巢式PCR方法检测非生殖器部位Bowen病HPV DNA

目的 建立一种巢式ＰＣＲ方法,探讨HPV DNA在非生殖器部位Bowen病中的检出率。方法 巢式PCR方法,用5对不同引物包括CN1FR、CN2FR、CN3FR、CN4FR以及CN5FR进行扩增。结果 通过ClustalX软件,将所设计的每对引物与已知HPV亚型的碱基序列逐一相比较,得知改良设计的引物可以使69种HPV亚型得到扩增,包括黏膜型HPV、皮肤型 HPV以及疣状表皮发育不良相关性 HPV;PCR反应体系的敏感度在10-2～10-3拷贝之间。41例非生殖器部位Bowen病的组织标本中,5例HPV DNA阳性,其中高危黏膜型3例（2例HPV16,1例HPV33）,皮肤型2例（HPV27和HPV76各1例）。结论 改良的巢式PCR方法具有较高的敏感性、特异性,部分非生殖器部位Ｂowen病发病与黏膜高危型ＨＰＶ感染相关。     

皮肤鳞状细胞癌中人乳头瘤病毒的检测

目的:检测皮肤鳞状细胞癌(SCC)皮损中的人乳头瘤病毒(HPV)并探讨其在SCC发病中的作用.方法:用原位杂交(ISH)、聚合酶链反应技术(PCR)检测SCC患者43例,健康者28例标本组织切片中的HPV6、11、16、18、31、33.结果:43例SCC中有2例肢端部位皮损呈HPV DNA阳性,检出类型为HPV16和HPV33.其中1例标本中HPV16和HPV33同时阳性.对照组全部为阴性.结论:HPV16等6种黏膜型HPV可能与肢端以外SCC的发生无相关性,而与肢端部位SCC可能有关联性.     

病毒性皮肤病

980603 14例鲍温样丘疹病皮损人类乳头瘤病毒DNA原位杂交的检测/纪华安（天津市长征医院病理室）…//中国皮肤性病学杂志.-1997,11（5）.-266 为探讨人类乳头瘤病毒（HPV）在鲍温样丘疹病（BP）皮损中的表达特点,应用地高辛标记的HPV 6B/11、16、、18型DNA探针,对14例BP患者皮肤和粘膜病理组织进行了原位杂交检测。结果:BP患者HPV6B/11阳性7例（50.0％）,其中强阳性3例     

原位杂交和PCR法检测鲍温病皮损中的人乳头瘤病毒

目的:检测鲍温病(BD)皮损中的人乳头瘤病毒(HPV)并探讨其在BD发病中的作用.方法:病例组BD皮损20例,其中女8例,男12例.对照组正常皮肤标本33例.用原位杂交(ISH)和PCR法检测组织切片中的HPV DNA,检测的类型为HPV6、11、16、18、31、33.结果:20例BD中仅有1例呈HPV DNA阳性,为HPV6型,系外阴部位皮损.手部、其他部位皮损组织均为阴性.对照组全部为阴性.结论:HPV16等6种黏膜型HPV可能并不参与生殖器外BD(EBD)的发病.     

鲍恩样丘疹病皮损中HPV16、18的检测

目的：探讨人乳头瘤病毒（HPV）16、18在鲍恩样丘疹病皮损中的感染情况。方法：采用杂交捕获试验筛选高危型HPV和低危型HPV阳性标本,对高危型HPV阳性标本经PCR技术检测其HPV16、18DNA阳性者。结果：45例鲍恩样丘疹病患者皮损中34（75.6%）例为高危型HPV阳性,在高危型HPV阳性标本中82.2%（28/34）为HPV16阳性,5.9%（2/34）为HPV18阳性,8.8%（3/34）HPV16、18均阳性,2.9%（1/34）未检测到HPV16及HPV18DNA。结论：多数鲍恩样丘疹病皮损中存在高危型HPV感染,尤其HPV16,故高危型HPV16感染与鲍恩样丘疹病的发生、发展密切相关。     

