Epidermodysplasia Verruciformis Human Papillomavirus Research Articles

Compound heterozygous CORO1A mutations in siblings with a mucocutaneous-immunodeficiency syndrome of epidermodysplasia verruciformis-HPV, molluscum contagiosum and granulomatous tuberculoid leprosy.

Coronin-1A deficiency is a recently recognized autosomal recessive primary immunodeficiency caused by mutations in CORO1A (OMIM 605000) that results in T-cell lymphopenia and is classified as T(-)B(+)NK(+)severe combined immunodeficiency (SCID). Only two other CORO1A-kindred are known to date, thus the defining characteristics are not well delineated. We identified a unique CORO1A-kindred. We captured a 10-year analysis of the immune-clinical phenotypes in two affected siblings from disease debut of age 7 years. Target-specific genetic studies were pursued but unrevealing. Telomere lengths were also assessed. Whole exome sequencing (WES) uncovered the molecular diagnosis and Western blot validated findings. We found the compound heterozygous CORO1A variants: c.248_249delCT (p.P83RfsX10) and a novel mutation c.1077delC (p.Q360RfsX44) (NM_007074.3) in two affected non-consanguineous siblings that manifested as absent CD4CD45RA(+) (naïve) T and memory B cells, low NK cells and abnormally increased double-negative (DN) ϒδ T-cells. Distinguishing characteristics were late clinical debut with an unusual mucocutaneous syndrome of epidermodysplasia verruciformis-human papilloma virus (EV-HPV), molluscum contagiosum and oral-cutaneous herpetic ulcers; the older female sibling also had a disfiguring granulomatous tuberculoid leprosy. Both had bilateral bronchiectasis and the female died of EBV+ lymphomas at age 16 years. The younger surviving male, without malignancy, had reproducibly very short telomere lengths, not before appreciated in CORO1A mutations. We reveal the third CORO1A-mutated kindred, with the immune phenotype of abnormal naïve CD4 and DN T-cells and newfound characteristics of a late/hypomorphic-like SCID of an EV-HPV mucocutaneous syndrome with also B and NK defects and shortened telomeres. Our findings contribute to the elucidation of the CORO1A-SCID-CID spectrum.

Detection of Human Papillomavirus DNA in Cutaneous Squamous Cell Carcinoma among Immunocompetent Individuals

The presence of certain types of human papillomavirus (HPV) is a known risk factor for the development of anogenital squamous cell carcinomas (SCCs). A similar association has been hypothesized for cutaneous SCCs, although, to our knowledge, no studies to date have combined sensitive HPV DNA detection techniques with epidemiologic data controlling for known risk factors to explore the association. We designed a case-control study examining HPV prevalence using highly sensitive PCR-detection assays in tissue samples from 85 immunocompetent patients with histologically confirmed SCCs and 95 age-matched individuals without a prior history of skin cancer. A standardized interview was administered to all study subjects to collect information pertaining to potential confounding variables. The overall detection rate of HPV DNA was high in case lesions (54%) and perilesions (50%) and in both sun-exposed normal tissue (59%) and non-sun-exposed normal tissue (49%) from controls. In comparing case tissue to control tissue, there was no differential detection of HPV DNA across various HPV species. However, HPV DNA from beta-papillomavirus species 2 was more likely to be identified in tumors than in adjacent healthy tissue among cases (paired analysis, odds ratio=4.0, confidence interval=1.3-12.0). The high prevalence of HPV DNA detected among controls suggests that HPV DNA is widely distributed among the general population. However, the differential detection of HPV beta-papillomavirus species in tumors among cases suggests that certain HPV types may be involved in the progression of cutaneous SCCs.

Epidermodysplasia verruciformis and human papilloma virus

Despite its rarity, epidermodysplasia verruciformis was addressed in depth in recent literature. Patients are afflicted by persistent human papillomavirus infections and develop cutaneous malignancies more frequently and younger than in the general population. The disease is therefore considered a model for a viral role in cutaneous oncogenesis, although implication is controversial. We focus on recent findings in genetics, highlight multiple viewpoints regarding the role of epidermodysplasia verruciformis-human papillomavirus in nonmelanoma skin cancer and other diseases, and discuss treatment strategies. Susceptibility loci for epidermodysplasia verruciformis were mapped and encoded protein functions are becoming better understood, but a unified genetic theory for epidermodysplasia verruciformis is lacking. Epidermodysplasia verruciformis-human papillomavirus, originally thought present only in epidermodysplasia verruciformis, is now considered ubiquitous, its role still being elucidated. Numerous therapies for epidermodysplasia verruciformis lesions were proposed, although there is no consensual first-line treatment strategy. Discoveries of novel mutations and further study of epidermodysplasia verruciformis-human papillomavirus in lesional and nonlesional skin of epidermodysplasia verruciformis patients and the general population may generate a cohesive theory regarding a viral role in cutaneous oncogenesis. Future understanding of the disease may yield an optimal approach to treating epidermodysplasia verruciformis patients.

‘HPV vulvitis’ revisited: frequent and persistent detection of novel epidermodysplasia verruciformis‐associated HPV genotypes

'Human papillomavirus (HPV) vulvitis' is a disputed entity where most studies examining for genital-mucosal (GM) HPV have been negative. Using degenerate and type specific primers for cutaneous (CU), GM and epidermodysplasia verruciformis (EV) HPV types, the prevalence of specific HPV types was investigated in biopsy specimens from 19 women with 'HPV vulvitis', seven with asymptomatic vulvar squamous papillomatosis (ASxVSP), and controls of vulvar fibroepithelial polyps (FEP) (15), vulvar condyloma (10) and normal vulva (NV) (10). HPV DNA/EV HPV/GM HPV/CU HPV were detected in 84/74/47/5% of vulvitis patients, 78/71/0/28% of ASxVSP, 47/20/20/7% of FEP, 10/10%/0/0 of NV and 100/0/100/10% of condyloma. Fourteen putatively novel HPV genotypes were detected in vulvitis and ASxVSP patients, but not in controls. The two most frequent novel EV HPV, Alb-4 and DL285, were detected in 9/19 (47%) and 5/19 (26%) of vulvitis cases and were persistently identified in serial biopsies. HPV co-infection and Alb-4 infection occurred significantly more frequently in vulvitis patients, particularly those complaining of 'burning' (62/62% vs. 17/7%, p < or = 0.004). Koilocytosis was identified significantly more frequently in vulvitis compared with non-condyloma controls (81% vs. 40%, p = 0.0001), and its presence correlated with detection of HPV DNA (r = 0.3, p = 0.002). The high frequency of novel EV HPV in HPV vulvitis and correlation of clinicopathologic findings with HPV DNA suggests that HPV vulvitis may indeed exist.

Epidermodysplasia verruciformis: a model for understanding the oncogenicity of human papillomaviruses.

The first evidence for the oncogenic potential of human papillomaviruses (HPVs) was obtained through the study of epidermodysplasia verruciformis (EV). This rare skin disease is characterized by disseminated, refractor, pityriasis versicolor-like lesions as well as flat wart-like lesions, and by the development of skin carcinomas in about 30% of the patients. EV is a multifactorial disease involving genetic, immunological and extrinsic (actinic) factors, in addition to infection with specific HPV types. A number of HPVs (at least 15 types) have been characterized in benign EV lesions. HPV DNA sequences are regularly detected in EV carcinomas but, in contrast to benign lesions, the types associated with cancers are usually restricted to HPV-5 and, less frequently, HPV-8, an HPV-5-related type. HPV-5 genomes are usually found as free monomeric or oligomeric DNA molecules in EV carcinomas, and frequently contain deletions. This is in contrast with HPV DNA sequences in genital cancers, which are often integrated into the host DNA. Evidence for the transcription of HPV-5 genomes in primary and metastatic EV carcinomas has recently been obtained. The available data indicate that HPV-5 and some HPV-5-related types have an oncogenic potential and play a role in the malignant transformation of EV lesions. Infection by these HPVs must be considered a major risk factor for the development of cancers in EV patients. EV HPV DNA sequences have only rarely been detected in premalignant or malignant lesions of the skin in the general population. This further stresses the role of genetic, immunological and extrinsic factors in the abnormal susceptibility of EV patients to a set of specific HPV types.

