Detection of human papillomavirus DNA in nongenital seborrhoeic keratoses

The histological similarities of seborrhoeic keratoses and common warts led to the investigation of the possible occurrence of human papillomavirus DNA (HPV-DNA) in a large number of nongenital seborrhoeic keratoses using the in situ hybridization technique. All specimens derived from normal skin (n=173) were negative for the applied HPV-DNA probe, whereas the HPV genome was detected in 34 of 173 seborrhoeic keratosis specimens (19.65%). Of 34 HPV-positive specimens, 15 contained types 6/11 and 14 types 31/33/35, and 5 showed no positive reaction to the applied types. These results suggest that a considerable percentage of nongenital seborrhoeic keratoses may be related to an HPV infection.Dedicated to Professor Dr. H. Ippen on the occasion of his 70th birthday     

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。     

Detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in nongenital seborrhoeic keratosis

BACKGROUND: DNA of epidermodysplasia verruciformis (EV)-associated human papillomaviruses (HPVs) has been widely detected in lesions of malignant skin tumours, benign tumours and other proliferative diseases of epithelial origin. OBJECTIVES: To investigate the presence of EV-associated HPV DNA in nongenital seborrhoeic keratosis (SK) and to elucidate the prevalence of distinct HPV genotypes. METHODS: We investigated HPV DNA in 55 nongenital SK biopsies, which were compared with 48 normal skin biopsies (healthy controls) using a nested polymerase chain reaction (PCR) using consensus primers CP65/CP70 and CP66/CP69. The positive PCR products were retracted and used to prepare recombination clones with T-vector. Distinct clones were analysed with endonucleases, and HPV genotypes were identified by direct sequencing. RESULTS: EV-associated HPV DNA was detected in 42 of 55 (76%) nongenital SK biopsies vs. only 13 of 48 (27%) healthy controls (chi2 = 22.087; P < 0.005). The prevalence was higher in patients with more than five lesions than in those with only one lesion (P < 0.05). Ten distinct HPV genotypes were detected in the nongenital SK biopsies: HPV 20, 23, 5, renal transplant recipient (RTR) X7, HPV 17, 37, 17b, RTRX4, RTRX4b and strain SK3. HPV 20 was found in 26 of 42 (62%) positive specimens, followed by HPV 23 (11 of 42, 26%) and HPV 5 (six of 42, 14%). Existence of multiple HPV genotypes was observed in 12 of 42 (29%) positive specimens. In healthy controls, five genotypes of EV-associated HPV (HPV 20, 23, 5, 17 and RTRX4) were detected, with the same predominant genotype of HPV 20 (five of 13, 38%). Several distinct HPV genotypes were found to coexist in four of 13 (31%) positive specimens. CONCLUSIONS: This study provides some evidence that EV-associated HPVs might play a part in the pathogenesis of nongenital SK.     

Detection of human papillomavirus type 2a DNA in verrucae vulgares by electron microscopic in situ hybridization

Electron microscopic in situ hybridization (EMISH) of common warts (verrucae vulgares) of the hands was performed using a biotinylated human papillomavirus type 2a (HPV-2a) DNA probe and immunogold labelling of ultrathin sections of 2% glutaraldehyde-fixed, Lowicryl K4M-embedded tissues. It was first established that the warts contained HPV-2a DNA by light microscopic in situ hybridization. The HPV-2a probe chiefly labelled cells in the horny, granular and upper spinous layers of the epidermis, and labelling decreased towards the basal cell layer. The gold particles were located precisely on the viral particles in the nuclei of granular cells. The lower limit of detection by EMISH was found to be the keratinocytes of the third cellular layer above the basal cells. These keratinocytes showed evidence of a viral cytopathic effect, suggesting that vegetative DNA replication in infected keratinocytes occurs at least as early as this level of the epidermis.Presented in part at the 91st Annual Meeting of the Japanese Dermatological Association, Chiba, April 1992     

Detection and sequences of human papillomavirus DNA in nongenital seborrhoeic keratosis of immunopotent individuals

