河南地区食管鳞癌和贲门腺癌与人乳头瘤病毒感染相关性的分析

目的:了解人乳头瘤病毒16型(HPV16)与河南食管癌高发地区食管鳞癌、贲门腺癌发生的关系.方法:利用聚合酶链式反应(PCR)对高发区食管鳞癌组织(44例)、贲门腺癌组织(18例)进行HPV-DNA检测.结果:食管鳞癌及贲门腺癌组织中均检测到HPV16 E6 DNA表达,但食管鳞癌HPV16 E6 DNA表达(84%,37/44)明显高于贲门腺癌(44%,8/18),P<0.01.食管鳞癌及贲门腺癌组织中,高危型HPV16 E6 DNA表达与患者年龄、性别、分化程度、浸润程度,淋巴结转移以及肿瘤分级无相关性,P值均＞0.05.结论:同一地区食管鳞癌和贲门腺癌均有不同程度HPV感染,提示HPV可能是两者共同相关致病危险因素,高危型HPV16感染可能在食管鳞癌和贲门腺癌发生中起重要作用.     

河北省磁县食管癌高发区食管鳞状细胞癌组织HPV检测及FHIT表达的研究

背景与目的:河北省磁县是食管癌高发区,有关人乳头状瘤病毒(humanpapillomavirus,HPV)感染和当地食管癌发生的关系尚无研究报道。本研究旨在探讨HPV感染在当地食管癌发生中的病因学意义,同时分析与环境致癌物密切相关的脆性组氨酸三联体基因(fragilehistidinetriad,FHIT)表达与HPV感染的关系。方法:收集河北省磁县食管癌高发区128例和非高发区24例食管鳞状细胞癌石蜡包埋组织,用PCR进行HPV检测,用免疫组化方法分析FHIT在蛋白水平上的表达情况,同时分析HPV感染和FHIT异常表达的关系。结果:PCR检测结果表明,河北省食管癌高发区食管鳞状细胞癌组织中HPV检出率为20.3%,略高于非高发区(8.3%),但两者差别无显著性(P>0.05)。免疫组化结果显示,食管癌高发区食管鳞癌组织中FHIT基因异常表达率(75.6%)明显高于非高发区食管鳞癌组织(54.2%,P<0.05)。食管癌组织中HPV感染和FHIT的异常表达之间无明显相关性。结论:河北省食管癌高发区食管癌组织FHIT的异常表达率明显高于非高发区病例。     

食管贲门双源癌患者癌组织中mdm2与Bcl-2和bax及p53蛋白的表达

目的:探讨同一个体食管贲门双源癌组织mdm2、Bcl-2、bax和p53蛋白变化特征及其意义。方法:采用免疫组化卵白素-生物素-过氧化物酶复合物(ABC)法和组织病理学方法,分析15例双源癌患者(同时发生食管鳞癌和贲门腺癌)mdm2、Bcl-2、bax和p53蛋白的表达状况。结果:15例食管和贲门双源癌患者食管鳞癌和贲门腺癌组织均出现不同程度的mdm2、Bcl-2、bax和p53蛋白的阳性表达,食管癌组织中免疫阳性率分别为67%(10/15)、87%(13/15)、73%(11/15)和73%(11/15);贲门癌组织中分别为67%(10/15)、80%(12/15)、80%(12/15)和67%(10/15),且免疫反应类型主要为弥漫型。mdm2、Bcl-2、bax和p53蛋白在食管和贲门双源癌肿瘤组织中一致性改变率分别为100%(15/15)、80%(12/15)、67%(10/15)和80%(12/15)。结论:食管和贲门双源癌存在较高的mdm2、Bcl-2、bax和p53蛋白表达一致性改变,提示食管贲门双源癌可能具有相似的发病因素和分子机制。     

