Arch Dermatol Res (2014) 306:93–95 DOI 10.1007/s00403-013-1424-8
CONCISE COMMUNICATION
Human beta papillomavirus DNA study in primary cutaneous squamous cell carcinomas and their corresponding metastases A. Toll • B. Lloveras • E. Masferrer • C. Ferra´ndiz-Pulido • V. Garcı´a-Patos • T. Gheit • R. M. Pujol • M. Tommasino
Received: 26 August 2013 / Revised: 10 October 2013 / Accepted: 17 October 2013 / Published online: 31 October 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract The association between beta human papillomavirus (HPV) types and cutaneous squamous cell carcinomas (cSCCs) is controversial. Several studies have found such an association, especially at early stages of carcinogenesis, but the presence of beta HPV types in aggressive cSCCs has only been reported in three patients previously. We aimed to search for beta HPV DNA in primary cSCCs and their corresponding lymph node metastases in a series of patients. The presence of DNA from 25 beta HPV types was determined using a multiplex PCR protocol in 35 primary cSCCs from 35 patients and their corresponding lymph node metastases. DNA from beta HPV types was detected in 9 % of primary cSCCs and in 13 % of metastases. No primary cutaneous SCC or lymphatic metastases A. Toll (&) R. M. Pujol Department of Dermatology, Hospital del Mar, Parc de Salut Mar, Pg/Marı´tim 25-29, 08003 Barcelona, Spain e-mail: [email protected] A. Toll Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Me`diques), Barcelona, Spain B. Lloveras Department of Pathology, Hospital del Mar, Parc de Salut Mar, Barcelona, Spain E. Masferrer Department of Dermatology, Hospital Universitari de Bellvitge, L0 Hospitalet de Llobregat, Barcelona, Spain C. Ferra´ndiz-Pulido V. Garcı´a-Patos Department of Dermatology, Hospital Universitari Vall d0 Hebron, Barcelona, Spain T. Gheit M. Tommasino International Agency for Research on Cancer, World Health Organization, 69372 Lyon, France
were found to share the same HPV DNA. These data suggest that beta HPV types do not play an etiopathogenic role in advanced stages of squamous cell carcinogenesis. Keywords Cutaneous squamous cell carcinoma Human papillomavirus Lymph nodes Metastases Aggressive
Introduction Mucosal high-risk (HR) human papillomavirus (HPV) types, e.g. HPV16, have been clearly associated with cervical cancer, other anogenital carcinomas and oropharyngeal cancers. In addition, the skin HPV types that belong to the genus beta of the HPV phylogenetic tree are suspected to play a role in the development of non-melanoma skin cancer (NMSC) [7]. Beta HPV types, e.g. HPV5 and 8, were first identified in individuals suffering from a genetic disorder called epidermodysplasia verruciformis (EV) [9]. Beta HPV types are highly ubiquitous in the normal population and, according to some authors, might also play a role in skin carcinogenesis in non-EV individuals [1]. In addition, impairment of the immune system in organ transplant recipients, which may favour viral infections, strongly increases the risk of developing NMSC [2]. Functional studies in in vitro and in vivo experimental models have highlighted the oncogenic properties of beta HPV types [14, 15]. However, analyses of NMSC specimens have not revealed the presence of viral transcripts [5]. In addition, the use of quantitative, type-specific PCR has shown that far \1 copy of HPV DNA per cell is present in NMSC specimens [16]. This scenario differs from that observed in cervical cancers, where at least one copy of mucosal HR HPV DNA is detected in each cancer cell [12].
123
94
Together, these findings suggest that beta HPV may play a role only at an initial stage of skin carcinogenesis. HPV16 detection in lymphatic metastases has been found to be concordant with that found in the corresponding primary oropharyngeal squamous cell carcinomas and may aid in finding the primary tumour in patients with cervical lymph node metastases of unknown origin [3]. A few small case series have addressed the study of beta HPV in primary cutaneous squamous cell carcinoma (cSCC) and their corresponding metastases [10, 11]. Ostrow et al. demonstrated the presence of HPV5 DNA in a cSCC and its subcutaneous metastasis in a patient with EV [11]. Nindl et al. observed the presence of identical HPV14, 21, and/or 36 variants in paired primary and metastatic tumours in two patients [10]. To further characterise the role of beta HPV types in skin carcinogenesis, we have determined the presence of viral DNA in cSCCs and their corresponding lymph node metastases. In case that HPV played a role in the induction of lymphatic metastases, one should expect to find HPV DNA in both types of lesions.