Prevalence of human papillomavirus in skin tumors from repair deficient xeroderma pigmentosum patients

The predisposition to skin cancers in childhood is the hallmark of xeroderma pigmentosum (XP), a rare autosomal recessive disorder, deficient in DNA repair and hypersensitive to ultraviolet irradiation. Human papillomavirus (HPVs), are common infections of the skin which are often found associated to benign lesions and non-melanoma skin cancers (NMSC), mainly squamous cell carcinomas (SCC) and basal cell carcinomas (BCC). Our study is the first to analyse 40 SCCs, 27 BCCs and nine normal skin biopsies from XP patients for HPV DNA which are found more frequently in SCCs (20/40) than in BCCs (4/27) or normal skin (2/9). The HPV spectrum includes 22 different epidermodysplasia verruciformis (EV) HPV types, which predominate in SCCs (48%) compared to BCCs (15%) and normal skin (22%). Our data, showing an association between EV HPV and SCCs from young XP patients is comparable to that found for NMSC from adult immunosuppressed organ transplant patients and raises the question of the importance of HPV infection in skin carcinogenesis.

Skin human papillomavirus type 38 alters p53 functions by accumulation of ΔNp73

The E6 and E7 of the cutaneous human papillomavirus (HPV) type 38 immortalize primary human keratinocytes, an event normally associated with the inactivation of pathways controlled by the tumour suppressor p53. Here, we show for the first time that HPV38 alters p53 functions. Expression of HPV38 E6 and E7 in human keratinocytes or in the skin of transgenic mice induces stabilization of wild-type p53. This selectively activates the transcription of deltaNp73, an isoform of the p53-related protein p73, which in turn inhibits the capacity of p53 to induce the transcription of genes involved in growth suppression and apoptosis. DeltaNp73 downregulation by an antisense oligonucleotide leads to transcriptional re-activation of p53-regulated genes and apoptosis. Our findings illustrate a novel mechanism of the alteration of p53 function that is mediated by a cutaneous HPV type and support the role of HPV38 and deltaNp73 in human carcinogenesis.

Human Papillomavirus Gene Expression in Cutaneous Squamous Cell Carcinomas from Immunosuppressed and Immunocompetent Individuals

Epidermodysplasia verruciformis (EV)-type human papillomavirus (HPV) DNA have been detected by PCR in squamous cell carcinomas (SCC) from both organ transplant recipients (OTR) and immunocompetent individuals. Their role in skin cancer remains unclear, and previous studies have not addressed whether the viruses are transcriptionally active. We have used in situ hybridization to investigate the transcriptional activity and DNA localization of HPV. EV-HPV gene transcripts were demonstrated in four of 11 (36%) OTR SCC, one of two (50%) IC SCC, and one of five (20%) OTR warts positive by PCR. Viral DNA co-localized with E2/E4 early region gene transcripts in the middle or upper epidermal layers. Non-EV cutaneous HPV gene transcripts were demonstrated in one of five (20%) OTR SCC and four of 10 (40%) OTR warts. In mixed infections transcripts for both types were detected in two of six (33%) cases. Our results provide evidence of EV-HPV gene expression in SCC; although only a proportion of tumors were positive, the similarly low transcriptional activity in warts suggests this is an underestimate. These observations, together with emerging epidemiological and functional data, provide further reason to focus on the contribution of EV-HPV types to the pathogenesis of cutaneous SCC.

Human Papillomaviruses Associated with Epidermodysplasia Verruciformis in Non-Melanoma Skin Cancers: Guilty or Innocent?

