The E7 protein of cutaneous human papillomavirus type 8 causes invasion of human keratinocytes into the dermis in organotypic cultures of skin.

Human papillomaviruses (HPV) have been implicated in the development of nonmelanoma skin cancer (NMSC). The molecular mechanisms by which these viruses contribute towards NMSC are poorly understood. We have used an in vitro skin-equivalent model generated by transducing primary adult human epidermal keratinocytes with retroviruses expressing HPV genes to investigate the mechanisms of viral transformation. In this model, keratinocytes expressing HPV genes are seeded onto a mesenchyme composed of deepidermalized human dermis that had been repopulated with primary dermal fibroblasts. Expression of the HPV8 E7 gene caused both an enhancement of terminal differentiation and hyperproliferation, but most strikingly, the acquisition of the ability to migrate and invade through the underlying dermis. The basement membrane integrity was disrupted in a time-dependent manner in areas of invading keratinocytes, as evidenced by immunostaining of its protein components collagen types VII, IV, and laminin 5. This was accompanied by the overexpression of extracellular matrix metalloproteinases MMP-1, MMP-8, and MT-1-MMP. These results suggest that the cutaneous HPV type 8 that is frequently found in NMSC of epidermodysplasia verruciformis patients may actively promote an invasive keratinocyte phenotype. These findings also highlight the importance of epithelial-extracellular matrix-mesenchymal interactions that are required to support cell invasion.

Increased prevalence of human papillomavirus in hairs plucked from patients with psoriasis treated with psoralen-UV-A.

BACKGROUND: Patients with psoriasis treated with psoralen-UV-A (PUVA) are at increased risk of skin cancer; however, the exact causes of this increased incidence are not well understood. It has been suggested that PUVA may increase expression of the tumorigenic agent human papillomavirus (HPV) in skin by directly stimulating virus replication, immune suppression, or both, thereby leading to skin cancer formation. OBJECTIVE: To determine whether HPV DNA prevalence in the skin is increased after long-term PUVA treatment. DESIGN: Screening for the presence of HPV sequences in DNA isolated from plucked body hairs of patients with psoriasis with a history of PUVA exposure and a history of skin cancer (group A), PUVA exposure and no history of skin cancer (group B), and no PUVA exposure and no history of skin cancer (group C). SETTING: University hospital. PATIENTS AND METHODS: Hair samples were obtained from 81 patients with psoriasis (56 men and 25 women; mean age, 52 years), including 16 in group A (mean number of PUVA exposures, 702), 35 in group B (mean number of PUVA exposures, 282), and 30 in group C. DNA was isolated from the hair samples and analyzed by polymerase chain reaction with the use of 2 nested primer systems specific for epidermodysplasia verruciformis-associated or related and genital or mucosal virus types, respectively. RESULTS: The rate of HPV DNA positivity was significantly higher in groups A (73% [11/15]) and B (69% [24/35]) than in group C (36% [10/28]) (A + B vs C, P =.009; chi(2) test; age adjusted). Conclusion The prevalence of HPV in the skin (hair follicles) is increased in patients with psoriasis who have a history of PUVA exposure.

Natural cell-mediated cytotoxicity of peripheral blood lymphocytes against target cells transfected with epidermodysplasia verruciformis-specific human papillomavirus type 8 L1 DNA sequences.

The aim of the present study was to characterise natural cell-mediated cytotoxicity against COS-7 cells transfected with potentially oncogenic HPV-8 L1 DNA sequences cloned in sense and antisense orientation and to evaluate their lysis by peripheral blood lymphocytes (PBL) from patients with epidermodysplasia verruciformis (EV), a rare disease associated with life-long infection by specific HPV types. COS-7 cells were transfected with HPV-8 Hinc II restriction fragment (nucleotide positions 5434-7654) cloned in sense (COS-L1S) and antisense (COS-L1A) orientation into pCB6 expression vector. Cytotoxic activity of isolated PBL against COS cell lines as well as K562 erythroleukaemic cells was evaluated by 51Cr-release assay. We found that lymphocytes responsible for natural lysis of COS and K562 cells are CD3-negative CD56-positive natural killer (NK) cells. Analysis of NK cell cytotoxic activity against different COS cell lines has revealed that lymphocytes from healthy subjects killed COS-L1S cells significantly more efficiently than wild COS-7 and COS-L1A cells. Significantly more efficient lysis of COS-L1S cells was also observed in EV patients. Thus, expression of HPV L1 renders target cells more susceptible to NK-mediated cytotoxicity that may enable more effective elimination of transformed cells.