Arch Dermatol Res (2014) 306:93–95 Table 1 Clinico-pathological variables of the patients and primary tumours included in the study Clinical
Results Thirty-five patients with primary cSCC who developed regional lymph node metastases were identified. Seventy paraffin-embedded samples, from 35 primary tumours and their corresponding lymph node metastases were retrieved. The mean age of the patients was 77, 25 years (range 46–95) and 74.3 % were men. Three of the 35 patients were immunosuppressed, and five of the skin
123
26/35
Age, year, mean (range)
77.25 (46–95)
Sun-exposed area
30/35
Tumour site Head and neck
29/35
Trunk
1/35
Extremities
5/35
Diameter (cm), mean (SD)
2.76 (1.88)
Immunosuppression
3/35
Recurrent cases Pathological
15/35
Depth (mm), mean (SD)
8.3 (4,8)
Desmoplasia
9/35
Differentiation Well
3/35
Moderately
25/35
Poorly
7/35
Perineural invasion
5/35
Vascular infiltration
2/35
T staginga
Methods Records from the Pathology Departments of three hospitals in Barcelona, Spain (Hospital del Mar, Hospital Universitari de Bellvitge and Hospital Vall d0 Hebron) were retrospectively searched to identify patients with cSCC who had developed metastatic spread between 2001 and 2011. Only those patients from whom both biopsy specimens (the primary tumour and metastases) were available were included in the study. Approval to conduct this study was obtained from the ethics committees of all the centres participating in the study, and in accordance with the guidelines of the Helsinki Declaration of 1975, as revised in 1983. We determined the presence of DNA from 25 beta HPV types, namely HPV5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96, using a multiplex PCR protocol combined with a Luminex-based assay, as previously reported [6].
Male gender
1
10/35
2
18/35
3
5/35
Unknown
2/35
SD Standard deviation a
T staging according to the seventh American joint Committee on Cancer classification
tumours were from non-sun-exposed anatomical sites. The clinico-pathological features of the evaluated patients are illustrated in Table 1. For the primary tumours, three DNA samples were found to be beta-globin negative and were excluded from the study. Of the 32 remaining cSCCs, only three (9.3 %) were positive for a single beta HPV infection, i.e. HPV22, 24, and 75. None of the corresponding metastases associated to these cases showed HPV positivity. All three beta HPV positive cases were from sunexposed anatomical sites in non-immunosuppressed patients. For the lymph node metastases, four specimens were found to be beta-globin negative and were not further analysed. Of the 31 remaining lymphatic metastases, four (13 %) were positive for a single beta HPV type, i.e. HPV5 (detected in two samples), 24, and 75. None of these cases showed HPV positivity in their corresponding primary cSCC.
Arch Dermatol Res (2014) 306:93–95
Discussion The prevalence of HPV DNA in NMSC from immunocompetent patients has been reported to range between 13 and 84 % [1]. Surprisingly, in this study beta HPV DNA was detected at a low rate, although a very sensitive assay was used for the detection of viral DNA. Beta HPV types may not be involved in NMSC, although such a conclusion is not in agreement with initial epidemiological studies, which consistently show a moderate positive association between HPV infection and a history of NMSC [1, 4, 8]. Moreover, we cannot exclude the possibility that metastatic cSCCs harbour lower HPV infection rates than nonmetastatic tumours. Independent studies have shown that beta HPV prevalence is higher in the precursor lesion, or actinic keratosis, than in the invasive cSCC, suggesting that the virus may play a role at early stages of carcinogenesis [13, 16]. Since it is well demonstrated that beta HPV E6 and E7 oncoproteins inhibit the DNA repair machineries, it is likely that beta HPV types facilitate the accumulation of ultraviolet-mediated DNA damage at early stages of tumour development and could be lost in invasive stages [16]. In this sense, Weissenborn et al. found that the HPV DNA load was undetectable by Q-PCR in eight metastases of cSCCs [16]. Moreover, our series mostly includes immunocompetent patients, which may harbour less HPVassociated cSCCs than immunosuppressed individuals. Quite surprisingly, we found HPV in four metastases in whose primary tumours no HPV DNA was detected. This may either reflect a technical limitation (false negative in primary tumours or false positive in the metastases) or a clonal selection. In any case, the latter scenario would seem clinically irrelevant in light of the few cases of metastases showing HPV positivity. Our study has some limitations, which should be stated. This was a retrospective study, with a small series. In addition, we did not compare our results with a nonmetastasizing series of tumours. In conclusion, irrespective of their hypothetical role in inducing epithelial malignancy in skin, beta HPV types do not appear to play a role in the maintenance of the malignant phenotype in advanced cSCC. Acknowledgments We thank Dr Karen Muller for editing and Tania Lobato for their technical support. This study was supported by Grants FIS PI10/00785, RD09/0076/ 00036, RD06/0020/0040, and PS09/00973 from Fondo de Investigacio´n Sanitaria (FIS), Instituto de Salud Carlos III, Ministerio de Sanidad, Fondo Europeo de Desarrollo Regional (FEDER), SAF201016089, Ministerio de Economı´a y Competitividad, Spain, from the AGAUR (2009 SGR 1409), the ‘‘Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC)’’, and by the European Commission, Grant HPV-AHEAD (FP7-HEALTH-2011282562).
