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Journal of Virological Methods 146 (2007) 1–4
Extension of the typing in a general-primer-PCR reverse-line-blotting system to detect all 25 cutaneous beta human papillomaviruses Ingo Nindl a,∗ , Anja K¨ohler a , Marc Gottschling a , Tobias Forschner a , Mandy Lehmann a , Chris J.L.M. Meijer b , Peter J.F. Snijders b , Eggert Stockfleth a a
Department of Dermatology, Venereology and Allergy, Charit´e, Skin Cancer Center Charit´e, University Hospital of Berlin, Charit´eplatz 1, D-10117 Berlin, Germany b Department of Pathology, Section of Molecular Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands Received 14 March 2007; received in revised form 22 May 2007; accepted 22 May 2007 Available online 29 June 2007
Abstract ␤-Papillomaviruses (PV) seem to be involved in the pathogenesis of cutaneous squamous cell carcinoma and its early stage actinic keratosis. In this study, typing was extended of a previously described consensus primer-mediated ␤- and ␥-cutaneous HPV PCR method followed by reverse-line-blotting (BGC-PCR/RLB) to detect all 25 known ␤-PV and to examine their prevalence in actinic keratosis. The typing format of the BGC-PCR assay was extended by adding hybridization probes of six ␤-PV (HPV 75, 76, 80, 92, 93, and 96) to the RLB system. Subsequently, tumor and normal skin tissues were collected from 75 patients with actinic keratosis, allowing typing for a total of 25 ␤- and 5 ␥-types. The analytical sensitivity was between 10 copies (HPV 75, 80, 92, 93, and 96) and 100 copies (HPV 76). Except for that of HPV 76, none of the added probes showed any cross-hybridization with other ␤-HPV. HPV DNA was detected in 45% of actinic keratosis and in 33% of normal skin by BGC-PCR, and at least one of the six added ␤-types was present in 19% of actinic keratoses and in 13% of normal skin. Six ␤-HPV types were added successfully to the typing format of the BGC-PCR/RLB system. The potential role of these types in the development of non-melanoma skin cancer awaits further studies. © 2007 Elsevier B.V. All rights reserved. Keywords: Actinic keratosis; ␤-Human papillomaviruses; General-primer PCR; Reverse-line-blotting
1. Introduction A causal relationship of genital human papillomaviruses (HPV) with the pathogenesis of cervical cancer has been established, and cutaneous HPV are considered to be involved in the development of non-melanoma skin cancer (IARC, 1995; Pfister, 2003; Akg¨ul et al., 2006). Mucosal HPV types are classified as ␣-PV infecting the mucosa of the genital and respiratory tract. Cutaneous HPV types infect the skin, belong to the ␤or ␥-PV genera, and are phylogenetically distant from ␣-PV (Gottschling et al., 2007). The first evidence for the involvement of cutaneous HPV in skin cancer was reported in patients with Epidermodysplasia verruciformis. The rare disease is char-
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acterized by extensive polymorphous warts that in about 30% of the patients convert into squamous cell carcinoma (Jabło´nska et al., 1972; Orth et al., 1978). ␤-HPV types are present in more than 90% of non-melanoma skin cancer in these patients and are referred to as Epidermodysplasia verruciformis-HPV types. HPV types of the ␤- and ␥-genera (␤/␥ cutaneous HPV types) were also detected at a high frequency in non-melanoma skin cancer of the general population, and the presence of ␤/␥ cutaneous HPV types in normal skin and hair follicles has been linked to an increased risk of squamous cell carcinoma (Harwood et al., 2004) and its early stage, actinic keratosis (Boxman et al., 2001). ␤/␥ Cutaneous HPV types prevent apoptosis after UV-radiation (Jackson and Storey, 2000). In epidemiological studies, moreover, these viruses display a high prevalence in actinic keratosis (85%) (Pfister et al., 2003), and a higher viral load in actinic keratosis, versus squamous cell carcinoma, has been reported (Weissenborn et al., 2005). These findings
I. Nindl et al. / Journal of Virological Methods 146 (2007) 1–4
suggest a role for HPV in the early stages of non-melanoma skin cancer. In the present study, a previously described general-primer polymerase chain reaction (PCR) reverse-line-blotting (RLB) typing system (Brink et al., 2005) was extended by adding RLB probes of 6 HPV types to detect all known 25 ␤-HPV types. Furthermore, this extended typing assay was used to examine actinic keratosis and normal skin of 75 immunocompetent patients. 2. Materials and methods 2.1. Clinical specimens Two punch biopsies were collected from each of 75 immunocompetent patients with actinic keratosis (57–88 years; mean age, 71 years). Specimens taken from the forehead (actinic keratosis) and the inner side of the upper arm (normal skin) were placed in liquid nitrogen and stored at −70 ◦ C. DNA was isolated by QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and the pellet was dissolved in 100 l AE buffer (Qiagen). For PCR experiments, 5 l of this solution was used.
