We investigated the role of β-HPVs in NMSC, using innovative methods to test population-based samples for the presence of HPV DNA belonging to all known sequenced types of the genus β. Although all 196 tumors tested for the current analysis were from unique patients, we do not have a confirmed record of prior NMSC as part of this study.
We tested the hypothesis that prevalence of HPV DNA differed between SCC and BCC samples and observed no significant difference between the two histologies overall. We did, however, find that SCC samples were more often infected with those types belonging to species 1 of the β genus, which includes HPV 5 and HPV 8, than in BCC samples. Although HPV 36 was detected most often within this species among SCC cases, the presence of HPV 24 differed most between the two histological groups. Interestingly, antibodies to HPV 24 (Feltkamp et al., 2003
) and HPV 24 DNA (Struijk et al., 2003
) have been positively associated with SCC in addition to HPV 24 DNA detected in the malignant lesions of epidermodysplasia verruciformis patients (Orth, 1987
; Gubinelli et al., 2003
). Furthermore, SCC tumors had a higher level of infectivity (positive for multiple types) by β-HPVs compared with BCC samples.
Nearly, all previous studies suggest an association between SCC and β-HPV DNA, yet comparison with these studies is difficult, in part, due to small sample sizes, and hence variable prevalence rates. Furthermore, studies used inconsistent methods that differ in both sensitivity and specificity (). For example Surentheran et al. (1998)
tested SCC tumors from six immunocompetent patients using PCR primer pairs lying within the open reading frame of the viral gene encoding the structural protein L1 and analyzing the sequence of the amplified gene fragment. But, recent investigations have shown that the L1 gene is less conserved than originally assumed (Iftner et al., 2003
). Consequently, studies (for example, Pfister et al., 2003
as well as Forslund et al., 2007
) using this methodology may have had limited ability to detect several HPV types (including, HPV 2–6, 8, 10, 11, 16, 31, 41, and 57).
Previous Studies of β-HPV DNA detection in NMSC tumor samples of immunocompetent individuals
An alternative approach is to use various PCR primer pairs lying within the most highly conserved, virus-specific E1 gene, which encodes an ATP-dependent DNA helicase. However, it has been recently reported that the primer pair CP4/CP5, used previously (for example, by Iftner and colleagues with 72 SCC and 18 BCC tumors), is not suitable for amplification due to interference in typing by sequencing (Tieben et al., 1993
). To avert this problem, we used an approach that involves PCR amplification of a small 114 bp nucleotide sequence from the HPV E1 open reading frame followed by a reverse hybridization step. The high degree of conservation of the viral E1 gene, as compared with that of the viral L1 gene, ensures that all 25 β-HPV types can be detected using a single amplicon with a sensitivity as high as 10 viral copies per cell. In comparison with PCR-based methods that have detection limits as high as 5 × 1015
, the analytical sensitivity of the primer set for each of the 25 β-HPV types in the reverse hybridization assay method is between 10 and 100 copies per PCR. Using this method, we found a substantial number of lesions (77.6% of BCCs and 83.7% of SCCs) from immunocompetent individuals tested positive for β-HPV. Natural HPV contamination (detection of passenger HPV types), although unlikely, cannot be fully excluded. Arguing against this is a study that examined this issue in eyebrow pluckings and observed unique HPV profiles among five individuals who shared a student household (de Koning et al., 2007
Using the reverse hybridization assay method, we detected nearly all β-HPV types in our study. In total, 24 of 25 known β-HPV types were detected; HPV 25 was not observed. Although HPV 25 has been previously found in the normal skin and flat wart-like lesions, the same studies also found no evidence of HPV 25 in any malignant skin tumors. Although no significant difference was found between SCC and BCC samples with regard to β-HPVs overall, there was a statistical difference when the types were limited to those belonging to species 1, emphasizing the potential importance of this highly related (70% nucleotide identity) subgroup of viruses.
In agreement with previous studies (Berkhout et al., 2000
; Karagas et al., 2006
), multiple β-HPV infections were detected in 67.3% of SCC samples, as compared with 50.0% of BCC lesions. Competition among different genotypes and the preferential use of a subset of PCR primers in the mix of broad-spectrum primers could have affected our results. On the other hand, if one HPV genotype is present in high molar excess over the other, the minor genotype could be out-competed and remain unidentified. The phenomenon is a common problem in broad-spectrum PCRs and can lead to an underestimation of the number of HPV genotypes within the same sample; however, we would expect any possible misclassification of the minor genotype to be non-differential by histology. Furthermore, the direct comparison of HPV profiles from matched normal skin to tumor profiles from each patient may strengthen our findings and help clarify which HPV types may be more important in tumor formation and/or progression.
HPVs encompass the most common “carcinogenic” viruses. Although a connection was discovered nearly a century ago, an etiological relationship between cutaneous HPV infection and skin cancers remains speculative. Our findings suggest that a large fraction of SCC and BCC tumors contain HPV DNA of the genus β-types, and specifically indicate a potential role of β1 and possibly other β-types in SCC occurrence.