Role of human papillomavirus in penile cancer,penile intraepithelial squamous cell neoplasias and in genital warts

Using PCR, the overall prevalence of human papillomavirus (HPV) DNA in penile carcinoma is about 40–45%, which is similar to the detection rate of HPV-DNA in vulvar carcinoma (50%). In analogy to vulvar cancer two different pathways of penile carcinogenesis seem to exist. In contrast to basaloid and warty penile cancers which are regularly HPV-associated (about 80–100%), only a part of keratinizing and verrucous penile carcinomas appear to be related with HPV (33–35%). Penile intraepithelial neoplasias comprising Bowen's disease, erythroplasia of Queyrat and bowenoid papulosis are precursor lesions of basaloid and warty carcinomas of the penis.Precursors of keratinizing carcinomas and verrucous carcinomas are not established. Whether lichen sclerosus and squamous-cell hyperplasia precede penile keratinizing carcinoma is a matter of discussion. Giant condylomata acuminata may precede the development of verrucous carcinomas in some cases. Since high risk HPVs are more frequently found in verrucous carcinomas than in giant condylomas, HPV typing may be a helpful diagnostic step to differentiate giant condyloma from verrucous carcinoma.     

Immunoperoxidase localization of papillomavirus antigen in cutaneous warts and bowenoid papulosis

Using the immunoperoxidase technique and a broadly cross-reactive antiserum which detects infection with any papillomavirus, papillomavirus capsid antigen was demonstrated in 25 of 48 cutaneous papillomas and in two of 38 cutaneous dysplasias. Both positive dysplastic lesions were diagnosed on histopathologic examination as bowenoid papulosis. Antigen-positive cutaneous warts were scattered in all age categories and at all body sites. In addition, there was little variation in antigen expression by morphologic type of wart. Antigen was localized in the nucleus of superficial epithelial cells. The amount of staining was variable, with some warts showing large numbers of stained nuclei. In other warts only isolated cells stained. In both cases of bowenoid papulosis, small foci of positively stained cells were observed. The finding of papillomavirus antigen in two of 21 cases of bowenoid papulosis is suggestive of an etiologic relationship between this lesion and a papillomavirus. An examination of antigen-negative cases of bowenoid papulosis for a papillomavirus genome will be necessary to confirm these results.     

Genital seborrheic keratoses are human papillomavirus-related lesions. A linear array genotyping test study

Controversy exists about the meaning of human papillomavirus (HPV) detection in seborrheic keratosis (SK). To clarify the pathogenic contributing role of HPV in the development of genital SK, we have studied 40 genital SKs, 20 extragenital SKs, and 20 non-SK genital lesions by polymerase chain reaction for HPV, using a Linear Array Genotyping test that detects 37 genital HPV types. Twenty-eight of the 40 genital SK specimens (70%) were positive for HPV. Twenty-seven of the 28 positive cases (96%) contained HPV6, one of them associated to HPV18 and HPV35 (4%), and the remaining lesion (4%) harbored HPV55. However, HPV was detected in only 2/20 extragenital SK samples (10%) and in 1/20 non-SK genital lesions (5%). Our results support a pathogenic relationship between HPV and genital SK by showing: 1) a high rate of virus detection in these lesions, with a strong predilection for HPV6, and 2) scarcity of genital HPV types in most of the remaining non-SK cutaneous genital lesions and in the extragenital SKs. HPV cannot be found in a minority of genital SKs using highly sensitive techniques, and therefore, other presently unknown factors may also be implied in the pathogenesis of these lesions.     

The presence of HPV types 6/11, 13, 16 and 33 in bowenoid papulosis in an HIV-positive male, demonstrated by DNA in situ hybridization.

A perianal bowenoid papulosis was examined for the presence of HPV types 2, 6, 11, 13, 16, 18, 32 and 33 by DNA in situ hybridization. Positive signals were seen for HPV types 6/11, 13, 16 and 33. HPV type 13 is strongly related to oral focal epithelial hyperplasia and has not been reported outside the oral cavity before.     

