trauma and loss of heterozygosity confronts the proposed pathologic role of chronic mucosal trauma in oral carcinogenesis

Chronic mucosal trauma is suggested as an additional etiologic risk factor for oral squamous cell carcinoma (OSCC), but there is a lack of experimental‐molecular data. If chronic trauma of the oral mucosa is carcinogenic, it should be associated with early genetic alterations seen during typical progression of OSCC, like loss of heterozygosity (LOH). We investigated LOH in the key chromosomal arms 3p, 9p and 17p in inflammatory fibrous hyperplasia associated with removable dental prosthesis and also in normal oral mucosa, by using the polymorphic microsatellite markers D3S1300 at 3p14.2, D9S1748 at 9p21, D17S1289 at 17p12 and D17S974 at 17p13 and capillary electrophoresis. LOH was detected in 2/15 (13%) fibrous hyperplasia samples similarly to other reactive and inflammatory lesions. None of the normal mucosa samples presented LOH. Our experimental‐molecular results do not support the hypothesis that trauma associated with dental prosthesis has an important role in oral carcinogenesis.     

Infrequent loss of heterozygosity of the major tumour suppressor genes in Indian oral cancers

The loss of heterozygosity (LOH) in tumour suppressor gene loci such as p53, retinoblastoma (rb) and adenomatous polyposis coli (apc) were analyzed in oral cancer tissues with matched controls by employing polymerase chain reaction based/restriction fragment length polymorphism (PCR-RFLP), variable number of tandem repeats (PCR-VNTR) analysis and microsatellite assay. The PCR-RFLP analysis showed an infrequent LOH in rb (17%), p53 (11%) and apc (10%) loci in these cases. The microsatellite assay also revealed only a low frequency of LOH in the microsatellite markers such as TP53 (25%), D5S505 (10%) and D3S1067 (0%) in the same samples. In contrast to the present study, similar studies from Western countries have reported a high frequency of LOH in p53, rb and apc genes in oral cancer tissues. The present preliminary study indicates that the gene aberration by LOH may be an insignificant mechanism in Indian oral cancers with respect to the tumour suppressor genes examined.     

P53,Ki-67蛋白在口腔鳞癌中的表达意义及与预后的关系

目的:研究细胞周期相关蛋白P53和核蛋白抗原Ki67在口腔鳞癌组织中的表达及其与预后的相关性,寻找预后判断的有效生物标志物。方法:用免疫组织化学EnVision染色法的方法检测随访资料完整的132例口腔鳞癌患者术后石蜡切片中P53、Ki67蛋白的表达,研究口腔鳞癌组织中P53、Ki67与病理特征的相关性及口腔鳞癌预后的相关性。结果:132例口腔鳞癌标本蛋白检测和统计分析结果表明,P53和Ki-67蛋白在口腔鳞癌中的高表达与患者细胞分化程度,肿瘤T分期和肿瘤原发灶有关(P＜0.05);P53蛋白的表达水平与口腔鳞癌术后生存率呈显著的相关性(P＜0.05);Ki67蛋白的表达水平与口腔鳞癌术后生存时间并无显著的相关性。结论:口腔鳞癌患者癌组织标本的P53表达水平与预后呈正相关,P53有望成为口腔鳞癌预后判断的间接性生物标志物。     

Loss of heterozygosity (LOH) in tumour suppressor genes in benign and malignant mixed odontogenic tumours

Although molecular alterations are reported in different types of odontogenic tumours, their pathogenesis remains to be established. Loss of heterozygosity (LOH) studies allow the identification of minimal regions of deletions of known or putative tumour suppressor genes, the losses of which may promote neoplastic growth. The purpose of this study was to investigate LOH in a set of odontogenic mixed tumours. Tumour suppressor gene loci on 3p, 9p, 11p, 11q and 17p chromosomes were analysed in five samples of ameloblastic fibroma (AF), three samples of ameloblastic fibro-odontoma (AFO) and three samples of ameloblastic fibrosarcoma (AFS). The most frequently lost genetic loci were p53 (17p13, 62%) and CHRNB1 (17p13, 55%). LOH at the chromosome regions 3p24.3, 9p22 and 9p22-p21 was identified only in AFS. No sample showed LOH at the chromosomal loci 3p21.2 and 11q13.4. For the region 9p22-p13, LOH occurred in one sample of AFO. The fractional allelic loss (FAL) was calculated for each sample. The mean FAL of the benign lesions (i.e. AF and AFO) was 22%, whereas the mean FAL of the malignant lesions (i.e. AFS) was 74.6%. In conclusion, our results show a higher FAL in AFS compared to its benign counterparts and reveal a different pattern of LOH of tumour suppressor genes in AFS, which may regulate changes in tumour behaviour.     

