Cutaneous HPV Research Articles

Biomolecular Dynamics of Nitric Oxide Metabolites and HIF1α in HPV Infection.

Viral infections cause oxygen deprivation, leading to hypoxia or anoxia in certain tissues. The limitation of mitochondrial respiration is one of the major events during hypoxia that induces alternative metabolic activities and increased levels of certain biomolecules such as nitric oxide (NO) metabolites. In this study, we aimed to investigate the role of NO metabolites and hypoxia in HPV infection. We included 36 patients with palmoplantar warts and 36 healthy subjects and performed serum determinations of NO metabolites (direct nitrite, total nitrite, nitrate, and 3-nitrotyrosine) and HIF1α, a marker of hypoxia. We found elevated serum levels in NO metabolites and HIF1α, and decreased direct nitrite/nitrate ratios in patients with warts versus controls. Additionally, we identified statistically significant positive correlations between NO metabolites and HIF1α levels, except for 3-nitrotyrosine. Our findings show that HPV infection causes hypoxia and alterations in NO metabolism and suggest a link between wart development and cellular stress. Our research could provide new insights for a comprehensive understanding of the pathogenesis of cutaneous HPV infections.

Inter-epitope spacer variation within polytopic L2-based human papillomavirus antigens affects immunogenicity

The human papillomavirus minor capsid protein L2 is being extensively explored in pre-clinical studies as an attractive vaccine antigen capable of inducing broad-spectrum prophylactic antibody responses. Recently, we have developed two HPV vaccine antigens – PANHPVAX and CUT-PANHPVAX- both based on heptameric nanoparticle antigens displaying polytopes of the L2 major cross-neutralizing epitopes of eight mucosal and twelve cutaneous HPV types, respectively. Prompted by the variable neutralizing antibody responses against some of the HPV types targeted by the antigens observed in previous studies, here we investigated the influence on immunogenicity of six distinct glycine-proline spacers inserted upstream to a specific L2 epitope. We show that spacer variants differentially influence antigen immunogenicity in a mouse model, with the antigen constructs M8merV6 and C12merV6 displaying a superior ability in the induction of neutralizing antibodies as determined by pseudovirus-based neutralization assays (PBNAs). L2-peptide enzyme-linked immunosorbent assay (ELISA) assessments determined the total anti-L2 antibody level for each antigen variant, showing for the majority of sera a correlation with their repective neutralizing antibody level. Surface Plasmon Resonance revealed that L2 epitope-specific, neutralizing monoclonal antibodies (mAbs) display distinct avidities to different antigen spacer variants. Furthermore, mAb affinity toward individual spacer variants was well correlated with their neutralizing antibody induction capacity, indicating that the mAb affinity assay predicts L2-based antigen immunogenicity. These observations provide insights on the development and optimization of L2-based HPV vaccines.

The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air-liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.

Evaluation of human papillomavirus DNA in colorectal cancer and adjacent mucosal tissue samples

BackgroundAlthough the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive.MethodsTo further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS).ResultsUsing Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205).ConclusionsOur findings indicate that HPV types belonging to the mucosal alpha, and the ‘cutaneous’ beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings.

Retraction Note: E6 proteins from diverse cutaneous HPV types inhibit apoptosis in response to UV damage.

Retraction Note: E6 proteins from diverse cutaneous HPV types inhibit apoptosis in response to UV damage.

Interaction of HPV16 and Cutaneous HPV in Head and Neck Cancer.

Human papillomavirus 16 (HPV16) is an established risk factor for Head and Neck Cancer (HNC). Recent reports have shown that genotypes from the beta (β) and gamma (γ) genera, also known as cutaneous HPV, can be found in the oral cavity, but their role is largely unidentified. We investigated the interaction between oral HPV16 and cutaneous HPV in HNC. We use data on incident HNC cases (n = 384) and frequency-matched hospital-based controls (n = 423) from the HeNCe Life study in Montreal, Canada. Participants were tested for alpha HPV and cutaneous genera using oral mouth rinse and brush samples. We used unconditional logistic regression to obtain adjusted odds ratios (aOR) and 95% confidence interval (CI) as a measure of the effect between HPV and HNC and assessed the interaction between HPV genotypes on the multiplicative and additive scales. Prevalence of HPV infection was higher among cases (73%) than controls (63.4%), with cases more likely to be coinfected with more than a single genotype, 52.9% vs. 43.5%, respectively. Infection with HPV16 alone had a strong effect on HNC risk aOR = 18.2 [6.2, 53.2], while infection with any cutaneous HPV, but not HPV16, appeared to have the opposite effect aOR = 0.8 [0.6, 1.1]. The observed effect of joint exposure to HPV16 and any cutaneous HPV (aOR = 20.4 [8.3, 50.1]) was stronger than the expected effect based on an assumption of independent exposures but was measured with considerable imprecision. While the point estimate suggests a positive interaction between HPV16 and cutaneous HPV, results were imprecise with relative excess risk due to interaction (RERI) = 2.4 [-23.3, 28.2]. There could be biologic interaction between HPV16 and genotypes from cutaneous genera, which warrants further investigation. Although cutaneous HPVs are not usually found in tumor tissues, they are cofactors that could interact with HPV16 in the oral cavity and thus strengthen the latter's carcinogenic effect.