95 Conflict of interest
None declared.
References 1. Aldabagh B, Angeles JG, Cardones AR, Arron ST (2012) Cutaneous squamous cell carcinoma and human papillomavirus: is there an association? Dermatol Surg 39:1–23 2. Bouwes Bavinck JN, Euvrard S, Naldi L et al (2007) Keratotic skin lesions and other risk factors are associated with skin cancer in organ-transplant recipients: a case-control study in The Netherlands, United Kingdom, Germany, France, and Italy. J Invest Dermatol 127:1647–1656 3. Desai PC, Jaglal MV, Gopal P (2009) Human papillomavirus in metastatic squamous carcinoma from unknown primaries in the head and neck: a retrospective 7 year study. Exp Mol Pathol 87:94–98 4. Farzan SF, Waterboer T, Gui J et al (2013) Cutaneous alpha, beta and gamma human papillomaviruses in relation to squamous cell carcinoma of the skin: a population-based study. Int J Cancer 133:1713–1720 5. Ganzenmueller T, Kluba Y, Yakushko et al (2012) Next-generation sequencing fails to identify human virus sequences in cutaneous squamous cell carcinoma. Int J Cancer 131:E1173– E1179. doi:10.1002/ijc.27581 6. Gheit T, Vaccarella S, Schmitt M et al (2009) Prevalence of human papillomavirus types in cervical and oral cancers in central India. Vaccine 27:636–639 7. Hall L, Struijk L, Neale RE, Feltkamp MC (2006) Re: human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin. J Natl Cancer Inst 98:1425–1426 8. Iannacone MR, Gheit T, Waterboer T et al (2013) Case-control study of cutaneous human papillomavirus infection in basal cell carcinoma of the skin. J Invest Dermatol 133:1512–1520 9. Jablonska S, Dabrowski J, Jakubowicz K (1972) Epidermodysplasia verruciformis as a model in studies on the role of papovaviruses in oncogenesis. Cancer Res 32:583–589 10. Nindl I, Koehler A, Meyer T (2006) Detection of human papillomavirus DNA in primary squamous cell carcinoma and metastases. Br J Dermatol 154:797–799 11. Ostrow RS, Bender M, Niimura M et al (1982) Human papillomavirus DNA in cutaneous primary and metastasized squamous cell carcinomas from patients with epidermodysplasia verruciformis. Proc Natl Acad Sci USA 79:1634–1638 12. Snijders PJ, Hogewoning CJ, Hesselink AT et al (2006) Determination of viral load thresholds in cervical scrapings to rule out CIN 3 in HPV 16, 18, 31 and 33-positive women with normal cytology. Int J Cancer 119:1102–1107 13. Vasiljevic N, Hazard K, Dillner J, Forslund O (2008) Four novel human betapapillomaviruses of species 2 preferentially found in actinic keratosis. J Gen Virol 89:2467–2474 14. Viarisio D, Mueller-Decker K, Kloz U et al (2011) E6 and E7 from beta HPV38 cooperate with ultraviolet light in the development of actinic keratosis-like lesions and squamous cell carcinoma in mice. PLoS Pathog 7:e1002125 15. Wallace NA, Robinson K, Howie HL, Galloway DA (2012) HPV 5 and 8 E6 abrogate ATR activity resulting in increased persistence of UVB induced DNA damage. PLoS Pathog 8:e1002807. doi:10.1371/journal.ppat.1002807 16. Weissenborn SJ, Nindl I, Purdie K (2005) Human papillomavirus-DNA loads in actinic keratoses exceed those in non-melanoma skin cancers. J Invest Dermatol 125:93–97
123
CONCISE COMMUNICATION
Human beta papillomavirus DNA study in primary cutaneous squamous cell carcinomas and their corresponding metastases A. Toll • B. Lloveras • E. Masferrer • C. Ferra´ndiz-Pulido • V. Garcı´a-Patos • T. Gheit • R. M. Pujol • M. Tommasino
