Beta human papillomaviruses infection and skin carcinogenesis

During the last decade, the worldwide incidence of keratinocyte carcinomas (KC) has increased significantly. They are now the most common malignancy, representing approximately 30% of all cancers. The role of ultraviolet (UV) radiation as a major environmental risk factor for skin cancers is well recognized. The aim of this review is to analyse the current understanding of the nature of beta‐human papillomavirus (HPV) and its association with KC and explore the implications for the management and prevention of these cancers. A comprehensive review of the literature on beta‐HPV and its association with KC was undertaken, the results reported in the form of a narrative review. A subgroup of HPV that infects the mucosal epithelia of the genital tract has been firmly associated with carcinogenesis. In addition, some HPV types with cutaneous tropism have been proposed to cooperate with UV in the development of KC. The first evidence for this association was reported in 1922 in patients with epidermodysplasia verruciformis (EV). Since then, epidemiological studies have highlighted the higher risk of skin cancer in patients with EV and certain cutaneous HPV types, and in vitro studies have elucidated molecular mechanisms and transforming properties of beta‐HPV. Furthermore, in vivo research conducted on transgenic mice models has shown the possible role of beta‐HPV in cutaneous carcinogenesis as a co‐factor with UV radiation and immunosuppression. There is good evidence supporting the role of beta‐HPV in the oncogenesis of KC. The high prevalence of beta‐HPV in human skin and the worldwide burden of KC makes the search for an effective vaccine relevant and worthwhile.     

A highly conserved nucleotide string shared by all genomes of human papillomaviruses

The nucleotide string TAAAACGAAAGT is the longest perfect homology shared by all sequenced human papillomavirus genomes. This nucleotide string, which was also found to be highly specific for human papillomavirus genomes, shares the same genomic position in all viral types (5 end of the E1 open reading frame) and putatively codes in every case for the same amino acids. One possible evolutionary model was used to estimate the probability of random occurrence of the nucleotide string in 10 human papillomavirus genomes. It assumed that the universal string had been subjected to the same mutation rate as the entire E1 open reading frame. The estimated probability was found to be very low, suggesting that the conservation of the string could not have resulted from random divergence and that its conservation among human papillomaviruses is likely to reflect the occurrence of biological constraints. It is speculated that this nucleotide string may be required to code for amino acids indispensable for the nuclear localization of E1-coded peptides or to bind cellular factors affecting viral replicative functions. Definitive evidence is expected to come from oligonucleotide-protein binding experiments and from site-directed mutagenesis of cloned HPV genomes. This motif, universal among human papillomaviruses, is being successfully used in the design of consensus primers from the early region.     

Prevalence of mucosal and cutaneous human papillomaviruses in different histologic subtypes of vulvar carcinoma.

Two independent pathways of vulvar carcinogenesis have currently been identified, one related to infection with mucosal human papillomaviruses (HPVs) and a second related to chronic inflammatory or autoimmune processes. The goal of the study was to examine a possible role of cutaneous HPVs from the beta genus in vulvar carcinogenesis and to evaluate the distribution of intratypic variants of HPV 16 in HPV 16-positive vulvar cancer. Consecutive cases of vulvar carcinoma were retrieved from the files and included the following histologic subtypes: keratinizing (n=21), basaloid (n=7), warty (n=1), mixed basaloid-warty (n=4), verrucous (n=4), keratoacanthoma (n=1), basal cell carcinoma (n=1). All tumors were microdissected and tested for 25 beta HPV types and 25 mucosal HPV types. Cases identified as positive for HPV 16 were further tested for intratypic variants. All cases were immunostained for p16INK4a. Beta HPVs were not detected in any of the tumor cases. Mucosal HPVs were detected in all but one basaloid/warty carcinomas; of these, nine cases (82%) were positive for HPV 16, including five European subtypes, one African subtype, one North American subtype and two indeterminate subtypes. Two of four verrucous carcinomas were positive for HPV 6. Mucosal HPVs were not detected in keratinizing carcinomas, keratoacanthoma and basal cell carcinoma. All cases of basaloid/warty carcinomas, but none of the remaining tumors, overexpressed p16INK4a protein. Our data do not support a role of beta HPVs in the pathogenesis of vulvar carcinoma. The study reaffirms the role of mucosal HPVs, in particular that of HPV 16, in the pathogenesis of basaloid and warty tumor subtypes. The HPV 16 intratypic variation showed correlation with patients' ethnic background. P16INK4a immunostaining seems to be a sensitive and specific marker of vulvar carcinomas positive for oncogenic mucosal HPVs.     

Comparative transforming potential of different human papillomaviruses associated with non-melanoma skin cancer

It is well established that high-risk human papillomaviruses (HPVs) that infect mucosal epithelia are the causative agents of cervical cancer. In contrast, the association of cutaneo-tropic HPV types with the development of non-melanoma skin cancer (NMSC) is less well defined. In this study, we have analysed the in vitro transforming potential of various cutaneous HPV types. Using oncogene cooperation assays with activated ras, we have shown that diverse cutaneous types, including 12, 14, 15, 24, 36 and 49, have significant transforming potential. Interestingly, most of this activity appears to be encoded by the E6 gene product. In contrast, the common HPV-10 exhibits no significant transforming potential in these assays. This difference may be a reflection of different patterns of cellular localization, with transforming E6s being nuclear and non-transforming being cytoplasmic. These results provide molecular support for a role of these viruses in the development of certain human malignancies.     