p63基因在食管鳞癌组织中的表达

目的:研究p63基因在食管癌和贲门癌组织中的表达情况。方法:应用免疫组织化学SP法检测贲门癌和食管癌p63基因蛋白的表达。结果:食管癌组织中p63表达阳性率为96.7%(29/30),贲门癌组织中p63基因蛋白免疫阳性表达率为10.4%(3/28),二者差异极显著(P<0.001)。在食管鳞癌组织中p63基因蛋白表达强度与癌组织的分化程度呈负相关(r=-0.383),与肿瘤的浸润呈正相关(r=0.577),与淋巴转移呈正相关(r=0.549),与年龄和性别无关(P>0.05)。结论:p63基因在食管鳞癌组织中呈高表达,表明可能参与致癌作用,而在贲门腺癌中的作用不明显,提示二者在发病的机理上可能存在差异。     

p16INK4A 和bcl-2 蛋白在CIN 和宫颈鳞癌中的表达

 目的 探讨p16INK4A和bcl-2在宫颈上皮内瘤（CIN）和宫颈鳞癌的表达状态及其在病理诊断中的意义。方法 采用免疫组化SP法对39例CIN、24例浸润性宫颈鳞癌和20例正常官颈组织进行p16INK4A和bck2检测。结果 正常宫颈组织不表达p16INK4A，CINⅠ阳性表达率很低并为局灶表达，但几乎所有的CINⅡ／Ⅲ和浸润性宫颈鳞癌均弥漫大量表达p16INK4A。正常宫颈组织只有基底细胞层表达bcl-2蛋白，CINⅢ和Ⅰ～Ⅱ期浸润癌bcl-2阳性细胞弥漫分布，阳性袁达率分别为53．8％和57．1％。Ⅲ～Ⅳ期浸润癌bcl-2阳性率下降，阳性细胞局限。结论 p16INK4A过表达与宫颈鳞癌恶性特性密切相关，检测p16INK4A有助于及时发现高级别CIN和宫颈鳞癌。     

食管鳞癌中hMLH1、E-cadherin、p16^INK4a基因启动子甲基化及其意义

背景与目的:目前认为抑癌基因启动子甲基化导致转录抑制是恶性肿瘤发生的重要机制之一,hgLH1、E-cadherin及p16INK4a基因在多种恶性肿瘤中都已被证实存在较高频率的甲基化.本研究通过检测食管鳞癌组织及癌旁组织中hMLH1、E-cadherin,p16INK4a基因启动子甲基化的发生情况,探讨hMLH1、E-cadherin、p16INK4a基因启动子甲基化在食管鳞癌发生发展中的作用.方法:采用酚-氯仿法提取105例食管鳞癌组织及癌旁组织的基因组DNA,应用甲基化特异性PCR对所提DNA进行hMLH1、E-cadherin、p16INK4a基因甲基化检测.采用EnVison免疫组织化学二步法对癌组织中上述3种基因蛋白表达进行检测.结果:癌组织中E-cadherin、hMLH1、p16INK4a基因启动子甲基化的阳性率分别为57.1%(60/105)、20.9%(22/105)和50.5%(53/105),而癌旁食管组织中相应的3个基因的甲基化率分别为10.5%(11/105)、1.9%(2/105)和7.6%(8/105),均显著低于癌组织.E-cadherin(P=0.021)及p16INK4a(P=0.026)基因甲基化与蛋白表达缺失密切相关,而hMLH1基因甲基化与蛋白表达无显著相关性.E-cadherin基因启动子甲基化与淋巴结转移有关(P=0.016),p16INK4a基因启动子甲基化与低分化癌有关性(P=0.024).hMLH1基因甲基化与各项临床病理特征均无关.结论:食管鳞癌中p16INK4a基因启动子甲基化与相应蛋白表达缺失密切相关,且在低分化癌中更多见;E-cadherin基因启动子甲基化与相应蛋白质表达缺失有相关性,并且有淋巴结转移多见的显著特征,这2个基因的甲基化位点与食管鳞癌密切相关.hMLH1基因甲基化可能并不直接参与食管鳞癌的发生、发展.     

