食管鳞癌HPV感染与病毒致癌基因E6的关系

目的：探讨人乳头瘤病毒（ＨＰＶ）与食管鳞状细胞癌的关系。方法：采用多重引物多聚酶链反应（ＰＣＲ）的免疫组化技术、对１０４例食管鳞癌进行ＨＰＶ ＤＮＡ和病毒癌基因Ｅ６蛋白检测。结果：ＨＰＶ ＤＮＡ阳性者占５０．９６％（５３／１０４），其中ＨＰＶ１６型ＤＮＡ４９．０６％（２６／５３），ＨＰＶ１８型 ＤＮＡ５．６％（３／５３），ＨＰＶ６／１１ ＤＮＡ７．５％（４／５３）；两上或三个类型的混合感染占３７．     

人乳头瘤病毒与P 53协同致膀胱移行细胞癌关系的研究

目的 研究人类乳头瘤病毒（ＨＰＶ）６、１１、１６和１８型及Ｐ５３与膀胱移行细胞癌的关系。方法 采用聚合酶链反应（ＰＣＲ）方法检测了７５例膀胱移行细胞癌组织中ＨＰＶ的感染,免疫组化ＳＰ法检测Ｐ５３蛋白表达情况。结果 膀胱移行细胞癌组织中ＨＰＶ６、１１、１６和１８的阳性率分别为６．７％（５／７５）,５．３％（４／７５）,３３．３％（２５／７５）和６．７％（５／７５）。低危型ＨＰＶ（６或１１）阳性率为９．３％（７／７５）,高危型ＨＰＶ（１６或１８）阳性率为３４．７％（２６／７５）。同一膀胱癌组织中两种以上（包括两种）ＨＰＶ亚型感染８例,占１０．６％。ＨＰＶ６、１６和１８型之间感染阳性率在肿瘤有无转移组中差异显著（Ｐ〈０．０５）,ＨＰＶ１６、１８的阳性率在肿瘤病理分级中差异有极显著性（Ｐ〈０．０１）。ＨＰＶ ＤＮＡ型别     

妊娠期人乳头瘤病毒的感染状况及母婴传播的研究

应用聚合酶链反应检测孕妇尖锐湿疣（ＣＡ）组织、无ＣＡ孕妇宫颈分泌物、外周血及羊水、新生儿咽分泌物、脐血中人乳头瘤病毒（ＨＰＶ）感染及母婴传播。结果表明：ＣＡ组织中ＨＰＶＤＮＡ阳性率为９０．３２％，以ＨＰＶ６／１１型为主；无ＣＡ孕妇宫颈分泌物中ＨＰＶＤＮＡ阳性率为３５．７１％，以ＨＰＶ１６／１８型为主；孕妇血中ＨＰＶＤＮＡ阳性率为５７．６９％；母婴间经产道垂直传播率为４４．４４％，血性经胎盘传播传播率为６０％。说明ＨＰＶ不仅存在于ＣＡ组织中，还存在于无ＣＡ孕妇生殖道及外周血中，母婴间传播途径除产道外，还有胎盘传播     

卵巢上皮性肿瘤p16抑癌基因突变与HPV感染的研究

采用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）技术，对同一卵巢上皮性肿瘤石蜡包埋组织中ｐ１６基因（第二外显子）突变及人乳头状瘤病毒（ＨＰＶ）感染进行相关性研究。并与正常卵巢组织进行对照。结果，２８例卵巢上皮性肿瘤组织中ｐ１６基因突变１５例，突变率为５３．６％（１５／２８），其中７例伴有ＨＰＶ１６型或ＨＰＶ１８型感染，占突变率的４６．７％。在卵巢上皮性肿瘤组ＨＰＶ１６、１８ＤＮＡ阳性率为５３．６％（１５／２８），对照组ＨＰＶ１６、１８ＤＮＡ阳性率为５．６（１／１８），二者比较有显著性差异。提示：卵巢上皮性肿瘤中ｐ１６基因突变与ＨＰＶ１６、１８型感染有关。ＨＰＶ１６、１８型感染与卵巢上皮肿瘤密切相关     

用聚合酶链反应检测食管癌组织中人乳头瘤病毒DNA

应用聚合酶链反应（ＰＣＲ）技术对汕头市区６８例食管癌的石蜡包埋标本进行人乳头瘤病毒（ＨＰＶ）ＤＮＡ序列检测，结果显示，ＨＰＶＤＮＡ总阳性率为６６．１８％（４５／６８），检出型别主要为ＨＰＶ６、１１、１６，检出率分别为２７．９４％、３６．７６％和２７．９４％，经统计学处理三型间无显著性差异；ＨＰＶ－１８及未定型别各占８．８２％。值得注意的是ＨＰＶ感染中多重感染占阳性病例的５３．３３％（２４／４５）。初步结果表明，汕头市食管癌高发区有较高的ＨＰＶ感染率，此与食管癌的发生，可能有密切关系。     