actinic keratoses epidermodysplasia verruciformis human papillomavirus in situ hybridization non-melanoma skin cancers organ transplant recipients squamous cell carcinomas ultraviolet radiation Non-melanoma skin cancers (NMSC), namely basal cell carcinomas (BCC) and squamous cell carcinomas (SCC), represent the most common form of malignancy in fair-skinned populations. Solar ultraviolet radiation (UVR), immunosuppression, and genetic constitution of the host are well-known risk factors for the development of NMSC. Epidermodysplasia verruciformis (EV), a rare genetic disease associated with a high risk of skin cancer, provided the first clue to a possible role of viruses in human skin carcinogenesis (Jablonska et al., 1972Jablonska S. Dabrowski J. Jakubowicz K. Epidermodysplasia verruciformis as a model in studies on the role of papovaviruses in oncogenesis.Cancer Res. 1972; 32: 583-589PubMed Google Scholar). Due to mutations in either of the related EVER1 and EVER2 genes, EV patients are abnormally susceptible to a specific group of related human papillomavirus genotypes (known as EV HPV) and to the oncogenic potential of some of these viruses (Orth, 1987Orth G. Epidermodysplasia verruciformis.in: Salzman N.P. Howley P.M. The Papovaviridae: The Papillomaviruses. Plenum Press, New York1987: 199-243Crossref Google Scholar; Ramoz et al., 2002Ramoz N. Rueda L.A. Bouadjar B. Montoya L.S. Orth G. Favre M. Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis.Nat Genet. 2002; 32: 579-581Crossref PubMed Scopus (311) Google Scholar). EV HPV cause widespread, inapparent infections in the general population (Antonsson et al., 2003Antonsson A. Erfurt C. Hazard H. et al.Prevalence and type spectrum of human papillomaviruses in healthy skin samples collected in three continents.J Gen Virol. 2003; 84: 1881-1886Crossref PubMed Scopus (152) Google Scholar). In EV patients, however, infection leads to persistent wart-like and macular skin lesions, and premalignant lesions and SCC develop on sun-exposed areas of the skin in about half of the patients. EV SCC harbor high copy numbers of episomal HPV genomes (HPV5 or occasionally HPV8, 14, 17, 20, or 47) and abundant transcripts of the E6 and E7 genes. Phenocopies of EV are exceptionally observed in immunosuppressed patients. EV is thus a rare experiment of nature that provides a model for studying the interaction among potentially oncogenic HPV genotypes, UVR, immunity, and genetic factors (Orth, 1987Orth G. Epidermodysplasia verruciformis.in: Salzman N.P. Howley P.M. The Papovaviridae: The Papillomaviruses. Plenum Press, New York1987: 199-243Crossref Google Scholar). EV HPV DNA sequences have only rarely been detected in cutaneous premalignant tumors and NMSC in non-EV patients by Southern blot hybridization, which is sensitive enough to detect one HPV genome per cell. Using highly sensitive PCR approaches, however, HPV DNA sequences were detected in 25%–65% of NMSC in immunocompetent individuals and in up to 90% of SCC in organ transplant recipients (OTR), a population at a greatly increased risk of developing SCC. Similar HPV detection rates were found for actinic keratoses (AK), the precursor lesion of SCC (reviewed inForslund et al., 2004Forslund O. Lindelöf B. Hradil E. et al.High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in stripped biopsies from the same tumors.J Invest Dermatol. 2004; 123: 388-394Crossref PubMed Scopus (132) Google Scholar). These data led researchers to consider a role for HPV, especially EV HPV types, in the development of NMSC (Orth, 2004Orth G. Human papillomaviruses and the skin: more to be learned.J Invest Dermatol. 2004; 123: XI-XIIICrossref PubMed Scopus (14) Google Scholar). Available epidemiological and seroepidemiological data provide some support to an association between the presence of EV HPV DNA in normal skin or eyebrow hairs or the seroreactivity to some EV HPV and an increased risk of AK and SCC (reviewed inPurdie et al., 2005Purdie K.J. Surentheran T. Sterling J.C. et al.Human papillomavirus gene expression in cutaneous squamous cell carcinomas from immunosuppressed and immunocompetent individuals.J Invest Dermatol. 2005; 125: 98-107Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar; Weissenborn et al., 2005Weissenborn S.J. Nindl I. Purdie K. et al.Human papillomavirus-DNA loads in actinic keratoses exceed those in non-melanoma skin cancers.J Invest Dermatol. 2005; 125: 93-97Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The link between EV HPV and the development of NMSC in immunocompetent and immunosuppressed individuals, however, remains unclear (reviewed inOrth, 2004Orth G. Human papillomaviruses and the skin: more to be learned.J Invest Dermatol. 2004; 123: XI-XIIICrossref PubMed Scopus (14) Google Scholar). EV HPV and non-EV cutaneous HPV types represent impressively diverse, ubiquitous viruses, which are highly prevalent in the normal skin of healthy adults (Antonsson et al., 2003Antonsson A. Erfurt C. Hazard H. et al.Prevalence and type spectrum of human papillomaviruses in healthy skin samples collected in three continents.J Gen Virol. 2003; 84: 1881-1886Crossref PubMed Scopus (152) Google Scholar). A great diversity of cutaneous HPV types, most frequently EV HPV or putative novel EV HPV-related types, is detected in AK and NMSC, and mixed infections are frequently observed in tumors of OTR patients. Furthermore, it has recently been shown that cutaneous HPV DNA is common on top of AK, SCC, and BCC but less prevalent (3.2–7.9-fold) in “stripped” tumors, suggesting that HPV DNA positivity may reflect, in part, contamination of the surface of the tumors by viral DNA or particles shed from infected healthy skin (Forslund et al., 2004Forslund O. Lindelöf B. Hradil E. et al.High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in stripped biopsies from the same tumors.J Invest Dermatol. 2004; 123: 388-394Crossref PubMed Scopus (132) Google Scholar). It should be also stressed that the tumor HPV DNA loads are usually low and that it remains unknown whether the viral sequences are transcriptionally active. The articles byWeissenborn et al., 2005Weissenborn S.J. Nindl I. Purdie K. et al.Human papillomavirus-DNA loads in actinic keratoses exceed those in non-melanoma skin cancers.J Invest Dermatol. 2005; 125: 93-97Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar and byPurdie et al., 2005Purdie K.J. Surentheran T. Sterling J.C. et al.Human papillomavirus gene expression in cutaneous squamous cell carcinomas from immunosuppressed and immunocompetent individuals.J Invest Dermatol. 2005; 125: 98-107Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, both published in this issue of the Journal, address these questions about the role of EV HPV in skin carcinogenesis. Weissenborn and colleagues determined HPV DNA loads by quantitative, type-specific, real-time PCR (Q-PCR) in a series of specimens of AK, NMSC, perilesional tissues, and SCC metastases. The majority of the samples were obtained from immunocompetent patients, and all of them had previously been found by PCR to harbor EV HPV types 5, 8, 15, 20, 24, or 36. For 40% of the 78 samples yielding conclusive results, including almost all metastases and half of the NMSC specimens, viral loads were beyond the detection limit (two HPV DNA copies in the assay). For the other specimens, HPV DNA viral loads ranged from 1 copy per 14,200 cell equivalents to 1 copy per 0.02 cell equivalents. An interesting finding was that the viral loads detected in AK were significantly higher than in SCC, whereas similar viral loads were observed for SCC, BCC, and perilesional tissues. Viral loads of 1 HPV DNA copy per less than 50 cells were found in 40% of 25 AK, but in only 8% of 13 BCC and 15% of 13 SCC. Only a few scattered, positive nuclei were detected by a very sensitive in situ hybridization (ISH) method in sections of NMSC and perilesional skin, in agreement with their similar low HPV DNA loads detected by Q-PCR. The localization of HPV DNA in AK was not investigated. The aims of the study of Purdie and colleagues were both to search for the presence of EV HPV DNA sequences in tumor cells and to determine whether these sequences were transcriptionally active in SCC (mostly OTR tumors) and in OTR warts. Specimens were previously found by PCR to harbor EV HPV types 5, 14, 15, 20, or 23, and further analyzed by DNA–DNA or RNA–RNA ISH. EV HPV DNA sequences were detected in 27.3% of 11 SCC found positive by PCR, whereas the 2 warts tested were found negative. Viral transcripts were detected in 38.5% of 13 SCC and 20% of five warts, mainly in the mid to upper epidermal layers. The four SCC illustrated showed positive areas, or clusters of positive cells, contrasting with the labeling pattern reported by Weissenborn et al. Nests of enlarged keratinocytes with pale stained cytoplasms, reminiscent of the cytopathic effect specific of EV HPV, were observed in the carcinoma in situ region of one OTR SCC. High amounts of HPV20 DNA and both early and late transcripts were detected in these cells. It seems likely that this case represents an EV phenocopy. Transcripts of the E2/E4 region (highly expressed during the late phase of the viral life cycle) were detected in the other specimens (including three SCC and one wart found negative for viral DNA by ISH), whereas no transcripts of the viral E6 and E7 oncogenes or L1 and L2 capsid genes were detected. Unfortunately, HPV DNA loads were not determined and viral transcripts were not characterized. This would have helped to appreciate the sensitivity and specificity of the ISH method. Their data led Weissenborn and colleagues to several conclusions. Although a passenger state cannot be excluded, the higher viral loads found in AK, compared with SCC, are compatible with a carcinogenic role of EV HPV in the early steps of tumor progression. As for NMSC, the presence of HPV does not seem to be required for the maintenance of the malignant state, and the similar low viral loads found in perilesional skin may suggest that HPV is more a passenger than a driver. Whether the presence of HPV in some tumor cells may have an impact on growth and invasion properties of the tumor, as proposed by the authors, would be worth considering. The study of Purdie and colleagues brings some evidence for EV HPV gene expression in a proportion of SCC, which may suggest an active rather than a latent or passenger status. The detection and characterization of viral E6 and E7 transcripts in tumor cells, however, remain to be achieved. Overall, the data reported in the two articles should stimulate further work on the contribution of EV HPV to the development of NMSC. When considering the possible implications of their findings, the authors refer to functional studies involving ectopic overexpression of E6 and E7 proteins of EV and non-EV cutaneous HPV genotypes (reviewed inStorey, 2002Storey A. Papillomaviruses: Death-defying acts in skin cancer.Trends Mol Med. 2002; 8: 417-421Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For instance, the E6 proteins of several cutaneous HPV have been shown to promote the degradation of the proapoptotic Bak protein induced in keratinocytes by UVR, and this antiapoptotic effect could favor the accumulation of UVR-induced mutations. A crucial question is whether the E6 and E7 proteins of EV HPV or non-EV cutaneous HPV (less frequently detected in NMSC) behave as oncoproteins when expressed in vivo, under the control of their own promoter. It should be kept in mind that infection in EV patients leads to the development of disseminated, widespread, persistent benign lesions of the skin. About 20 specific HPV types may be characterized in these lesions, and patients are usually infected by more than one genotype (up to 11). The abundant replication of these viruses leads to a specific cytopathic effect, and ISH experiments usually detect a single EV HPV genotype in each lesion. Upon their third or fourth decade, about half of the patients start developing AK and Bowen's carcinoma in situ, which evolve into invasive NMSC (mostly SCC). In contrast with the great diversity of EV HPV observed in benign lesions, EV carcinomas are predominantly associated with HPV5 or, in single cases, with HPV8, 14,17, 20, or 47, and high levels of transcripts of the E6/E7 region are detected. This indicates that only a subset of EV HPV is endowed with an oncogenic potential. EV cancers usually arise on sun-exposed areas of the skin and a syncarcinogenic role of HPV5 and UVR is supported by the frequent mutations of the p53 gene in premalignant and malignant EV tumors (Padlewska et al., 2001Padlewska K. Ramoz N. Cassonnet P. et al.Mutation and abnormal expression of the p53 gene in the viral skin carcinogenesis of epidermodysplasia verruciformis.J Invest Dermatol. 2001; 117: 935-942Crossref PubMed Google Scholar). EV HPV cause widespread asymptomatic infections in the general population (Antonsson et al., 2003Antonsson A. Erfurt C. Hazard H. et al.Prevalence and type spectrum of human papillomaviruses in healthy skin samples collected in three continents.J Gen Virol. 2003; 84: 1881-1886Crossref PubMed Scopus (152) Google Scholar). Recent studies have disclosed that EV HPV infections are under the control of two genes of unknown function, EVER1 and EVER2, which are mutated in EV patients (Ramoz et al., 2002Ramoz N. Rueda L.A. Bouadjar B. Montoya L.S. Orth G. Favre M. Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis.Nat Genet. 2002; 32: 579-581Crossref PubMed Scopus (311) Google Scholar). Furthermore, an EV-like syndrome has been found to occur frequently after hematopoietic stem-cell transplantation in patients with a severe combined immune deficiency caused by common γc cytokine receptor subunit or JAK-3 deficiency. This may suggest that γc/JAK-3 signaling in keratinocytes could have a role in EV HPV immunity (Laffort et al., 2004Laffort C. Le Deist F. Favre M. et al.Severe cutaneous papillomavirus disease after haemopoietic stem-cell transplantation in patients with severe combined immune deficiency caused by common γc cytokine receptor subunit or JAK-3 deficiency.Lancet. 2004; 363: 2051-2054Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Focusing not only on HPV but also on the genes controlling these viruses should provide clues to our understanding of the role of EV HPV in the skin carcinogenesis process in non-EV patients.