Inverse modulation of intraepithelial Langerhans' cells and stromal macrophage/dendrocyte populations in human papillomavirus-associated squamous intraepithelial lesions of the cervix

Ninety-four cervical biopsies from normal tissue to high-grade squamous intraepithelial lesion (SILs) were examined for the presence of intraepithelial Langerhans' cells and subpopulations of stromal macrophages/dendrocytes by immunohistochemistry using anti-S100,-L1, -CD68 and -factor XIIIa antibodies. Human papillomavirus (HPV) detection was performed in all cases by using first a mixture of DNA probes for 14 HPV types commonly found in anogenital biopsies at low stringency conditions (T _m -40°C) and by reanalyzing the tissues at high stringency (T _m - 10°C) with HPV 6/11, 16/18 and 31/33/35 biotinylated probe cocktails and individual digoxigenin-labelled probes. SILs and metaplastic tissues were significantly associated with a depletion of S100-positive intraepithelial Langerhans' cells when compared with normal epithelium. In contrast, there was a significant increase in L1-positive stromal macrophages in SIL biopsies compared with normal or metaplastic cervix. A significantly higher density of CD68-positive macrophages was also observed in high-grade SILs compared with normal or metaplastic biopsies and with low-grade SILs. The density of factor XIIIa-positive dendrocytes was found to be higher in SILs compared with metaplastic tissues and in high-grade SILs when compared with normal cervical biopsies. No specific relationship was found between the densities of these cells and the HPV type detected in SILs separated into low grade and high grade. The significance of this inverse modulation of intraepithelial Langerhans' cells and stromal macrophages/dendrocytes in normal and SIL biopsies is discussed in relation to HPV infection and malignant transformation.     

外阴Bowen样丘疹病15例临床病理分析

目的 探讨Bowen样丘疹病的临床病理特征、鉴别诊断及病因。方法 对15例Bowen样丘疹病进行临床和病理分析并随访。采用多重引物PCR检测12例Bowen样丘疹病上皮组织中HPV DNA，用免疫组化SP法检测15例HPV抗原及p53蛋白。结果 15例Bowen样丘疹病患者全为女性，年龄21～40岁(平均29．5岁)。病变外观为色素性扁平丘疹，组织学与Bowen病相似，但异型性较小。12例病变组织HPV6、11、16、18型病毒基因检测均为阴性，对照组检测阳性。广谱HPV抗原检测阳性2例(2／15)，p53蛋白阳性2例(2／15)。结论 Bowen样丘疹病的诊断应该结合典型临床特征，该病属良性病变，但伴有重度非典型增生病例可视为癌前病变，发病与HPV感染的关系尚待肯定。     

Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

A new method was developed for detection of human papillomavirus (HPV) by loop-mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real-time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type-specific. In order to evaluate the reliability of HPV type-specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV-6 DNA and HPV-11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV-16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real-time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV-11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA.     

外阴鲍文祥丘疹病的诊断与误诊分析

目的：探讨鲍文祥丘疹病的临床与病理特征以及误诊原因。方法：回顾分析22例鲍文祥兵疹病的论断、治疗情况，并进行鉴别诊断。结果：22例分别误诊为鲍文病、尖锐湿疣、黑色素痣或恶性质黑色素瘤等，通过病理协助诊断，全部确诊为鲍文样丘疹病。经治疗后好转，随访2年未见复发。结论：鲍文样丘疹病易与鲍文病疾病相混淆，诊断要结合临床形态观察和病理组织学检查，才能准确诊断和治疗鲍文样丘诊病，该病有自愈倾向。     

Epithelial to mesenchymal transition in cutaneous squamous cell carcinoma is correlated with COX-2 expression but not with the presence of stromal macrophages or CD10-expressing cells