Role of TP53 in the progression of pre-malignant and malignant oral mucosal lesions. A follow-up study of 144 patients

Background:  Prediction of progression from pre-malignant oral mucosal lesions to malignancy, or recurrence of an existing oral squamous cell carcinoma (OSCC), is an important clinical problem in oral medicine.
Methods:  This study presents a follow-up of a study published in 2002. Samples from 54 patients with OSCC, 45 with oral lichen planus (OLP) and 45 with hyperkeratosis (clinically leukoplakia), diagnosed between 1987 and 1996, were analysed for TP53 protein expression and TP53 mutation. Follow-up was 11–17 years for OSCC (mean 13.3), 12–22 years for OLP (mean 15.9) and 12–17 years for hyperkeratosis (mean 14.5).
Results:  Of the 54 OSCC patients, 28 experienced recurrent disease, 21 died of OSCC, 22 died of other causes. Of the 14 OSCC patients with mutated TP53 ( n  = 11), the cancer recurred in eight (57%) and in 20/39 (51%) without mutation. Expression of TP53 protein was significantly associated with reduced overall survival. Among OLP patients, nine were TP53- mutated out of 31 tested. One TP53- mutated OLP patient developed OSCC in a different site. Of the hyperkeratosis patients, three were mutated of 22 tested. One hyperkeratosis patient (non-mutated) developed OSCC in the same site.
Conclusion:  TP53 mutations can exist in benign oral mucosal lesions for many years without progression to malignancy. No association was found between TP53 protein expression or TP53 mutation and recurrence of OSCC or disease-related survival. Overall survival was reduced in patients with positive TP53 protein expression.     

Association of aberrant p53 and p21WAF1 immunoreactivity with the outcome of oral verrucous leukoplakia in Taiwan

The expression of p53 and p21WAF1 in 53 oral verrucous leukoplakias (OVLs), mostly non-dysplastic lesions, was investigated to ascertain the role of such events in malignant conversion. Immunohistochemical analysis revealed aberrant p53 and p21WAF1 immunoreactivity in 51% (27 cases) and 75% (40 cases), respectively. After an average follow-up period of three and a half years, histopathological examination revealed that 22 (42%) cases had developed oral squamous cell carcinoma (OSCC), 14 (26%) cases had undergone recurrence, and 17 (32%) cases were free of disease. The oncogenic potential of this subset of premalignant lesions warrants attention. A significant difference in the frequency of OSCC progression/recurrence was noted in lesions bearing aberrant immunoreactivity of either p53 (93% vs 42%; P=0.00008) or p21WAF1 (80% vs 32%; P=0.002) in comparison with lesions without immunoreactivity. This study suggested that the aberrant immunoreactivity of p53 and p21WAF1 may represent important alterations of OVL and could affect the outcome of this lesion.     

p21WAF1/CIP1 expression is a marker of poor prognosis in oral squamous cell carcinoma

BACKGROUND: Previous research on the prognostic relevance of p21(WAF1/CIP1) in oral squamous cell carcinomas (OSCC) yielded inconclusive and contradictory data. OBJECTIVES: To investigate the prognostic significance of p21(WAF1/CIP1) expression, its relationship to p53 accumulation, proliferation-associated proteins Ki-67 and cyclin D1 in relation to survival and clinicopathological features in OSCC. METHODS: Surgical specimens taken from 106 randomly selected patients were studied by immunohistochemistry. Expression of the protein of interest was correlated with clinical data. RESULTS: p21(WAF1/CIP1) expression was found in 61.3% of OSCCs. Expression of p21(WAF1/CIP1) significantly correlated with tumor size (P = 0.005), lymph node involvement (P = 0.002), clinical stage (P < 0.001), and tumor site (P = 0.002). Patients with tumors showing p21(WAF1/CIP1) immunopositivity had decreased 2-year survival (P = 0.018). Expression of p21(WAF1/CIP1) was not related to age, gender, risk factors (tobacco, alcohol), dental status, or tumor differentiation grade. The p21(WAF1/CIP1) expression positively correlated with proliferation-related variables Ki-67 (P = 0.010) and cyclin D1 (P < 0.001), but not with p53 expression. CONCLUSIONS: The expression of p21(WAF1/CIP1) was found to be associated with poorer prognosis and tumor aggressivity in OSCC.