The Role of p53 Codon 72 and Human Papilloma Virus Status of Cutaneous Squamous Cell Carcinoma in the Swedish Population

The arginine variant of the p53 codon 72 polymorphism as well as anogenital and epidermodysplasia verruciformis (EV) types of human papilloma virus (HPV) are suggested to confer increased risk for developing cutaneous squamous cell carcinoma (SCC). In this pilot study, we analysed the p53 codon 72 genotype distribution in 106 microdissected samples from normal and tumour tissues of 53 cases of cutaneous SCC and 96 controls from Sweden. Both normal and tumour samples from cases of SCC were screened for anogenital and EV HPV. The p53Arg allele was not associated with the development of cutaneous SCC. Anogenital HPV (44%) was more prevalent than EV HPV (12%). Data also indicate that anogenital HPV is more common in tumour samples, but HPV infection was not identified as a significant risk factor for developing SCC. The presence of anogenital HPV, but not EV HPV might be a risk factor for development of cutaneous SCC.

TP53 mutations in squamous-cell carcinomas of the conjunctiva: evidence for UV-induced mutagenesis.

Squamous cell carcinoma of the conjunctiva is associated with sun exposure and often occurs in HIV-positive individuals. We have analysed TP53 mutations in 21 cases of squamous cell carcinoma and 22 controls with benign conjunctival lesions from a region (Uganda, Africa) with a high prevalence of heavy sun exposure and HIV infection. TP53 mutations were detected in 11 cases (52%) and 3 controls (14%). Seven of the mutations (6 in cases and 1 in controls) were CC-->TT transitions, a molecular signature of mutagenesis by solar UV rays. A similar prevalence (56%) of TP53 mutations was found in 18 squamous cell carcinoma cases positive for epidermodysplasia verruciformis human papillomavirus types. The prevalence of CC-->TT transitions reported here is the highest observed in any cancer type and matches that of skin cancers in subjects with xeroderma pigmentosum, an inherited disease with hypersensitivity to UV damage. These results confirm at the molecular level the causal role of solar UV rays in the aetiology of squamous cell carcinoma of the conjunctiva and suggest that infection with epidermodysplasia verruciformis types of human papillomavirus may act as a cofactor to increase the sensitivity of conjunctiva cells to UV-induced mutagenesis.

High Prevalence of Cutaneous Human Papillomavirus DNA on the Top of Skin Tumors but not in “Stripped” Biopsies from the Same Tumors

Genomes of human papillomaviruses (HPV) are common in biopsies from non-melanoma skin cancers but are also found on healthy skin and it is possible that HPV positivity in tumor biopsies by PCR may merely reflect contamination of the lesion surface. To investigate this issue, 229 immunocompetent patients were tested for HPV DNA in swab samples collected on top of skin tumors and in biopsies of the same tumors, obtained after stripping with tape to remove superficial layers. HPV DNA was detected on top of 69% (159 of 229) of the lesions, and in 12% (28 of 229) of the stripped biopsies (p<0.001). The difference was seen for all four types of tumors studied. Seborrheic keratosis had 79% (34 of 43) HPV positivity on top of lesions versus 19% (eight of 43) in biopsies; actinic keratosis had 83% (38 of 46) HPV positivity on top versus 11% (five of 46) in biopsies; basal cell carcinoma had 63% (69 of 109) on top versus 8% (nine of 109) in biopsies and squamous cell carcinoma had 58% (18 of 31) on top versus 19% (six of 31) in biopsies. HPV DNA is common in superficial layers of lesions, but is not necessarily present throughout tumors.

Increased risk of skin cancer associated with the presence of epidermodysplasia verruciformis human papillomavirus types in normal skin.

Human papillomaviruses (HPVs) are found in normal skin and in benign and malignant skin conditions. Epidermodysplasia verruciformis (EV) HPV types are those most plausibly linked to the development of squamous cell carcinomas of the skin. To assess the risk of nonmelanoma skin cancer (NMSC) associated with the presence of EV HPV in normal skin in immunocompetent (IC) individuals and renal transplant recipients (RTRs). Using a degenerate and nested polymerase chain reaction technique, HPV DNA was sought in 124 normal skin samples from sun-exposed and nonsun-exposed sites, from 39 IC individuals and 38 RTRs, both with and without NMSC. Data were analysed using the Mantel-Haenszel test and by logistic regression analysis. HPV DNA was detected in 58/67 (87%) and 20/57 (35%) samples from renal transplant and IC patients, respectively. There was no difference in either the prevalence or spectrum of HPV types found in sun-exposed and nonsun-exposed normal skin. However, there was significant association between NMSC and the presence of EV HPV DNA. Multivariate analysis provided an odds ratio of 6.41 (95% confidence interval 1.79-22.9) for the association of EV HPV DNA in normal skin (irrespective of site) and NMSC status, even after stratifying for patient group and adjusting for the clustering effect of multiple sampling. Conversely, there was no association between skin cancer status and the presence of cutaneous or mucosal HPV types in either sun-exposed or nonsun-exposed skin. HPV DNA is widespread in normal adult skin, particularly in transplant patients. In our study, the presence of EV but not cutaneous HPV DNA in normal skin was significantly associated with NMSC status and may prove to be of predictive value for skin cancer risk. These data provide reason to focus on EV HPV types as causal agents in skin cancer.

Epidermodysplasia verruciformis human papillomavirus types and carcinoma of the conjunctiva: a pilot study.

A total of 21 squamous-cell carcinoma of the conjunctiva (SCC) and 22 control subjects had conjunctival samples tested for human papillomavirus (HPV) types using PCR-based assays. Epidermodysplasia verruciformis HPV types were found in 86% of SCC cases and 36% of control subjects (Odds ratio=12.0), suggesting a role of HPVs in the aetiology of SCC.

Epidermodysplasia verruciformis human papillomavirus types are frequently detected in skin lesions associated with HIV infection

Human papillomavirus (HPV) is one of the more common viral causes of mucocutaneous lesions in HIV positive individuals. The incidence of cutaneous warts in adult and paediatric HIV infection is estimated to be up to 27%. These warts are often multiple, large, rapidly growing, refractory to treatment, and may result in considerable morbidity. Epidermodysplasia verruciformis (EV)-like presentations have been reported and epidermal dysplasia progressing to in situ carcinoma has also been reported within the warts of HIV positive individuals. There is evidence that immune status may influence the HPV types harboured by skin lesions. However, there are relatively few studies in which the spectrum of HPV types present in HIV-associated cutaneous lesions have been analysed. In this study we present the results of HPV typing from a series of lesion from HIV infected individuals.

Possible involvement of epidermodysplasia verruciformis human papillomaviruses in the immunopathogenesis of psoriasis: a proposed hypothesis.

We have shown previously in psoriasis a very high prevalence of epidermodysplasia verruciforms-associated human papillomavirus 5 (EVHPV5) DNA and antibodies to human papillomavirus 5 (HPV5) virus-like particle (VLP) L1, and we suggested that this benign hyperproliferative disorder could be a reservoir for EVHPVs. Here we provide new data confirming the expression of EVHPVs in psoriasis and present our hypothesis on their possible involvement in the immunopathogenesis of the disorder. The new important finding was detection by radioimmunoprecipitation assay of a very high prevalence of antibodies to E6/E7 HPV5 oncoproteins, known to enhance keratinocyte proliferation. More recently, EV genes were identified, EVER1 and EVER2, whose mutations are responsible for epidermodysplasia verruciformis. Eidermodysplasia verruciforms-associated human papillomaviruses are harmless to the general population as a result of genetic restriction, which in psoriasis appears to be partly alleviated, and this may allow the viral gene expression. We hypothesize that induction of keratinocyte proliferation in psoriasis by various stimuli initiates the EVHPV life cycle with expression of early (E6/E7) and late (L1) viral proteins. The early proteins may, in turn, enhance the keratinocyte proliferation, and the late proteins could serve as target for specific-B- and T-cell-mediated responses. Immune responses against the viral antigens in the epidermis may result in the chemoattraction of leukocytes and Munro abscess formation, as well as in production of the psoriatic process. The novel immunomodulatory therapies could also inhibit immune responses against EVHPV proteins, leading to decreased cytokine production, keratinocyte proliferation and EVHPV expression. Thus the beneficial effect of these therapies is not discordant with the propose hypothesis of possible involvement of EVHPVs in the immunopathogenesis of psoriasis.

A broad spectrum of human papillomavirus types is present in the skin of Australian patients with non-melanoma skin cancers and solar keratosis.