Received: 26 August 2013 / Revised: 10 October 2013 / Accepted: 17 October 2013 / Published online: 31 October 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract The association between beta human papillomavirus (HPV) types and cutaneous squamous cell carcinomas (cSCCs) is controversial. Several studies have found such an association, especially at early stages of carcinogenesis, but the presence of beta HPV types in aggressive cSCCs has only been reported in three patients previously. We aimed to search for beta HPV DNA in primary cSCCs and their corresponding lymph node metastases in a series of patients. The presence of DNA from 25 beta HPV types was determined using a multiplex PCR protocol in 35 primary cSCCs from 35 patients and their corresponding lymph node metastases. DNA from beta HPV types was detected in 9 % of primary cSCCs and in 13 % of metastases. No primary cutaneous SCC or lymphatic metastases A. Toll (&) R. M. Pujol Department of Dermatology, Hospital del Mar, Parc de Salut Mar, Pg/Marı´tim 25-29, 08003 Barcelona, Spain e-mail: [email protected] A. Toll Cancer Research Program, IMIM (Institut Hospital del Mar d’Investigacions Me`diques), Barcelona, Spain B. Lloveras Department of Pathology, Hospital del Mar, Parc de Salut Mar, Barcelona, Spain E. Masferrer Department of Dermatology, Hospital Universitari de Bellvitge, L0 Hospitalet de Llobregat, Barcelona, Spain C. Ferra´ndiz-Pulido V. Garcı´a-Patos Department of Dermatology, Hospital Universitari Vall d0 Hebron, Barcelona, Spain T. Gheit M. Tommasino International Agency for Research on Cancer, World Health Organization, 69372 Lyon, France
were found to share the same HPV DNA. These data suggest that beta HPV types do not play an etiopathogenic role in advanced stages of squamous cell carcinogenesis. Keywords Cutaneous squamous cell carcinoma Human papillomavirus Lymph nodes Metastases Aggressive
Introduction Mucosal high-risk (HR) human papillomavirus (HPV) types, e.g. HPV16, have been clearly associated with cervical cancer, other anogenital carcinomas and oropharyngeal cancers. In addition, the skin HPV types that belong to the genus beta of the HPV phylogenetic tree are suspected to play a role in the development of non-melanoma skin cancer (NMSC) [7]. Beta HPV types, e.g. HPV5 and 8, were first identified in individuals suffering from a genetic disorder called epidermodysplasia verruciformis (EV) [9]. Beta HPV types are highly ubiquitous in the normal population and, according to some authors, might also play a role in skin carcinogenesis in non-EV individuals [1]. In addition, impairment of the immune system in organ transplant recipients, which may favour viral infections, strongly increases the risk of developing NMSC [2]. Functional studies in in vitro and in vivo experimental models have highlighted the oncogenic properties of beta HPV types [14, 15]. However, analyses of NMSC specimens have not revealed the presence of viral transcripts [5]. In addition, the use of quantitative, type-specific PCR has shown that far \1 copy of HPV DNA per cell is present in NMSC specimens [16]. This scenario differs from that observed in cervical cancers, where at least one copy of mucosal HR HPV DNA is detected in each cancer cell [12].
123
94
Together, these findings suggest that beta HPV may play a role only at an initial stage of skin carcinogenesis. HPV16 detection in lymphatic metastases has been found to be concordant with that found in the corresponding primary oropharyngeal squamous cell carcinomas and may aid in finding the primary tumour in patients with cervical lymph node metastases of unknown origin [3]. A few small case series have addressed the study of beta HPV in primary cutaneous squamous cell carcinoma (cSCC) and their corresponding metastases [10, 11]. Ostrow et al. demonstrated the presence of HPV5 DNA in a cSCC and its subcutaneous metastasis in a patient with EV [11]. Nindl et al. observed the presence of identical HPV14, 21, and/or 36 variants in paired primary and metastatic tumours in two patients [10]. To further characterise the role of beta HPV types in skin carcinogenesis, we have determined the presence of viral DNA in cSCCs and their corresponding lymph node metastases. In case that HPV played a role in the induction of lymphatic metastases, one should expect to find HPV DNA in both types of lesions.
