人类乳头瘤病毒与宫颈糜烂癌变的相关性研究

目的 :探讨人类乳头瘤病毒与宫颈糜烂癌变的相互关系。方法 :阴道镜下对 116例宫颈糜烂 ,12例宫颈癌及 36例正常宫颈分别钳取活组织 ,用聚合酶链反应对活组织进行HPV公共引物及HPV16、HPV18型特异引物检测。结果 :2 9.31%的宫颈糜烂患者HPV阳性 ,正常宫颈阳性率仅 11.11% (P <0 .0 5) ,宫颈糜烂Ⅰ度、Ⅱ度、Ⅲ度及宫颈癌中HPV16是最常见亚型 ,其阳性率分别为 9 38%、18 90 %、2 5 53%、75 0 % ,宫颈颗粒型或乳突型糜烂与宫颈癌强相关 (OR =5 56,95%CI =1 2 1～ 2 5 4 9)。“高危”HPV DNA检出率随宫颈鳞柱上皮异位程度增加而升高。结论 :宫颈糜烂的病因可能与持续感染“高危”HPVs有关。     

人乳头瘤病毒16型转化基因在不同阶段宫颈上皮病变组织中的分布及基因变异特点

目的 研究人乳头瘤病毒(HPV)16 E6、E7和E5基因在湖北地区不同阶段宫颈上皮病变患者组织中的分布以及E6、E7基闪的变异特点.方法 从124例宫颈癌、17例宫颈上皮内瘤变(CIN) Ⅰ+CINⅡ级、23例CIN Ⅲ级和36例慢性宫颈炎患者活检或手术切除标本中提取组织DNA,用HPV16 E6、E7和E5特异性引物进行PCR扩增,对部分扩增的 E6 和 E7 产物片段进行测序分析.结果 在官颈炎、CIN Ⅰ+CINⅡ级、CINⅢ级和宫颈癌组织中,E6基因的阳性率分别为25.0%、29.4%、60.9%和76.6%;E7基因的阳性率分别为16.7%、41.2%、43.5%和61.3%:E5 基因的阳性率分别为5.6%、5.9%、30.4%和40.3%.E6、E7和E5基因在不同阶段宫颈上皮病变组织中的阳性率差异均有统计学意义(均P<0.01).在80例官颈癌测序组织中,有47例发生E6基因178位点的T→C突变,突变率为58.8%,相应氨基酸由天冬氨酸(Asp)改变为谷氨酸(Glu);而在20例宫颈炎和22例CINⅠ～Ⅲ级测序组织中,E6基因178位点的突变率分别为25.0%和31.8%.在30例宫颈癌测序组织中,有21例发生E7基因647位点的A-G突变,突变率为70.0%,相应氨基酸由天冬酰胺(Asn)改变为丝氨酸(Ser);而在20例宫颈炎和22例CINⅠ～Ⅲ级测序组织中,E7基因647位点的突变率分别为35.0%和40.9%.结论 HPV16 E6、E7和E5基因与宫颈癌的发生和发展有高度的相关性.但E5基因在不同阶段官颈上皮病变中可能存在不同程度的缺失.中国湖北地区流行的HPV16病毒株可能为HPV16亚洲型变异株.     

宫颈肿瘤组织高危型HPV新分型检测方法的建立及应用

摘 要：［目的］ 建立一种新型宫颈肿瘤组织高危型人乳头瘤病毒（HPV）检测及分型方法,并评价其临床应用。［方法］ 基于我国人群流行的5种高危型HPV（HPV 16、18、31、33、51）的 E6/E7早期基因序列,设计特异性引物,以明确HPV型别的宫颈癌细胞株及宫颈肿瘤组织为模板,进行PCR检测,结合基因测序验证引物的特异性。进一步优化条件,建立高危型HPV多重PCR检测方法,检测65例宫颈肿瘤组织标本,与临床上使用的流式荧光杂交HPV检测法比较,评价其敏感性和特异性。［结果］ 成功建立可同时检测5种高危型HPV的多重PCR检测方法,对65例宫颈肿瘤组织进行高危型HPV检测,检出率为72.3%,明显高于临床流式荧光杂交检测法的检出率（32.31%）（χ2=3.86,P<0.05）。［结论］ 基于HPV E6/E7早期基因的高危型HPV多重PCR检测方法是特异、灵敏的高危型HPV检测方法。     

