Prediction of conserved microRNAs from skin and mucosal human papillomaviruses

Eight human papillomavirus (HPV) types including four cutaneous HPV types (HPV-5, HPV-8, HPV-20 and HPV-38) and four mucosal HPV types (HPV-6, HPV-11, HPV-16 and HPV-18) were selected for this miRNA study. Pre-miRNAs were predicted using a computer programme, and the conserved mature miRNAs were compared to currently known miRNAs. Predicted HPV miRNAs related to miR-466, -467 and -669 were common and specific to the mucosal HPV types. Northern blot hybridization confirmed a predicted miRNA in HPV-positive cervical cancer cell lines encoded by mucosal HPVs. HPV-38 was predicted to express an miRNA conserved to human let-7a and the expression of let-7a, in HPV-38-positive non-melanoma skin cancer (NMSC) biopsies was 10-fold higher than those with HPV-positive (for other types except HPV-38) and HPV-negative NMSCs, suggesting that let-7a expression might be related to HPV-38 infection. Potential gene targets of the predicted miRNA that may aid HPV in infection and pathogenesis were also analysed.     

Prevalence of human papillomavirus DNA in different histological subtypes of cervical adenocarcinoma

The prevalence of human papilloma virus (HPV) DNA in different histological subtypes of cervical adenocarcinoma and related tumors was examined using formalin-fixed, paraffin-embedded tissue samples from 105 primary cervical adenocarcinomas and adenosquamous carcinomas. Broad-spectrum HPV DNA amplification and genotyping was performed with the SPF10 primer set and line probe assay (LiPA), respectively. HPV DNA was detected in 82 of 90 (91%) mucinous adenocarcinomas, encompassing endocervical, intestinal, and endometrioid histological subtypes, and in nine of nine adenosquamous tumors (100%). HPV DNA was not detected in any nonmucinous adenocarcinomas including clear cell, serous, and mesonephric carcinomas (0/6). The most common viral types detected in adenocarcinoma were HPV 16 (50%) and HPV 18 (40%), followed by HPV 45 (10%), HPV52 (2%), and HPV 35 (1%). Multiple HPV types were detected in 9.7% of the cases. In conclusion, mucinous adenocarcinomas and adenosquamous carcinomas of the cervix demonstrate a very high prevalence of HPV DNA, similar to that reported for cervical squamous cell carcinoma. Only rare histological variants of cervical adenocarcinoma seem unrelated to HPV infection.     

Detection of mucosal type human papillomavirus in cutaneous squamous cell carcinoma in Iran

Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer. The oncogenic role of human papilloma virus (HPV) in cutaneous SCC has been suggested by several studies performed on immunosuppresed patients. However, the role of mucosal type HPV in SCC patients with normal immunity has not been studied extensively. Sixty skin biopsies from immunocompetent SCC patients and 60 benign skin specimens were evaluated for mucosal type HPV DNA using polymerase chain reaction (PCR) and immunohistochemistry (IHC). Mucosal type HPV DNA was detected in 18 of 60 cases (30%) and in 7 of 60 controls (11.6%) using PCR. HPV immunostain was positive in 16 of 60 cases (26.6%) and in 15 of 60 controls (25%). Mixed infection with HPV 18, 11, 6 was found in half of the SCC cases. The most prevalent subtype was HPV 18 followed by HPV 6 and 11. The frequency of HPV DNA was significantly elevated in our cases compared to controls (P value <0.01, OR = 16.8, 95% CI: 3.3–74.9). Our findings suggest an association of mucosal type HPV, especially HPV 18, with skin SCC in Iranian patients with normal immunity.     

Prevalence of human papillomaviruses in lung carcinomas: a study of 218 cases.

High-risk human papillomaviruses (HPV) are largely implicated in the carcinogenesis of cervical carcinomas. Their role in bronchopulmonary carcinomas is still unclear. In the present study, we have explored 218 fresh frozen lung tumours for the presence of HPV with the Roche line blot assay and for the expression of mRNAs encoding E6 oncoprotein in HPV positive tumours. Only four samples were positive for HPV detection, one poorly differentiated squamous cell carcinoma and three large cell carcinomas. E6 mRNA was undetectable in these four samples. Our data confirm the low prevalence of HPV in lung carcinomas in Western European countries and do not plead in favour of a carcinogenic role for HPV in these carcinomas.     