Epithelial to mesenchymal transition (EMT) is an intricate process by which epithelial cells loose epithelial characteristics and acquire a mesenchymal-like phenotype. EMT and cyclooxygenase 2 (COX-2) expression are related to tumor invasion and metastasis. The tumor microenvironment plays a major role in tumor progression and the induction of EMT. Here, we investigated the relationship between EMT and COX-2 expression as well as tumor-associated macrophages (TAM) and CD10-positive stromal cells during the development of cutaneous squamous neoplastic lesion. We performed immunohistochemical staining for vimentin, E-cadherin, β-catenin, COX-2, CD68, and CD10 in 41 cases of squamous cell cancers (SCC), 20 of Bowen's disease, 30 of actinic keratosis, and 30 samples of normal skin. SCC cells showed significantly increased vimentin expression and reduced expression of membranous E-cadherin and β-catenin compared with cells in precursor lesions and in normal skin. COX-2 expression was also markedly increased in SCC cells. E-cadherin expression was positively correlated with β-catenin expression and inversely correlated with COX-2 expression in SCC cells. The number of TAM and CD10-positive stromal cells increased from the normal skin to precursor lesions and SCC cells. The number of TAM and of CD10-positive stromal cells did not correlate with the expression of E-cadherin, β-catenin, COX-2, and vimentin in SCC cells. We suggest that cutaneous SCC cells show EMT, which appears to be correlated with COX-2 expression but not with stromal CD10 expression and TAM.     

Analysis of benign and malignant urogenital tumors for human papillomavirus infection by labelling cellular DNA

A total of 268 biopsies from the genital region was screened for the presence of human papillomavirus DNA. The specimens included carcinoma of the vulva, vagina, cervix, corpus uteri, ovaries and penis, and Bowen's carcinomas, Bowenoid papuloses, Bowen's disease, cervical intraepithelial neoplasias (CIN I to III), Buschke-Löwenstein tumors, a cervical polyp, decidua, endometrium and histologically normal biopsies. Of 45 carcinomas, 18 contained either HPV 16 and/or 18 and 3 HPV 6-related sequences.In a few individual biopsies double or even triple infections were noted. Unusual was the presence of HPV 2-related DNA in one biopsy from Bowen's disease, whereas 2 condylomata acuminata contained HPV 3-related DNA and one contained HPV DNA related to a group of epidermal HPV's found in epidermodysplasia verruciformis lesions.     

Immunohistology of human thymomas

We analyzed lymphocyte surface markers in seven thymomas. In six mixed thymomas, the staining patterns were similar for T11, OKT8, OKT6, OKT4, Coulter clone T4, and Leu 3a/3b. Staining patterns for Ia and keratin showed a dendritic pattern. Occasional S 100-positive interdigitating dendritic cells were identified. In three patients with associated myasthenia gravis, no significant differences in staining patterns were identified. A different pattern was seen in a patient with hypogammaglobulinemia, red blood cell aplasia, and a spindle cell thymoma: T11-, T8-, and T6-positive cells each comprised 70% to 80% of the tumor; but, cells of the helper/inducer phenotype differed when measured with OKT4 antibody (0%) vs Coulter clone T4 antibody (60%) and Leu 3a/3b (60%). This unusual phenotype, which was present on both tumor cells and peripheral T cells, appears related to the antigenic polymorphism of the T4 antigen and is believed to have no functional significance. Importantly, this discrepancy among commercial antibodies has significant implications in the general use of these reagents in clinical evaluations.     

Two newly identified human papillomavirus types (HPV 40 and 57) isolated from mucosal lesions

Two new papillomaviruses, HPV 40 and HPV 57, were isolated from a PIN lesion and an inverted papilloma of the maxillary sinus, respectively. HPV 40 showed a 13% homology to HPV 7 by reassociation kinetics and HPV 57 showed a 17% homology to HPV 2 and 25% homology to HPV 27. Hybridization of the DNA of these papillomaviruses to a wide variety of different tumor biopsies revealed that HPV 40 was present in a few genital condylomata acuminata as well as in bowenoid lesions. HPV 57 DNA was present in an oral wart, a genital condyloma acuminatum, and verrucae vulgares lesions from two immunosuppressed patients.     