高危型HPV感染在宫颈癌前病变、宫颈癌中的临床意义

目的:分析高危型HPV感染在宫颈癌前病变、宫颈癌发生和发展中的关系,为宫颈病变的筛查和HPV疫苗的选择提供理论依据。方法:选择来我院妇科肿瘤中心就诊的1 197例患者作为研究对象,根据病理检查结果将病例分为慢性宫颈炎组（212例）、LSIL组（142例）、HSIL组（484例）和宫颈癌组（359例）,检测各组高危型HPV感染的情况,分析不同组HPV的阳性率及HPV亚型的分布。结果:慢性宫颈炎组 HPV 感染阳性率为9.43%,LSIL组为78.87%,HSIL组为92.15%,宫颈癌组为97.77%,各组间阳性率两两比较差异有统计学意义（P<0.001）。各组中,高危HPV单一感染率均大于多重感染率,差异有统计学意义（P<0.001）。慢性宫颈炎组以HPV52、HPV16、HPV58为主要亚型（阳性率分别为 3.77%、2.83%和 1.41%）,LSIL组以HPV52、HPV16、HPV58为主要亚型（阳性率分别为 23.94%、21.13%和 16.90%）,HSIL组以HPV16、HPV52、HPV58为主要亚型（阳性率分别为33.06%、29.34%和 19.42%）,宫颈癌组以HPV16、HPV58、HPV52为主要亚型（阳性率分别为 65.18%、20.89%和 10.86%）。本地区HSIL及宫颈癌人群感染率最高的5种亚型是HPV16、HPV18、HPV33、HPV52、HPV58。结论:宫颈病变程度越严重,HPV阳性率越高,不同程度宫颈病变HPV感染的亚型分布有差异。高危型HPV检测对本地区宫颈病变的早期筛查及HPV疫苗的接种有重要的指导意义。     

子宫颈上皮内瘤样病变和子宫颈癌组织中Smad2/3和HPV16 E7的表达及意义

背景与目的:Smads蛋白是转化生长因子-β(transforming growth factor-β,TGF-β)超家族信号转导的下游信号蛋白,与多种肿瘤的发生密切相关;人乳头状瘤病毒(human papillomavirus,HPV)感染是宫颈癌的重要致癌因素,但目前对两者在宫颈癌发病过程中相互关系的研究尚不充分.本研究拟检测不同宫颈病变组织中Smad2/3和HPV16 E7蛋白的表达,探讨HPV感染和Smad2/3蛋白变化在宫颈癌形成和演进进程中的相互关系.方法:采用免疫组织化学SP法检测20例宫颈慢性炎症、30例宫颈上皮内瘤样病变(cervical intraepithelial neoplasia,CIN)和30例宫颈癌组织中Smad2/3和HPV16 E7的表达情况,比较其在各种病变中表达水平的差异.结果:Smad2/3蛋白在慢性宫颈炎、CIN和宫颈癌组织中的阳性率分别为50.0%、73.3%和93.3%,宫颈癌组分别与慢性宫颈炎组和CIN组比较,差异有统计学意义(P＜0.05),慢性宫颈炎组与CIN Ⅲ级组相比,差异有统计学意义(P＜0.05).HPV16 E7蛋白阳性率在慢性宫颈炎、CIN和宫颈癌组织中分别为60.0%、66.7%和83.3%,各组间两两比较差异无统计学意义(P＞0.05).Smad2/3阳性率与宫颈癌临床分期、病理类型、组织学分级和淋巴结转移无关(P＞0.05).Smad2/3与HPV16 E7表达呈正相关(r=0.271,P=0.015).结论:Smad2/3蛋白在宫颈癌组织中表达升高,可能在宫颈癌的发生过程中起重要作用;HPV感染与Smad2/3蛋白在宫颈癌组织中表达升高密切相关.     