Detection and typing of human papillomaviruses in mucosal and cutaneous biopsies from immunosuppressed and immunocompetent patients and patients with epidermodysplasia verruciformis: a unified diagnostic approach.

AIM: To develop a unified diagnostic approach for the detection of human papillomavirus (HPV) DNA in skin and mucosal biopsies from both immunocompetent and immunosuppressed individuals using a degenerate polymerase chain reaction (PCR) method. METHODS: The sensitivity and specificity of three published degenerate primer sets (HVP2/B5 and F14/B15; MY09/MY11; CP62/69 outer and CP65/68 nested primer pairs) were evaluated in PCR reactions with serial dilutions of 12 representative cloned HPV types. This combination of primers was then used to detect HPV DNA in 49 benign and malignant lesions of cutaneous and mucosal origin from immunosuppressed, immunocompetent, and epidermodysplasia verruciformis (EV) patients, and compared with detection rates using single primer sets alone. RESULTS: The observed sensitivity of MY09/MY11 and CP62/69 + CP65/68 was high for mucosal and EV HPV types, respectively. The sensitivity of all primer sets for cutaneous types was low, but nonetheless the use of this combination of primers allowed HPV DNA detection in all of the benign warts analysed. Several mixed infections were also identified. A high prevalence of HPV DNA was similarly detected in squamous cell carcinomas from immunocompromised patients; the HPV types found were exclusively EV related. CONCLUSIONS: The use of a combined degenerate primer PCR approach considerably improves HPV DNA detection over individual primer sets and allows detection of mixed infections. The findings may help explain the discrepancies in published reports relating to HPV DNA detection in benign and malignant skin lesions. Further modifications to this method are in progress which should significantly improve comprehensive HPV detection and typing for diagnostic purposes.     

Proteomic analysis reflects different histologic subtypes of epithelial ovarian cancer

Ovarian carcinoma is a significant cause of cancer mortality in women worldwide. As a heterogeneous disease, distinct clinical and molecular characteristics exist among different histologic subtypes. With the developments in proteomics, surfaced-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is sensitive enough to detect minute quantity of proteins from serum or microdissected cryostat sections. Herein we hypothesized that differentially expressed protein profiles exist in ovarian carcinomas with distinct histologic subtypes. Compared with endometrioid carcinoma, two peaks were significantly higher in serous carcinoma with the m/z of 3622 Da and 4778 Da, reinforcing the need to treat different histologic subtypes of ovarian cancer as different disease entities. Better understanding of these potential diagnostic and therapeutic biomarkers followed with proof-of-target effect will finally contribute to rational combinations of novel therapy and improve individual ovarian cancer patient outcome.     

The role of human papillomaviruses in the pathogenesis and histologic classification of precancerous lesions of the cervix

It is clear that the relation between HPV infection and cervical neoplasia is more complex than initially realized. Preliminary molecular virologic data suggest preferential distributions of low- and high-risk HPV types in CIN that tend to correlate with the morphologic appearance. Thus, mild and moderate dysplasias (CIN I and II) contain a diverse distribution of HPV types, including a minority that have a high risk of malignant potential. HPV, therefore, appears to play a major role as a promoter. Neoplastic transformation is probably determined by specific HPV types but, in addition, requires initiation by some other carcinogenic stimulus, e.g., HSV II, cigarette smoking. Despite numerous studies, performed during the past 30 years, the long-term behavior of dysplasia remains uncertain. The natural history of HPV-associated lesions is unknown. Until this information is available, it is recommended that the conventional dysplasia--CIS or CIN nomenclature be used. The presence of associated viral changes can be considered and added to the diagnosis, e.g., "moderate dysplasia (CIN II) with evidence of papillomavirus infection." Treatment should be the same for all intraepithelial lesions, regardless of the presence of morphologic evidence of HPV. In the future, it may be necessary to modify the classification of precancerous lesions of the cervix if it is shown that a specific HPV type induces a characteristic morphologic alteration or that the HPV type, in and of itself, has greater prognostic significance. Until then, confusion will be minimized and management optimized if the conventional dysplasia--CIS or CIN nomenclature is employed.     