Sequence divergence yet conserved physical characteristics among the E4 proteins of cutaneous human papillomaviruses

Human papillomavirus (HPV) types 1, 2, and 4 together comprise the major cause of cutaneous papillomas in the general population. We have aligned the genomes of these three viruses by partial sequence analysis, and have sequenced the E4 open reading frames (ORFs) of HPV 2 and HPV 4. After expression as beta-gal fusion proteins in bacteria, antibodies raised to the putative E4 gene-products of both virus types were used to identify the native E4 proteins in naturally occurring tumors. At the primary amino acid sequence level, the E4 protein of HPV 2 was found to be most homologous with those of HPV 6 and 11 and was not closely related to those of HPV 1 or 4. Although the E4 ORF represents a region of weak homology amongst papillomaviruses, the E4 encoded proteins showed significant conservation in their physical characteristics. Like those of HPV 1, the E4 proteins of both HPV 2 and HPV 4 were found to be composed of a major low-molecular-weight doublet (16.5/18K for HPV 2, 20/21K for HPV 4, c.f. 16/17K for HPV 1) along with minor high-molecular-weight species, which probably represent dimers of the smaller proteins, (33K for HPV 2, 40K for HPV 4, c.f. 32/34K for HPV 1). The E4 products of all three virus types were multiply charged, and exhibited a characteristic migration pattern following alkaline urea gel electrophoresis. Although the levels of E4 expression in tumors induced by the different virus types was very different, this was found to correlate closely with the level of virus production characteristic of each virus type. In all three cases, E4 proteins were found to be primarily cytoplasmic, and to be associated with the distinctive cytoplasmic inclusion granules characteristic of each virus type. The poor sequence conservation between the E4 protein of HPVs 1, 2, and 4, taken alongside the ability of these viruses to infect similar histological sites, suggests that E4 may not be involved in determining tissue specificity. Our results suggest conserved physical characteristics (acidic, multiply charged, ability to form dimers) and similar site of expression may be the important factors for E4 function.     

Biology of human papillomaviruses

Human papillomaviruses (HPVs) cause squamous cancers of epithelial surfaces, of which genital cancers are the most common. In this article we have attempted to describe the properties and functions of the viral proteins of HPV type 16, a common cause of genital cancers, and have tried to suggest how their expression may lead to a dysregulated cell which may become malignant. These viruses are attempting to replicate in terminally differentiating keratinocytes and must stimulate G1 to S-phase progression for the replication of their genome. As part of the successful completion of replication and assembly of infectious virus particles, the virus needs at least partial differentiation to occur. Therefore, at the same time as differentiation is occurring, the nuclei of infected cells are in S-phase. While the mechanisms of action of the viral proteins are not completely understood, researchers are making progress and this article strives to bring together the conclusions from some of this work.     

Immunologic surveillance and human papillomaviruses

Papillomaviruses, the cause of warts, are the oldest known human tumor viruses. Here Holger Kirchner reviews the evidence for immunosurveillance against human papillomaviruses and concludes that surveillance mechanisms may also operate against the tumors that these viruses induce.     

Typing of human papillomaviruses in condylomata acuminata from Greece

DNA samples from recurrent condylomata acu-minata biopsies of Greek males and females were examined for the presence of human papil-lomavirus (HPV) DNA using high-stringency Southern blot hybridization analysis. Of the twenty-six biopsies, 25 were positive for the HPV 6/11-related DNA sequences, and when further analyzed with the polymerase chain reaction (PCR) the HPV-negative biopsy was also positive for HPV 6/11 DNA. Nineteen specimens were further characterized based on their Pstl restriction endonuclease hybridization pattern. Twelve biopsies were positive for HPV 6a, one biopsy was positive for HPV 11a, and one biopsy was positive for HPV 6c DNA. Three specimens contained HPV 6/11 related DNA that gave an unusual Pstl pattern, and one specimen appeared to represent a multiple HPV infection containing HPV 6/11- and HPV 31/35/39-related sequences. Finally, one sample contained a mixture of HPV 6a DNA and an HPV 6a-like genome. Biopsies were also taken from adjacent apparently normal tissue, 0.5 cm away from the lesion, in 19 of the patients. Only one of these was found to be positive for HPV 6a DNA by Southern blot analysis. © 1994 Wiley-Liss, Inc.     

Oncogenic activities of human papillomaviruses

Infectious etiologies for certain human cancers have long been suggested by epidemiological studies and studies with experimental animals. Important support for this concept came from the discovery by Harald zur Hausen's group that human cervical carcinoma almost universally contains certain “high-risk” human papillomavirus (HPV) types. Over the years, much has been learned about the carcinogenic activities of high-risk HPVs. These studies have revealed that two viral proteins, E6 and E7, that are consistently expressed in HPV-associated carcinomas, are necessary for induction and maintenance of the transformed phenotype. Hence, HPV-associated tumors are unique amongst human solid tumors in that they are universally caused by exposure to the same, molecularly defined oncogenic agents, and the molecular signal transduction pathways subverted by these viral transforming agents are frequently disrupted in other, non-virus-associated human cancers.     