detection of 19 of 25 ␤ and 5 of 7 ␥-cutaneous HPV types (BGCPCR), amplifying a 72-bp L1 fragment (Brink et al., 2005). Briefly, each PCR included three negative controls and three positive controls containing 10, 100, and 1000 copies of HPV 8 plasmid DNA, in a background of 100 ng human placental DNA, per reaction. Typing was performed with 24 HPV specific 5 amino-linked oligonucleotides by RLB. The typing format of the BGC-PCR/RLB system was extended by adding 6 hybridization oligonucleotides specific for HPV 75, 76, 80, 92, 93, and 96. Oligonucleotides, approximately 20 bases in length, were designed with the melting temperature near 55 ◦ C (Table 1). Selected oligonucleotides were subjected to a database search [NCBI Blast Searches (http://www.ncbi.nlm.nih.gov/BLAST/)] to verify their specificity. The quality of each clinical specimen was examined by ␤-globin PCR, as described previously (Nindl et al., 2004), and only PCR positive specimens were included in the present study. All clinical specimens were analyzed twice on different days, and HPV types were only scored if both experiments revealed consistent results. 3. Results and discussion 3.1. Sensitivity and speciﬁcity of the extended BGC-PCR/RLB system
2.2. HPV genome plasmids Serial dilutions of HPV genome plasmids (HPV 75, 80, 92, 93, 96, and subcloned HPV 76, and HPV 4, 8, 12, 15, and 21), in a background of 100 ng human placental DNA, were used to examine the sensitivity and specificity of the extended BGCPCR/RLB system. 2.3. BGC-PCR/RLB system and extension of HPV typing HPV detection and typing was performed as described previously, using a consensus primer-mediated PCR method for the
The minimal number of mismatches between the consensus primers and sequences of the 6 added types (HPV 75, 76, 80, 92, 93, and 96) was 0–4 for the forward primers and 3–5 for the reverse primers (Table 1). Analytical sensitivity of the extended BGC-PCR/RLB system was determined by testing serial dilutions of HPV genomes (HPV 75, 80, 92, 93, 96, and subcloned HPV 76) in a background of 100 ng human placental DNA per reaction (Fig. 1). By RLB analysis of BGC-PCR amplicons, the analytical sensitivity was found to be between 10 (HPV 75, 80,
Table 1 Oligonucleotide sequences and minimal number of mismatches of the extended BGC-PCR/RLB system to detect HPV 75, HPV 76, HPV 80, HPV 92, HPV 93, and HPV 96 GenBank accession no.
Sequence (5 → 3 )
Hybridization probe of the RLB Nt (bases)
NC 004500 (AF531420) AY382778
HPV 93 HPV 96
NC 005134 (AY382779)
AAT CCA AAT ATA TTG GAA AAT TG AAT TCC AAT ATA TTA GAA AAT TG ATG CAG GTA TTT TAG AAG AG TCC AAA TAT TTT GGA GGA CT CTC ACT GCT AGA GGA ATG GCA GAC ATT TTA GAA GAT TG
Tm (salt adjusted) (◦ C)
Consensus primer (Brink et al., 2005) Minimal number of mismatches* with HPV types
No. of mismatches with the best matching primers Forward primer(s)
4 and 76 (3)
4, 14, 20, 21, and 75 (4)
8 and 12 (5)
21 and 80 (3)
BGC-PCR, ␤/␥ cutaneous HPV polymerase chain reaction; RLB, reverse-line-blotting; HPV, human papillomavirus; Nt, nucleotides; Tm , melting temperature; ORF, open reading frame. * Minimal number of mismatches are shown in parentheses.
I. Nindl et al. / Journal of Virological Methods 146 (2007) 1–4
Fig. 1. Reverse-line-blot (RLB) of the 6 ␤ HPV types (75, 76, 80, 92, 93, and 96), to determine analytical sensitivity. The sensitivity of the extended BGC-PCR/RLB system was determined by serial dilutions of the cloned HPV genomes (105 , 104 , 103 , 102 , 10 and 1 viral copy/ies) in 100 ng human placental DNA per reaction. In each experiment three negative controls (nc, 37–39) in a background of 100 ng human placental DNA were included. The reactions were scored either negative or positive, and the sensitivity is shown at the top of each HPV type.