食管鳞癌中p16基因启动子区甲基化及其表达

 目的探讨食管鳞癌（ESCC）p16基因甲基化的状况及其表达与食管鳞癌临床病理特征之间的关系。方法采用甲基化特异性PCR方法（MSP）分别检测75例食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用Envision免疫组化法检测食管癌组织及癌旁组织的p16蛋白的表达。结果75例标本中，食管癌组织、癌旁组织和切缘细织p16基因甲基化率分别为41．3％（31／75）、13．3％（10／75）和6．67％（5／75）。癌组织和癌旁组织P16蛋白的阳性表达率分别为29．3％（22／75）和56．7％0（17／30）。31例癌组织p16基因甲基化阳性标本中有2例（6．4％）检测到P16蛋白的表达，而44例癌组织p16基因甲基化阴性标本中有20例（45．5％）检测到P16蛋白的表达。食管癌组织p16基因甲基化率显著高于癌旁组织和切缘组织（P〈0.01），P16蛋白表达与p16基因甲基化呈负相关。p16基因启动子区甲基化与食管癌的组织学分级、肿瘤部位无明显相关，与临床分期、淋巴转移密切相关。结论p16基因甲基化在食管癌发生发展中起着重要作用，食管鳞癌的分期和淋巴结转移与p16基因甲基化之间有密切关系。     

慢性乙型肝炎病毒感染与p16INK4A基因启动子甲基化关系的研究

目的探讨慢性乙型肝炎病毒(HBV)感染对p16INK4A基因启动子甲基化的影响及其在HBV相关肝细胞癌(HCC)形成中的作用。方法选取23例HBV相关HCC癌及癌旁组织、25例慢性乙型肝炎肝穿刺组织,采用甲基化特异性PCR(MSP)检测p16INK4A基因启动子的甲基化状态;免疫组化EnVision二步法测定肝组织内HBsAg、HBcAg、HBeAg和HBx蛋白的表达;荧光实时定量PCR检测肝组织HBV DNA含量;PCR扩增和直接测序检测HBV x基因变异。结果癌组织p16INK4A基因启动子甲基化阳性率(47.83%)明显高于癌旁组织(17.39%),慢性乙型肝炎患者p16INK4A基因启动子甲基化阳性率(36.00%)与癌组织、癌旁组织差异无统计学意义。在癌旁组织和慢性乙型肝炎, p16INK4A基因启动子甲基化者HBx蛋白表达中位数分别为3.000和0.250,明显高于非甲基化者(0.500和0.000),但在癌组织中,HBx蛋白的表达与p16INK4A基因启动子甲基化状态无关。HBsAg、HBcAg、组织HBV DNA含量和x基因突变均与p16INK4A基因启动子甲基化状态无关。结论在癌前病变中,p16INK4A基因启动子甲基化与HBx蛋白高表达有关,HBx蛋白可能通过诱导p16INK4A基因启动子甲基化而使该抑癌基因失活,在HBV相关HCC形成中起重要作用。     

高发区食管癌患者p16基因甲基化及其表达的比较研究

目的:比较p16基因甲基化在食管癌高发区河南林州(北方组)和广东揭阳(南方组)之间的异同,探讨p16基因甲基化在不同气候环境条件下的两地食管鳞癌(ESCC)发生中的作用。方法:采用甲基化特异性PCR方法(MSP)分别检测两地食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用EnVision免疫组化法检测两地食管癌组织及癌旁组织的p16蛋白的表达。结果:南方组75例标本中,食管癌组织、癌旁组织和切缘组织p16基因甲基化率分别为41.3%(31/75)、13.3%(10/75)和6.67%(5/75);癌组织和癌旁组织p16蛋白的阳性表达率分别为29.3%(22/75)和56.7%(17/30);31例p16基因甲基化阳性标本中有2例(6.4%)检测到p16蛋白的表达,而44例p16基因甲基化阴性标本中有20例(45.5%)检测到p16蛋白的表达。北方组65例标本中,食管癌组织、癌旁组织、切缘组织p16基因甲基化率分别为52.3%(34/65)、16.9%(11/65)和7.69%(5/65);食管癌组织和癌旁组织p16蛋白的阳性表达率分别为32.3%(21/65)和66.7%(20/30);34例p16基因甲基化阳性标本中有4例(11.8%)检测到p16蛋白的表达,而31例p16基因甲基化阴性标本中有17例(54.8%)检测到p16基因的表达。两地组内癌组织p16甲基化率均显著高于癌旁组织和切缘组织,p16蛋白表达与p16基因甲基化呈负相关(P<0.01)。两地同类组织比较p16甲基化率和p16蛋白表达率均无显著性差异(P>0.05)。结论:p16基因异常甲基化后功能失活可能是南、北两地食管癌癌变过程的重要事件,在我国南北环境气候条件不同的两地高发区食管癌的发生中均起着重要的作用。本研究为环境因素和p16基因功能之间的生物学关联在食管癌的发生中提供了一定的证据。     