Filaggrin expression in cutaneous and mucosal human papillomavirus induced lesions

A series of 32 human papillomavirus induced lesions derived from epidermis and mucosa was studied for the modulation of filaggrin-profilaggrin (F-PF) expression according to the degree of virus infection as compared to normal skin and mucosa biopsies. This investigation was carried out on frozen sections using indirect immunofluorescence for filaggrin detection and group specific viral antigen and by in situ hybridization with biotinylated probes for viral DNA detection and typing. The 9 cutaneous warts showed an increase of F-PF expression in upper layer cells as compared to normal epidermis, which could be related to the high production of virus (viral antigen and HPV types 1 or 2). The 5 condyloma acuminata displayed also an enhanced expression of these components which was located in several upper layers but virus infection was confirmed in 2 of them with HPV types 6, 11 or 16. The 6 laryngeal papillomas exhibited a granular reactivity pattern for F-PF in suprabasal cell layers with an increase in the upper layers; viral antigen was found in 4 cases and HPV DNA types 6, 11 or 16 were detected in 4 specimens. Conversely among 12 cervical intraepithelial neoplasia, F-PF was expressed only in very superficial layers in few cases, without any correlation with the DNA detection (6, 11 or 16, 18). Taken together these data are suggestive of an intense expression of F-PF in benign lesions which can replicate the virus and a discrete or an absent expression of these components in premalignant or malignant lesions.     

Prevalence of high- and low-risk oncogenic human papillomaviruses in patients with external genital pathology

The paper presents the results of examining 49 patients with genital papillomas, vulvar and vaginal leukoplakia, dysplasia, and cancer. The findings may suggest that human papillomavirus plays an important role in the development of vulvar and vaginal lesions and reconsider the importance of high- and low-risk oncogenic genotypes in the development of benign neoplasms, precancerous conditions, and malignant tumors of the vulva and vagina.     

Development of a general-primer-PCR-reverse-line-blotting system for detection of beta and gamma cutaneous human papillomaviruses

The beta and gamma genera of papillomaviruses consist of epidermodysplasia verruciformis-related human papillomaviruses (HPVs) and phylogenetically related cutaneous HPVs. Here, we have developed a consensus primer PCR assay and reverse line blot typing system coupled thereto (referred to as beta and gamma cutaneous HPV PCR [BGC-PCR]) for detection and typing of 24 beta and gamma HPVs (HPV types 4, 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 48, 49, 50, 60, and 65). Because the HPV-specific PCR products are only 72 bp in size, the system is suitable for formalin-fixed, paraffin-embedded specimens and other samples in which the DNA is of suboptimal quality. This system was able to detect and type as little as 100 ag to 1 fg HPV DNA per reaction (depending on the HPV type) in a background of 100 ng human DNA without any cross-reactivity between the tested types. Beta and gamma HPVs were detected in DNA extracted from plucked eyebrow hairs of 31 of 34 renal transplant recipients. In addition, formalin-fixed, paraffin-embedded specimens from nonmelanoma skin tumors of renal transplant recipients (n = 25) and immunocompetent individuals (n = 15) scored BGC-PCR positive in 21 and 6 cases, respectively, with HPV type 5 (HPV5) and HPV8 being the predominant types. The data indicate that this method can be a valuable, user-friendly tool for the detection and typing of cutaneous HPV in clinical specimens and may have implications for future monitoring of vaccines or alternative treatment modalities for diseases caused by these cutaneous HPVs.     

