周期素依赖性激酶4抑制因子基因启动子甲基化与口腔颊癌发生发展的关系

目的探讨p16基因启动子甲基化在口腔颊癌发生发展中的作用.方法对颊黏膜白斑10例、颊癌30例的新鲜标本采用MSP、Western blot杂交分析p16甲基化和p16蛋白表达情况.结果颊癌p16甲基化率高于颊部白斑（P＜0.05）;颊癌有颈淋巴结转移组p16甲基化率高于无转移组（P＜0.5）;颊癌p16蛋白失表达与pl6基因甲基化呈正相关（P＜0.01）.结论启动子甲基化是导致p16基因失活的主要方式,与颊癌的发生、转移有密切关系.     

P16蛋白在颊癌及癌前病变中的表达及临床意义

目的:探讨P16在口腔颊癌中的表达特点及其与临床病理和预后的关系。方法:采用免疫组化和图像分析技术,检测32例颊黏膜白斑和扁平苔藓,30例颊癌组织中P16蛋白的表达水平,并将结果与临床分期、转移等指标进行统计分析。结果:正常颊黏膜鳞状上皮和上皮单纯性增生中均有P16蛋白表达为100%(10/10,22/22);上皮异常增生中P16蛋白的表达率为90.0%(9/10);颊癌P16的表达率为40.0%(12/30),颊癌P16的表达率比上皮异常增生明显降低(P(0.01)。P16缺失表达的患者绝大多数处于临床Ⅲ、Ⅳ期(16/18)。临床Ⅲ、Ⅳ期病例P16蛋白表达水平比Ⅰ、Ⅱ期者明显降低(P(0.01);有颈淋巴结转移组P16蛋白阳性表达率低于无转移组(P(0.05)。结论:P16蛋白的表达与颊癌临床分期及颈淋巴结转移有关;P16蛋白缺失时,颊癌预后较差。P16可作为临床评估颊癌浸润、转移和预后的参考指标。     

口腔白斑和鳞癌中p16基因甲基化的研究

目的　探讨p16基因甲基化在口腔黏膜白斑癌变中的作用。方法　采用甲基特异性PCR技术检测了 10例正常口腔黏膜、20例白斑上皮单纯增生、11例白斑上皮轻度异常增生、10例白斑上皮中度异常增生、9例白斑上皮重度异常增生、10例鳞癌Ⅰ级、12例鳞癌Ⅱ级、8例鳞癌Ⅲ级标本的p16基因甲基化情况。结果　上述各组的p16基因甲基化率分别为 0、5%、18%、10%、22%、50%、42%和 63%。p16基因甲基化率与口腔黏膜组织恶性度显著正相关,并与p16蛋白缺失表达率呈显著正相关关系(rp=0 777 , rs=0 960, P<0 001)。结论　p16基因甲基化状态可作为白斑癌变和口腔鳞癌诊断的分子生物学重要标志之一。     

p73在口腔黏膜白斑和口腔鳞癌中的表达

目的：观察p73在口腔黏膜白斑和口腔鳞癌中的表达,了解p73在口腔鳞癌发生过程中的变化规律。方法：白斑病理标本20例,口腔鳞状细胞癌病理标本31例,另取正常口腔黏膜10例,采用免疫组化检测方法观察p73的表达。应用SPSS11.0软件包对实验结果进行χ^2检验和单因素方差分析。结果：口腔白斑和口腔鳞癌病变组织中p73的表达率分别为65%和90.3%,均显著高于正常黏膜组（20%）,且随组织恶性程度增高呈明显上升趋势,正常黏膜上皮组、白斑组与鳞状细胞癌3组间比较,差异有统计学意义（P〈0.05）。结论：p73的表达与口腔鳞癌的癌变过程密切相关,可作为口腔白斑癌变的标志物。     

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.     