慢性宫颈炎患者宫颈中人乳头瘤病毒基因的检测

应用聚合酶链反应技术检测正常妇女与慢性宫颈炎患者宫颈人乳头瘤病毒感染情况的结果显示，南京市慢性宫颈炎患者ＨＰＶ－ＤＮＡ总阳性率达５８．６％；ＨＰＶ６，１１，１６，１８型阳性率分别为２９．３％，３０．７％，２８．６％和３４．３％，而正常妇女中ＨＰＶ总阳性率为１９．４％，４个型别ＨＰＶ阳性率为９．７％，１６．１％，３．２％和３．２％，明显低于宫颈炎患者。如皋市慢性宫颈炎患者ＨＰＶ－ＤＮＡ总阳性率高达８     

斑点分子杂交检测HPV感染与宫颈癌的关系

采用ＤＮＡ－ＤＮＡ分子杂交技术，对经病理组织学确诊的慢性宫颈炎，宫颈癌和正常宫颈的宫颈活检组织中ＨＰＶ６，ＨＰＶ１１，ＨＰＶ１６，ＨＰＶ１８型ＤＮＡ进行同源序列检测，结果表明ＨＰＶ６，ＨＰＶ１１，ＨＰＶ１６，ＨＰＶ１８型ＤＮＡ的检出率在正常宫颈均为０；在慢性宫颈炎分别为１６．０９％，１２．６４％，１１．４９％，３．４５％；在宫颈癌组分别为３．９６％，１．９８％，４６．５３％，７．９２％。在宫颈癌组     

人乳头状瘤病毒不同型别与宫颈病变的相关性研究

目的探讨人乳头状瘤病毒（ＨＰＶ）不同型别与宫颈病变性质的关系。方法应用ＰＣＲ技术和原位杂交方法对６１例宫颈上皮内瘤（ＣｅｒｖｉｃａｌｉｎｔｒａｅｐｉｔｈｅｌｉａｌＮｅｏｐｌａｓｉａＣＩＮ）和１２例宫颈鳞癌（ＳＣＣ）进行ＨＰＶ６Ｂ／１１、１６、１８ＤＮＡ检测。结果ＰＣＲ检测结果显示ＨＰＶ６、１１主要分布于低度鳞状上皮内病变（６１９％）和一部分ＣＩＮⅡ中（２０％），而在ＣＩＮⅢ和ＳＣＣ中检测不到；ＨＰＶ１６、１８的检出率随ＣＩＮ级别增高而增加，在ＳＣＣ中高达８３３％。原位杂交结果显示在低度鳞状上皮内病变中，地高辛（Ｄｉｇ）标记的ＨＰＶ６Ｂ／１１、１６、１８ＤＮＡ杂交物质在核中均呈细颗粒状，为“游离型”。上述杂交阳性信号形态亦出现于ＣＩＮⅡ的所有ＨＰＶ６Ｂ／１１及部分ＨＰＶ１６、１８型感染中，而ＣＩＮⅢ和宫颈鳞癌及部分ＣＩＮⅡ中，其杂交阳性信号均为非颗粒状的“整合型”。结论低度鳞状上皮内病变是以ＨＰＶ６、１１低危型为主的多型别病毒的繁殖性感染，ＣＩＮⅢ和宫颈鳞癌为ＨＰＶ１６、１８高危型病毒的整合型感染，而在ＣＩＮⅡ中存在着ＨＰＶ６，１１和ＨＰＶ１６，１８的繁殖性感染及ＨＰＶ１６，１８的整合型感染     

人喉癌组织中人乳头瘤病毒DNA的检测

目的为探讨喉癌与人乳头瘤病毒（ＨＰＶ）感染的关系和ＨＰＶ在喉癌中基因组型的分布与表达。方法应用聚合酶链反应技术（ＰＣＲ）制备非放射性探针标记物－地高辛标记ＨＰＶ共有引物探针,对１４６例喉不同病变的新鲜组织标本（喉癌６８例,喉其它病变４８例,正常喉组织３０例）,进行ＨＰＶ６,１１,１６,１８,３１,３３,３５,４２,５８共９型ＨＰＶＤＮＡ感染的检测;阳性者用多重引物ＰＣＲ方法分型。结果喉癌ＨＰＶ感染阳性率４５．６％（３１／６８）,喉癌颈转移淋巴结组织阳性率２０．０％（３／１５）,喉癌前病变阳性率１１．８％（２／１７）,声带息肉阳性率６．３％（１／１６）,１５例癌旁及１５例癌周正常喉组织均为ＨＰＶＤＮＡ阴性。ＨＰＶＤＮＡ型别分布在喉癌中以ＨＰＶ１６、１８型为主,喉良性病变中以ＨＰＶ６、１１型为主。结论喉癌发生与ＨＰＶ感染有关。     