Genotyping of 30 kinds of cutaneous human papillomaviruses by a multiplex microfluidic loop-mediated isothermal amplification and visual detection method

BackgroundHuman papillomaviruses (HPVs), a group of non-enveloped small viruses with double-stranded circular DNA which lead to multiple skin diseases such as benign warts, are commonly seen in clinics. The current HPV detection systems aim mainly at mucosal HPVs, however, an efficient clinical approach for cutaneous HPVs detection is lacking.ObjectivesTo establish a rapid detection system for cutaneous HPVs using a colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue (HNB) dye in combination with microfluidic technology.MethodsL1 DNA sequences of the 30 cutaneous HPVs were chemically synthesized, and LAMP primers against L1 DNA were designed with use of an online LAMP designing tool. Isothermal amplification was performed with use of a water bath and the amplification results were inspected with the naked eye. Using PCR sequencing as a control method, the specificity and sensitivity of the new detection system were obtained by detecting clinical samples.ResultsThe lower detection limit of the LAMP assay was 107 viral DNA copies/μl when tested on synthesized L1 DNA sequences, which was better than the conventional PCR. Compared to PCR sequencing, the sensitivity of HPV27, HPV2, HPV1, HPV57, HPV3, HPV4, HPV7 and HPV75 genotypes detections were 100%, whereas the specificity was 34.55, 45.12, 95.83, 98.59 and 97.62% respectively, when tested on clinical samples.ConclusionsThe new cutaneous type HPV detection system is characterized by both a good sensitivity and specificity compared to conventional methods.

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.

Single-cell RNA sequencing reveals cell type-specific HPV expression in hyperplastic skin lesions

Human Papillomavirus infection is highly prevalent worldwide. While most types of HPV cause benign warts, some high-risk types are known to cause cervical cancer, as well as cancer of the oral cavity and head and neck. Persistent cutaneous HPV infection can be particularly problematic in patients with chronic immunosuppression, for example following organ transplantation. Due to unknown mechanisms, these patients may develop numerous warts, as well as present with a dramatically increased skin cancer prevalence.Despite an association between HPV persistence in the epidermis and excessive wart or squamous cancer development, the molecular mechanisms linking immunosuppression, HPV expression and excessive epidermal proliferation have not been determined, largely due to low-sensitivity methodology to capture rare viral transcription events. Here, we use single-cell RNA sequencing to profile HPV-positive skin lesions from an immunosuppressed patient that were found to express the alphapapillomavirus HPV78 in basal keratinocytes, suprabasal keratinocytes and hair follicle stem cells. This method can be applied to detect and investigate HPV transcripts in cutaneous lesions, allowing mechanistic links between immunosuppression-induced HPV life cycle and epidermal hyperproliferation to be uncovered.

Mucosal and cutaneous Human Papillomavirus seroprevalence among adults in the prevaccine era in Germany — Results from a nationwide population-based survey

BackgroundHuman Papillomavirus (HPV) vaccination of girls was introduced in Germany in 2007. However, data on the distribution of vaccine-relevant HPV types in the general population in Germany in the prevaccine era are limited. MethodsSerum samples collected during the German National Health Interview and Examination Survey 1998 (GNHIES98), a nationally representative study including men and women aged 18–79 years, were tested for antibodies to 19 mucosal and cutaneous HPV types. Multivariable regression models were developed to identify associations between demographic and behavioral characteristics and HPV seropositivity. ResultsOf the 6517 serum samples tested, almost a quarter was seropositive for at least one of the nine HPV vaccine types with no clear age-pattern. HPV-6 and HPV-59 were the most common mucosal types, while HPV-1 and HPV-4 were the most common cutaneous HPV types. Factors independently associated with HPV-16 seroprevalence were seropositive to other sexually transmitted infections and lifetime number of sex partners, as well as urbanity (only among females). ConclusionsPrevalence of naturally acquired antibodies to HPV types which can be prevented by vaccination is high in both sexes and all age groups. These data can serve as baseline estimates to evaluate the population-level impact of the current vaccination strategy.