抑癌基因PTEN在口腔颌面部鳞状细胞癌中的突变与缺失

目的：探讨抑癌基因PTEN在口腔颌面部鳞状细胞癌发生、发展过程中的作用。方法：应用聚合酶链反应(PCR)及克隆、基因测序的方法来分析口腔颌面部鳞状细胞癌及正常黏膜组织中PTEN基因第5、第6及第8外显子有无突变及缺失。结果：通过对PTEN基因三个外显子的克隆测序，均未发现有突变及缺失。结论：在DNA水平抑癌基因PTEN在口腔鳞状细胞癌的发生过程中不起作用。     

High Frequency of p53 Gene Mutations in Oral Squamous Cell Carcinoma

目的 :研究口腔鳞状细胞癌发生发展过程中p5 3抑癌基因 5 - 8外显子的突变。方法 :采用非同位素PCR -SSCP -EB染色技术。结果 :30例口腔鳞癌组织标本中 ,16例检测出p5 3基因点突变。结论 :口腔鳞癌中p5 3基因点突变率为 5 3.3% ,p5 3基因突变与口腔鳞癌的发生发展关系密切     

涎腺腺样囊性癌细胞株p16基因缺失、突变及表达意义

目的　研究 p16基因在涎腺腺样囊性癌细胞株中的缺失和突变等结构变化及其表达之间的关系。方法　应用聚合酶链反应 (PCR)和 PCR-单链构象多态性 (SSCP)对涎腺腺样囊性癌细胞株 ACC- 2和高转移细胞克隆 ACC- M进行 p16基因缺失、突变的检测 ,应用免疫组化方法检测 p16基因在细胞株中的蛋白表达。结果　涎腺腺样囊性癌细胞株 ACC- 2检测 p16基因阳性 ,高转移细胞克隆 ACC- M缺失 ,两个细胞克隆均无点突变 ,ACC- 2的p16蛋白表达阳性 ,而 ACC- M蛋白表达阴性。结论　 p16基因在高转移涎腺腺样囊性癌克隆中的缺失 ,表明 p16基因在涎腺腺样囊性癌的演进和转移中具有抑癌作用。     

颊癌P16及P53蛋白表达与临床病理的相关研究

目的探讨P16和P53在口腔颊癌中的表达特点及其与临床病理的关系。方法采用免疫组化和图像分析技术，检测32例颊粘膜白斑和扁平苔藓，30例颊癌和10例正常颊粘膜组织中P16和P53蛋白的表达水平。并将结果与病理分级、临床分期、癌转移等指标进行统计分析。结果正常颊粘膜、单纯性增生、不典型增生、颊癌中P16表达的阳性率分别为：100％、100％、90％、40％；P53表达的阳性率依次为：0、4，5％、25．5％、53．3％。颊癌P16的表达率比上皮不典型增生明显降低（P〈0．05）。颊癌P53的表达率比上皮单纯性增生明显升高（P〈0．05）。临床Ⅰ、Ⅱ期病例中P16蛋白表达水平高于Ⅲ、Ⅳ期的病例（P〈0.01）。无颈淋巴结转移病例中，P16蛋白表达水平高于有转移的病例（P〈0.05）。病理分级为Ⅰ级的病例，P53蛋白表达水平明显低于Ⅱ、Ⅲ级的病例（P〈0．01）。临床Ⅰ、Ⅱ期病例中，P53蛋白表达水平低于Ⅲ、Ⅳ期者（P〈0．05）。无颈淋巴结转移病例中，P53蛋白表达水平低于有转移的病例（P〈0.05）。P16阳性组中，P53的表达显著低于P16阴性组（P〈0．05）。结论颊癌早期P53蛋白有过度表达。P16低表达和P53高表达时提示颊癌预后较差。P16和P53蛋白的联合检测可作为临床评估颊癌浸润、转移和预后的参考指标。     

TRAIL在口腔鳞状细胞癌和白斑中的表达及意义

目的：研究肿瘤坏死因子相关凋亡诱导配体（TNF-related apoptosis inducing ligand,TRAIL）在口腔鳞状细胞癌（OSCC）组织及白斑中的表达及意义。方法：采用免疫组化Maxvision两步法检测54例口腔鳞状细胞癌、30例口腔黏膜白斑、22例正常口腔黏膜组织中TRAIL的表达水平。结果：TRAIL在口腔鳞状细胞癌组织和黏膜白斑中的表达水平均低于其在正常口腔黏膜中的表达水平（P〈0.05）,分别为33.3%、56.7%、86.4%,其在癌组织与白斑中表达水平的差异无统计学意义。TRAIL在高分化癌组织中的表达水平高于中低分化组（P〈0.05）。TRAIL在不同临床分期组间及淋巴结转移与非转移组间表达水平的差异无统计学意义。结论：TRAIL表达的抑制可能与口腔鳞状细胞癌及口腔黏膜白斑的发生有关,并且TRAIL可能参与了初始阶段的正常黏膜向癌组织的转化。TRAIL在口腔鳞状细胞癌中的表达水平与癌组织的分化程度相关。     