The presence of inhibitory RNA elements in the late 3'-untranslated region is a conserved property of human papillomaviruses

Here we have tested the inhibitory activity of the late untranslated region (UTR) of nine different human papillomavirus (HPV) types representing three different genera and six different species. These HPVs include both low-risk and high-risk types. We found that the late UTR of the various HPVs all displayed inhibitory activity, although they inhibited gene expression to various extent. The late UTR from the two distantly related HPV types 1 and 16, which are two different species that belong to different genera, each interacted with a 55 kDa protein. This protein cross-linked specifically to both HPV-1 and HPV-16 late UTR, although it bound more strongly to HPV-16 than to HPV-1, which correlated with the higher inhibitory activity of the HPV-16 late UTR. Mutagenesis experiments revealed that inactivation of two UGUUUGU motifs in the HPV-16 late UTR or two UAUUUAU motifs in the HPV-1 late UTR resulted in loss of binding of p55. In summary, these results demonstrate that the presence inhibitory elements encoding PuU(3-5)Pu-motifs in the HPV late UTR is a conserved property of different HPV types, species and genera, and suggest that these elements play an important role in the viral life cycle.     

Improved amplification of genital human papillomaviruses

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.     

Bead-based multiplex genotyping of human papillomaviruses

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.     

The biological properties of E6 and E7 oncoproteins from human papillomaviruses

More than 100 different human papillomavirus (HPV) types have been isolated so far, and they can be sub-grouped in cutaneous or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts. A sub-group of human mucosal HPVs, referred to as high-risk HPV types, is responsible for approximately 5% of all human cancers, which represents one-third of all the tumours induced by viruses. Epidemiological and biological studies have shown that HPV16 is the most oncogenic type within the high-risk group. Emerging lines of evidence suggest that, in addition to the high-risk mucosal HPV types, certain cutaneous HPVs are involved in skin cancer. HPV-associated cancers are intimately linked to HPV persistence and the accumulation of chromosomal rearrangements. The products of the early genes, E6 and E7, of the high-risk mucosal HPV types play a key role in both events. Indeed, these proteins have developed a number of strategies to evade host immuno-surveillance allowing viral persistence, and to alter cell cycle and apoptosis control, facilitating the accumulation of DNA damage/mutations. Often, the two oncoproteins target the same cellular pathways with different mechanisms, showing a strong synergism in promoting cellular transformation and neutralizing the immune response. Here, we review most of the findings on the biological properties and molecular mechanisms of the oncoproteins E6 and E7 from mucosal and cutaneous HPV types.     

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.     

A new PCR-based assay amplifies the E6-E7 genes of most mucosal human papillomaviruses (HPV)

We established a new assay to detect the E6-E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene.     

An overview of human papillomaviruses and current vaccine strategies

Cervical cancer is one of the most common cancers in women worldwide, particularly in developing countries. The viral origin of cervical cancer has been proven beyond any reasonable doubt. Persistent infection with certain subsets of human papillomaviruses is recognized as a necessary cause for the development of cervical cancer. Persistence of oncogenic HPVs, immunodeficiency, high HPV viral load and cofactors like smoking, multiple sex partners and poor nutrition predispose to cervical cancer. Prophylactic vaccines using HPV virus-like particles containing capsid protein L1 have shown protection against disease in animals and are currently undergoing clinical trials. Therapeutic vaccines using HPV E6 and E7 proteins are also being investigated for their ability to remove residual infection.     

Human papillomaviruses and non-melanoma skin cancer: basic virology and clinical manifestations

Human papillomaviruses (HPV) infect cutaneous and mucosal epithelia and induce benign and malignant lesions. Non-melanoma skin cancer (NMSC), encompassing basal cell carcinoma and squamous cell carcinoma (SCC), is the most frequent cancer in the Caucasian population, and the incidence has increased dramatically worldwide. Ultraviolet (UV) radiation is a major risk factor for NMSC, and cutaneous HPV is also considered to play an active role during the pathogenesis of these cancers. The first evidence for the involvement of HPV in NMSC was reported in patients with Epidermodysplasia verruciformis (EV). HPV types detected in skin tumours of these patients are referred to as EV/cutaneous HPV types belonging to the beta- and gamma-papillomaviruses (PV). Epidemiological studies have shown a higher risk of several EV/cutaneous HPV types for NMSC. Furthermore, in vitro and animal models show transforming properties of some PV types. The anti-apoptotic activities, and the delay of DNA repair mechanism caused by some EV/cutaneous HPV E6 proteins in response to UV-induced mutations, may lead to the persistence of DNA-damaged keratinocytes. Thus, specific EV/cutaneous HPV types as co-factors in association with UV-radiation and the immune system seem to be involved in the early pathogenesis of cutaneous SCC.