A deceitful case of an inguinal adenophlegmon as a presenting complaint for underlying metastatic cutaneous squamous cellcarcinoma

Squamous cell carcinoma is the second most common primitive skin cancer known to be responsible for the majority of deaths from skin cancer excluding melanoma. Through this presentation, we illustrate an unusual case of squamous cell carcinoma revealed by a superinfected lymph node metastasis mimicking an adenophlegmon. A 58-year-old patient presented to the emergency room with an inguinal bulge evolving for 3 months, gradually increasing in size, recently becoming painful. The clinical examination revealed a red warm and painful crural swelling. No intertoes space intertrigo or any other lower-extremity infections were found. The blood tests showed a biological inflammatory syndrome: WBC =11000/mm3, CRP=119mg/L. An abdominal CT scan concluded to an inguinal adenophlegmon. We decided then to proceed to surgery. In view of the suspicious tissular aspect found peroperatively, a biopsy was performed. The histological examination leaded to the diagnosis of squamous cell carcinoma. This case strongly suggests that underlying malignancy must be considered in front of every adenophlegmon whithing patients having cutaneous HPV induced lesions (red and yellow circle) even if the clinical examination is negative for of an invasive tumor and the usual surgical procedure of incision and drainage must be prohibited. The primary tumor, as in the case of our patient, may develop on bowenoid papulosis (yellow circle); which is characterized by multiple, small skin colored to reddish brown papules, primarily occurring on the genitalia of young adults. The right management in this case should be based on surgical lymph node dissection with or without adjuvant radiation therapy.

Beta and gamma human papillomaviruses in anal and genital sites among men: prevalence and determinants

Data regarding the anogenital distribution of and type-specific concordance for cutaneous β- and γ-HPV types in men who have sex with women is limited and geographically narrow. Knowledge of determinants of anogenital detection of cutaneous HPV types in different regions is needed for better understanding of the natural history and transmission dynamics of HPV, and its potential role in the development of anogenital diseases. Genital and anal canal samples obtained from 554 Russian men were screened for 43 β-HPVs and 29 γ-HPVs, using a multiplex PCR combined with Luminex technology. Both β- and γ-HPVs were more prevalent in the anal (22.8% and 14.1%) samples than in the genital (16.8% and 12.3%) samples. Low overall and type-specific concordance for β-HPVs (3.5% and 1.1%) and γ-HPVs (1.3% and 0.6%) were observed between genital and anal samples. HIV-positive men had higher anal β- (crude OR = 12.2, 95% CI: 5.3–28.1) and γ-HPV (crude OR = 7.2, 95% CI: 3.3–15.4) prevalence than HIV-negative men. Due to the lack of genital samples from the HIV-positive men, no comparison was possible for HIV status in genital samples. The lack of type-specific positive concordance between genital and anal sites for cutaneous β- and γ-HPV types in heterosexual men posits the needs for further studies on transmission routes to discriminate between contamination and true HPV infection. HIV-positive status may favor the anal acquisition or modify the natural history of cutaneous HPV types.

Identification of C/EBPα as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes.