多肿瘤抑制基因1在口腔黏膜癌前病变和鳞癌中的变异

目的　检测多肿瘤抑制基因 1(multipletumorsuppressor 1,MTS1)基因在口腔黏膜癌前病变和鳞癌中的表达和变异 (纯合性缺失和突变 )情况 ,以期发现MTS1在口腔黏膜恶变发生发展中的作用。方法　采用免疫组化SP法检测MTS1的表达情况 ;同时采用PCR、SSCP技术检测相同标本中MTS1基因exon1和exon2的纯合性缺失和突变情况。结果　口腔癌前病变中MTS1基因全部表达 ,无基因缺失和突变。鳞癌中MTS1的表达阳性率 6 0 % ;有 10例发生exon1和 (或 )exon2的纯合性缺失 ,4例基因突变 ,总变异率为 31.1% (14 / 4 5 ) ;伴有局部淋巴结转移的鳞癌的基因变异率为5 7.1% ,不伴有淋巴结转移的为 8.3% ,两者间差异有显著性 (P <0 .0 5 )。结论　MTS1基因在口腔癌前病变中无改变 ,不能作为检测口腔癌前病变的生物学指标 ;MTS1基因改变在口腔鳞癌的发生、发展中起一定作用     

The correlations between alteration of p16 gene and clinicopathological factors and prognosis in squamous cell carcinomas of the buccal mucosa

J Oral Pathol Med (2012) 41 : 463–469 Objective: To evaluate relationships between the alteration of p16 gene and the clinical status and prognosis of the patients with squamous cell carcinoma of the buccal mucosa. Methods: Thirty buccal cancers were included in the analysis. Deletion analysis was performed by PCR. Point mutation analysis was used by PCR‐SSCP and direct sequencing. Methylation‐specific PCR methods were adopted for the evaluation of p16 methylation. The correlation between alteration of p16 gene and clinicopathological factors buccal cancer was evaluated by Fisher’s exact test. Kaplan–Meier and Cox regression were used to investigate the relationship between p16 alteration and survival time. Results: The frequency of p16 alteration was 63.3% in buccal carcinomas. P16 deletion was associated significantly with tumor size (P = 0.01). P16 point mutation was associated significantly with differentiation (P = 0.006). P16 methylation was associated significantly with nodes metastasis (P = 0.027). The overall survival rate of 30 buccal carcinomas was 53.3%. The Log‐rank test (P = 0.021) and univariate Cox regression analysis (P = 0.030) revealed that p16 methylation was significantly associated with the overall survival rate. Multivariate analysis showed that p16 deletion, p16 mutation, and p16 methylation were not statistically significant. Conclusions: The alterations of p16 gene may play a major role in malignancy and development and metastases of buccal carcinoma and may be an excellent marker of aggressive clinical behavior. P16 methylation has a prognostic value in buccal carcinoma but not an independent prognosis factor. P16 point mutation and p16 deletion have not prognostic significance in buccal carcinoma.     

涎腺腺样囊性癌p16基因失活机制的研究

目的　研究p16基因在涎腺腺样囊性癌中的失活机制。方法　收集山东大学齐鲁医院及口腔医院口腔 颌面外科涎腺腺样囊性癌新鲜标本53例,应用PCR、聚合酶链反应-单链构象多态性分析(PCR-SSCP)及甲基化特 异性PCR(MSP)技术检测涎腺腺样囊性癌中p16基因的纯合性缺失、突变及甲基化。结果　53个病例中16例 (30·2%)纯合性缺失,4例(7·5%)突变,26例(49·1%)高甲基化。结论　p16基因在涎腺腺样囊性癌中的主要失活 机制是启动子高甲基化和纯合性缺失,而基因突变比较少见。     

口腔颌面部鳞癌组织中p16基因变异的初步研究

目的 检测口腔颌面部鳞癌中p16基因的缺失和突变，以了解p16基因在口腔颌面部恶性肿瘤发生发展过程中的作用。方法 应用聚合酶链反应－单链构象多态性分析（PCR－SSCP）技术，检测33例原发性口腔颌面部鳞癌的石蜡标本，将p16基因的变异频率与口腔颌面部鳞癌的临床分期，组织学分级及淋巴结转移进行统计学分析。结果 p16基因的缺失率为21％，点突变率为6％，变异频率为27％。临床Ⅰ、Ⅱ、Ⅲ、Ⅳ期每两期之间无显著性差异（P＞0．05）；而临床Ⅰ、Ⅱ期和Ⅲ、Ⅳ期之间有显著性差异（P＜0．05）。组织学分级和有，无淋巴结转移之间p16基因变异无显著性差异（P＞0．05）。结论 p16基因的缺失和突变是口腔颌面部鳞癌中常见的分子事件，它的失活在口腔颌面部鳞癌的发生发展中起着重要作用，说明其变异与肿瘤的临床分期密切相关，随着肿瘤临床进程的发展，其变异频率增高。研究未发现其变异与肿瘤组织学分级和淋巴结转移之间存在相关关系。     