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     

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.     

Possible identification of third stromal component in extraadrenal paraganglioma: myofibroblast in fibrous band and capsule

Sustentacular and dendritic cells are known as the stromal components of extraadrenal paraganglioma. We identified a third stromal component in such a case. A 66-year-old Japanese woman complained of abdominal pain. The tumor was discovered near the right adrenal gland in the retroperitoneum. Histologically, the tumor consisting of round to oval neoplastic cells with eosinophilic cytoplasm proliferating with a “zellballen” pattern. Sustentacular cells were positive for S-100. Dendritic cells positive for HLA-DR were seen among the neoplastic nests. Additionally, many alpha-smooth muscle actin (ASMA)-positive and hcaldesmon-negative stromal cells, namely, myofibroblasts, were distributed in the capsule and fibrous band. Ultrastructurally, myofibroblasts contained many myofilaments and dense bodies in the cytoplasm. Finally, we identified the third stromal component, namely, myofibroblasts, in the extraadrenal paraganglioma. These myofibroblasts may play a role in the stromal response of host against neoplasm or the regulation of tumor growth.     

Vulvar carcinomas: search for sequences homologous to human papillomavirus and herpes simplex virus DNA

Ten cases of intraepithelial carcinoma, five with Bowenoid features and five with early invasion, and ten cases of invasive vulvar carcinoma were examined by in situ hybridization and Southern blot analysis using DNA probes for human papillomavirus (HPV) types 6, 11, 16, 18 and 31. HPV DNA was detected in 90% of the intraepithelial cases and in 10% of the invasive cases. All positive cases showed the presence of DNA of HPV type 16. The cases with intraepithelial lesions revealed a strong correlation between the presence of HPV type 16 DNA, cigarette smoking habit, other potential cofactors such as herpes simplex (HSV) DNA sequences and the use of contraceptive drugs, and clinicopathologic features of Bowen's type in situ squamous cell carcinoma. Similar associations were not observed among the cases with invasive disease. While HPV-16 is associated with differentiated Bowenoid type vulvar intraepithelial neoplasia, which appears to be the most common form of early carcinoma of the vulva, the same association was not seen with respect to advanced vulvar invasive squamous cell carcinoma.     

Imiquimod 5% cream: a new treatment for Bowen's disease

Bowen's disease (BD) is a squamous cell carcinoma in situ. Recent studies suggest that human papilloma virus plays an important role in the development of BD. We investigated whether imiquimod 5%, a topical immune response modifier, is an effective treatment for BD in five immunocompetent patients. The lesions were one genital and four extragenital. The frequency of application varied from three times weekly up to twice daily, and treatment duration ranged from 8-24 weeks. Four patients achieved clinical and histological cure. The patient with the genital lesion gained an important reduction in size and infiltration, which enabled surgical removal of the remaining lesion with good functional and cosmetic result. Our results suggest that topical imiquimod 5% is an effective treatment for BD through its viral and antitumor effects.     

Cystosarcoma phylloides of the breast and its mimics. An immunohistochemical and ultrastructural study