宫颈癌患者HPV16型E6蛋白的表达纯化及血清抗体检测

背景与目的:人乳头瘤病毒16型(humanpapillomavirustype16,HPV16)是宫颈癌组织中最常见的高危HPV,其相应蛋白的血清抗体与宫颈癌的发生发展相关。本研究构建HPV16E6重组表达载体并表达纯化获得HPV16E6重组蛋白,用于检测不同人群血清相应抗体,初步探讨本地区HPV16E6血清抗体反应与宫颈癌的相关性。方法:将HPV16E6基因与pRSET-A融合表达载体连接,获得E6表达重组体,转化大肠杆菌BL21(DE3)并用异丙基硫代-$-D-半乳糖苷(isopropylthio-$-D-galactoside,IPTG)诱导表达。表达的包涵体变性后经Ni柱纯化,复性并经活性鉴定后,用以包被ELISA板,检测正常女性、慢性宫颈炎患者和宫颈癌患者血清抗体。同时采用荧光偏振方法分型检测宫颈癌组织HPVDNA。结果:pRSET-16E6表达重组体的工程菌经IPTG诱导后可表达Mr24×103的HPV16E6组氨酸融合蛋白,表达量占菌体蛋白的22.3%。表达形式为包涵体,重组蛋白纯度达95%以上,其活性经ELISA法证实。80例正常女性、46例慢性宫颈炎和32例宫颈癌患者血清抗体阳性率分别为5.0%、6.5%和31.2%,宫颈癌患者HPV16E6血清抗体阳性率显著高于正常人(P<0.002)及慢性宫颈炎患者(P<0.01),而正常人与慢性宫颈炎患者间的差异无显著性。32例宫颈癌患者癌组织中,HPVDNA阳性率90.6%,HPV16DNA阳性率46.9%。HPV16DNA阳性组血清HPV16E6抗体阳性率(46.7%)高于阴性组(17.6%),但两组间的差异无显著性(P>0.05)。结论:在pRSET-A/BL21中表达获得的HPV16E6融合蛋白,可用于宫颈癌相关HPV的血清学研究;宫颈癌患者HPV16E6血清抗体阳性率明显高于正常人和慢性宫颈炎患者。     

HLA-DR 抗原及HPV16/18E6蛋白在宫颈癌的表达及其相互关系

目的:通过检测HLA-DR抗原及HPV16/18E6蛋白在宫颈癌组织中的表达情况,探讨由HLA-DR抗原介导的免疫应答在感染高危型HPV至发展为宫颈癌过程中的作用机制.方法:采用免疫组织化学SP法检测宫颈癌、CIN及正常宫颈组织中HLA-DR抗原及HPV16/18E6的表达情况.结果:HLA-DR抗原的阳性表达率在宫颈癌、CIN、正常宫颈组织中分别为 81.8%、73.3%、37.5%,而HPV16/18E6蛋白的阳性表达率则分别为75.8%、60.0%、37.5%,两者在三组中的表达均有显著差异 (P均﹤0.05).HLA-DR抗原及HPV16/18E6蛋白在宫颈癌组织中的阳性表达率随临床分期、分化程度变化及淋巴结是否转移而不同,但其差异无显著意义 (P均﹥0.05).结论:HLA-DR抗原递呈病毒抗原给T细胞引起的免疫应答可能在宫颈癌的发生发展过程中起着重要的作用.     