Patients suffering from Epidermodysplasia verruciformis (EV), a rare inherited skin disease, display a particular susceptibility to persistent infection with cutaneous genus beta-human papillomavirus (beta-HPV), such as HPV type 8. They have a high risk to develop non-melanoma skin cancer at sun-exposed sites. In various models evidence is emerging that cutaneous HPV E6 proteins disturb epidermal homeostasis and support carcinogenesis, however, the underlying mechanisms are not fully understood as yet. In this study we demonstrate that microRNA-203 (miR-203), a key regulator of epidermal proliferation and differentiation, is strongly down-regulated in HPV8-positive EV-lesions. We provide evidence that CCAAT/enhancer-binding protein α (C/EBPα), a differentiation-regulating transcription factor and suppressor of UV-induced skin carcinogenesis, directly binds the miR-203 gene within its hairpin region and thereby induces miR-203 transcription. Our data further demonstrate that the HPV8 E6 protein significantly suppresses this novel C/EBPα/mir-203-pathway. As a consequence, the miR-203 target ΔNp63α, a proliferation-inducing transcription factor, is up-regulated, while the differentiation factor involucrin is suppressed. HPV8 E6 specifically down-regulates C/EBPα but not C/EBPβ expression at the transcriptional level. As shown in knock-down experiments, C/EBPα is regulated by the acetyltransferase p300, a well-described target of cutaneous E6 proteins. Notably, p300 bound significantly less to the C/EBPα regulatory region in HPV8 E6 expressing keratinocytes than in control cells as demonstrated by chromatin immunoprecipitation. In situ analysis confirmed congruent suprabasal expression patterns of C/EBPα and miR-203 in non-lesional skin of EV-patients. In HPV8-positive EV-lesions both factors are potently down-regulated in vivo further supporting our in vitro data. In conclusion our study has unraveled a novel p300/C/EBPα/mir-203-dependent mechanism, by which the cutaneous HPV8 E6 protein may expand p63-positive cells in the epidermis of EV-patients and disturbs fundamental keratinocyte functions. This may drive HPV-mediated pathogenesis and may potentially also pave the way for skin carcinogenesis in EV-patients.

Prevalence of cutaneous beta and gamma human papillomaviruses in the anal canal of men who have sex with women

BackgroundData regarding anal cutaneous HPV detection among HIV-positive and HIV-negative persons largely relies on studies among men who have sex with men in limited geographical settings. Understanding the distribution, determinants, and potential human health effects of anal cutaneous HPV types among men who have sex with women (MSW) is important. MethodsAnal canal swab samples from 415 Russian MSW (384 HIV-negative and 31 HIV-positive) were tested for 43 β-HPVs and 29 γ-HPVs, using a multiplex PCR combined with Luminex technology. Resultsβ-HPV was detected in 24.4% and γ-HPV in 15.9% of anal samples of all Russian MSW. In total, 34 β-HPV and 19 γ-HPV types were detected, with the most commonly detected β-HPV types being 110, 22 and 124 and the most common γ-HPV types being 95, 132 and 50. For both genera, being HIV-positive at the time of testing was a significant determinant of detection (74.2% for β-HPVs and 48.4% for γ-HPVs compared to 20.1% and 12.5% in HIV-negative MSW, respectively). ConclusionsA wide spectrum and moderate prevalence of anal β-HPV and γ-HPV types was found in our MSW study sample, suggesting that routes other than penile-anal intercourse may be important in cutaneous HPV transmission.

Downregulation of Toll-Like Receptor 9 Expression by Beta Human Papillomavirus 38 and Implications for Cell Cycle Control.

Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKβ) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1), decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

A coding variant in TMC8 (EVER2) is associated with high risk HPV infection and head and neck cancer risk.

HPV infection is a causal agent in many epithelial cancers, yet our understanding of genetic susceptibility to HPV infection and resultant cancer risk is limited. Epidermodysplasia Verruciformis is a rare condition of extreme susceptibility to cutaneous HPV infection primarily attributable to mutations in TMC6 and TMC8. Genetic variation in the TMC6/TMC8 region has been linked to beta-type HPV infection and squamous cell carcinoma of the skin, cervical cancer, HPV persistence and progression to cervical cancer. Here, we have tested the hypothesis that the common TMC8 SNP rs7208422 is associated with high-risk HPV infection and risk of head and neck squamous cell carcinoma (HNSCC). Seropositivity to the HPV L1 protein (HPV16, 18, 11, 31, 33, 35, 45, 52, 58) was measured in 514 cases and 452 population-based controls. Genotype was significantly associated with seropositivity to HPV18 L1 (OR TT vs AA = 0.48, 95% CI = 0.22–0.99) and borderline significantly associated with HPV16 L1 (OR TT vs AA = 0.58, 95% CI = 0.22–1.17). There was a consistent inverse association between TMC8 genotype and infection with other HPV types, including statistically significant associations for HPV31 and HPV52. Consistent with these results, the variant T genotype was associated with a reduced risk of HNSCC (ORAT: 0.63, 95% CI 0.45–0.89, ORTT: 0.54, 95% CI 0.36–0.81), even among subjects seronegative for all HPV types (ORAT: 0.71, 95% CI 0.45–1.11, ORTT: 0.54, 95% CI 0.31–0.93). Our data indicate that common genetic variation in TMC8 is associated with high-risk HPV infection and HNSCC etiology.