p73第2外显子第4和14位基因多态性与人乳头状瘤病毒易感性的相关性研究

【摘要】 目的 探讨人乳头状瘤病毒（HPV）易感性与p73基因第2外显子第4和14位碱基多态性的相关性。 方法 收集南京地区尖锐湿疣患者83例、Bowen样丘疹病患者11例的皮损和血样,以在性病门诊进行健康体检的性病高危人群150例为阴性对照,采用直接测序法检测皮损中感染的HPV-DNA序列,并在Genbank Blast上进行比对,鉴定HPV型别,抽提尖锐湿疣和Bowen样丘疹病患者以及阴性对照组的血样DNA,以shP73引物对p73基因片段进行扩增和测序,检测患者和阴性对照组的p73第2外显子第4和14位基因的多态性,分析HPV易感性和宿主p73基因多态性之间的相关性。 结果 83例尖锐湿疣患者皮损中HPV-6型35例占42.2%,HPV-11型34例占41.0%;11例Bowen样丘疹病患者皮损中HPV-16型5例（5/11）,HPV-6型3例（3/11）。p73基因多态性检测,83例尖锐湿疣患者血样测序阳性83例,11例Bowen样丘疹病患者血样测序阳性11例,对照组150例血样测序阳性132例。与对照组相比,三种基因型中,A4T14/A4T14 基因型感染HPV患尖锐湿疣（OR = 4.89,95% CI = 1.50 ~ 15.91）或Bowen样丘疹病（OR = 7.11,95% CI = 1.144 ~ 44.20）的风险更高。G4C14/G4C14基因型与其他两种基因型相比,感染HPV后患Bowen样丘疹病的风险较低（OR = 0.16,95% CI = 0.04 ～ 0.65）。 结论 p73第2外显子携带A4T14基因增加了Bowen样丘疹病和尖锐湿疣的患病风险,但其第4和14位碱基的多态性与罹患高危型与低危型HPV之间并无明显的相关性。     

MDM2蛋白和MDM2 mRNA在尖锐湿疣中的表达

目的 研究尖锐湿疣中MDM2蛋白及MDM2 mRNA的表达情况,探讨MDM2在尖锐湿疣增殖及癌变中的作用。方法 分别采用免疫组化染色及原位杂交方法检测外阴尖锐湿疣和正常人皮肤组织中MDM2蛋白及mRNA的表达情况,同时用PCR法检测人乳头瘤病毒(HPV)型别。结果 32例外阴尖锐湿疣中MDM2蛋白阳性表达18例(56.25%),MDM2 mRNA阳性表达22例(68.75%)。MDM2蛋白与mRNA共同阳性表达14例。PCR检测示尖锐湿疣皮损中HPV6/11型28例(87.5%),HPV16/18型4例(12.5%)。在18例MDM2蛋白阳性表达的标本中,HPV6/11型15例,HPV16/18型3例。在22例MDM2 mRNA阳性表达的标本中,HPV6/11型18例,HPV16/18型4例。而正常人皮肤中MDM2蛋白及MDM2 mRNA均未见表达。结论 MDM2的异常表达可能与尖锐湿疣过度增殖及癌变有关。     

双功能氧化酶2在尖锐湿疣患者皮损中的表达及与HPV型别关系的研究

目的:检测尖锐湿疣(CA)患者皮损中是否表达双功能氧化酶2(Duox2),并分析其与HPV6/11,16/18型别之间的关系,以初步探讨其与CA发病之间的可能联系。方法:用PCR-荧光探针法检测40例CA患者皮损及对照组(20例,正常皮肤组织)中Duox2的mRNA表达水平并比较,聚合酶链反应(PCR)检测及鉴定CA组织中人乳头瘤病毒(HPV)感染类型:HPV6/11、HPV16/18。结果:CA组患者皮损中Duox2 mRNA表达水平明显高于对照组(t=-0.24,P=0.007)。40例患者中仅HPV6/11阳性23例,仅HPV 16/18阳性8例,HPV6/11DNA阳性组Duox2 mRNA表达水平明显低于HPV16/18DNA阳性组(t=4.92,P=0.001)。结论:CA患者中,HPV病毒感染特别是HPV16、18病毒会引起Duox2的水平增高。     

原位杂交检测鲍温样丘疹病及Bowen病中HPV16 DNA

采用生物素标记的核酸探针原位杂交技术,检测了30例鲍温样丘疹病、15例Bowen病中16型人乳头瘤病毒(HPV16)感染的组织定位及染色模式。结果显示,30例鲍温样丘疹病中,HPV16阳性13例;15例Bowen病中,HPV16阳性6例。鲍温样丘疹病、Bowen病中HPV16感染累及棘细胞全层;主要为核内团块状着色。Bowen病中,HPV16感染尚累及基底层细胞且存在HPV的点状染色模式。提示鲍温样丘疹病与Bowen病比较,HPV16感染的组织定位及染色模式均有所不同。     