BACKGROUND: The etiology of seborrhoeic keratosis (SK) is unknown. Its clinical and histopathological similarities to verrucae vulgaris and condyloma acuminatum prompted us to examine whether human papillomavirus (HPV) is present in SK lesions. In the present study, HPVs were frequently detected from genital lesions or hair follicle in immunocompromised host. OBJECTIVE: We analyzed 104 nongenital SK specimens diagnosed by clinical and histopathological examinations for HPV DNA in immunopotent individuals. METHOD: We analyzed SK specimens for HPV DNA using in situ hybridization (ISH), polymerase chain reaction (PCR), Southern blot hybridization, and sequencing of viral DNA of PCR-amplified fragments. And we also examined virion, which is the capsid protein of HPV in ISH-positive specimens by immunochemical examination. We identified eight mucosal and two cutaneous type HPVs. RESULT: ISH revealed that 30 of 104 (28.8%) SK samples contained HPV DNA. All ISH-positive specimens were demonstrated virion in the nuclei of the epidermal keratinocytes. PCR analysis showed that 87 (83.7%) samples contained HPV-18, 81 (77.9%) HPV-6, and 73 (70.2%) contained both HPV-18 and -6. The incidence of HPV-1 (7.7%) and HPV-2 (14.4%) was relatively low. All 20 normal controls were negative for HPV DNA by ISH but seven were positive by PCR sequencing. CONCLUSION: Our results suggest that HPV, possibly coinfection with HPV-6 and -18 and unknown type(s) of HPV, plays an important role in the pathogenesis of SK.     

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.     

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

应用地高辛标记的 HPV6B/11、16、18型 DNA探针对 14例鲍温样丘疹病进行原位杂交检测。结果显示 14例中 7例 HPV 6B/11和 18型阳性 ,两者反应强度相似 ,即强阳性 3例 ,弱阳性 4例 ,而6B/11、18型阳性同时伴 16型阳性的仅 3例 ,且 16型反应较弱 ,表明有半数鲍温样丘疹病的发病与 6B/11、18型 HPV感染有关     

Detection of human papillomavirus type 56 in Bowen's disease involving the nail matrix

Background As Bowen’s disease of the nail apparatus is quite rare, there have been only a few reports on the prevalence of human papillomavirus (HPV) infection in this condition. Objectives The purpose of this study was to clarify the association of HPV with this disease involving the nail apparatus. Methods Five patients with Bowen’s disease of the nail apparatus were investigated clinically, virologically and histologically. Total DNAs extracted from excised skin lesions were analysed using polymerase chain reaction (PCR) for the presence of HPV DNA and the amplified products were subjected to DNA sequence analyses. Histological localization of HPV DNA was examined by in situ hybridization. Results In three of five patients, HPV was detected by PCR amplification, and subsequent sequence analyses of the PCR products showed the sequences of HPV type 56. A common clinical feature of the three HPV‐positive patients was longitudinal melanonychia. In contrast, the two HPV‐negative patients presented with a convex nail deformity and a periungual ulcerative lesion. In two of three positive cases, there was a silent point mutation in the L1 gene of each HPV. In the remaining one case, the nucleotide sequence was consistent with the consensus sequence of HPV 56. Sequence analyses of the E6 gene revealed the infection of different variants of HPV 56 among the three cases. The viral genomes were located in keratinocyte nuclei upon in situ hybridization. Conclusions HPV 56 may be involved in the carcinogenesis of Bowen’s disease affecting the nail matrix with longitudinal pigmentation.     

Nestin expression in Bowen's disease and Bowen's carcinoma associated with human papillomavirus

Human papillomaviruses (HPVs) have been detected in lesions of Bowen's disease (BD) and Bowen's carcinoma (BC); the invasive tumor retains the cytological characteristics of BD. Previous reports suggest that nestin-expressing hair follicle stem cells are undifferentiated and pluripotent, and nestin expression in some tumors indicates poor differentiation and high grade of malignancy. We identified HPV-DNA in BD (n=25) and BC (n=23) by in situ hybridization (ISH) analysis with INFORM(?) HPV III (Ventana Medical Systems. AZ, USA) and determined nestin expression by indirect immunohistochemical staining with anti-nestin polyclonal antibody (IBL, Gunma, Japan). We detected HPV-DNA in 68% of BD and in 87% of BC. In BD, 13 cases demonstrated the punctuate pattern, and four showed nestin expression. In BC, 19 cases showed the punctuate pattern and 16 showed nestin expression. HPV-DNA integrates into the host genome, and this is observed as the punctuate pattern on ISH. The nestin expression was statistically high in group of BC than BD (P<0.01). These results therefore suggest that HPV-DNA integrated in the genome of tumor cells of these diseases and contributed to malignant alteration. From the standpoint of tumorigenesis, BC might represent one type of poorly differentiated, high-grade squamous cell carcinoma.     