RCAS1在宫颈癌组织中的表达及其与HPV16感染的关系

背景与目的:表达在SiSo细胞上的受体结合肿瘤抗原(receptor-binding cancer antigen expressed on SiSo cells,RCAS1)在多种肿瘤组织中呈高表达,并与肿瘤逃避免疫监视有关.本研究检测RCAS1蛋白在宫颈癌组织中的表达及其与HPV16感染的相关性,并探讨其临床意义.方法:采用免疫组化SP(streptavidin-peroxidase)法,分别检测71例宫颈癌、76例宫颈上皮内瘤样病变(CIN)及20例正常宫颈上皮组织中RCAS1蛋白与HPV16 E7蛋白的表达,并分析两者的表达与临床病理因素的关系.结果:宫颈癌组织中,RCAS1蛋白主要表达于癌细胞膜和/或细胞浆,HPV16 E7蛋白主要表达于癌细胞核.正常宫颈上皮组织不表达RCAS1蛋白,CIN与宫颈癌组织中RCAS1蛋白的表达率分别为39.47%和77.46%,HPV16 E7蛋白的表达率分别为0.05%、28.94%和61.97%,提示随着宫颈病变恶性程度的进展,RCAS1与HPV16 E7表达均逐渐增强(P＜0.05).低分化宫颈癌组中RCAS1表达显著高于高、中分化宫颈癌组(P=0.002),但与患者年龄、临床分期及组织学分型无关(P＞0.05);HPV16 E7在鳞癌组中的表达显著高于腺癌组(P=0.000),但与患者年龄、临床分期、组织学分级无关(P＞0.05).RCAS1的表达与HPV16感染在宫颈癌中的表达呈正相关(r=0.780,P=0.000).结论:RCAS1基因在宫颈癌组织中表达增强,RCAS1表达强度与宫颈癌恶性程度相关;RCAS1阳性的宫颈癌组织中存在HPV16感染.     

半定量PCR检测子宫颈组织中人乳头瘤病毒16型DNA的研究

目的 :探讨子宫颈组织中人乳头瘤病毒 16型 (HPV16 )E6基因的含量与疾病严重程度的关系。方法 :用半定量PCR检测 2 0例慢性宫颈炎 ,6例宫颈非典型增生 ,18例宫颈癌组织中HPV16E6基因的含量。结果 :宫颈非典型增生及宫颈癌组织中E6基因的含量显著高于慢性宫颈炎 (P <0 0 1) ;宫颈癌及宫颈非典型增生的差异无显著性 (P >0 0 5 )。结论 :HPV16E6基因的含量随着宫颈疾病严重程度增加而增加 ,定量检测HPV16DNA的含量可能作为监测宫颈癌高危人群的一种方法     

半定量PCR检测子宫颈组织中人乳头瘤病毒16型DNA的研究

目的：探讨子宫颈组织中人乳头瘤病毒16型（HPV16）E6基因的含量与疾病严重程度的关系。方法：用半定量PCR检测20例慢性宫颈炎，6例宫颈非典型增生，18例宫颈癌组织中HPV16E6基因的含量。结果：宫颈非典型增生及宫颈癌组织中E6基因的含量显著高于慢性宫颈炎（P＜0．01）；宫颈癌及宫颈非典型增生的差异无显著性（P＞0．05）。结论：HPV16E6基因的含量随着宫颈疾病严重程度增加而增加，定量检测HPV16 DNA的含量可能作为监测宫颈癌高危人群的一种方法。     

HPV presence precedes abnormal cytology in women developing cervical cancer and signals false negative smears

In a retrospective case-control study, we investigated high-risk HPV DNA presence by general primer GP5+/6+ PCR in the last normal cervical smear in the patient archives (i.e. baseline smear) of 57 women who later developed cervical cancer. Also, normal cervical smears of 114 age-matched control women were analysed. High-risk HPV DNA was detected in 37 of the 57 (65%) baseline smears of the case women, and 7 (6%) of 114 smears of the control women (OR 28, 95% Cl 11-72). The HPV positive subsequent smears and cervical cancer biopsies of the case women contained the same HPV type as was detected in the baseline smear. After cytological revision, the baseline smears of 48 case women (84%) were reclassified as abnormal, 33 (69%) of which scored high-risk HPV DNA positive. Ultimately, an undisputable normal baseline smear was found in only 10 case women. In 7 (70%) of them this smear was HPV positive, whereas only 7 (7%) of 104 revised, undisputable normal smears of control women were high-risk HPV positive (OR 32, 95% Cl 6.8-153). The results showed that (1) high-risk HPV presence precedes abnormal cytology in women who develop cervical cancer, and (2) high-risk HPV testing signals false-negative smears of women at risk of cervical cancer.     