Human papillomavirus (HPV) may play a role in the pathogenesis of non-melanoma skin cancer (NMSC) in epidermodysplasia verruciformis (EV) patients, but in the general population no specific HPV types have been associated with these lesions. Objectives To examine the spectrum of HPV types present in the skin and tumours of Australian patients with NMSC or solar keratosis (SK). Biopsies from tumours, and cotton swab samples of perilesional skin and buttock skin from each of 59 Australian patients with basal cell carcinoma (BCC), squamous cell carcinoma (SCC) or SK were tested for HPV DNA by polymerase chain reaction (PCR) using HPV consensus (FAP) primers and by type-specific primers for HPV 38 and candidate HPV 92. The identification of HPV type from consensus PCR was performed by sequencing and comparison with GenBank. In total, 49 of 59 (83%) patients harboured HPV DNA, which was detected in 28 of 64 (44%) biopsies, 48 of 64 (75%; P < 0.001) perilesional swabs and 36 of 59 (61%; P = 0.04) buttock swabs. Forty-five different HPV types/putative types were detected: 15 were previously characterized HPV types, 17 were earlier described putative types and 13 were new putative types. In addition, six subtypes and four variants of HPV sequences were identified. HPV types within the B1 group (EV HPV types) were found in 26 of 64 (40%) lesions, 44 of 64 (69%) perilesional swabs and 35 of 59 (59%) buttock swabs. HPV 38 was detected in 23 of 59 (39%) patients, and was found in seven of 16 (43%) SKs, but was less common in SCCs [three of 23 (13%); P = 0.037] and BCCs [four of 25 (16%); P = 0.056]. Candidate HPV 92 was found in seven of 59 (12%) patients. A broad spectrum of HPV types, the majority from the B1 group, was found in skin of Australian patients with skin tumours. HPV 38 was found significantly more often in SK than in SCC. However, the role of cutaneous HPV infection in the pathogenesis of NMSC remains elusive.

Do epidermodysplasia verruciformis human papillomaviruses contribute to malignant and benign epidermal proliferations?

The aim of this review is to present new data on epidermodysplasia verruciformis (EV) and EV human papillomaviruses (HPVs), regarded previously as specific to the disease. Recently introduced highly sensitive molecular methods for virologic studies allow detection of EV HPVs in non-EV populations. In this article, we present the most recent findings on EV and EV HPVs, which shed new light on a possible contribution of EV viruses to malignant and benign epidermal proliferation. We discuss the significance of EV HPV DNA detection in premalignant cutaneous lesions and nonmelanoma skin cancers; however, direct evidence for the causative role of EV HPV is still not available. In psoriasis, a high frequency of EV HPV-5 and other EV HPVs in the skin and the presence of specific HPV-5 antibodies strongly suggest expression of EV HPV proteins in this extensive epidermal proliferation. Epidermodysplasia verruciformis HPV-5 may also be transiently expressed in epidermal repair processes, whereas in psoriasis there is a continuous epidermal proliferation that could result in persistent viral expression. A potential contribution of EV HPVs to the pathogenesis of psoriasis is also supported by the recently disclosed co-localization of susceptibility loci for psoriasis and EV in the same region of chromosome arm 17qter; however, specific genes for both conditions are still not identified.

Human papillomaviruses and non-melanoma skin cancer

Epidemiological and experimental studies have overwhelmingly confirmed human papillomaviruses as important causal agents in anogenital carcinogenesis. A role for human papillomaviruses has also been proposed in a diverse range of other malignancies, and particular interest has focused on non-melanoma skin cancer, the commonest malignancy in fair-skinned populations worldwide. Although the evidence for this is considerably less convincing than for anogenital cancer, important epidemiological and functional data have emerged over the past year that have furthered our understanding of the possible contribution of human papillomaviruses to skin cancer. Epidemiological human papillomavirus DNA detection studies have shown associations with non-melanoma skin cancer, but have also emphasized the ubiquity of epidermodysplasia verruciformis human papillomavirus types in normal skin, hair follicles and benign hyperproliferative disorders, as have seroepidemiological approaches. Functional investigations have demonstrated mechanistically relevant interactions between the virus and ultraviolet radiation, host cytokines and cellular proteins including p53 and the pro-apoptotic protein Bak. Taken together, these data have advanced our understanding of the contribution of human papillomaviruses to malignant transformation in cutaneous keratinocytes, but further research is required before a causal association between human papillomaviruses and skin cancer is reliably confirmed.

Association Between Epidermodysplasia Verruciformis-Associated Human Papillomavirus DNA in Plucked Eyebrow Hair and Solar Keratoses