High prevalence of epidermodysplasia verruciformis-associated human papillomavirus DNA in actinic keratoses of the immunocompetent population.

Skin cancers in both immunosuppressed and immunocompetent populations are associated with epidermodysplasia verruciformis human papillomavirus (EV-HPV) DNA. However, little is known about the prevalence of EV-HPVs in actinic keratoses in immunocompetent individuals. Actinic keratoses from 114 patients were classified as low-grade ( n=76) or high-grade ( n=38) according to the extent of histological atypia. HPV DNA was amplified from 54 frozen and 60 paraffin-embedded biopsy specimens by nested polymerase chain reaction (PCR) with several consensus and type-specific primers. PCR products were sequenced for typing. These results were compared with HPV detection in skin cancers ( n=20) and Bowen's disease ( n=18). A broad spectrum of EV-HPV types including oncogenic HPV5 and HPV8 and partially characterized sequences were detected in actinic keratoses and cutaneous cancers. In actinic keratoses a higher prevalence of EV-HPV DNA was found in frozen tissues than in formalin-fixed tissues (85% vs 67%). There was no difference between the low- and high-grade actinic keratoses either in terms of EV-HPV DNA prevalence or the results of serological study using HPV8 virus-like particles. The detection rate of EV-HPVs was lower in skin cancers and Bowen's disease. This would suggest involvement of EV-HPVs in the early stages of cutaneous oncogenesis.

Human papillomavirus-positive tonsillar carcinomas: a different tumor entity?

Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyer's tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site ( P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per beta-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading ( P=0.008) and alcohol consumption ( P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.

Evidence for the association of human papillomavirus infection and cutaneous squamous cell carcinoma in immunocompetent individuals.

OBJECTIVE: The aim of our study was to evaluate human papillomavirus (HPV) infection as a risk factor for cutaneous squamous cell carcinoma (SCC) in immunocompetent individuals. DESIGN: Hospital-based case-control study. SETTING: Referral center for dermatologic diseases for central and southern Italy. PARTICIPANTS: Consecutive patients with histologically confirmed cutaneous SCC (n = 46) and control subjects (n = 84) chosen by frequency matching (age and sex) among patients admitted with unrelated diseases. MAIN OUTCOME MEASURE: Infection with epidermodysplasia verruciformis-related HPV types, blindly assessed by serologic testing (viruslike particle enzyme-linked immunosorbent assay). Information was obtained on known potentially confounding risk factors (family history, history and signs of sun exposure, and pigmentary traits) and on history of HPV-related lesions and diseases, assessed by interview and examination by a dermatologist. RESULTS: Positive serologic findings for HPV type 8 were associated with SCC (odds ratio, 3.2; 95% confidence interval, 1.3-7.9) independently of other risk factors, whereas positive serologic findings for HPV type 15 were negatively associated with SCC (odds ratio, 0.4; 95% confidence interval, 0.2-0.9). Other variables significantly associated with the tumor were family history of skin cancer, professional or recreational sun exposure, light eye color, high number of solar keratoses and seborrheic keratoses on the body surface, and residency in radon-emitting buildings. CONCLUSIONS: Positive serologic findings for HPV type 8 are associated with SCC occurrence in immunocompetent individuals. Viral infection could act as a cofactor in the tumor development, along with genetic predisposition, solar radiation, and other environmental exposures. If confirmed, these findings could open new perspectives for treatment and prevention of SCC.

Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene E6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8.

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3' part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs "CCAAC" (nt 57-73) and M29 (nt 84-112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P(175). In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.

Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus.

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.