Detection of human papillomavirus (HPV) DNA in oral squamous cell carcinomas by in situ hybridization and polymerase chain reaction

Summary A series of 156 formalin-fixed, paraffin-embedded biopsies from 40 patients with surgically-treated oral squamous cell carcinomas was analysed for the presence of human papillomavirus (HPV) infection by histopathological evaluation, in situ DNA hybridization and polymerase chain reaction (PCR). Epithelial changes suggesting a HPV lesion within, or adjacent to, the carcinoma lesions were found in 16 out of 40 patients (40%). Morphological signs of a flat HPV lesion were found in four cases (10%), those of inverted type in three cases (7.5%), and those of papillary type in nine cases (22.5%). HPV DNA was demonstrated in one of the lesions by in situ hybridization with biotin-labelled DNA cocktail probe containing HPV types 6, 11, 16 and 18. With the PCR technique, samples from 11 (27.5%) of the 40 patients proved to contain HPV DNA. Of these, HPV 6 was demonstrated in one case, HPV 16 in ten cases and HPV 18 in one case. HPV DNA was exclusively detected in the biopsies showing carcinoma tissue or its adjacent precancer lesions. No viral DNA was found in the biopsies derived from the tumour-free resection margins. These results provide further evidence to support the concept of HPV involvement in the aetiology of oral squamous cell carcinomas, most probably acting synergistically with other carcinogens.     

人乳头瘤病毒与皮肤Bowen病的相关性

目的 探讨人乳头瘤病毒(HPV)与皮肤Bowen病发病的相关性.方法 41例皮肤Bowen病患者皮损以及48例健康对照皮肤,采用多对引物,应用巢式PCR进行HPV DNA检测,同时应用半定量PCR进行病毒定量,对HPV DNA阳性标本进一步采用原位杂交方法分析组织内病毒的分布状况.结果 41例皮肤Bowen病患者皮损HPV DNA阳性检出率为12%(5例),其中黏膜型3例(2例HPV16,1例HPV33),病毒定量相当于101～103拷贝,原位杂交显示在多数肿瘤细胞核中有广泛阳性表达,而肿瘤邻近的正常组织无信号表达;皮肤型2例(HPV27和HPV76各1例),原位杂交均无阳性表达.此外,对照组中有1例检出HPV DNA,属于疣状表皮发育不良相关型HPV23,检出率为2.1%,与皮肤Bowen病皮损中HPV检出率比较,差异无统计学意义.2例患者皮肤型病毒定量较低,与正常对照组中检出的1例HPV23相似,均相当于10-2～10-3拷贝.结论 某些皮肤Bowen病的发病与黏膜型HPV密切相关.     

Bowen's disease of the feet. Presence of human papillomavirus 16 DNA in tumor tissue

A 36-year-old black man with bilateral squamous cell carcinoma in situ of the feet is described. The DNA hybridization analysis performed on the tumor tissue demonstrated the presence of human papillomavirus 16 DNA. Human papillomavirus 16 has been detected repeatedly in genital carcinomas, and evidence is mounting that it may play an important role in the development of malignancy.     

Detection of human papillomavirus type 16 in Bowen's carcinoma of the toe

Background Human papillomavirus (HPV) is known to cause cervical cancer. Because it has been detected in lesions of Bowenoid papulosis, Bowen’s disease, and Bowen’s carcinoma, HPV infection has been implicated in the pathogenesis of these diseases. Methods A 44‐year‐old man was diagnosed clinicopathologically with Bowen’s carcinoma of the right great toe. He developed multiple organ metastases and died. We examined HPV DNA in skin biopsy specimens from the primary and skin metastatic lesions by polymerase chain reaction (PCR) and in situ hybridization (ISH). The PCR assay was carried out using primer sets specifically designed for detecting the E6 and E7 genes of the HPV types associated with malignancy. Purified and cloned PCR products were subjected to DNA sequence analysis. The ISH studies used INFORM^® HPV III probes. Results We found HPV DNA in specimens from both the primary and the skin metastatic lesions. DNA sequencing detected HPV type 16. We compared the base sequences of viral DNA from the primary and metastatic lesions. Point mutations of the base sequences of the E6 and E7 genes were observed in viral DNA from metastases but not in that from primary lesions. The E6 gene had mutated from G to A in the 383rd base sequence, causing a Glu‐to‐Lys amino acid change. Results of ISH showed punctuate signals in the nuclei of tumor cells. Conclusions We suspect an association between HPV 16 infection and the development of this malignant occurrence.     

Polymerase chain reaction for producing biotinylated human papillomavirus DNA probes for in situ hybridization.

