HPV5b variant in a neoplastic lesion of an Italian patient affected by epidermodysplasia verruciformis

ARTICLE

Epidermodysplasia verruciformis (EV) is a rare dermatosis that is associated with cutaneous cancers in about half of the patients [1]. EV can be defined as a genetic disorder that determines a lifelong infection of the skin by human Papillomaviruses (HPV) [2]. The EV-HPVs are harmless for the general population and have been found in skin cancers of immunosuppressed patients [3]. These findings suggest that defective immune surveillance can be responsible for both the EV-HPV infection persistence and the EV-HPV associated cancers. The cutaneous lesions of EV patients are benign at first, usually starting in early childhood and resembling plane warts of the hands and face. Carcinomas develop after a long latent period and frequently arise in skin exposed to sunlight, evoking not only DNA damages but also a local or systemic immunosuppression [4]. The benign lesions contain different EV HPVs; whereas HPV types 5, 8, and, much less frequently, types 14, 17, 20 and 47 can be detected in the tumours [1]. Variants of HPV5 have been recognized on the basis of the genetic heterogeneity of the E6 open reading frame (ORF), indicating an extensive variability similar to that reported for the widespread HPV16 variants [5]. To address the question of the genetic variability and stability of the variants, it is important to have data from different geographical areas. In view of the rarity of the EV patients, we report here on a case of classic EV associated with cutaneous cancers in which we detected the presence and expression of the HPV5b variant. This variant has been already detected in a Japanese [6] and a Polish patient [5]. Our findings reinforce the hypothesis that this variant is stable.

Case report

A 51-year-old Caucasian man was referred for the development of new cutaneous lesions on the right thorax. The patient reported the appearance of reddish papules similar to flat-warts on the dorsum of the hands at the age of 7-8 months and thereafter on the trunk. At the age of 10 years, the patient was hospitalised for the appearance of new lesions on trunk, face and arms. First evidence of malignant transformation was at the age of 23 when an epithelioma on the forehead was surgically removed. Thereafter, the patient underwent surgery for a bowenoid carcinoma on the back at the age of 42 and for a squamous cell carcinoma in an area of actinic keratosis on the internal corner of left eye three years later.

The clinical examination revealed the presence of two little, contiguous, red, dome-shaped papular lesions with a rough surface on the right clavicular area (Fig. 1). On the trunk and arms, widespread reddish, pigmented or depig-mented flat warty lesions, somewhat scaly, of different size were observed (Fig. 2); some of these lesions were pityriasis versicolor-like.

Two skin biopsy specimens were taken from the dome-shaped lesion of the right clavicular area and from a flat warty lesion on the right hand dorsum. Multiple paraffin sections were stained with hematoxylin and eosin. One of the two clavicular lesions revealed the presence of an epithelial neoplasm with solid pattern and minimal keratin differentiation. This neoplasm showed cyto-architectural disorder with some large, atypical cells with clumped chromatin and mitotic figures (Fig. 3). These features were consistent for a carcinoma in situ (Bowen's disease). The flat lesion on the hand back showed acanthosis, hyperkeratosis and hypergranulosis with downward proliferation of the rete ridges. In the upper levels of the epidermis swollen cells with blue-gray pale cytoplasm and nuclear pleomorphism were detected; these features were consistent for EV-specific lesions (Fig. 4).

Paraffin embedded sections of 20 nm thickness from the histological preparations were dewaxed, ethanol dehydrated and utilized for the DNA extraction, as previously described [7]. RNA extraction was carried out by a commercial kit (AMBION, Austin, TX, USA), according to the manufacturer's instructions.