应用原位PCR技术检测阴茎癌组织中人乳头瘤病毒DNA

目的：研究人乳头瘤病毒（ＨＰＶ）与阴茎癌的关系。方法：应用原位ＰＣＲ技术对４６例阴茎癌组织中的ＨＰＶ１６和ＨＰＶ１８ＤＮＡ进行检测。结果：３３例阴茎癌中检测到了ＨＰＶＤＮＡ（７１．７％），其中２９例ＨＰＶ１６ＤＮＡ阳性（６３．０％），６例ＨＰＶ１８ＤＮＡ阳性（１３．０％），２例ＨＰＶ１６和ＨＰＶ１８ＤＮＡ均阳性，４例ＨＰＶ１８ＤＮＡ阳性而ＨＰＶ１６阴性；２例淋巴结转移癌，３例癌旁不典型增生组织和１例癌旁增生组织ＨＰＶ１６ＤＮＡ阳性。结论：阴茎癌与ＨＰＶ１６、ＨＰＶ１８感染有密切关系，原位ＰＣＲ技术是一项敏感性高、特异性强的技术。     

Penile/anal condylomas and squamous cell cancer

Summary Acuminate condylomas from the penis (n=17) and anus (six cases), three anal/penile giant condylomas, anal Bowen's disease (four cases), and intraanal squamous cell carcinomas with associated condylomatous changes (10 cases) including two verrucous carcinoma were studied for human papillomavirus (HPV) infections with nick translated, biotinylated cDNA probes for HPV 6, 11, 16 and 18. In addition, six cases of flat white penile lesions designated as lichen sclerosus et atrophicus were examined.Reannealed complementary DNA strands were detected in situ with either immunoenzyme or immunogold protocols.The in situ hybridizations resulted in 1/6 positive penile lichenoid lesions, 12/17 positive penile acuminate condylomas, 6/6 positive anal acuminate condylomas (including two condylomas with cellular atypias), 2/3 positive giant condylomas, 1/4 positive anal bowenoid lesions, and 4/10 positive keratinized squamous cell carcinomas, two of them being verrucous carcinomas. All penile/anal condylomas and two giant condylomas harboured HPV 6 and/or 11 DNA.The five positive carcinomas (carcinoma in situ/invasive cancer) contained HPV 6 and/or 11 in two cases (including the verrucous carcinomas), and HPV 16 and/or 18 in three cases (one carcinoma in situ, two invasive carcinomas).Recurrent malignancies were seen in one case to harbour the same HPV type as the primary lesions (HPV 16). In one particular patient, a double infection with HPV 16 and HPV 18 was demonstrated in distantly located malignant tumours. Our study confirms the restrictions and the value of non-isotopic hybridization methods applied to archival tissues, and extends the knowledge on the presence and distribution of HPV infections at anogenital sites.This study was supported by the Deutsche Forschungsgemeinschaft (Lo 285/2-4) and the Hamburger Stiftung zur Förderung der Krebsbekämpfung     

In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia

The authors examined 18 cervical intraepithelial neoplasms (CIN) for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and DNA-DNA in situ hybridizations for HPV DNA sequences and compared the epithelial distribution of HPV 16 DNA sequences with HPV 6/11 sequences in selected condylomas. Fifteen of the 18 CIN lesions contained HPV 16 DNA as determined by Southern blot hybridization. With the use of biotinylated HPV 16 DNA probes, 10 of the 18 were positive by in situ hybridization, 9 of which were also positive by Southern blot hybridization. In situ hybridization to HPV 16 probes was found primarily in areas of CIN which contained either maturation or koilocytotic atypia, although in two cases hybridizing sequences were detected in superficial cells from epithelium with no discernible maturation. Staining in both condylomas and CIN lesions varied in distribution and intensity. However, in some CIN lesions staining from cell to cell varied considerably. This greater variability in staining appeared to correlate with greater morphologic variations which characterize CIN, and which may influence greater variation in HPV DNA replication. Thus, some differences in patterns of hybridization for HPV DNA between CIN and condylomas may be explained by morphologic differences in the two classes of lesions. Differences in viral gene expression between condylomas and CIN and their relationship to morphologic findings remain to be clarified.     

Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.     