Epidermodysplasia-verruciformis-associated human papillomavirus DNA has been demonstrated in squamous cell carcinomas and plucked hair from immunocompetent patients and renal transplant recipients. This study investigated the association between infection with epidermodysplasia-verruciformis-associated human papillomavirus, identified by the detection of viral DNA in plucked eyebrow hairs, and solar keratoses. These lesions are strongly predictive of squamous cell carcinoma. In a cross-sectional study 518 individuals were enrolled from a randomly selected sample of a subtropical Australian community. Epidermodysplasia-verruciformis-associated human papillomavirus DNA in eyebrow hair was detected using a nested polymerase chain reaction specific for epidermodysplasia-verruciformis-associated human papillomavirus types. Epidermo dysplasia-verruciformis-associated human papillomavirus DNA was present in 121 (49%) of 245 men and 116 (44%) of 262 women. There was a strongly significant increase in epidermodysplasia-verruciformis-associated human papillomavirus infection with age (p < 0.00001), with prevalences of 29% in the 25–39 y age group, 42% at 40–59 y and 65% in the 60–79 y age group. Among men there was a strong association between epidermodysplasia-verruciformis-associated human papillomavirus and solar keratoses with an odds ratio, adjusted for age, skin color, and occupational sun exposure, of 3.40 (95% confidence interval, 1.77–6.53). No such association was found among women [odds ratio 1.03 (95% confidence interval 0.59–1.77, after adjustment for the same factors)]. Differences in occupational sun exposure and smoking histories could not explain these apparently different associations between epidermodysplasia-verruciformis-associated human papillomavirus infection and solar keratoses in men and women. In conclusion, epidermodysplasia-verruciformis-associated human papillomavirus infection is associated with solar keratoses in men suggesting that epidermodysplasia-verruciformis-associated human papillomavirus infection, in conjunction with sex specific factors (like androgens), may be involved in neoplastic changes of keratinocytes. Epidermodysplasia-verruciformis-associated human papillomavirus DNA has been demonstrated in squamous cell carcinomas and plucked hair from immunocompetent patients and renal transplant recipients. This study investigated the association between infection with epidermodysplasia-verruciformis-associated human papillomavirus, identified by the detection of viral DNA in plucked eyebrow hairs, and solar keratoses. These lesions are strongly predictive of squamous cell carcinoma. In a cross-sectional study 518 individuals were enrolled from a randomly selected sample of a subtropical Australian community. Epidermodysplasia-verruciformis-associated human papillomavirus DNA in eyebrow hair was detected using a nested polymerase chain reaction specific for epidermodysplasia-verruciformis-associated human papillomavirus types. Epidermo dysplasia-verruciformis-associated human papillomavirus DNA was present in 121 (49%) of 245 men and 116 (44%) of 262 women. There was a strongly significant increase in epidermodysplasia-verruciformis-associated human papillomavirus infection with age (p < 0.00001), with prevalences of 29% in the 25–39 y age group, 42% at 40–59 y and 65% in the 60–79 y age group. Among men there was a strong association between epidermodysplasia-verruciformis-associated human papillomavirus and solar keratoses with an odds ratio, adjusted for age, skin color, and occupational sun exposure, of 3.40 (95% confidence interval, 1.77–6.53). No such association was found among women [odds ratio 1.03 (95% confidence interval 0.59–1.77, after adjustment for the same factors)]. Differences in occupational sun exposure and smoking histories could not explain these apparently different associations between epidermodysplasia-verruciformis-associated human papillomavirus infection and solar keratoses in men and women. In conclusion, epidermodysplasia-verruciformis-associated human papillomavirus infection is associated with solar keratoses in men suggesting that epidermodysplasia-verruciformis-associated human papillomavirus infection, in conjunction with sex specific factors (like androgens), may be involved in neoplastic changes of keratinocytes. epidermodysplasia verruciformis epidermodysplasia-verruciformis-associated human papillomavirus Cutaneous squamous cell carcinomas and basal cell carcinomas, together called nonmelanoma skin cancers, are the most common malignant tumors among white populations (Harvey et al., 1989Harvey I. Shalom D. Marks R.M. Frankel S.J. Non-melanoma skin cancer.Br Med J. 1989; 299: 1118-1120Crossref PubMed Google Scholar;Goldberg, 1996Goldberg L.H. Basal cell carcinoma.Lancet. 1996; 347: 663-667Abstract PubMed Scopus (97) Google Scholar;Marks, 1996Marks R. Squamous cell carcinoma.Lancet. 1996; 347: 735-738PubMed Scopus (106) Google Scholar). Solar keratoses are benign skin tumors predominantly found on the face, backs of hands, and forearms, sites that are chronically exposed to the sun (Frost and Green, 1994Frost C.A. Green A.C. Epidemiology of solar keratoses.Br J Dermatol. 1994; 131: 455-464Crossref PubMed Scopus (233) Google Scholar). On rare occasions solar keratoses are precursors of squamous cell carcinoma and they share the same risk factors (Frost and Green, 1994Frost C.A. Green A.C. Epidemiology of solar keratoses.Br J Dermatol. 1994; 131: 455-464Crossref PubMed Scopus (233) Google Scholar;Frost et al., 1998Frost C.A. Green A.C. Williams G.M. The prevalence and determinants of solar keratoses at a subtropical latitude (Queensland, Australia).Br J Dermatol. 1998; 139: 1033-1039Crossref PubMed Scopus (85) Google Scholar). Exposure to sunlight is generally considered to be the most important environmental risk factor for both solar keratoses and cutaneous squamous cell carcinoma (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1730) Google Scholar;Marks, 1996Marks R. Squamous cell carcinoma.Lancet. 1996; 347: 735-738PubMed Scopus (106) Google Scholar). The high prevalence of human papillomavirus (HPV) DNA detected in solar keratoses and squamous cell carcinomas of immunosuppressed patients suggests a potential role for HPV infection in the etiology of these lesions (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar;Shamanin et al., 1996Shamanin V. zur Hausen H. Lavergne D. et al.Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and non-immunosuppressed patients.J Natl Cancer Inst. 1996; 88: 802-811Crossref PubMed Scopus (292) Google Scholar;Pfister and ter Schegget, 1997Pfister H. ter Schegget J. Role of HPV in cutaneous premalignant and malignant tumors.Clin Dermatol. 1997; 15: 335-347Abstract Full Text PDF PubMed Scopus (115) Google Scholar). HPV are small double-stranded DNA viruses found in a wide variety of proliferative lesions of epithelial origin. The earliest evidence for the involvement of specific HPV types in human skin cancer originated from studies in patients suffering from the rare hereditary disease epidermodysplasia verruciformis (EV) (Orth et al., 1980Orth G. Favre M. Breitburd F. Epidermodysplasia verruciformis: a model for the role of papillomaviruses in human cancer.in: Essex M. Todaro G. zur Hausen H. Viruses in Naturally Occurring Cancers. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 259-282Google Scholar). These HPV types and phylogenetically related types are commonly referred to as EV–HPV types and include HPV 5, 8, 9, 12, 14, 15, 17, 19–25, 36–38, and 47. As well as being detected in solar keratoses and squamous cell carcinoma, EV–HPV DNA has also been found in plucked hair from both immunosuppressed and immunocompetent persons (Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Crossref PubMed Scopus (278) Google Scholar), in skin scrapings and biopsies from psoriatic patients (Favre et al., 1998Favre M. Orth G. Majewski S. Baloul S. Pura A. Jablonska S. Psoriasis: a possible reservoir for human papillomavirus type 5, the virus associated with skin carcinomas of epidermodysplasia verruciformis.J Invest Dermatol. 1998; 110: 311-317Crossref PubMed Scopus (146) Google Scholar;Weissenborn et al., 1999Weissenborn S.J. Hopfl R. Weber F. Smola H. Pfister H.J. Fuchs P.G. High prevalence of a variety of epidermodysplasia verruciformis-associated human papilloma-viruses in psoriatic skin of patients treated or not treated with PUVA.J Invest Dermatol. 1999; 113: 122-126Crossref PubMed Scopus (59) Google Scholar); and from patients with bullous disease (Favre et al., 2000Favre M. Majewski S. Noszczyk B. Maienfisch F. Pura A. Orth G. Jablonska S. Antibodies to human papillomavirus type 5 are generated in epidermal repair processes.J Invest Dermatol. 2000; 114: 403-407Crossref PubMed Scopus (74) Google Scholar). These findings indicate that EV–HPV are seen not only in certain groups of skin cancer patients, but are widely distributed among the general population. To date, the role of EV–HPV in the pathogenesis of solar keratoses and squamous cell carcinoma and also in psoriasis is unclear. Recently, we successfully described a noninvasive technique, plucking hairs, to obtain biologic samples from all individuals (cases and controls) in a nested case–control study in order to determine their EV–HPV status. Recently, we detected the same EV–HPV types in multiple lesions from the same individual collected over time (Berkhout et al., 2000Berkhout R.J.M. Bouwes Bavinck J.N. ter Schegget J. Persistance of human papillomavirus DNA in benign and (pre) malignant skin lesions from renal transplant recipients.J Clin Microbiol. 2000; 38: 2087-2096PubMed Google Scholar) and in hair samples from different body sites collected at the same time point (Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Crossref PubMed Scopus (278) Google Scholar). This suggests that viral DNA isolated from hairs might be a marker of HPV infections elsewhere on the body. As squamous cell carcinoma and solar keratoses commonly occur on the face, eyebrow hairs were chosen to identify those individuals infected with EV–HPV. Participants were drawn from a much larger community study of skin cancer in Nambour, Queensland (Boxman et al., 2000Boxman I.L.A. Russell A. Mulder L.H.C. Bouwes Bavinck J.N. ter Schegget J. Green A. Case-control study in a subtropical Australian population to assess the relation between non-melanoma skin cancer and epidermodysplasia verruciformis human papillomavirus DNA in plucked eyebrow hairs.Int J Cancer. 