DNAs were subject to PCR amplification with CP 65-70 primers that amplify a large number of cutaneous and EV associated HPVs [8]. Both samples from the thorax and hand showed an amplified band corresponding to the presence of HPV sequences. To ascertain the occurrence of the specific types more frequently associated with carcinoma, DNA samples were also amplified with specific primers for the E6 region located between nt 209 to 229 (5'-GGAGCCGAACACCAACAGAA-3') and nt 424 to 444 (5'-CGACAGCACGC AAACACACA-3') for the HPV5 and between nt 527 to 547 (5'-GGTGTCAAAA CTGCTTGTCA-3') and nt 604 to 624 (5'-CCTTTCCAGCCT CCTCTAAC-3') for HPV8, respectively. As shown in the left panel of Figure 5, only primers for HPV5 were able to produce a band of the predicted length in ethidium bromide stained agarose gel. Southern blot hybridization with enzyme linked internal probe confirmed the detection of HPV5 sequences. The transcriptional activity of the virus was also evaluated by analyzing total RNA extracted from the histological preparation by RT-PCR. Briefly, RNA was reverse transcribed in cDNA by a commercial kit (Gene Amp, Roche Molecular Systems Inc., NJ, USA) utilizing random exhamers as primers. The cDNA was subjected to nested PCR with the above-mentioned E6/HPV5 primers. The first step of amplification consisted of 30 cycles with each cycle running 1 min at 95° C, 1 min at 55° C and 2 min at 72° C. The second step of amplification was performed on 5 microl of the first step PCR, in the same conditions. As shown in the right panel of figure 5, samples contained HPV5 E6 transcripts. The PCR amplified products of 235 bp from patient's DNA were subjected to direct automated sequencing. Sequence analysis showed a perfect homology to the sequences of the E6 region of the HPV5b variant [5-6]. The same sequence was present in both samples from thorax and hand.

Discussion

EV is a genodermatosis with onset between 5-10 years of age. The first symptom suggestive of EV is the dissemination on the trunk, neck, and face of lesions resembling flat warts, more red or brownish than common warts. Twenty-thrity years from the appearance of the first benign lesions, malignant conversion starts in more than 50% of the patients. We report here a typical case of EV with a very early onset of the disease and the development of skin tumors from the twenties. The clinical features with the widespread plane warts and the pytiriasis versicolor-like lesions together with the onset in early childhood and the clinical evolution are pathognomonic of EV. The specific histological findings confirm the clinical diagnosis [9].

A key aspect of EV disease is the infection by EV types of HPV. Types 5 and 8 seem to be more closely related to the conversion to malignant lesions. The well-known appearance of tumour lesions in the skin surface exposed to sunlight indicates that UV can influence the clinical course of the disease by inducing DNA damage and immunosuppression [10]. One of the hypotheses is that UV may induce mutations in the HPV genome within the lesions. Genetic mutations of HPV5 have been detected in different patients worldwide and, in the literature, sequences of many variants have been published [11]. These sequence mutations may represent stable variants of the virus that could be more closely related to the oncogenic potential of each particular type. As EV is a rare disease it is important to obtain data not only about the type of HPV involved but also about the specific variant. By this analysis the oncogenic potential of a single variant may be ascertained. At present, no conclusive data about the major association with oncogenic features can be withdrawn, as the same variants have been isolated from both benign and malignant lesions of the same or different patients. The identification of the HPV5b variant in our typical case of EV adds new data about this variant that was first isolated in a Japanese patient and thereafter in a Polish one. The identity of our sequence with that published clearly indicates that the HPV5b is a stable variant, which may be found in the neoplastic lesions of EV patients [1, 5, 6]. The oncogenes of EV-viruses appear to be E6, E7 and E2. The HPV 5, like other 'high risk' EV-viruses, differs from related HPV types in the transforming activity of the E6 gene [12]. In benign lesions, E6 expression is usually under tight control as demonstrated by the detection of rare E6 transcripts in a few cells within the superficial layers of the epidermis [13]. The RT-PCR assay employed in our experiments is unable to detect differences in the expression level of E6 mRNA transcripts and therefore we cannot draw any conclusion about the hypothesis of a lack of cellular control and active involvement of this viral protein in the development and/or maintenance of the neoplasia. Nevertheless, the presence of E6 transcripts in both benign and neoplastic lesions of our patient indicates that the HPV5 DNA sequences are transcriptionally active.

CONCLUSION

In conclusion, our case report indicates that the same HPV variants can be detected in different geographical areas and therefore, as hypothesized by Deau et al. [5], some cellular genes may exert a selection pressure so strong as to induce the emergence of this HPV5b infectious variant.

Acknowledgements

Federica Poggiali is a recipient of a fellowship from Lega Italiana Tumori. Work partially supported by Biotechnology Project CNR and Ministry of Health.

Article accepted on 28/5/01

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