食管鳞癌及贲门腺癌中分泌型卷曲相关蛋白4基因启动子区甲基化状态的联合检测

目的:检测食管鳞癌(esophageal squamous cell cancer,ESCC)和贲门腺癌(gastric cardia adenocarcinoma,GCA)中分泌型卷曲相关蛋白4(secreted frizzled related protein 4, SFRP4)的基因甲基化状态,探讨其与食管鳞癌和贲门腺癌发生的关系.方法:应用甲基化特异性PCR(methylation-specific PCR,MSP)及RT-PCR的方法检测49例食管鳞癌及58例贲门腺癌中SFRP4基因的甲基化状态及其mRNA表达情况,应用免疫组织化学法检测Wnt通路中心因子β-catenin蛋白的表达情况,并分析其与临床病理参数间的关系.结果: 在食管鳞癌及贲门腺癌中,SFRP4基因的甲基化率分别为42.6%(21/49)和72.4%(42/58),均明显高于癌旁非肿瘤组织(P<0.01).在贲门腺癌中SFRP4基因的高甲基化与肿瘤患者的临床分期相关,与病理分级无关;而在食管鳞癌中,该基因的甲基化与各临床病理指标均无关(P>0.05).SFRP4基因在食管鳞癌及贲门腺癌中mRNA阳性表达率分别为69.3%(34/49)和44.8%(26/58),均明显低于癌旁非肿瘤组织(P<0.05),且肿瘤组织中mRNA表达与该基因的甲基化状态明显相关(P<0.01).通路中心因子β-catenin蛋白在食管鳞癌及贲门腺癌中的异质表达率分别为65.3%(32/49)和86.2%(50/58),均明显高于癌旁非肿瘤组织(P<0.01),且其异质表达与SFRP4基因的甲基化状态相关(P<0.05).结论:SFRP4基因的高甲基化状态可能是引起贲门癌及食管癌发生的共同分子机制之一,并有可能通过Wnt/β-catenin信号转导通路发挥作用. 检测SFRP4基因甲基化状态对于贲门腺癌的预后评估有一定参考价值.     

Hypermethylation of tumor suppressor genes (p16INK4A, p14ARF and APC) in adenocarcinomas of the upper gastrointestinal tract

Aberrant promoter methylation is an important mechanism for gene silencing. In the present study, 50 Barrett's esophagus-associated esophageal adenocarcinomas (ADC), 50 cardiac ADC and 50 gastric ADC were investigated by means of methylation-specific real-time PCR for hypermethylation in the tumor suppressor genes APC, p16(INk4A) and p14(ARF). Additionally, expression of p16(INK4A) protein in the carcinomas was assessed using immunohistochemistry. Marked differences in hypermethylation were found between esophageal, cardiac and gastric ADC in the APC gene (78% vs. 32% vs. 84%) and in the p16(INK4A) gene (54% vs. 36% vs. 10%). Hypermethylation of p14(ARF) was absent from esophageal ADC and present infrequently in cardiac (2%) and gastric ADC (10%). Complete loss of p16(INK4A) protein expression was detectable in 45% of all tumors and was significantly associated with hypermethylation of the p16(INK4A) gene (p<0.0001, chi(2)-test). Our results suggest that hypermethylation of p16(INK4A) and APC are frequent findings in esophageal, cardiac and gastric ADC. Additionally, the data point to a tumor specific methylation pattern in upper gastrointestinal ADC.     

Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas

Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV-DNA and the use of p16(INK4A) overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridization (FISH) and p16(INK4A)-specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16(INK4A) accumulation. Only 5 out of 48 HPV-negative tumors showed p16(INK4A) immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non-smoking patients with HPV-containing TSCC show a remarkably better disease-specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16(INK4A) overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV-positive tumors show a favorable prognosis as compared to those with HPV-negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non-smokers differ from those occurring in smokers, the former being related to HPV 16 infection.     

p16INK4A Expression in Squamous Cell Carcinomas of the Vagina and the Vulva in Tunisian Women

Background: The role of p16INK4A expression in uterine cervix cancer is well established. In the remainingfemale lower genital tract cancers, the importance of p16INK4A up-regulation is less clear. In our study, we analyzedthe role of p16INK4A expression and HPV infection in carcinomas of the vulva and the vagina in Tunisian women.Materials and Methods: We conducted a retrospective study of 30 carcinomas including 15 vulvar squamouscell carcinomas (SCCs) and 15 vaginal SCCs. Immunohistochemistry was used to determine p16INK4A expression.HPV detection and typing was by in situ hybridization. Results: p16INK4A expression was detected in 86.7% ofvaginal SCCs with a strong and diffuse immunostaining in 60% of cases, and also in 73.3% of vulvar SCCswith focal immunoreactivity in 53.3% The association between p16INK4A expression and HPV infection wassignificant in vaginal SCCs (p=0.001) but not vulvar SCCs (p>0.05). Conclusions: p16INK4A expression could beused as a useful marker for HPV positivity in vaginal SCCs similar to that described in uterine cervix cancers.However, our data support the presence of 2 different mechanisms for p16INK4A expression in HPV-related andHPV-unrelated vulvar carcinomas.     

Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan

Inactivation of p16INK4a gene through promoter hypermethylation has been frequently observed in non small cell lung cancer; however, various studies have shown a controversial correlation between p16INK4a hypermethylation and cigarette smoking. Our recent report showed that human papillomarvirus (HPV) 16/18 infections were associated with the development of nonsmoking female lung cancer in Taiwan and we further speculated that HPV infection may be linked with p16INK4a hypermethylation. To verify the influence of environmental exposure, including cigarette smoking, environmental carcinogen exposure and HPV infections on p16INK4a hypermethylation, tumors from 162 lung patients, including 67 smoking males, 41 nonsmoking males and 58 nonsmoking females, were subjected to p16INK4a hypermethylation analysis by methylation-specific PCR. As the results showed, p16INK4a hypermethylation was detected in 40 (59.7%) of 67 smoking male, 15 (36.6%) of 41 nonsmoking male and 35 (60.3%) of 58 nonsmoking female lung tumors. This result seemed to reveal that gender and cigarette smoking both possess an equal influence on p16INK4a hypermethylation. This result also led to a speculation that HPV infection may promote p16INK4a hypermethylation in nonsmoking female lung cancer patients. From our data, p16INK4a hypermethylation frequency in nonsmoking female lung tumors with HPV infection was as high as 70% (30 of 43) compared to those without HPV infection (33%; 5 of 15). In fact, the correlation between HPV infection and p16INK4a hypermethylation was only observed in nonsmoking female lung tumors (p = 0.017), but not in smoking male or nonsmoking male lung tumors. Moreover, the reverse correlation between p16INK4a immunostaining and p16INK4a promoter hypermethylation was also only observed in nonsmoking female lung tumors. These results strongly suggested that the involvement of HPV infection in lung tumorigenesis of nonsmoking female cancer patients in Taiwan may be mediated at least in part through the increase of hypermethylation to cause p16INK4a inactivation.     