2000; 86: 118-121Crossref PubMed Scopus (83) Google Scholar). A nonsignificant negative association between the presence of EV–HPV DNA in eyebrow hair and basal cell carcinoma was found and a nonsignificant positive association with squamous cell carcinoma, the estimated relative risk of developing squamous cell carcinoma among HPV-infected individuals being 2.00 (95% confidence interval 0.50–8.00). Given the shortcomings of a previous study due to limited numbers of cases with squamous cell carcinomas (n = 25), we decided to investigate the association between the presence of EV–HPV DNA in eyebrow hair and the more prevalent solar keratoses, in a larger cross-sectional study. The purpose of this study was to establish the prevalence of EV–HPV DNA in eyebrow hair, to examine factors that may be associated with EV–HPV infection, and to address the question of whether EV–HPV infection is associated with the presence of solar keratoses. In 1986, 2095 residents of the township of Nambour in Queensland, Australia, who were randomly selected from the electoral roll (enrollment is compulsory) attended a skin cancer survey (Green et al., 1988Green A. Beardmore G. Hart V. Leslie D. Marks R. Staines D. Skin cancer in a Queensland population.J Am Acad Dermatol. 1988; 19: 1045-1052Abstract Full Text PDF PubMed Scopus (179) Google Scholar). In 1992, 1626 of these participants agreed to be part of a skin cancer prevention trial involving randomization to sunscreen and/or β-carotene (Green et al., 1994Green A. Battistutta D. Hart V. et al.The Nambour Skin Cancer and Actinic Eye Disease Prevention Trial: design and baseline characteristics of participants.Control Clin Trials. 1994; 15: 512-522Abstract Full Text PDF PubMed Scopus (87) Google Scholar,Green et al., 1999Green A. Williams G. Neale R. et al.Daily sunscreen application and betacarotene supplementation in prevention of basal-cell and squamous-cell carcinomas of the skin: a randomised controlled trial.Lancet. 1999; 354: 723-729Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). Half of the 1250 participants attending the 1996 skin examinations were invited to participate in the present cross-sectional study and 518 persons took part with their informed consent. All received whole-body skin examinations by experienced dermatologists in 1992, 1994, and 1996. At the 1996 skin examination, dermatologists counted and mapped the positions of all solar keratoses present on 13 body sites. Those with one or more solar keratoses at any site were defined as having prevalent solar keratoses. As well, about eight to 10 hairs were plucked from eyebrows by one of the investigators or a trained assistant using a disposable pair of tweezers and gloves for each individual. Only hairs that contained hair follicles were collected and samples were stored at -70°C until analysis. Standard questionnaires were used to obtain data on known or suspected risk factors for solar keratoses, including skin color, hair color, eye color, tanning ability, occupational sun exposure at baseline, lifetime and recent sunburn histories and smoking. Hair samples were lyzed in L6 buffer for 1 h. One portion of the buffer was supplemented with diatoms (Boom et al., 1990Boom R. Sol C.J. Salimans M.M. Jansen C.L. van Wertheim-Dillen P.M. van der Noordaa J. Rapid and simple method for purification of nucleic acids.J Clin Microbiol. 1990; 28: 495-503Crossref PubMed Google Scholar) and incubated for a further hour. DNA was isolated according to a method described inBoom et al., 1990Boom R. Sol C.J. Salimans M.M. Jansen C.L. van Wertheim-Dillen P.M. van der Noordaa J. Rapid and simple method for purification of nucleic acids.J Clin Microbiol. 1990; 28: 495-503Crossref PubMed Google Scholar. The remaining portion of L6 buffer was stored at - 70°C. A nested polymerase chain reaction (PCR) method was used to detect EV–HPV DNA. The degenerate primers of the first PCR (CP62/CP70a) (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar;Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Crossref PubMed Scopus (278) Google Scholar) and nested PCR (CP65/CP69a) (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar;Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Crossref PubMed Scopus (278) Google Scholar) were located in the L1 open reading frame and designed to detect the subgroup of known EV–HPV types. Eight to 10% of the extractable DNA was used as input to the first step PCR. All PCR reactions were performed as described byBoxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Crossref PubMed Scopus (278) Google Scholar. A negative control (water) was included between each set of two DNA samples and processed in the same way as the hair samples throughout DNA preparation and both PCR steps. No negative controls were positive for EV–HPV. To determine the efficacy of the nested PCR, 10-fold dilutions of DNA of a plasmid containing HPV15 were used as positive controls. The detection limit for this plasmid was 0.1 fg, representing about 10 molecules of HPV DNA. HPV typing of 89 positive PCR samples confirmed that only EV–HPV DNA was amplified by the PCR used (Boxman et al., 2000Boxman I.L.A. Russell A. Mulder L.H.C. Bouwes Bavinck J.N. ter Schegget J. Green A. Case-control study in a subtropical Australian population to assess the relation between non-melanoma skin cancer and epidermodysplasia verruciformis human papillomavirus DNA in plucked eyebrow hairs.Int J Cancer. 2000; 86: 118-121Crossref PubMed Scopus (83) Google Scholar). In addition, because none of the negative controls were positive in the PCR, no further sequence analyses were performed in our study. Calculations were performed with the statistical software package of the SAS Institute Inc. (Cary, NC). The data were analyzed using Student's t test and the chi-square test. Odds ratios were estimated from logistic regression. Multivariate logistic regression analysis was used to adjust for confounders. Factors that were considered were age, sex, tanning ability, skin color, occupational sun exposure, smoking, and trial design factors [daily sunscreen application (vs discretionary sunscreen application) and of β-carotene (vs placebo)] (Green et al., 1999Green A. Williams G. Neale R. et al.Daily sunscreen application and betacarotene supplementation in prevention of basal-cell and squamous-cell carcinomas of the skin: a randomised controlled trial.Lancet. 1999; 354: 723-729Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). In addition, Mantel–Haenszel weighted odds ratios were calculated to adjust for age groups by using Epi-Info (version 6.04b, Centers for Disease Control & Prevention, U.S.A. and World Health Organization, Geneva, Switzerland). Among the 518 individuals who agreed to participate there were five whose samples were lost during transport or storage, and of the remaining 513 individuals, the presence of solar keratoses was scored in 507 individuals. Analyses were restricted to these 507, of whom 245 (48%) were male. The mean age of men taking part was 53.0 y, and of women, 53.1 y. Characteristics of persons with and without solar keratoses are presented in Table I. Persons with solar keratoses were significantly older, were more often male, had fairer skin, less often had an outdoor occupation, and had more frequently developed skin cancer. There was a strong association between the presence of solar keratoses and the presence of EV–HPV in plucked eyebrow hair (p < 0.00001).Table ICharacteristics of persons with and without solar keratosesPersons without solar keratosesaMean (± SD) age in years of (i) persons without solar keratoses: 47.4 (± 11.4), and (ii) persons with solar keratoses: 57.8 (± 11.8). Difference: 10.4 y (8.37; 12.43). n (%)Persons with solar keratosesaMean (± SD) age in years of (i) persons without solar keratoses: 47.4 (± 11.4), and (ii) persons with solar keratoses: 57.8 (± 11.8). Difference: 10.4 y (8.37; 12.43). n (%)Odds ratio (95% confidence interval)NumberbNumbers do not always add to the total due to missing information.231276Age categorized 25–3967 (29.0)22 (8.0)1 40–59128 (55.4)135 (48.9)3.21 (1.82; 5.71) 60–7936 (15.6)119 (43.1)10.07 (5.25; 19.45)Sex Female140 (60.6)122 (44.2)1 Male91 (39.4)154 (55.8)1.94 (1.34; 2.81)Tanning ability Tan only29 (12.6)24 (8.7)1 Burn then tan169 (73.2)197 (71.6)1.41 (0.76; 2.61) Always burn33 (14.3)54 (19.6) 1.98 (0.93; 4.28)Skin color Olive/brown31 (13.4)14 (5.1)1 Medium102 (44.1)102 (37.1)2.21 (1.06; 4.67) Fair98 (42.4)159 (57.8)3.59 (1.74; 7.50)Occupational sun exposure Mainly indoors119 (51.5)94 (34.2)1 In and outdoors76 (32.9)109 (39.6)1.82 (1.20; 2.76) Mainly outdoors36 (15.6)72 (26.2)2.53 (1.52; 4.23)Smoking Nonsmokers136 (59.4)151 (55.3)1 Ex-smokers66 (28.8)94 (34.4)1.28 (0.85; 1.93) Current smokers27 (11.8)28 (10.3)0.93 (0.50; 1.73)Skin cancercSCC, squamous cell carcinoma; BCC, basal cell carcinoma. No skin cancer187 (80.9)137 (49.6)dOdds ratios were not calculated because of blank values in some of the groups. SCC only015 (5.4) SCC + BCC09 (3.3) BCC only15 (6.5)54 (19.6) Not characterized29 (12.6)61 (22.1)EV–HPV in eyebrow hair Negative148 (64.1)122 (44.2)1 Positive83 (35.9)154 (55.8)2.25 (1.55; 3.28)a Mean (± SD) age in years of (i) persons without solar keratoses: 47.4 (± 11.4), and (ii) persons with solar keratoses: 57.8 (± 11.8). Difference: 10.4 y (8.37; 12.43).b Numbers do not always add to the total due to missing information.c SCC, squamous cell carcinoma; BCC, basal cell carcinoma.d Odds ratios were not calculated because of blank values in some of the groups. Open table in a new tab Characteristics of persons with and without EV–HPV infection are presented in Table II. The overall prevalence of EV–HPV infection was 49.4% (121 of 245) for men and 44.3% (116 