No evidence that human papillomavirus is responsible for the aggressive nature of recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma

TO THE EDITOR

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin disease caused by mutations in the gene encoding type VII collagen (Christiano et al., 1993). The condition is characterized by skin fragility, trauma-induced skin blistering and chronic non-healing wounds (Mellerio et al., 2007). Patients with RDEB are at strongly increased risk of developing aggressive cutaneous squamous cell carcinoma (SCC) which is the cause of death by age 45 years in 70% of individuals with the most severe form of RDEB (Fine et al., 2009). Potential similarities between the microenvironment of non-healing wounds and mucosal epithelia where human papillomaviruses (HPV) have been shown to induce the development of SCC (Zur Hausen, 2009) led us to investigate whether HPV infection could be responsible for the increased incidence and aggressive nature of RDEB SCC. The involvement of HPV in the development of cutaneous SCC remains controversial except in the rare genodermatosis epidermodysplasia verruciformis (EV) where up to 60% of patients develop SCC containing high copy numbers of beta-PV (β-PV) (Harwood and Proby, 2002) which_may be restricted to a minority of tumor cells (Dell’Oste et al., 2009). The possibility that HPV are also involved in RDEB SCC has not been addressed.

HPV are a diverse group of small double-stranded DNA viruses that infect squamous epithelial cells. The two largest genera are alpha-PV (α-PV), comprising all HPV genotypes found in mucosal lesions as well as many of those associated with benign skin warts, and β-PV, containing HPV types which have most frequently been associated with EV and cutaneous SCC (de Villiers et al., 2004). Because vaccines against prevalent high risk mucosal/genital HPV (HPV16/18) are available and effective at preventing cancer development (Zur Hausen, 2009), we tested RDEB SCC for the presence of 18 high risk α-PV types using the digene HPV genotyping reverse hybridization assay (RHA) detection kit (Qiagen, Leiden, The Netherlands). We tested DNA prepared from 21 separate SCC isolated from 12 RDEB patients as well as DNA from 39 organ transplant recipient (OTR) SCC and 18 immunocompetent (ICP) SCC collected for a separate study (Purdie et al., 2009) with 6 vulval SCC samples included as positive controls. All DNA were isolated from frozen tissue collected after informed consent and in accordance with Helsinki guidelines following research ethics committee approval. All cutaneous SCC from RDEB and non-RDEB individuals were negative, whereas 9 different α -PV types were detected in the vulval SCC samples.

Next we tested the presence of beta-PV using the RHA kit skin (β) HPV detection system (Diassay, Rijswijk, The Netherlands) (de Koning et al., 2006). In addition to the cutaneous SCC samples described we tested 7 peri-tumoral skin samples from 5 of our SCC RDEB patients and normal uninvolved skin from 11 RDEB patients who had not yet developed SCC. As before, all DNA were isolated from frozen tissue. Data are summarized in Table 1. The overall detection rate of β-PV in RDEB SCC was 90% compared with 74% and 78% for OTR SCC and ICP SCC respectively, with similar frequencies observed in RDEB peri-tumoral and normal skin. As β-PV infection has been shown to be prevalent at low copy number in non-tumour bearing normal skin and hair follicles, a role for these viruses is perhaps more plausible in lesions with higher viral load (Feltkamp et al., 2008). We next examined viral load for β-PV using quantitative PCR (Q-PCR) as described (Weissenborn et al., 2010), with input cell equivalents determined by normalization to β-globin. HPV β types were chosen for investigation on the basis of their frequent detection in at least 2 of the 3 groups of SCC samples (Table 1). Data are summarized in Table 2. Only 25-38% of the RHA-positive SCC samples analyzed were above the detection threshold for Q-PCR, suggesting that the initial RHA detection method was extremely sensitive. The Q-PCR detection efficiency in RDEB SCC was not significantly different from that reported in a previous study of predominantly ICP SCC (Weissenborn et al., 2005) where 46% of samples showed detectable β-PV viral load (p=0.248, Fisher’s exact test). Furthermore, the relative viral load in the two studies were very similar, with all SCC showing comparatively low overall HPV-DNA copy numbers of less than one per five cell equivalents. It must be emphasized however that these data do not exclude the possibility of a considerably higher viral load in a subset of tumor cells, as recently demonstrated for EV SCC (Dell’Oste et al., 2009). With the exception of 2 HPV8-positive samples where viral load were comparable, the RDEB peri-tumoral and normal skin samples examined were below the Q-PCR detection threshold. These combined data imply that high copy number HPV infection is not a general feature of RDEB skin, despite its unique microenvironment. In particular, RDEB SCC does not appear significantly different from other cutaneous SCC in overall prevalence of HPV infection or viral load.