Cystosarcoma phylloides of the breast is a tumor composed of breast ducts and a cellular stromal component that can be benign or malignant. The origin of the stromal cells is controversial. We undertook an immunohistochemical and ultrastructural study of 11 cases of cystosarcoma phylloides to assess the histogenesis of the stromal component. By light microscopy, 4 were diagnosed as benign, and 7 were diagnosed as malignant. Antibodies to vimentin, desmin, actin, high- and low-molecular-weight keratins, and S100 protein were used for immunohistochemical staining. In the 4 benign cases of cystosarcoma phylloides, the stromal cells stained positively only for vimentin. In the malignant tumors, the spindle cell component stained for vimentin in all the cases. In addition, the malignant stromal cells coexpressed desmin in two cases and keratin and S 100 protein in another case. By electron microscopy the stromal component in the benign case and in two of five malignant cases was composed of a mixture of fibroblasts and myofibroblasts. The entire neoplastic stroma in two other malignant cases showed features of smooth-muscle differentiation, whereas in another case all the stromal cells showed myoepithelial differentiation. Thus, in benign and malignant cystosarcoma phylloides, the stromal component consists of a mixture of fibroblasts and myofibroblasts. Leiomyosarcomas and myoepitheliomas can mimic malignant cystosarcoma phylloides, but immunohistochemistry and electron microscopy can differentiate these entities. This is important since their biologic behavior is different.     

Myoepitheliomas and myoepithelial adenomas of salivary gland origin. Immunohistochemical evaluation of filament proteins, S-100 alpha and beta, glial fibrillary acidic proteins, neuron-specific enolase, and lactoferrin

Immunohistochemical identification of keratin proteins (TK, KL1 and PKK1), vimentin, myosin, S-100 protein (using polyclonal antiserum) and S-100 alpha and beta subunits, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), lactoferrin, and lysozyme was made in myoepitheliomas, myoepithelial adenomas, and clear cell adenomas of salivary gland origin. Myoepithelioma cells were divided into two types: plasmacytoid cells, which showed great heterogeneity in terms of keratins and S-100 alpha and beta proteins and a lack of GFAP, NSE, lactoferrin, and lysozyme in most the cells, and fibrous and dendritic tumor cells, which displayed variable staining for keratin and S-100 alpha and beta proteins. Myoepithelial adenomas were composed of small-, intermediate-, and large-sized spindle cells that showed irregular positive reactions for keratins and S-100 alpha and beta. Immunohistochemical deposition of S-100 protein was restricted strongly to the dendritic cells present in hyalinous and myxomatous areas. Clear cell adenomas revealed uniformly slight staining of keratins and S-100 proteins, and negative staining or rarely positivity for GFAP, NSE, lactoferrin, and lysozyme. When the immunohistochemical deposition of these proteins was compared between normal glands and myoepithelial tumors, heterogeneity of expression of keratins, S-100 proteins, GFAP, and NSE was notable in the tumors. Progenitor cells of several kinds of myoepithelioma were suggested to be intercalated reserve cells, which are thought to be the same cell that gives rise to pleomorphic adenoma of salivary glands.     

Association of HPV infection and clearance with cervicovaginal immunology and the vaginal microbiota

Cervical human papillomavirus (HPV) infection may increase HIV risk. Since other genital infections enhance HIV susceptibility by inducing inflammation, we assessed the impact of HPV infection and clearance on genital immunology and the cervico-vaginal microbiome. Genital samples were collected from 65 women for HPV testing, immune studies and microbiota assessment; repeat HPV testing was performed after 6 months. All participants were HIV-uninfected and free of bacterial STIs. Cytobrush-derived T cell and dendritic cell subsets were assessed by multiparameter flow cytometry. Undiluted cervico-vaginal secretions were used to determine cytokine levels by multiplex ELISA, and to assess bacterial community composition and structure by 16S rRNA gene sequence analysis. Neither HPV infection nor clearance were associated with broad differences in cervical T cell subsets or cytokines, although HPV clearance was associated with increased Langerhans cells and HPV infection with elevated IP-10 and MIG. Individuals with HPV more frequently had a high diversity cervico-vaginal microbiome (community state type IV) and were less likely to have an L. gasseri predominant microbiome. In summary, HPV infection and/or subsequent clearance was not associated with inflammation or altered cervical T cell subsets, but associations with increased Langerhans cells and the composition of the vaginal microbiome warrant further exploration.