of 262) for women. There was a strongly significant increase in EV–HPV infection with age (p < 0.00001), with prevalences of 29% in the 25–39 y age group, 42% at 40–59 y, and 65% in the 60–79 y age group. Occupational sun exposure was significantly associated and sex, tanning ability, skin color, and smoking were not statistically significantly associated with EV–HPV infection (see Table II).Table IICharacteristics of persons with and without HPV infectionPersons without HPV infectionaMean age (± SD) in years of (i) persons without HPV infection: 49.2 (± 11.4) and (ii) persons wth HPV infection: 57.5 (± 12.7). Difference: 8.3 (6.20; 10.40) years. n (%)Persons with HPV infectionaMean age (± SD) in years of (i) persons without HPV infection: 49.2 (± 11.4) and (ii) persons wth HPV infection: 57.5 (± 12.7). Difference: 8.3 (6.20; 10.40) years. n (%)Odds ratio (95% confidence interval)NumberbNumbers do not always add to the total due to missing information.270237Age categorized 25–3963 (23.3)26 (11.0)1 40–59153 (56.7)110 (46.4)1.74 (1.01; 3.03) 60–7954 (20.0)101 (42.6)4.53 (2.49; 8.30)Sex Female146 (54.1)116 (48.9)1 Male124 (45.9)121 (51.1)1.23 (0.85; 1.77)Tanning ability Tan only26 (9.7)27 (11.4)1 Burn then tan197 (73.2)169 (71.3)0.83 (0.45; 1.53) Always burn46 (17.1)41 (17.3)0.86 (0.41; 1.80)Skin color Olive/brown23 (8.6)22 (9.3)1 Medium104 (38.7)100 (42.2)1.01 (0.50; 2.01) Fair142 (52.8)115 (48.5)0.85 (0.43; 1.67)Occupational sun exposure Mainly indoors132 (49.1)81 (34.2)1 In and outdoors88 (32.7)97 (40.9)1.80 (1.18; 2.73) Mainly outdoors49 (18.2)59 (24.9)1.96 (1.20; 3.22)Smoking Nonsmokers157 (58.6)130 (66.5)1 Ex-smokers80 (29.8)80 (34.2)1.21 (0.80; 1.81) Current smokers31 (11.8)24 (10.3)0.93 (0.50; 1.74)Skin cancercSCC, squamous cell carcinoma; BCC, basal cell carcinoma. No skin cancer183 (67.8)141 (59.5)1 SCC only3 (1.1)12 (5.1)5.19 (1.33; 23.64) SCC + BCC5 (1.9)4 (1.7)1.04 (0.23; 4.55) BCC only28 (10.4)41 (17.3)1.90 (1.09; 3.33) Not characterized51 (18.9)39 (16.5)0.99 (0.60; 1.63)Solar keratoses Absent148 (54.8)83 (35.0)1 Present122 (45.2)154 (65.0)2.25 (1.55; 3.28)a Mean age (± SD) in years of (i) persons without HPV infection: 49.2 (± 11.4) and (ii) persons wth HPV infection: 57.5 (± 12.7). Difference: 8.3 (6.20; 10.40) years.b Numbers do not always add to the total due to missing information.c SCC, squamous cell carcinoma; BCC, basal cell carcinoma. Open table in a new tab From the outset our analyses showed that there was a strong association between EV–HPV infection and solar keratoses among men but no such association among women, and thus the association between HPV infection and solar keratoses have been presented separately for men and women. To calculate the association between HPV infection and solar keratoses, we adjusted for age, skin color, and occupational sun exposure. No adjustments were made for past or contemporary skin cancer history, because solar keratoses are potential precursors of squamous cell carcinoma. Among men there was a strong association between presence of EV–HPV in plucked eyebrow hair and presence of solar keratoses with a crude odds ratio of 4.03 (95% confidence interval 2.22–7.33). The pooled odds ratio adjusted for age groups by use of the Mantel–Haenszel test was 2.65 (95% confidence interval 1.40–5.11) Table III. After adjustment for age, skin color, and occupational sun exposure by use of a multivariate model, the odds ratio was 3.40 (95% confidence interval, 1.77–6.53). No such association was found among women, who had a crude odds ratio related to the presence of solar keratoses of 1.36 (95% confidence interval 0.81–2.29) Table III, which was reduced to 1.03 (95% confidence interval 0.59–1.77) after adjustment for age, skin color, and occupational sun exposure by use of a multivariate analysis. Although individuals participating in this study were randomly selected from a much larger skin cancer prevention trial involving randomization to sunscreen and/or β-carotene (Green et al., 1994Green A. Battistutta D. Hart V. et al.The Nambour Skin Cancer and Actinic Eye Disease Prevention Trial: design and baseline characteristics of participants.Control Clin Trials. 1994; 15: 512-522Abstract Full Text PDF PubMed Scopus (87) Google Scholar,Green et al., 1999Green A. Williams G. Neale R. et al.Daily sunscreen application and betacarotene supplementation in prevention of basal-cell and squamous-cell carcinomas of the skin: a randomised controlled trial.Lancet. 1999; 354: 723-729Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar), adjustment for these interventions had no effect on the association between solar keratoses and HPV infection (data not shown).Table IIIRisk of solar keratoses dependent on the presence of HPV DNA in plucked eyebrow hair stratified according to age groupsPersons without solar keratoses n (%)Persons with solar keratoses n (%)Odds ratioPooled odds ratioaAdjustment for age groups, using Mantel–Haenszel weighted odds ratio.Men All agesHPV neg.65 (71.4)59 (38.3)11HPV pos.26 (28.6)95 (61.7)4.03 (2.22; 7.33)2.65 (1.40; 5.11) 25–39 yHPV neg.29 (85.3)8 (53.3)1HPV pos.5 (14.7)7 (46.7)5.07 (1.05; 25.97) 40–59 yHPV neg.32 (68.1)35 (47.3)1HPV pos.15 (31.9)39 (52.7)2.38 (1.04; 5.50) 60–79 yHPV neg.4 (40.0)16 (25.0)1HPV pos.6 (60.0)49 (75.0)2.04 (0.42; 9.74)Women All agesHPV neg.83 (59.3)63 (51.6)11HPV pos.57 (40.7)59 (48.4)1.36 (0.81; 2.29)1.11 (0.63; 1.94) 25–39 yHPV neg.22 (67.7)4 (57.1)1HPV pos.11 (33.4)3 (42.9)1.50 (0.21; 10.22) 40–59 yHPV neg.49 (60.5)37 (60.7)1HPV pos.32 (39.5)24 (39.3)0.99 (0.48; 2.07) 60–79 yHPV neg.12 (46.2)22 (40.7)1HPV pos.14 (53.8)32 (59.3)1.25 (0.44; 3.55)a Adjustment for age groups, using Mantel–Haenszel weighted odds ratio. Open table in a new tab In the Nambour population (Queensland, Australia), the presence of EV–HPV DNA in plucked eyebrow hair was clearly associated with the presence of solar keratoses. Age was strongly associated with both the presence of EV–HPV infection in eyebrow hair and solar keratoses. This study data confirm these associations. We speculate that individuals are gradually persistently infected with a large range of cutaneous HPV types during their lifetime. Nevertheless, increasing age only partly explains the association between HPV infection and solar keratoses (see Table III). Surprisingly, the association between HPV infection and solar keratoses was observed among men but not women. An intriguing question is why. As the overall EV–HPV prevalence in men and women is similar, other factors than HPV infection per se may be important for this difference between the genders. The higher level of occupational sun exposure and smoking among men did not explain this apparent difference. Thus, if this sex-specific association is confirmed in this study or by others, new sex-specific factors (such as androgens) will need to be identified that can explain the difference between men and women regarding the association between EV–HPV infection and solar keratoses. The pathogenesis of solar keratoses and the subsequent squamous cell carcinomas is a complex interplay between immunologic, genetic, environmental, and possibly hormonal factors. The observed association between solar keratoses, EV–HPV infection and age might indicate that elderly patients not only more frequently develop solar keratoses but also have a less effective immunologic response against EV–HPV infections. On the other hand in addition to sun exposure, EV–HPV infection may play a direct part in the pathogenesis of solar keratoses. Sun exposure might directly activate functions of the viral genes (Purdie et al., 1999Purdie K.J. Pennington J. Proby C.M. Khalaf S. de Villiers E.M. Leigh I.M. Storey A. The promoter of a novel human papillomavirus (HPV77) associated with skin cancer displays UV responsiveness, which is mediated through a consensus p53 binding sequence.EMBO J. 1999; 18: 5359-5369Crossref PubMed Scopus (84) Google Scholar) or sun-exposure may enhance HPV replication in the host by inducing (local) immunosuppression and/or inactivation of keratinocyte growth regulating host genes (e.g., p53) (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1730) Google Scholar). Recently, it was found that the E6 early protein from a range of cutaneous HPV types (including the EV–HPV type 5) effectively inhibits apoptosis by abrogation of Bak in response to ultraviolet damage, which might be important for malignant progression of HPV-containing lesions (Jackson and Storey, 2000Jackson S. Storey A. Eb proteins from diverse cutaneous HPV types inhibit apoptosis in response to UV damage.Oncogene. 2000; 19: 592-598Crossref PubMed Scopus (175) Google Scholar;Jackson et al., 2000Jackson S. Harwood C. Thomas M. Banks L. Storey A. Role of Bak in UV-induced apoptosis in skin cancer and abrogation by HPV E6 proteins.Genes Dev. 2000; 14: 3065-3073Crossref PubMed Scopus (276) Google Scholar). As EV–HPV is widely distributed, as shown in the present and other studies (Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Crossref PubMed Scopus (278) Google Scholar,Boxman et al., 1999Boxman I.L.A. Mulder L.H.C. Russell A. Bouwes Bavinck J.N. Green A. ter Schegget J. Human papillomavirus type 5 is commonly present in immunosuppressed and immunocompetent individuals.Br J Dermatol. 1999; 141: 246-249Crossref PubMed Scopus (51) Google Scholar), we hypothesize that individuals who are infected by EV–HPV are at an increased risk of developing solar keratoses and squamous cell carcinomas, possibly by preventing ultraviolet-induced apoptosis. Molecular biologic studies are required to clarify the mechanisms by which the interplay between EV–HPV genes, ultraviolet exposure, and keratinocyte cell growth and differentiation takes place. This study was supported by the Dutch Cancer Foundation (UvA 95-994) and the National Health and Medical Research Council of Australia (grant no. 922608). We also thank the study participants and their doctors for their participation and support. Nambour Skin Cancer Prevention Study comprised the following collaborators: G. Williams, R. Neale, V. Hart, D. Leslie, P. Parsons, G.C. Marks, P. Gaffney, D. Battistutta, and C. Lang