Table 1.

Percentage samples positive for β-HPV by reverse hybridization assay with multiple infection indicated.

Sample Group	RHA- positive	Types most frequently detected	Number of types detected1
			1	2	3	4	5
OTRbSCC (n=39)	74% (29)	23 (9) 15 (7) 5 (4) 93 (4)	31% (9)	28% (8)	21% (6)	3% (1)	17% (5)
ICcSCC (n=18)	78% (14)	5(2) 8 (2) 9 (2) 24 (2)	43% (6)	36% (5)	7% (1)	7% (1)	7% (1)
RDEB SCC (n=21)	90% (19)	23 (6) 12 (4) 15(4) 8 (3) 24 (3)	37% (7)	37% (7)	11% (2)	5% (1)	11% (2)
RDEB peri- lesional skin; patients with SCC (n=7)	71% (5)	17 (4) 8 (2) 23 (2) 24 (2)	40% (2)	20% (1)	-	-	40% (2)
RDEB normal skin; patients without SCC (n=11)	82% (9)	23 (8) 49 (5) 17 (3)	33% (3)	-	56% (5)	11% (1)	-

Abbreviations: HPV, human papillomavirus; IC, immunocompetent; OTR, organ transplant recipient; RDEB, recessive dystrophic epidermolysis bullosa; RHA, reverse hybridization assay; SCC, squamous cell carcinoma.

¹Shown as a proportion of RHA-positive samples.

Numbers in parentheses indicate number of samples positive.

Table 2.

Analysis of viral load for HPV types 5, 8, 15, 23 and 24 in each sample group.

Sample Group1	Q-PCR positive
	Total	HPV52	HPV8	HPV15	HPV23	HPV24
OTR SCC (n=13)	38% (5)	75% (3 of 4); 1 per 97,1 per 883, 1 per 4702	50% (1 of 2); 1 per 4445	14% (1 of 7); 1 per 176	11% (1 of 9); 1 per 30	33% (1 of 3); 1 per 1407
IC SCC (n=7)	29% (2)	50% (1 of 2); 1 per 320	50% (1 of 2); 1 per 80	0 (0)	0(1)	0(2)
RDEB SCC (n=12)	25% (3)	50% (1 of 2); 1 per 674	66% (2 of 3); 1 per 15, 1 per 393	0(4)	17% (1 of 6); 1 per 9	33% (1 of 3); 1 per 79
RDEB peri- lesional skin; patients with SCC (n=3)	67% (2)	0(0)	100% (2 of 2); 1 per 38, 1 per 58	0(0)	0(2)	0(2)
RDEB normal skin; patients without SCC (n=8)	0%	0(0)	0(0)	0(0)	0(8)	0(0)

Abbreviations: HPV, human papillomavirus; IC, immunocompetent; OTR, organ transplant recipient; Q-PCR, quantitative PCR; RDEB, recessive dystrophic epidermolysis bullosa; RHA, reverse hybridization assay; SCC, squamous cell carcinoma.

¹Number of samples positive for types 5, 8, 15, 23 or 24 by RHA: note that figures are not additive due to the presence of multiple infections.

²Viral loads were calculated per cell equivalent by normalizing to beta globin.

To our knowledge the possible contribution of HPV infection to RDEB-SCC pathogenesis is previously unreported. Although our numbers are small, 21 cases of RDEB SCC is a significant series for this rare disease and compares favorably with other examples of analysis in this tumor group (23 RDEB SCC examined for p53 immuno-staining (Slater et al., 1992) and 25 RDEB SCC analyzed for matrix metalloproteinase (MMP) immuno-staining (Kivisaari et al., 2008) for example. The use of highly sensitive PCR methodology to detect relatively ubiquitous viral DNA sequences together with confounding factors such as type of tissue analysed have yielded conflicting data on HPV prevalence in OTR versus ICP individuals, actinic keratosis versus SCC, and normal skin (Harwood and Proby, 2002; Weissenborn et al., 2005; Feltkamp et al., 2008; Mackintosh et al., 2009). Nevertheless, combined viral load data have shown that in contrast to α-PV and cervical cancer, β-PV presence is not necessary in each tumor cell for maintenance of the malignant phenotype, although this does not preclude a role for HPV in cutaneous SCC. Our results from RDEB SCC lie within the range of other cutaneous SCC data and show no evidence of HPV contribution to the increased incidence and aggressive nature of RDEB SCC.