液基细胞学联合 HPV mRNA 检测对宫颈癌的诊断价值

目的：探索高危型 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 两种检测方法分别联合宫颈薄层液基细胞学检查（TCT）应用于宫颈癌早期诊断的临床意义。方法：选择2013年1月至2014年12月我院妇科门诊就诊行 TCT 检查的259例检测标本,对高危型 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 进行检测,结合 xb 细胞病理学分级和组织病理学诊断进行统计分析。结果：纳入研究的259例患者中 HPV E6／E7 mRNA 检测阳性率为35．1％（91／259）,HPV E6／E7 DNA 检测阳性率为52．1％（135／259）。NILM、ASCUS、LSIL、HSIL 四种细胞病理学分级 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 检出率分别为23．5％、29．4％、64．3％、66．7％和32．4％、52．5％、61．9％、83．3％。对于正常组织,HPV E6／E7 mRNA 的敏感度、准确度和阳性预测值均低于HPV E6／E7 DNA（P ＜0．05）,但特异度和阴性预测值高于 HPV E6／E7 DNA（P ＜0．001）。对于 CIN1组织, HPV E6／E7 mRNA 的敏感度、特异度和阳性预测值均低于 HPV E6／E7 DNA（P ＜0．05）,但准确度和阴性预测值高于 HPV E6／E7 DNA（P ＜0．001）。在 CIN2和 CIN3组织中,HPV E6／E7 mRNA 的准确度和阴性预测值均高于 HPV E6／E7 DNA（P ＜0．001）,HPV E6／E7 mRNA 的敏感度、特异度、阳性预测值与 HPV E6／E7 DNA 无统计学差异（P ＞0．05）。结论：高危型 HPV E6／E7 mRNA 检测能更准确地反映病毒感染后的活化状态,HPV宫颈薄层液基细胞学检查联合高危型 HPV E6／E7 mRNA 检测可提高宫颈癌早期筛查的准确性。     

Evaluation of Primers and PCR Performance on HPV DNA Screening in Normal and Low Grade Abnormal Cervical Cells

High risk human papillomaviruses (HR-HPVs) are associated with increased risk of normal cervical cellsdeveloping to dysplasia and cervical carcinoma. Therefore, HR-HPV DNA testing can predict an endpoint ofcervical carcinogenesis that is earlier than the development of cervical abnormalities. Not only the sensitivity ofmethods but also the amount of HPV DNA are very important and might be parameters to distinguish HPVdetection. In this study, we evaluated the effects of primer sets and the polymerase chain reaction (PCR)performance with low viral load samples with normal cervical cytology (140 samples) and mild dysplasia (140samples) using two consensus primers MY09/MY11 and GP5+/6+. The PCR was performed with single andnested PCR. Positive samples with both primer sets were then HPV genotyped by dot blot hybridization. Resultsshowed higher sensitivity of single PCR using primer GP5+/GP6+ than primer MY09/MY11. HPV DNA wasdetected in 15% (21 of 140)and 20.7% (29 of 140) of normal cervical samples, respectively. For mild dysplasiasamples, HPV DNA was detected in 37.1% (52 of 140) with MY09/MY11 and 50% (70 of 140) using GP5+/GP6+. In normal cervical samples, the positivity rate was increased to 38.5% (54 of 140) by nested PCR usingprimer GP5+/6+, but only 2 mild dysplasia samples that were negative by single GP5+/6+ were positive by autonestedPCR. These results suggested that, in low viral load samples, the sensitivity of HPV DNA detectiondepends not only on primer sets but also PCR performance. HPV 16 was the most common in mild dysplasiasamples (20.8%), whereas HPV type 58 was found in 11.1%. This study suggested that nested PCR might benecessary for HPV DNA detection in cervical samples of women participating in cervical cancer screening.     

A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for detection of cervical precancer and cancer.