尖锐湿疣、鲍温样丘疹病及鲍温病皮损中人乳头瘤病毒感染与P16蛋白表达的关系

目的：探讨鲍温样丘疹病（BP）、鲍温病（BD）及尖锐湿疣（CA）皮损中高、低危人乳头瘤病毒（HPV）感染对P16蛋白表达的影响。方法：用荧光定量PCR（FQ－PCR）方法检测30例CA病损及12例正常人皮肤粘膜中HPV6／11、HPV16／18DNA；用原位杂交方法检测30例BP、15例BD中HPV16DNA；用免疫组化方法检测上述组织中P16蛋白、Ki-67蛋白的表达。结果：在HPB感染皮损中，P16蛋白、Ki-67蛋白的表达均增高（P＜0．05），且在HPV16阳性的BP、BD中的表达高于HPV6／11感染的CA（P＜0．05）；P16蛋白表达与Ki-67蛋白表达呈正相关（P＜0．05）。结论：HPV感染皮损中，P16蛋白的过度表达可能是机体对HPV感染引起细胞异常增殖的一种反馈调节；P16蛋白表达的不同可能是由于高、低危HPV引起的Rb蛋白功能失活不同。     

Human papillomaviruses of the mucosal type are present in some cases of extragenital Bowen's disease

BACKGROUND: Bowen's disease in the genital area is generally considered to be caused by mucosal high-risk human papillomaviruses (HPVs). However, the detection rate and spectrum of HPVs in extragenital Bowen's disease are various and it is not clear to what extent HPV is involved in its pathogenesis. OBJECTIVES: To assess the degree of association of HPV in extragenital cases by examining detection rates, types, quantities and localization of HPV. METHODS: A polymerase chain reaction (PCR) approach that we had previously established, which can give sensitive detection of a broad range of HPVs from cutaneous [including epidermodysplasia verruciformis-related HPVs (EV-HPVs)] to mucosal HPVs, was applied to samples from 41 patients with extragenital Bowen's disease and normal skin samples from 48 individuals. Semiquantitative L1-PCR and tyramide-based in situ hybridization (ISH) were also employed for positive cases. RESULTS: HPVs belonging to the mucosal high-risk group were detected in three patients with Bowen's disease (7%; two HPV 16 and one HPV 33), with 10(1)-10(3) copy equivalents per diploid amount of cellular DNA. They were distributed among most nuclei of tumour cells but in none of the cells of adjacent normal skin. HPVs belonging to the cutaneous group were detected in two patients (5%; HPV 27 and HPV 76) at 10(-2)-10(-3) copy equivalents, the same level as in a normal skin specimen positive for type 23 EV-HPV. No positive signals were observed by ISH. CONCLUSIONS: HPVs belonging to the mucosal high-risk group may participate in the development of extragenital Bowen's disease.     

Detection of human papillomavirus in cutaneous extragenital Bowen's disease in immunocompetent patients

INTRODUCTION: A specific link between human papillomavirus (HPV) types 16, 18, 31, and 33 and genital carcinomas and between HPV type 5 and cutaneous extragenital carcinomas in patients with epidermodysplasia verruciformis and renal transplant has been previously found. The aim of this prospective study was to detect HPV in cases of cutaneous extragenital Bowen's disease (BD) from non-immunosuppressed patients. PATIENTS AND METHODS: Twelve cases of cutaneous extragenital BD or Bowen's carcinoma (BC), seen in the period 1994-1996 and confirmed by histologic examination, were included in the study. Tissue sections were studied by in situ hybridization with a mixture of HPV DNA probes and specific HPV DNA probes. In addition, study on fresh materiel from 1995 included: Southern blot hybridization with various usual HPV probes (6, 11, 16, 18, 31, 33, 35, 39, 42), polymerase chain reaction (PCR) with hybridization using consensus HPV probes and probes specific for HPV types 6, 11, 16, 18 and 33. In positive samples with conventional PCR, in situ PCR with probes specific for HPV types 6/11 and 16 was performed on tissue sections. RESULTS: In situ hybridization was negative in all the cases. Southern blot hybridization was negative in our 9 studied cases. Three cases studied by consensus PCR were positive. PCR with specific HPV probes revealed positivity on two of these cases: HPV 6 in one, and HPV 16 in another. In situ PCR was positive with a mixed 6/11 HPV probe in the third positive consensus PCR case. DISCUSSION: Our study revealed the presence of HPV in 3 out of 12 cases of cutaneous extragenital BD and BC. HPV type 16, found in BC of skull, was the most usually found type in the literature. HPV types 6/11, detected in 2 cases, were rarely found in cutaneous extragenital BD and BC and these results are in favor of the oncogenic effect of these virus types. In our study, in situ hybridization and Southern blot hybridization were negative in all the cases; HPV was only found in 3 cases by conventional PCR and in 1 case by in situ PCR. The low range of detection of HPV in cutaneous extragenital BD may be due to the used methods, to difficulties related to sampling and/or to a low number of copies of the HPV genoma.     