New generic primer system targeting mucosal/genital and cutaneous human papillomaviruses leads to the characterization of HPV 115, a novel Beta-papillomavirus species 3

We explored the cutaneotropic HPV genetic diversity in 71 subjects from Argentina. New generic primers (CUT) targeting 88 mucosal/cutaneous HPV were designed and compared to FAP primers. Overall, 69 different HPV types/putative types were identified, being 17 of them novel putative types. Phylogenetic analysis of partial L1 sequences grouped 2 novel putative types in the Beta-PV, 14 in the Gamma-PV and 1 in the Mu-PV genera. CUT primers showed broader capacity than FAP primers in detecting different genera/species and novel putative types (p < 0.01). Using overlapping PCR, the full-length genome of a Beta-PV putative type was amplified and cloned. The new virus, designated HPV 115, encodes five early genes and two late genes. Phylogenetic analysis indicated HPV 115 as the most divergent type within the genus Beta-PV species 3. This report is the first providing data on cutaneous HPVs circulating in South America and expands our knowledge of the Papillomaviridae family.     

Three novel papillomaviruses (HPV109, HPV112 and HPV114) and their presence in cutaneous and mucosal samples

To expand our knowledge of the genomic diversity of human papillomaviruses (HPVs), we searched for new HPVs in squamous cell carcinomas of the skin (SCC) and seemingly HPV-negative, otherwise typically HPV-associated lesions. We describe the characterization of three novel HPV types. HPV109 was isolated from an SCC, HPV112 from a condyloma and HPV114 from a low-grade cervical lesion. Pairwise alignment of the L1 sequences classified HPV114 to genus alpha species 3, whereas HPV112 defined a new species in the genus gamma. HPV109 had uncertain classification because of a low and about equal similarity in the L1 gene (between 60% and 65%) to different genera. Type-specific real-time PCRs of cervical samples, a majority from women with low grade atypical cytology, (n = 2856) and various cutaneous samples (n = 538), found HPV114 in 1.7% (48/2856) of the genital samples, whereas both HPV109 and 112 were rare viruses found at high viral loads only in their index samples.     

Human papillomaviruses of different types in precancerous lesions of the uterine cervix: histologic, immunocytochemical and ultrastructural studies

In a prospective study of 34 women with abnormal Papanicolaou smears, biopsy and cervicovaginal lavage specimens were analyzed for the presence of human papillomaviruses (HPVs) by Southern blot analysis and probes for HPVs 6, 11, 16, and 18. In 22 of the 23 patients with cervical lesions (96%), HPV DNA was identified in one or more specimens. All patients in whom HPV DNA was found had either koilocytotic or dysplastic lesions on biopsy or Papanicolaou smear. Immunocytochemical demonstration of HPV in biopsy samples was associated with the presence of large amounts of HPV DNA and with the ultrastructural identification of viral particles. The presence of HPV DNA in cervical biopsy specimens was limited to discrete geographic areas of the cervix with histologic abnormalities. Although HPV 16 and other related HPV types were found in all cases of severe cervical intraepithelial neoplasia, the type of HPV present in a given specimen could not be predicted on the basis of morphologic, immunocytochemical, or electron microscopic findings. It is concluded that virtually all dysplastic lesions of the cervix contain HPV DNA, that HPV is thus likely to be a major etiologic agent in the pathogenesis of cervical dysplasia, and that histopathologic features are not predictive of HPV type.     

Oncogenic human papillomaviruses and ploidy in cervical lesions.

AIM: To compare ploidy measurements obtained on tissue sections of selected low and high grade squamous intraepithelial lesions containing oncogenic HPV (types 16, 18 or 33) detected by in situ hybridisation (ISH) or PCR. METHODS: DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of eight lesions exhibiting atypical squamous cells or squamous atypia and 53 low and 63 high grade squamous intraepithelial lesions in which HPV had been detected by ISH or PCR. RESULTS: Aneuploidy was strongly associated with the presence of oncogenic HPV, being detected in 50% of lesions with squamous atypia and 75.5% of the low and 95.2% of the high grade squamous intraepithelial lesions. The multiploid profile was highly associated with high grade lesions and with the pattern of HPV DNA integration. CONCLUSIONS: The presence of aneuploidy is strongly suggestive of the presence of oncogenic HPV types. Combining the detection of HPV by ISH and PCR with DNA image cytometry may provide the pathologist and the physician with important prognostic information about low grade lesions, especially when these lesions have a multiploid DNA profile and contain oncogenic HPV.