PURPOSE: To evaluate carcinogenic human papillomavirus (HPV) mRNA for E6 and E7 mRNA detection on clinical specimens to identify women with cervical precancer and cancer. EXPERIMENTAL DESIGN: We evaluated a prototype assay that collectively detects oncogenes E6/E7 mRNA for 14 carcinogenic HPV genotypes on a sample of liquid cytology specimens (n=531), masked to clinical data and to the presence of HPV genotypes detected by PGMY09/11 L1 consensus primer PCR assay. RESULTS: We found an increasing likelihood of testing positive for carcinogenic HPV E6/E7 mRNA with increasing severity of cytology (P(Trend) < 0.0001) and histology (P(Trend) < 0.0001), with 94% of cervical intraepithelial neoplasia grade 3 (CIN3) histology cases (46 of 49) and all five cancer cases testing positive for carcinogenic HPV E6/E7 mRNA. Overall, fewer specimens tested positive for carcinogenic HPV E6/E7 mRNA than for carcinogenic HPV DNA (P<0.0001, McNemar's chi(2) test), especially in women with 相似文献   

高危型HPV E6/E7 mRNA检测对宫颈高级别病变的预警分流价值

目的:通过比较检测HPV E6/E7 mRNA和HPV DNA两种HPV检测方法对宫颈高级别病变的检出能力,探讨HPV E6/E7 mRNA检测在宫颈癌筛查中的预警分流价值。方法:采集2016年7月至2017年6月就诊于我院接受宫颈癌筛查的1 515例女性宫颈脱落细胞,利用转录介导扩增法检测高危型HPV E6/E7 mRNA（n=505）和PCR+膜杂交法检测HPV DNA（n=1 010）,以组织病理学诊断为金标准,比较两种检测方法对宫颈高级别病变检出率。结果:利用HPV E6/E7 mRNA检测组阴道镜转诊率32.69%（17/52）、HPV DNA检测组阴道镜转诊率10.62%（12/113）,差异有统计学意义（P＝0.001）;HPV E6/E7 mRNA检测组CIN2+的检出率42.42%;HPV DNA组 CIN2+的检出率28.16%,差异有统计学意义（P＝0.034）。结论:与HPV DNA检测方法相比,HPV E6/E7 mRNA检测对CIN2+病变预警分流价值较高。     

通用引物SPF1/GP6++与SPF1/GP6+聚合酶链式反应检测多型别人乳头瘤病毒敏感度的比较

目的 对比通用引物SPF1/GP6++与SPF1/GP6+ PCR两种方法检测人乳头瘤病毒（HPV）的敏感度和感染型别范围。方法 以包含HPV16全长DNA序列的质粒为模板,应用HPV L1基因型别特异性引物进行PCR扩增,获得15种型别HPV模拟靶基因序列并克隆入pEASY-T1载体,将梯度稀释的重组质粒掺入50 ng正常人基因组DNA,模拟各型别HPV感染的待测样本,对比SPF1/GP6+和SPF1/GP6++两组通用引物的检测敏感度。进一步在68例人宫颈癌组织DNA样本中进行对比验证。结果 与SPF1/GP6+PCR比较,SPF1/GP6++ PCR对HPV11,31,34,39,51,52,53,56,58,61和66型别的检测敏感度提高了1到2个数量级,对于HPV16,18,33和35常见感染型别的检测敏感度相似。应用SPF1/GP6++ PCR检测宫颈癌组织HPV感染的总阳性率为98.5%（67/68）,检测到11例双重感染和2例三重感染;而应用SPF1/GP6+ PCR检测的总阳性率为95.6%（65/68）,仅检测到7例双重感染。结论 在SPF1/GP6+ PCR基础上建立了SPF1/GP6++ PCR方法,并证实SPF1/GP6++ PCR具有更高的检测敏感度和更广的HPV型别检测范围。     

A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears

The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the beta-globin gene appeared to be superior for the GTC-based assays. Using competitive beta-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 x 10(5) to 3 x 10(5) genome equivalents in smears containing 5 x 10(5) to 20 x 10(5) nucleated cells, indicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3-6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 +/- 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears.     