Detection of human papillomavirus DNA in cell scrapes and formalin-fixed, paraffin-embedded tissue of the uterine cervix by filter in situ hybridisation

Filter in situ hybridisation (FISH) was used to detect the presence of DNA of human papillomavirus (HPV) types 6/11 or 16/18 in cell scrapes (CYTOFISH) and formalin-fixed, paraffin-embedded biopsies (HISTOFISH) taken from the uterine cervices of 19 women. Paraffin tissue sections collected for HISTOFISH were either digested with pepsin or lysed with alkali/Triton X-100. The digest or lysate of the tissue sections and cell scrapes were applied to nylon or nitrocellulose membranes for nucleic acid hybridisation using 32P-labeled HPV-DNA probes. CYTOFISH and HISTOFISH were compared directly by taking samples for each method from the cervices of the same women. Of 19 women examined by colposcopy, cytology, and histology, eight were assessed as normal and 11 had evidence of a cervical disorder and/or the presence of HPV infection. Whereas no HPV-DNA was detected in the normal cases, the presence of HPV-DNAs was detected by both CYTOFISH and HISTOFISH in 11 cases with histological evidence of HPV infection and/or dysplasia. In these HPV positive cases, eight contained HPV 16/18, two HPV 6/11, and one a mixed infection of HPV 6/11/16/18. The high correlation between the results of CYTOFISH and HISTOFISH shows that formalin-fixed, paraffin-embedded cervical biopsies are suitable specimens for the detection and typing of HPV-DNA by FISH. Both CYTOFISH and HISTOFISH should facilitate studies on the prevalence and distribution of HPVs and their association with neoplasia.     

Detection and localization of human papillomavirus in penile condylomas and squamous cell carcinomas using in situ hybridization with biotinylated DNA viral probes

Human papillomavirus (HPV) has been previously demonstrated in male genital neoplasms using Southern blot hybridization (SBH) and in situ hybridization with radiolabeled probes (ISH-R). In this study we used in situ hybridization with biotinylated DNA viral probes (ISH-B), a technique that can be applied to routinely collected and processed tissue. Thirty cases of exophytic penile condyloma acuminatum and nine cases of invasive squamous cell carcinoma of the penis were examined for the presence of HPV using ISH-B for HPV types 6, 11, 16, 18, 31, and 33. HPV DNA was found in 25 of 30 (83%) penile condylomas; HPV type 6 in 13 (43%); and HPV type 11 in 12 (40%). Slight cross-reactivity between HPV types 6 and 11 was noted. None of the condyloma cases was positive for HPV types 16, 18, 31, or 33. One of the nine patients with squamous cell carcinoma of the penis was positive for HPV 16. In situ hybridization with biotinylated DNA viral probes is a highly sensitive method for detecting and localizing HPV in penile condylomas. This method, however, may not be as sensitive as SBH for detecting HPV in invasive penile squamous cell carcinomas.     

Ultrastructural visualization of human papillomavirus DNA in verrucous and precancerous squamous lesions.

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.     

Ultrastructural Visualization of Human Papillomavirus DNA in Verrucous and Precancerous Squamous Lesions

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dotlike positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA. Acta Pathol Jpn 41: 757-762, 1991.     

Genital human papilloma virus infection in Oslo studied by dot blot DNA hybridization and the polymerase chain reaction.

Samples from patients with genital condyloma acuminata or with cervical condylomas and/or dysplasia and from women without cytological/clinical evidence of cervical affection were examined by dot blot DNA hybridization or the polymerase chain reaction (PCR). The PCR was much more sensitive than dot blot, more than doubling the human papilloma virus (HPV) findings. HPV DNA, mainly HPV 6/11, was detected in 18 of 19 biopsies of condyloma acuminata, whereas HPV 16 was most frequently detected in the 21 cervices (76%) with condyloma and/or dysplasia. HPV 16 was detected in eight of 103 cervical smears with no signs of infection. The prevalence of HPV 16 in cervical samples was somewhat higher than expected. This suggests that, in Oslo, HPV 16 is a common HPV type in women with cytologically normal cervices. HPV 18 was relatively rare and was detected only in combination with other HPVs.     

用聚合酶链反应检测结肠癌人乳头状瘤病毒基因

目的　应用聚合酶链反应,检测人乳头状瘤病毒(Humanpapillomavirus,HPV)基因与结肠癌及癌区周边组织的相关性。方法　将结肠镜检获取的72例活检标本进行病理检测,其中结肠癌46例,非癌26例(结肠癌周边组织标本10例),标本用蜡块包埋与固定液两种方法固定,用聚合酶链反应(PCR)特定DNA片段体外扩增法。结果　结、直肠癌人乳头状瘤毒基因检测阳性率434%,结、直肠癌周边组织阳性率10%,非癌组织阳性率为0%。结论　癌区组织基因(HPVs)检测率较高,与非癌对照组相比,差异有显著性(P<0.05),癌组织中HPVs主要以HPV1633型和HPV18型为主,统计学分析表明结、直肠癌的发生、发展与HPV感染有一定的相关性,尤以HPV16型关系更为密切。