Expression of cell-cycle proteins p53, p21 (WAF-1), PCNA and Ki-67 in benign, premalignant and malignant skin lesions with implicated HPV involvement.

A series of 120 biopsies from benign (verruca vulgaris and keratoacanthoma), premalignant (actinic keratosis and extragenital Bowen's disease) and malignant (squamous cell carcinoma) skin lesions were studied immunohistochemically for the expression of cell-cycle proteins p53, p21 (WAF-1), PCNA and Ki-67. The presence of human papillomavirus (HPV) DNA in these samples had been analysed previously using in situ hybridization (ISH) and PCR. Moderate to intense expression of both PCNA and Ki-67 was present in most of the lesions studied. PCNA staining was extensive in the epidermis underneath the layers where abundant HPV DNA staining was shown in HPV DNA-positive verrucas. In keratoacanthomas, p21 and PCNA expression remained low, despite intense p53 expression. In actinic keratosis, only half of the specimens showed overexpression of p53 associated with moderate or intense expression of PCNA. In extragenital Bowen's lesions, all these cell-cycle markers were overexpressed, but in squamous cell carcinomas, they were heterogeneously expressed and showed no correlation with tumour differentiation. Our results suggest a mechanism by which HPV can reactivate the host genes (leading to cell proliferation) to support its own DNA replication. Also p21 might start keratinocyte differentiation in areas where HPV DNA replication starts. Cell proliferation remained active in actinic keratosis and Bowen's lesions, emphasizing the precancer character of these lesions in contrast with the benign nature of keratoacanthoma and verruca vulgaris.     

Oncogenic mucosal human papillomaviruses in Bowen's disease of the hands

INTRODUCTION: The association between genital Bowen Disease (BD) and human papillomavirus (HPV) especially HPV-16 infection is well known, but it is more rarely related to extragenital BD. The aim of this study was to detect the presence of oncogenic HPV infection in the BD of the hands considering the histology and the presence of oncogenic HPV detected by in situ hybridization (ISH) and polymerase chain reaction (PCR). MATERIAL AND METHODS: Eleven formalin-fixed and paraffin-embedded samples of BD of the hands were selected. We looked for koilocytosis using standard histological procedure. ISH was performed using genomic HPV DNA probes types 16, 18 and 33 labeled with digoxigenin. Next, with a new amplification system using biotinyled tyramide (Kit Dako Gen Point K620) was used to detect hybrids. Otherwise, Baay's HPV type specific primers (type 16, 18, 31 et 33) were chosen for PCR. RESULTS: Koilocytosis were observed in all cutaneous samples. HPV 16 was detected in 9/11 cases (82 p. 100): 2/10 with ISH and 9/11 with PCR. DISCUSSION: We report here the largest series of BD of the hands, associated with HPV type 16 infection. This high rate (82 p. 100) compared to the other series is linked to the high sensitivity of PCR and is increased by the choice of Baay's oligonucleotide primers. Besides, the low HPV rate (2/10) by ISH, is similar to those obtained in the other series. Although ISH is less sensitive than PCR, the morphological study was useful to argue the pathogenicity of HPV. Finally, the high prevalence of genital oncogenic HPV on the hands, plaid in the scratching hypothesis resulting in autoinoculation from HPV lesions in the genital region.     