Human Papillomavirus Genotype Distribution and E6/ E7 Oncogene Expression in Turkish Women with Cervical Cytological Findings

Background: Infection with certain human papillomavirus (HPV) genotypes is the most important riskfactor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPVinfection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women withdifferent cervical cytological findings in Mersin province, Southern Turkey. Materials and Methods: A total of476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in thestudy. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNAexpression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENSEasyQ®HPV v1.1). Results: Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR.The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL(6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were,in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%).Conclusions: Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Becauseof the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the roleof mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive andcytology negative. These epidemiological data will be important to determine the future impact of vaccinationon HPV infected women in our region.     

Antibodies against high‐risk human papillomavirus proteins as markers for invasive cervical cancer

Different human papillomavirus (HPV) genes are expressed during the various phases of the HPV life cycle and may elicit immune responses in the process towards malignancy. To evaluate their association with cervical cancer, antibodies against proteins from HPV16 (L1, E1, E2, E4, E6 and E7) and HPV18/31/33/35/45/52/58 (L1, E6 and E7) were measured in serum of 307 invasive cervical cancer cases and 327 controls from Algeria and India. Antibody response was evaluated using a glutathione S‐transferase‐based multiplex serology assay and HPV DNA detected from exfoliated cervical cells using a GP5+/6+‐mediated PCR assay. Among HPV16 DNA‐positive cases, seroprevalence of HPV16 antibodies ranged from 16% for HPV16 E1 to 50% for HPV16 E6 and all were significantly higher than controls. Seroprevalence of E6, E7 and L1 antibodies for HPV18 and for at least one of HPV31/33/35/45/52/58 were also higher in cases positive for DNA of the corresponding type (50% and 30% for E6 of HPV18 and HPV31/33/35/45/52/58 combined, respectively). E6 and E7 antibodies were rarely found in controls, but cross‐reactivity was evident among cancer cases positive for DNA of closely phylogenetically‐related HPV types. E6 or E7 antibodies against any of the eight HPV types were detected in 66.1% of all cervical cancer cases, as compared to 10.1% of controls. E6, and to a lesser extent E7, antibodies appear to be specific markers of HPV‐related malignancy. However, even among cases positive for the same type of HPV DNA, approximately one‐third of cervical cancer cases show no detectable immune response to either E6 or E7.     

Detection of Human Papillomavirus DNA in Routine Cervical Scraping Samples: Use for a National Cervical Cancer Screening Program in a Developing Nation

Background: Human papillomavirus is a well-established cause of the development of a variety of epitheliallesions in the cervix. However, as yet, incorporation of HPV testing into cervical cancer screening either as anadjunct or stand alone test is limited due to its cost. We therefore here ascertained the presence and type specificityof human papilloma virus (HPV) DNA in routine cervical scrapings. Materials and Methods: Cervical scrapingswere collected from women attending clinics for routine Pap smear screening. HPV-DNA was detected by PCRusing MY09/11 and GP5+/GP6+ primer sets and genotyping was accomplished by cycle-sequencing. Results: Atotal of 635 women were recruited into the study with mean±SD age of 43±10.5 years. Of these 92.6% (588/635)were reported as within normal limits (WNL) on cytology. The presence of HPV infection detected by nestedMY/GP+-PCR was 4.4% (28/635). The overall prevalence of high-risk HPV (HR-HPV) in abnormal Pap smearswas 53.8% (7/13). HPVs were also seen in 3.1% (18/588) of smears reported as WNL by cytology and 5.9%(2/34) in smears unsatisfactory for evaluation. Conclusions: The overall percentage of HPV positivity in routinecervical screening samples is comparable with abnormal findings in cytology. Conventional Pap smear ‘missed’a few samples. Since HPV testing is expensive, our results may provide valuable information for strategisingimplementation of effective cervical cancer screening in a country with limited resources like Malaysia. If Papsmear coverage could be improved, HPV testing could be used as an adjunct method on cases with ambiguousdiagnoses.