1. Human papillomavirus (HPV) associated skin diseases Baki Akgül Institute of Virology University of Cologne Germany Skin Tumour Laboratory Cancer Research UK, London, UK

2. HPVs are small DNA tumour viruses. HPVs can be subdevided in cutaneous and mucosal types based on their anatomical site of infection. mucosal types (alpha types: HPV16, 18) are associated with cervical cancer The association between HPV and skin cancer was first identified in patients with the rare disease Epidermodysplasia verruciformis (EV) EV patients have a predisposition to infection with EV types or beta-HPV types) and develop non-melanoma skin cancer (NMSC), mainly SCCs, at sun-exposed body sites. These skin cancers frequently harbour the oncogenic types HPV5 or HPV8. HPV and skin cancer De Villiers et al., Virology, 2004

3.  Recent epidemiological studies have shown that HPVs are also found in SCCs of the general population.  27-85% of SCC positive compared to normal skin where 15-35% of samples were positive for HPV  Remarkable is the even higher prevalence of beta-HPV (85%) in premalignant actinic keratoses (Pfister et al., 2003). Beta-HPV prevalence in skin tumours Akgül et al., J. Pathol., 2006

4. Beta-HPV loads in skin tumours  However, viral DNA load in skin tumours is low; a single HPV copy can be detected in only 10 - 10000 dysplastic cells, confirmed by in situ hybridization and quantitative PCR).  The HPV-DNA loads in actinic keratoses,however, exceeds those in NMSC, which suggests a particular involvement of HPV in the early stages of cutaneous oncogenesis. SCC AK 1 HPV copy per >500 cells 23 32 1 HPV copy per 50-500 cells 8 8 1 HPV copy per 5-50 cells 15 28 1 HPV copy per <5 cells 0 12 SCC, HPV15 (Quant. PCR: 1 HPV copy per 708 cells) In situ hybridization quantitative PCR Weissenborn et al., JID, 2005

5. Biochemical studies  Since cell transforming activities of EV-HPV (beta-HPV) proteins differ from the alpha-HPV types (HPV16):  The aim of our biochemical study was to analyze effects of the oncogenic HPV8 early genes E2, E6, E7 on human keratinocytes in organotypic skin cultures (raft- cultures) because of the fact that the papillomavirus life cycle is linked to the differentiation of the skin. Akgül et al., J. Pathol, 2006

6. Use of collagen type 1 gels, repopulated with 3T3 mouse fibroblasts Boxman et al., 2001 Organotypic skin cultures primary dermal fibroblasts on reticular dermis primary cutaneous keratinocytes (expressing HPV8 genes) on papillary dermis Akgül et al. De-epidermised human dermis

7. HPV8-E7 causes invasion of keratinocytes into the dermis control HPV8 E7HPV8 E6/E7 HPV8 E7 Akgül et al., Cancer Res., 2005

8. Control HPV8 E7 21 days Basement membrane is disrupted in HPV8-E7 cultures Localization of type VII collagen also lack of: Collagen type IV Laminin V Control HPV8 E7 Akgül et al., Cancer Res., 2005

9. MMP-1MMP-8MT1-MMP Control HPV8 E7 Control HPV8 E7 Control HPV8 E7 MMP expression in HPV8-E7 cultures Akgül et al., Cancer Res., 2005

10. Transgenic mice model HPV8 trangenic mouse with benign skin tumour transgene  Generation of a transgenic mouse with the complete early region of HPV8 under control of the keratin-14 promoter.  99,4% of HPV8-transgenic mice developed single or multifocal benign tumors, characterized by varying degrees of epidermal dysplasia  SCC developed in 6% of animals  Skin tumours developed without any further treatment with physical or chemical carcinogens. Schaper et al., Cancer Res., 2005

11. Immunhistochemical detection of MMP in mouse SCC negative control MT1-MMP staining negative control MMP-13 staining Akgül et al., Exp. Dermatol. 2005

12. Conclusions  In contrast to cervical cancer, the presence of HPV is probably not mandatory for maintenance of the malignant phenotype of skin cancer cells.  Low DNA loads, a small number of HPV-positive cells and higher loads in actinic keratoses are compatible with a role of cutaneous HPVs as a carcinogen in early phases of non-melanoma skin cancer development.  The induction of human keratinocyte invasion by HPV8-E7 and the spontaneous development of skin cancers in HPV8 transgenic mice provided the first experimental evidences for a role of HPV in NMSC development.

13. Acknowledgements Skin Tumour Laboratory, Cancer Research UK Alan Storey Harshad Navsaria Irene Leigh Charlotte Proby Catherine Harwood Institute of Virology, University of Cologne Herbert Pfister Regina Pfefferle Sönke Weissenborn Department of Dermatology, Cologne Cornelia Mauch Thomas Krieg