Polymerase chain reaction (PCR) was used to produce biotinylated DNA probes for human papillomavirus (HPV) types 16 and 18. The specificity and sensitivity of the probes were tested with in situ hybridization to detect HPV DNA in cervical biopsies or cell lines (CaSki, SiHa, and HeLa). The Gene Amp DNA Amplification kit (Perkin-Elmer Cetus, Norwalk, CT) was used to perform PCR according to the manufacturer's instructions, except that dTTP was substituted by different concentrations of biotinylated dUTP (bio-11-UTP). As the template DNA, the DNA extracted either from CaSki or HeLa cells was used. The reaction mixture was taken through up to 40 cycles of amplification in a Perkin-Elmer Cetus Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT). The highest yield was achieved when the concentrations of dTTP and biotinylated dUTP were 150 microM and 50 microM, respectively. In situ hybridization results compatible with those obtained with biotinylated or radioactively labelled whole genomic HPV DNA probes were demonstrated when primers from E6, E7, and L1 ORF of the HPV 18 were used to produce the biotinylated probe by PCR. With HPV 16, the positive signals were always weaker with the PCR probe than with the whole genomic probe. Overall, the PCR probes might have a lower sensitivity than the whole genomic probes. The background stain was always stronger with the PCR probes than with the whole genomic probes, especially with HPV 16 probes. There does not seem to be a clear correlation between the sensitivity of PCR probes and the size or nucleotide content of the probe.     

Clinical manifestations of human papillomavirus infection in nongenital sites.

Our knowledge of warts dates thousands of years. Most warts represent no more than a transient infection in the hands and feet of children and adults. With the relatively recent medical advances permitting the prolonged survival of immunocompromised hosts, however, HPV-induced lesions have become an important problem. In these patients, lesions represent a recurring, intractable infection that predisposes the patient to the development of skin cancer. Such problems have been appreciated for some time in patients with EV. Newer laboratory techniques have led to an increasing number of clinical entities linked with an HPV cause in the nonimmunosuppressed host. Although evidence incriminating HPV as a causative factor for genital cancers of the cervix and the skin continues to mount, such evidence for nongenital Bowen's disease, certain squamous cell carcinomas of the skin, keratoacanthomas, and other tumors of the skin also has begun to grow. It is to be hoped that continued advances in molecular biologic techniques will further delineate the relationship between HPV and these conditions, lead to an HPV classification scheme that is more utilitarian from a clinical point of view, and ultimately lead to improved treatment.     

Office therapy for human papillomavirus infection in nongenital sites.

The many different treatment possibilities for the eradication of warts provide evidence that no single method that is completely effective has been found. Although the various methods described herein are usually successful therapies for warts, they are all associated with treatment failures and side effects. Until the perfect cure for warts is discovered, the physician must evaluate every wart carefully before deciding on a course of action.     

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。     

In situ DNA hybridization analysis of human papillomavirus (HPV) sequences in benign oral mucosal lesions

Summary A series of 144 surgically treated benign oral mucosal lesions were analysed using an in situ DNA hybridization technique with ³⁵S-labeled human papillomavirus (HPV) DNA probes to demonstrate the DNA of HPV types 6, 11, 13, and 16, in routinely processed, paraffin-embedded biopsy specimens. These lesions and an additional 62 benign oral mucosal biopsy specimens (total, 206 specimens) were also assessed by the indirect immunoperoxidase (IP-PAP) technique to detect the expression of HPV structural proteins (viral antigens). A total of 54/206 (26.2%) lesions were observed to express HPV antigens, being found in 45/92 (48.9%) of the squamous cell papillomas/condylomas, in 1/54 fibrous hyperplasias, in 1/8 true fibromas, and in 7/8 (87.5%) of the focal epithelial hyperplasia (FEH) lesions. Of the HPV DNA-positive lesions, 15/45 (33.3%) expressed HPV antigens, the expression not being related to any particular HPV type. HPV DNA sequences were found in 45/144 (31.3%) of the lesions. HPV DNA was present with the highest frequency in FEH (83.3%), papillary hyperplasia (28.6%), fibrous hyperplasia (24.4%), and true fibromas (14.3%). The most frequent HPV type was HPV 11, representing 37.8% of the DNA-positive lesions. HPV 13 DNA, previously regarded as specific to FEH, was disclosed as a single HPV type in seven cases, and as a double infection by HPV 11 and 13 in an additional three cases, including all five morphologically distinct entities. Noteworthy is the discovery, of the high-risk HPV type 16 DNA in 17.8% of the DNA-positive lesions, four papilloma/condyloma lesions, three fibrous hyperplasias, and one FEH. The results confirm the previously reported evidence regarding HPV involvement in oral mucosal lesions. The implications of these findings are discussed in terms of the epidemiology, HPV etiology, and proper classification of the oral mucosal lesions, with special emphasis on the discovery of the high-risk HPV type 16 in the benign lesions as well as in oral cancer. The use of the in situ DNA hybridization as a powerful tool in detecting the specific HPV DNA sequences in routinely processed oral biopsy specimens is strongly recommended.