Arch Dermatol Res (2014) 306:93–95 Table 1 Clinico-pathological variables of the patients and primary tumours included in the study Clinical
Results Thirty-five patients with primary cSCC who developed regional lymph node metastases were identified. Seventy paraffin-embedded samples, from 35 primary tumours and their corresponding lymph node metastases were retrieved. The mean age of the patients was 77, 25 years (range 46–95) and 74.3 % were men. Three of the 35 patients were immunosuppressed, and five of the skin
123
26/35
Age, year, mean (range)
77.25 (46–95)
Sun-exposed area
30/35
Tumour site Head and neck
29/35
Trunk
1/35
Extremities
5/35
Diameter (cm), mean (SD)
2.76 (1.88)
Immunosuppression
3/35
Recurrent cases Pathological
15/35
Depth (mm), mean (SD)
8.3 (4,8)
Desmoplasia
9/35
Differentiation Well
3/35
Moderately
25/35
Poorly
7/35
Perineural invasion
5/35
Vascular infiltration
2/35
T staginga
Methods Records from the Pathology Departments of three hospitals in Barcelona, Spain (Hospital del Mar, Hospital Universitari de Bellvitge and Hospital Vall d0 Hebron) were retrospectively searched to identify patients with cSCC who had developed metastatic spread between 2001 and 2011. Only those patients from whom both biopsy specimens (the primary tumour and metastases) were available were included in the study. Approval to conduct this study was obtained from the ethics committees of all the centres participating in the study, and in accordance with the guidelines of the Helsinki Declaration of 1975, as revised in 1983. We determined the presence of DNA from 25 beta HPV types, namely HPV5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96, using a multiplex PCR protocol combined with a Luminex-based assay, as previously reported [6].
Male gender
1
10/35
2
18/35
3
5/35
Unknown
2/35
SD Standard deviation a
T staging according to the seventh American joint Committee on Cancer classification
tumours were from non-sun-exposed anatomical sites. The clinico-pathological features of the evaluated patients are illustrated in Table 1. For the primary tumours, three DNA samples were found to be beta-globin negative and were excluded from the study. Of the 32 remaining cSCCs, only three (9.3 %) were positive for a single beta HPV infection, i.e. HPV22, 24, and 75. None of the corresponding metastases associated to these cases showed HPV positivity. All three beta HPV positive cases were from sunexposed anatomical sites in non-immunosuppressed patients. For the lymph node metastases, four specimens were found to be beta-globin negative and were not further analysed. Of the 31 remaining lymphatic metastases, four (13 %) were positive for a single beta HPV type, i.e. HPV5 (detected in two samples), 24, and 75. None of these cases showed HPV positivity in their corresponding primary cSCC.
Arch Dermatol Res (2014) 306:93–95
Discussion The prevalence of HPV DNA in NMSC from immunocompetent patients has been reported to range between 13 and 84 % [1]. Surprisingly, in this study beta HPV DNA was detected at a low rate, although a very sensitive assay was used for the detection of viral DNA. Beta HPV types may not be involved in NMSC, although such a conclusion is not in agreement with initial epidemiological studies, which consistently show a moderate positive association between HPV infection and a history of NMSC [1, 4, 8]. Moreover, we cannot exclude the possibility that metastatic cSCCs harbour lower HPV infection rates than nonmetastatic tumours. Independent studies have shown that beta HPV prevalence is higher in the precursor lesion, or actinic keratosis, than in the invasive cSCC, suggesting that the virus may play a role at early stages of carcinogenesis [13, 16]. Since it is well demonstrated that beta HPV E6 and E7 oncoproteins inhibit the DNA repair machineries, it is likely that beta HPV types facilitate the accumulation of ultraviolet-mediated DNA damage at early stages of tumour development and could be lost in invasive stages [16]. In this sense, Weissenborn et al. found that the HPV DNA load was undetectable by Q-PCR in eight metastases of cSCCs [16]. Moreover, our series mostly includes immunocompetent patients, which may harbour less HPVassociated cSCCs than immunosuppressed individuals. Quite surprisingly, we found HPV in four metastases in whose primary tumours no HPV DNA was detected. This may either reflect a technical limitation (false negative in primary tumours or false positive in the metastases) or a clonal selection. In any case, the latter scenario would seem clinically irrelevant in light of the few cases of metastases showing HPV positivity. Our study has some limitations, which should be stated. This was a retrospective study, with a small series. In addition, we did not compare our results with a nonmetastasizing series of tumours. In conclusion, irrespective of their hypothetical role in inducing epithelial malignancy in skin, beta HPV types do not appear to play a role in the maintenance of the malignant phenotype in advanced cSCC. Acknowledgments We thank Dr Karen Muller for editing and Tania Lobato for their technical support. This study was supported by Grants FIS PI10/00785, RD09/0076/ 00036, RD06/0020/0040, and PS09/00973 from Fondo de Investigacio´n Sanitaria (FIS), Instituto de Salud Carlos III, Ministerio de Sanidad, Fondo Europeo de Desarrollo Regional (FEDER), SAF201016089, Ministerio de Economı´a y Competitividad, Spain, from the AGAUR (2009 SGR 1409), the ‘‘Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC)’’, and by the European Commission, Grant HPV-AHEAD (FP7-HEALTH-2011282562).
