Previous studies have proposed β-HPV as a potential causative agent in cuSCC, citing the presence of viral DNA in tumor tissue, but these have not definitively proved an epidemiologic association or evaluated any particular mechanism of transformation. We used whole transcriptome sequencing to test the hypothesis that HPV is required for the maintenance of cuSCC through expression of viral oncoproteins. Transcriptome sequencing revealed a complete absence of HPV mRNA in these tumors, similar to paired normal skin. This stood in stark contrast to the abundant HPV messages detected in cervical SCC and its derivative HeLa cell line. Our results in fact, contradict the hypothesis that expression of viral oncogenes is required for maintenance of cuSCC.
Periungual SCC represents a special site on the cutaneous epithelium. These tumors are associated with high-risk α-HPV(Alam et al., 2003
; Kreuter et al., 2009
; Moy et al., 1989
), which has been reported as episomal, and in a single case, integrated(Sanchez-Lanier et al., 1994
; Theunis et al., 1999
). In our control periungual SCC, β-HPV8 and FA51.2 DNA were detected along with α-HPV16. This tumor contained 46.9 α-HPV16 copies per cell but only 0.3 β-HPV8 copies per cell. HPV16 mRNA reads represented 0.06% of the transcriptome, but no β-HPV mRNA reads were detected. Taken together, this control specimen supports our impression of α-HPV as the driver and β-HPV as a mere passenger in periungual SCC.
As in previous studies(Antonsson et al., 2000
; Asgari et al., 2008
; Berkhout et al., 2000
; Berkhout et al., 1995
; Boxman et al., 1997
; de Koning et al., 2007
; de Koning et al., 2009
; Forslund et al., 2007
; Forslund et al., 2003a
; Forslund et al., 2003b
; Harwood et al., 2000
; Hazard et al., 2007
; Shamanin et al., 1994
; Shamanin et al., 1996
), β-HPV DNA was detectable by nested PCR in 30% of SCC, but was also found in a comparable proportion (28%) of normal skin samples. Moreover, we found extremely low viral loads in tumors that were positive for the viral genome. In all but 3 tumor samples, the viral load was less than 1 copy per cell. Importantly, the 3 contradictory samples all came from a single renal transplant recipient with multiple KAs of the lower leg and may reflect a unique feature of that case. Use of PCR and sequencing allowed identification of a broad range of HPV types although the multiplicity of infection may be limited by the number of clones sequenced. Alternate methods for β-HPV detection such as line blots and microarrays allow simultaneous detection of types but are limited in the types detected. While DNA from other HPV types may be present in these samples, this does not alter the conclusion of this study. The low copy number of β-HPV DNA, combined with the absence of virally-derived oncogenic messages, strongly suggest that β-HPV transcription is not required for tumor maintenance.
Our data were consistent with previous evidence that β-HPV merely colonizes the skin. Immunosuppression and older age were associated with a higher prevalence and viral load of β-HPV, consistent with prior studies(Boxman et al., 2001
; de Koning et al., 2009
; Struijk et al., 2003
). These phenomena likely reflected the role of the immune system and age-related immune senescence in controlling epidermal colonization with HPV rather than explaining the increased incidence of SCC in OTRs and older patients. The prevalence of HPV DNA in tape-stripped biopsies is far lower than that on the surface, further supporting a passenger role(Forslund et al., 2004
). Support for β-HPV as a passenger also comes from a study of tumors from patients with xeroderma pigmentosum, in which prevalence of viral carriage increases with age and is very low in tumors from children(Luron et al., 2007
). A reversed relationship in which SCC somehow results in the presence or increase of β-HPV DNA or antibodies is possible, although further investigation would be required to substantiate this.
Insertional mutagenesis is another mechanism of viral oncogenesis; this mechanism has been described for oncoretroviruses but not for DNA viruses. High-risk α-HPV types can integrate into the host genome but require continual expression of the viral E6 and E7 proteins for their oncogenic activities(Dyson et al., 1989
; Scheffner et al., 1990
; Werness et al., 1990
). The recently described Merkel cell polyomavirus (MCV), another small DNA oncovirus, also integrates into the host genome(Feng et al., 2008
), but continued expression of the MCV truncated large T antigen is similarly required for carcinogenesis(Houben et al., 2010
). In contrast, there are no reports of β-HPV integration into the genome of cuSCC. The low viral loads of β-HPV in cuSCC reported here further indicate that, even if β-HPV had integrated, only at most only a small proportion of the genomes within any tumor could contain integrated β-HPV, casting doubt upon integration as a carcinogenic mechanism.
It has also been suggested that β-HPV might play a role in induction but not maintenance of cuSCC (based on higher viral load of HPV in precancerous actinic keratoses versus primary SCC, metastatic tumor, or perilesional skin(Weissenborn et al., 2005
)). This may occur by interfering with cellular DNA repair or apoptosis following UV-irradiation, creating a pool of genomically unstable cells at risk of oncogenic transformation. Our study was not designed to address this hypothesis. But it should be noted that such a hypothesis would represent a substantial departure from the role played by α-HPV in mucosal SCC, and from the carcinogenic mechanisms known to be employed by other families of DNA tumor viruses in general. Therefore, the most straightforward interpretation of our data is that the sporadic and low-level presence of β-HPV genomic DNA in these tumors, unaccompanied by evidence of active viral gene expression, most likely represents colonization rather than an etiologic association.