高危型HPV负荷量和p16INK4A蛋白监测评估宫颈锥切术治疗CIN

[目的]评价高危型人乳头状瘤病毒HPV负荷量的检测和p16INK4A蛋白的表达在预测宫颈上皮内瘤变(CIN)宫颈锥切术后残存病变或复发中的意义.[方法]回顾性分析142例2008年10月至2010年12月因CIN行宫颈锥形切除术治疗患者的临床资料.所有患者均于宫颈锥形切除术前6个月以内和术后6～12个月进行HPV负荷量检测,并采用免疫组化方法检测HPV DNA阳性患者宫颈细胞中p16INK4A蛋白表达.[结果]宫颈锥切术前,随着CIN级别的上升,HPV负荷量以及p16INK4A蛋白表达均明显增强(P＜0.05).但在宫颈锥切术后,HPV负荷量和p16INK4A蛋白表达明显降低,宫颈锥切术前和术后两者之间差异有统计学意义(P＜0.05).[结论] HPV负荷量持续增高和p16INK4A蛋白持续呈强阳性是宫颈锥切术后发生残存病变或复发的高危因素,在监测HPV负荷量的同时检测p16INK4A蛋白的表达,对判断宫颈锥切术后发生残存病变或复发有重要意义.     

Nuclear co-expression of p14ARF and p16INK4A in uterine cervical cancer-derived cell lines containing HPV

The Papanicolaou test (Pap) has been responsible for a significant reduction of cervical cancer-related morbimortality. In order to increase its sensitivity and specificity new markers have been studied and incorporated to cytological and histological methods for diagnosis for cervical cancer, such as p16INK4A that has been considered the immunocytochemical marker of choice for detection of HPV related cancers. We considered that p14ARF could be a complementary marker in order to improve the accuracy of cytological diagnosis because its genetic proximity to p16INK4A. We performed a systematic analysis of several putative cervical cancer markers in order to evaluate their performance in the detection of malignancy, in comparison with p16INK4A and p14ARF, using immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses. Most markers were non-specific and could not discriminate HPV infected cancer cell lines from other non HPV malignant. In contrast, nuclear co-expression of p16INK4A and p14ARF was observed only in HPV-transformed cancer cell lines. Notably, in C-33A cervical cancer cells (HPV negative), p14ARF was present in the nucleoli, but p16INK4A was conspicuously absent from the nuclei of these cells. We conclude that both markers; p16INK4A and p14ARF are complementary and should be evaluated jointly in order to improve the accuracy of cytological diagnosis of cervical cancer.     

Methylation Status of P16Ink4a in Human Papillomavirus-Associated Cancer of Oral Cavity and Oropharynx in Northeastern Thailand

Background: Over-expression of p16INK4a protein is a biomarker for human papillomavirus (HPV)-associated cervical cancer. However, absence of p16INK4a protein expression in HPV-associated cancer of the oral cavity and oropharynx has been reported. Among a number of possible reasons for this is methylation, which is frequently noted in the promoter region of p16INK4a and is associated with silencing of the gene and disease severity. Methods: We investigated the relationships between p16INK4a protein expression, HPV infection and methylation status of the p16INK4a promoter in cancers of the oral cavity and oropharynx. Fifty-three formalin-fixed paraffin-embedded (FFPE) cancer tissue samples from the oral cavity (49 cases) and oropharynx (4 cases) were studied. P16INK4a protein expression was determined using immunohistochemical staining (IHC). Additional oral tissues lacking squamous intraepithelial lesions (SILs), and cervical tissues with high-level SILs, were used as negative and positive controls, respectively. High-risk HPV infection was detected using HPV E6/E7 mRNA in situ hybridization. Methylation status of the p16INK4a promoter was investigated using sodium bisulfite treatment and methylation-specific PCR (MS-PCR).Results: HPV infection was found in 40.8% (20/49) and 50.0% (2/4) of oral cavity and oropharynx cancers, respectively. Promoter methylation of p16INK4a occurred in 73.6 % of all cases and differed significantly in frequency between HPV-positive (90.9%, 20/22) and HPV-negative (61.3%, 19/31) samples. Expression of p16INK4a was found in 35.8% (19/53) and commonly detected in samples with p16INK4a unmethylation (79.5%). Interestingly, the silencing of p16INK4a (64.2%, 34/53) was significantly associated with methylation status (91.2%, 31/34), especially in HPV-infected samples in which the p16INK4a promoter was methylated (52.9%, 18/34). Conclusions: This result demonstrated high frequency of p16INK4a promoter methylation status in HPV-associated HNSCC subsets that could influence the silent p16INK4a expression and might promote disease severity.     