95 Conflict of interest
None declared.
References 1. Aldabagh B, Angeles JG, Cardones AR, Arron ST (2012) Cutaneous squamous cell carcinoma and human papillomavirus: is there an association? Dermatol Surg 39:1–23 2. Bouwes Bavinck JN, Euvrard S, Naldi L et al (2007) Keratotic skin lesions and other risk factors are associated with skin cancer in organ-transplant recipients: a case-control study in The Netherlands, United Kingdom, Germany, France, and Italy. J Invest Dermatol 127:1647–1656 3. Desai PC, Jaglal MV, Gopal P (2009) Human papillomavirus in metastatic squamous carcinoma from unknown primaries in the head and neck: a retrospective 7 year study. Exp Mol Pathol 87:94–98 4. Farzan SF, Waterboer T, Gui J et al (2013) Cutaneous alpha, beta and gamma human papillomaviruses in relation to squamous cell carcinoma of the skin: a population-based study. Int J Cancer 133:1713–1720 5. Ganzenmueller T, Kluba Y, Yakushko et al (2012) Next-generation sequencing fails to identify human virus sequences in cutaneous squamous cell carcinoma. Int J Cancer 131:E1173– E1179. doi:10.1002/ijc.27581 6. Gheit T, Vaccarella S, Schmitt M et al (2009) Prevalence of human papillomavirus types in cervical and oral cancers in central India. Vaccine 27:636–639 7. Hall L, Struijk L, Neale RE, Feltkamp MC (2006) Re: human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin. J Natl Cancer Inst 98:1425–1426 8. Iannacone MR, Gheit T, Waterboer T et al (2013) Case-control study of cutaneous human papillomavirus infection in basal cell carcinoma of the skin. J Invest Dermatol 133:1512–1520 9. Jablonska S, Dabrowski J, Jakubowicz K (1972) Epidermodysplasia verruciformis as a model in studies on the role of papovaviruses in oncogenesis. Cancer Res 32:583–589 10. Nindl I, Koehler A, Meyer T (2006) Detection of human papillomavirus DNA in primary squamous cell carcinoma and metastases. Br J Dermatol 154:797–799 11. Ostrow RS, Bender M, Niimura M et al (1982) Human papillomavirus DNA in cutaneous primary and metastasized squamous cell carcinomas from patients with epidermodysplasia verruciformis. Proc Natl Acad Sci USA 79:1634–1638 12. Snijders PJ, Hogewoning CJ, Hesselink AT et al (2006) Determination of viral load thresholds in cervical scrapings to rule out CIN 3 in HPV 16, 18, 31 and 33-positive women with normal cytology. Int J Cancer 119:1102–1107 13. Vasiljevic N, Hazard K, Dillner J, Forslund O (2008) Four novel human betapapillomaviruses of species 2 preferentially found in actinic keratosis. J Gen Virol 89:2467–2474 14. Viarisio D, Mueller-Decker K, Kloz U et al (2011) E6 and E7 from beta HPV38 cooperate with ultraviolet light in the development of actinic keratosis-like lesions and squamous cell carcinoma in mice. PLoS Pathog 7:e1002125 15. Wallace NA, Robinson K, Howie HL, Galloway DA (2012) HPV 5 and 8 E6 abrogate ATR activity resulting in increased persistence of UVB induced DNA damage. PLoS Pathog 8:e1002807. doi:10.1371/journal.ppat.1002807 16. Weissenborn SJ, Nindl I, Purdie K (2005) Human papillomavirus-DNA loads in actinic keratoses exceed those in non-melanoma skin cancers. J Invest Dermatol 125:93–97
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