92, 93, and 96) and 100 (HPV 76) viral copies per reaction. Except for that of HPV 76, none of the RLB probes showed cross-hybridization with the tested types (HPV 4, 8, 12, 15, 21, 75, 76, 80, 92, 93, and 96), which have the minimal number of mismatches with these 6 hybridization probes (Table 1). Only the HPV 76 probe showed a weak cross-hybridization with high copy numbers of HPV 75 (equal to or greater than 104 viral copies; Fig. 1). Overall, the number of hybridization probes of the BGCPCR/RLB system was expanded successfully to detect 30 ␤/␥ cutaneous HPV, including all 25 known ␤ types. The analytical sensitivity of the BGC-PCR assay to detect 24 ␤/␥ cutaneous HPV types is between 10 and 1000 viral copies, and the method is highly specific (Brink et al., 2005). In this study the sensitivity of the 6 new ␤-HPV types was high and similar to the previously reported sensitivity. Moreover, the lack of crosshybridization with different ␤/␥ cutaneous HPV types, with the exception of HPV 76, showed a high specificity of the extended BGC-PCR/RLB system, at least for the currently known HPV types. 3.2. Prevalence of the six β-HPV types in actinic keratosis HPV DNA of 24 ␤/␥ cutaneous types was detected in 45% of actinic keratosis samples and in 33% of normal skin biopsies from 75 immunocompetent patients using the BGC-PCR/RLB
system (Table 2). At least one of the six additional ␤-HPV types (HPV 75, 76, 80, 92, 93, and 96) was present in 19% of actinic keratosis and in 13% of normal skin biopsies. In 10 patients, the new HPV types were present both in actinic keratosis and normal skin samples, and in four patients, only the skin tumor was HPV positive for one of these types. The highest prevalence in actinic keratosis among the 6 additional ␤-HPV types was found for HPV 80 (10%), followed by HPV 93 (5%) and HPV 75, 92, and 96 (1% each). Altogether, HPV DNA of 30 ␤/␥ cutaneous types was detected in 47% of actinic keratosis and 37% of normal skin samples (Table 2). ␤/␥ Cutaneous HPV types were detected in 85% of 114 immunocompetent patients with actinic keratosis (Pfister et al., 2003). Lower prevalences of 36% were reported in 14 immunocompetent patients with actinic keratosis (Meyer et al., 2001), in 68% of actinic keratosis samples from 56 immunosuppressed renal transplant recipients (Berkhout et al., 2000), and in 47% of 75 immunocompetent patients with actinic keratosis in the present study. These partially conflicting results indicate that the precise prevalence still has to be determined based on a standard procedure to avoid bias from sample collection and/or the detection system. Thus, all cutaneous types are necessary to be included in future studies that may have importance to evaluate the role of individual ␤/␥ cutaneous HPV types in skin cancer. Overall, the extended BGC-PCR/RLB detection system is applicable for large epidemiological studies analyzing the risk
Table 2 Prevalence of 24 ␤/␥ cutaneous and 6 additional ␤-HPV types detected in actinic keratosis and normal skin from 75 immunocompetent patients Clinical specimens
Actinic keratosis (75) Normal skin (75)
24 ␤/␥ cutaneous HPV types (Brink et al., 2005)
6 additional ␤-HPV types 75
34 (45%) 25 (33%)
7.5(1×) a (10%) 5.5(1×) a (7%)
1 (1%) 0
3.5(1×) a (5%) 3.5(1×) a (5%)
1 (1%) 1 (1%)
HPV, human papillomavirus. a The number of double infections is indicated in subscript parentheses.
Total of 6 ␤-HPV types
Total of 30 ␤/␥ cutaneous HPV types
14 (19%) 10 (13%)
35 (47%) 28 (37%)
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of ␤/␥ cutaneous HPV types for cutaneous squamous cell carcinoma and its early stage, actinic keratosis. Acknowledgements We are grateful to E.-M. de Villiers (DKFZ, Heidelberg, Germany) for providing plasmid clones for HPV 4, 8, 75, 76, and 80, to O. Forslund (Lund University, Malm¨o, Sweden) for providing plasmid clones for HPV 92, 93, and 96, and to G. Orth (Institut Pasteur, Paris, France) for providing plasmid clones for HPV 12, 15, and 21. This work was supported by a grant of the DKH, Germany (grant no. 70-2588), and by the ROTRF, Switzerland (grant no. 590944305). References Akg¨ul, B., Cooke, J.C., Storey, A., 2006. HPV-associated skin disease. J. Pathol. 208, 165–175. Berkhout, R.J., Bouwes Bavinck, J.N., Ter Schegget, J., 2000. Persistence of human papillomavirus DNA in benign and (pre)malignant skin lesions from renal transplant recipients. J. Clin. Microbiol. 38, 2087–2096. Boxman, I.L., Russell, A., Mulder, L.H., Bouwes Bavinck, J.N., Ter Schegget, J., Green, A., 2001. Association between Epidermodysplasia verruciformisassociated human papillomavirus DNA in plucked eyebrow hair and solar keratoses. J. Invest. Dermatol. 117, 1108–1112. Brink, A.A., Lloveras, B., Nindl, I., Heideman, D.A., Kramer, D., Pol, R., Fuente, M.J., Meijer, C.J.L.M., Snijders, P.J.F., 2005. Development of a generalprimer-PCR-reverse-line-blotting system for detection of beta and gamma cutaneous human papillomaviruses. J. Clin. Microbiol. 43, 5581–5587. ´ Bravo, Gottschling, M., Stamatakis, A., Nindl, I., Stockfleth, E., Alonso, A., I.G., 2007. Multiple evolutionary mechanisms drive papillomavirus diversification. Mol. Biol. Evol. 24, 1242–1258.
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