Alterations of p16/MTS1 gene in oral squamous cell carcinomas from Taiwanese

To determine the alterations of the p16/MTS1 gene in oral squamous cell carcinoma (OSCC), we examined in Taiwanese patients the mutation, deletion and methylation of p16/MTS1 in primary OSCCs associated mostly with betel quid (BQ)/tobacco use. Among 110 tumors undergoing mutational analyses, seven (6%) showed mutations in exon 2 or the intron 1/exon 2 splice site. All but one mutation disrupted the encoded proteins. Base transitions represented the vast majority (6/7) of the mutations identified in BQ/tobacco consuming subjects. It was noted that 15/56 (27%) tumors examined by restriction fragment methylation analysis revealed a significant level of methylation in different loci of exon 1 as compared with the respective non-cancerous tissue. Mutation of p16/MTS1 was exclusively identified in carcinomas of buccal mucosa, whereas methylation of the p16/MTS1 promoter region occurred preferentially in carcinomas of the tongue (54%) rather than at other sites (22%). Homozygous deletion was not found in 56 paired samples examined, nor was hemizygous deletion indicated in 12 informative cases. The results indicated aberrant methylation and mutation as the molecular abnormality of p16/MTS1 in the OSCC from Taiwanese.     

Alterations of p16INK4a tumour suppressor gene in mucoepidermoid carcinoma of the salivary glands

Mucoepidermoid carcinoma (MEC) is common in the salivary glands, but alterations of the p16(INK4a) tumour suppressor gene are largely unknown. The aim of this study was to analyse p16(INK4a) gene alterations in MEC, and evaluate their significance for carcinogenesis. Thirty-eight salivary glands with MEC and six normal salivary glands were studied for p16(INK4a) alterations. In the MEC-affected group, there were 23.7% (9/38) and 13.2% (5/38) cases of homozygous deletion, and 5.3% (2/38) and 2.6% (1/38) cases of point mutation in p16(INK4a) exon 1 and exon 2, respectively. Hypermethylation of the p16(INK4a) gene promoter was found in 13 cases (13/38, 34.2%). Alterations of the p16(INK4a) gene were not found in the normal salivary glands. These findings suggest that the main mechanisms of inactivation of the p16(INK4a) gene in MEC of the salivary glands are promoter hypermethylation and homozygous deletion.     

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 Immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.     

The correlation between alteration of p16 gene and clinical status in oral squamous cell carcinoma

The purpose of the study was to evaluate the presence of alteration of the tumor suppressor gene p16 and to correlate these changes with the clinical status of the patients in oral squamous cell carcinoma. Forty-eight oral squamous cell carcinomas were included in the analyses. Deletion analysis was performed by the polymerase chain reaction (PCR). Mutation analysis was restricted to exon 1 and exon 2 of the p16 gene, previously shown to have a high incidence of mutations. The sequences containing exon 1 and exon 2 were amplified by PCR and screened with a single-strand conformation polymorphism (SSCP) technique. Samples showing band shifts in SSCP were sequenced by PCR direct sequencing. Western blots were used to detect the protein expression of the p16 gene, and the results were evaluated with regard to their biological relevance in correlation with clinicopathological factors. Seven (14.6%) deletions were found; 5 (10.4%) mutations were discovered and located in different codons; 26 (54%) specimens had no p16 protein expression; in 11 specimens with p16 deletion or mutation, p16 protein could not be detected. One mutation was non-sense. The p16 gene alterations showed no relationship with location and clinical stage of cancer; however, a close relationship between p16 alterations and cancer metastasis to neck lymph node was found. The alteration rate gradually elevated from well to poorly differentiated grades. We perceive two results. First, the alterations of the p16 gene are common in oral squamous cell carcinoma. Second, the alterations of the p16 gene may attribute to the metastatic behavior or histological grade of cancer cells.