Premalignant lesions and cancers of the skin in the general population: evaluation of the role of human papillomaviruses

To evaluate the role of human papillomaviruses (HPV) in the development of premalignant lesions and cancers of the skin in the general population, 314 biopsies obtained from 227 patients with benign neoplasms, premalignant lesions, and cancers of the skin and from 25 patients with squamous cell carcinoma of the lip were analyzed by Southern blot hybridization. DNA probes specific for various cutaneous and genital HPV types were used in hybridizations conducted under nonstringent or stringent conditions. HPV DNA sequences were only detected in eight specimens obtained from six patients: HPV 34 in one case of periungual Bowen's disease, HPV 36 and an as yet uncharacterized HPV in two cases of actinic keratosis, HPV 20 in one case of basal cell carcinoma, an as yet unrecognized HPV in one case of squamous cell carcinoma, and HPV 16 in one case of squamous cell carcinoma of the lip. None of the specimens of cutaneous horn and keratoacanthoma contained detectable HPV DNA. In contrast, HPV DNA sequences, mostly HPV 16, were detected in 13 of 23 cases of anogenital Bowen's disease and invasive Bowen's carcinoma. HPV DNA sequences were not detected in 90 cutaneous samples further analyzed by the polymerase chain-reaction technique, using amplification primers that contain conserved sequences among the genomes of HPV. These results strongly suggest that the known HPV types play only a minor role, if any, in skin carcinogenesis in the general population.     

Failure to demonstrate human papillomavirus (HPV) involvement in Bowen’s disease of the skin

To assess the role of human papillomavirus (HPV) in the aetiology of extragenital Bowen’s disease (EBD), a series of 91 cases were analysed using in situ hybridization (ISH) with whole genomic DNA probes of HPV types 1, 2, 4, 6, 7, 11, 12, 15, 16, 18, 26, 36, 37, 39, 42, 55 and 59. No HPV DNA was found in any of the samples tested. A polymerase chain reaction (PCR) was also used to analyse 37 of the 91 samples, using both the consensus primers MY09/MY11 which amplify a large number of HPV types from the L1 region and the degenerate primers CP65/CP70, which amplify the complete set of epidermodysplasia verruciformis (EV) HPV types. All the cases were also negative in the PCR. The results suggest that EBD is not HPV-related, at least in immunocompetent patients. Received: 12 March 1996     

Human papillomavirus and extragenital in situ carcinoma

BACKGROUND: The relation between human papillomavirus (HPV) and extragenital Bowen's disease (BD) is controversial. METHODS: This study used in situ hybridisation to evaluate the rate of HPV in extragenital cutaneous BD and investigated possible relations with immune status and exposure of skin to light. RESULTS: HPV DNA was detected in 58% of 69 samples from 50 patients. The percentage of HPV detection was not significantly higher in exposed (55%) than unexposed areas (65%), and no difference in HPV rate was found between immunosuppressed and immunocompetent patients. CONCLUSION: Thus, this study confirms the high rate of HPV detection in extragenital cutaneous BD and suggests that there is no apparent relation concerning exposed areas and immune status.     

Human papillomavirus type 58 in Bowen's disease of the elbow

Human papillomavirus (HPV) can be detected in skin lesions of Bowen's disease, particularly on the fingers, and its genotype is associated with mucosal/genital types of HPV. We report herein an 85-year-old woman who had HPV-associated Bowen's disease on her elbow. HPV-58 DNA was detected in the lesion by polymerase chain reaction with restriction fragment length polymorphism and by Southern blot hybridization. In situ hybridization revealed numerous hybrid cells in the nuclei of the upper epidermis and stratum corneum of Bowen's disease. A high-risk type of mucosal HPV-58 DNA is associated with Bowen's disease in this case, suggesting that HPV-related Bowen's disease is not always restricted to genital or finger lesions.     

No evidence of human papillomavirus DNA in actinic keratosis

Human papillomaviruses (HPVs) are involved in premalignant and malignant skin diseases as well as in a variety of benign cutaneous and mucosal lesions. Actinic keratosis (AK) is a common premalignant disease. Its association with HPV infection has recently been evaluated in a few studies, but the results are contradictory. For further assessment of the role of HPVs in AK, a series of 100 paraffin-embedded biopsy specimens taken from subjects with AK were studied for the presence of HPV types 1, 2, 3, 4, 5, 7, 12, 15, 26, 36, 37 and 59 DNA using in situ hybridization (ISH) under high stringency conditions (Tm -10°C). All specimens were definitely negative for all biotinylated HPV DNA probes tested. One-fifth of the specimens were studied using the polymerase chain reaction (PCR) with general primers to confirm the negative results. All cases were also negative in the PCR. Our results suggest that HPVs are not directly involved in the aetiology of AK.