Cutaneous HPV and skin cancer

Papillomaviruses (HPVs) are small non-enveloped icosahedral viruses that infect the keratinocytes of skin and mucosa. The cutaneous HPV types are represented mainly by the beta and gamma genera, which are widely present in the skin of normal individuals. More than 40 beta-HPV types and 50 gamma-HPV types have been isolated, and these numbers are continuously growing. The main cause of non-melanoma skin cancer is exposure to ultraviolet radiation (UVR). However, cutaneous HPVs that belong to the beta genus may act as a co-carcinogen with UVR. The association between beta-HPVs and skin cancer was first reported in patients with epidermodysplasia verruciformis (EV), who frequently develop cutaneous squamous cell carcinoma (SCC) on sun-exposed areas. Isolation of HPVs from the lesions suggested that HPVs might act as a co-carcinogen with UVR in EV patients. Beta-HPVs may also play a role in cutaneous SCC in immunocompromised non-EV and in immunocompetent individuals. Several studies have reported an association of viral DNA and/or antibodies to beta HPV types with SCC. Interestingly, HPV prevalence and viral load decrease during skin carcinogenesis, being significantly higher in actinic keratosis than in SCC, suggesting that the virus may play a role in the early stages of tumour development (the "hit-and-run" hypothesis). Concordantly, in vivo and in vitro studies have shown that E6 and E7 from certain cutaneous HPV types display transforming activities, further confirming their potential role in carcinogenesis.

Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the potential connection between some beta HPVs and cancer.

Prospective seroepidemiologic study on the role of Human Papillomavirus and other infections in cervical carcinogenesis: Evidence from the EPIC cohort

To evaluate prospectively the association between serological markers of selected infections, including HPV, and risk of developing cervical cancer (CC) and precancer, we performed a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) study that included 184 cases of invasive CC (ICC), 425 cases of cervical intraepithelial neoplasia (CIN) grade 3 or carcinoma in situ (CIS), and 1,218 matched control women. At enrollment participants completed lifestyle questionnaires and provided sera. Subjects were followed-up for a median of 9 years. Immunoassays were used to detect serum antibodies to Human Herpes Virus 2 (HHV-2), Chlamydia trachomatis (CT), Chlamydia pneumoniae, L1 proteins of mucosal and cutaneous HPV types, E6/E7 proteins of HPV16/18, as well as to four polyomaviruses. Adjusted odds ratios (OR) [and 95% confidence intervals (CI)] for CIN3/CIS and ICC risk were respectively: 1.6 (1.2-2.0) and 1.8 (1.1-2.7) for L1 seropositivity to any mucosal HPV type, 1.0 (0.4-2.4) and 7.4 (2.8-19.7) for E6 seropositivity to HPV16/18, 1.3 (0.9-1.9) and 2.3 (1.3-4.1) for CT seropositivity, and 1.4 (1.0-2.0) and 1.5 (0.9-2.6) for HHV-2 seropositivity. The highest OR for ICC was observed for HPV16 E6 seropositivity [OR = 10.2 (3.3-31.1)]. Increasing number of sexually transmitted infections (STIs) was associated with increasing risk. Non-STIs were not associated with CC risk. In conclusion, this large prospective study confirms the important role of HPV and a possible contribution of CT and HHV-2 in cervical carcinogenesis. It further identifies HPV16 E6 seropositivity as the strongest marker to predict ICC well before disease development.

Improved detection of human papillomavirus harbored in healthy skin with FAP6085/64 primers

FAP59/64, FAP6085/6319, and CUT primer sets were designed for detecting cutaneous HPV and have been used in many clinical and epidemiology studies. The FAP6085/64 primer set was first evaluated in this study and the FAP6085/64 combination was found to be much more sensitive than all three original primer sets by using HPV plasmids as a template. To confirm further the effectiveness of the FAP6085/64 primer set in human DNA templates, 90 palmar exfoliated cell DNA samples were used to detect the cutaneous HPV by both the FAP59/64 and FAP6085/64 primer sets. The overall proportion of HPV detection in those skin samples was 77.8% (70/90) using FAP6085/64, as compared to 55.6% (50/90) using FAP59/64. The FAP6085/64 primer set was also applied in a population based study. The proportion of HPV detection was 73.96% (2076/2807) in skin samples collected from healthy individuals, and a total of 336 different PV types were found. Sixty (17.9%) of them were fully characterized HPV types, 127 (37.8%) were putative HPV types which had been described previously, 149 (44.3%) were novel putative HPV types, and two animal PVs were also detected. These results suggest that the FAP6085/64 primer set was sensitive and effective for detection of cutaneous HPV in healthy skin samples.