Expression of p16INK4a and MIB-1 in relation to histopathology and HPV types in cervical adenocarcinoma

A total of 101 primary cervical adenocarcinomas were analyzed for the presence of p16INK4a and MIB-1 expression in correlation with the presence of 'high-risk' types of human papillomavirus (HR-HPV) and clinical outcome.We found that adenocarcinoma grading showed a significant negative correlation to p16INK4a levels (p=0.001): i.e. we found less intense p16 staining in poorly differentiated tumors than in more highly differentiated tumors as well as a highly significant correlation between HPV infection and higher levels of p16INK4a staining (p=0.00), which was similar for different HPV-types. Tumor suppressor protein p16INK4a levels were higher in HPV positive than in HPV negative tumors. Higher levels of the proliferation marker MIB-1 were associated with poorer outcome. Higher MIB-1 levels were seen in tumors with a lower grade and higher stage at diagnosis. Moreover, MIB-1 levels seem to be higher in tumors due to infection with HPV 16 and 18 compared with HPV 45. MIB-1 may be a helpful marker in grading adenocarcinoma: a high level of expression of MIB-1 indicates a low grade of tumor, whereas high expression of p16INK4a indicates a highly differentiated of the tumor. Thus, immunostaining for p16INK4a appears to be a useful diagnostic tool for cervical adenocarcinoma.     

Expression of p16(INK4a) in relation to histopathology and viral load of 'high-risk' HPV types in cervical neoplastic lesions

A total of 91 cervical archival biopsy series were analysed for the presence and viral load of 'high-risk' types of human papillomavirus (HR-HPV), and p16(INK4a) expression. The women had various degrees of CIN (cervical intraepithelial neoplasia). HPV 16 was the most prevalent type found, at 47% frequency. The frequency of HPV 16 increased with increasing immunoreactivity to p16(INK4a), from 39% to 44% at cases scored low to medium, to 65% at high reactivity. Thirty (33%) of the samples had negative p16(INK4a) analysis results, but were positive for HR-HPV. There was no significant correlation between viral load and the level of p16(INK4a) expression, while the grade of CIN correlated to such expressions. Thus, p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and might complement the HPV analysis in a clinical prognostic procedure in order to find women at risk for cervical cancer.     

Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer

The CDKN2A locus on human chromosome 9p21 encodes two tumor suppressors, p14(ARF) and p16(INK4A), which enhance the growth-suppressive functions of the retinoblastoma (Rb) and the p53 proteins, respectively. Conversely, the E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPVs) causally associated with carcinogenesis of the uterine cervix contributes to tumor development by inactivating p53 and Rb. Nevertheless, a correlation between expression of p14(ARF)/p16(INK4A) and HPV infection in uterine cervix is less clear. To clarify this, we examined 25 cervical cancers and 11 normal uterine cervixes. HPV was detected in 21 of 25 cervical cancers (84%) and their subtype was determined by PCR-RFLP. Quantitative real-time RT-PCR assays showed overexpression of p14(ARF) mRNA in all 21 HPV-positive cases (100%). p16(INK4A) mRNA was overexpressed in 17 cases of the HPV-positive cases (81%). In four HPV-negative cancers, reduced expression of p14(ARF) mRNA was detected in two cases (50%) and reduced p16(INK4A) mRNA in three cases (75%). Our data indicate that the overexpression of p14(ARF) and p16(INK4A) strongly associates with HPV-positive cervical cancers and that reduced expression of p14(ARF) and p16(INK4A) correlates with HPV-negative cervical cancers. These findings may indicate that impaired p14(ARF) and p16(INK4A) mRNA expression contribute to tumor development in HPV-negative cervical cancers by failure to support p53 and Rb instead of their inactivation by HPV E6 and E7.