Pietro Donati; Luca Muscardin; Ada Amantea; Francesca Paolini; Aldo Venuti

doi:10.1684/ejd.2009.0629

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Detection of HPV-15 in painful subungual tumors of incontinentia pigmenti: successful topical therapy with retinoic acid

European Journal of Dermatology. Volume 19, Numéro 3, 243-7, May-June 2009, Therapy

DOI : 10.1684/ejd.2009.0629

Summary

Auteur(s) : Pietro Donati, Luca Muscardin, Ada Amantea, Francesca Paolini, Aldo Venuti , Laboratory of Cutaneous Histopathology, Institute of Dermatology S. Maria and San Gallicano, Via Chianesi, 53, 00144 Rome, Italy, Laboratory of Virology, Regina Elena Cancer Institute Via delle Messi d’Oro 156 00158 Rome Italy.

Illustrations

ARTICLE

Auteur(s) : Pietro Donati¹, Luca Muscardin¹, Ada Amantea¹, Francesca Paolini², Aldo Venuti²

¹Laboratory of Cutaneous Histopathology, Institute of Dermatology S. Maria and San Gallicano, Via Chianesi, 53, 00144 Rome, Italy
²Laboratory of Virology, Regina Elena Cancer Institute Via delle Messi d’Oro 156 00158 Rome Italy

accepté le 3 Decembre 2008

Incontinentia pigmenti (IP) is an X-linked dominant disorder, which occurs in female patients and is lethal for male foetuses. The mutated gene is the inhibitor of kappa light polypeptide gene enhancers in B-cells, kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential modulator (NEMO). This gene, located in the Xq28 chromosomal region, regulates the NF-kb signaling pathway. The altered NF-kb activity inhibits the production of chemokines, adhesion molecules and cytokines, leading to an increase cellular sensitivity to apoptosis [1].

In the skin the disease typically evolves in four stages: an inflammatory (vesiculobullous) stage, a verrucous stage, a pigmented stage and the fourth and final hypopigmented-atrophic stage [2]. The majority (80%) of patients are affected by multiple ectodermal and mesodermal defects. Among the ectodermal defects nail dystrophy is a rare manifestation, occurring in 7% of patients [3, 4]. In a more recent review of 40 cases of IP, “nail dystrophy” was present in 10% of cases [5]. Given the very low numbers of IP reported in literature, firm data concerning the incidence of subungual tumors in IP patients cannot be extrapolated. Subungual painful tumors and onycodystrophy could be different manifestations of IP nail defects.

We discuss a typical case of IP with subungual tumors (STIP) with a short review on the occurrence of these tumors in IP. For the first time the presence of Human Papillomavirus (HPV) type 15 was detected in a tumoral subungual lesion and topical therapy with retinoic acid cured the lesions.

Case report

Three years ago a 31-year-old Caucasian woman noticed the onset of a subungual nodule on the third finger of the left hand, with a painful symptomatology and, subsequently, other nodules appeared under other nails. The family anamnesis revealed that she had four brothers and seven sisters, all without dermatological diseases. After birth she suffered a cutaneous vesiculobullous eruption, mostly localized on the inferior limbs, where now it is possible to see hypopigmented linear lesions (figure 1A). These clinical manifestations were not referred to as IP, and were treated as an aspecific dermatitis, which resolved after a short time. The clinical examination revealed: i) a partial anodontia of the right mandibular arcade (figure 1B); ii) blindness of the right eye (figure 1C); iii) an hyperkeratotic growth and destruction of the nail plate with associated onycolysis in the third finger of the left hand; iv) a hyperkeratotic growth under the nail plate, which was conserved with partial onycolysis, on the fourth finger of the right hand (figure 1D). The patient complained of a continuous painful symptomatology in these lesions, clinically a squamous neoplasia was suspected for the lesion of the third finger of the left hand and biopsies of the nail bed were performed from both lesions. The histological examination of the nail plate showed an acanthotic epithelium with pseudoepitheliomatous features and the presence of numerous dyskeratotic and necrotic keratinocytes (figure 2A). The granular layer contained clumps of keratohyaline granules with hypergranulosis, and the presence of a few large vacuolated cells (koilocyte-like cells) was detected in the more superficial part of the malpighian layer.

In addition to the histological examination, consecutive paraffin embedded sections were de-waxed by xylene/ethanol treatment and utilized for the DNA/RNA extraction according to standard procedures. PCR-grade DNA was extracted from both lesions whereas PCR-grade RNA was obtained only from the biopsy of the third finger of left hand, because the RNA in the other biopsy was completely degraded and no amplification was revealed with primers for housekeeping genes (beta-actin and beta-globin).

DNA was analyzed by PCR for the presence of HPV utilizing the primers of Berkhout et al. [6]. Sequence DNA analysis of the amplified products showed the presence of a DNA sequence overlapping that of the HPV 15. Total RNA was also tested by RT-PCR using the One step commercial kit (Invitrogen, Milan, Italy) with specific primers for the E7 gene of HPV 15. The presence of a band corresponding to the product of the HPV 15 E7 amplified product was revealed and confirmed by sequence analysis (figure 2B), demonstrating that the virus was transcriptionally active.

The diagnosis of painful subungual tumors in Incontinentia Pigmenti was made on the basis of the histological examination with the presence of dyskeratotic cells, the discovery of achromic lesions on the inferior limbs and the presence of other somatic defects (dental and ocular abnormalities). A topical therapy with 0.05% retinoic acid cream, twice a day, was prescribed. After one month of therapy the patient referred an improvement of the painful sensation and after six months a definitive clinical resolution was achieved with both a decrease of the tumor burden and also pain release (figure 3).

Discussion

Our case is a typical IP in the fourth stage; the diagnosis was achieved in adult life on the basis of ocular and dental abnormalities, achromic lesions on the legs and the presence of painful subungual tumors. The differential diagnosis of STIP (table 1) must be made among viral warts, epidermoid cysts, squamous cell carcinoma and subungual keratoacanthoma [4, 7-10]. The clinical and histological features of all these diseases allow the differential diagnosis with STIP. Only keratoacanthoma shares very similar clinical (i.e. multiplicity and spontaneous regression) and histological aspects (i.e. dyskeratotic and koilocyte-cell), but the different age of onset, the gender and, finally, the other associated symptoms, can differentiate the two diseases. To date, 14 cases of STIP have been described in the literature and the reported clinical/histological features indicate that in our patient a correct diagnosis of STIP was made (table 2) [3, 4, 9-16]. Clinically all the reported cases had hypopigmented lesions on the limbs and, in four patients, a partial alopecia was described. Ocular abnormalities were present in four patients and dental defects were recorded in ten patients. The histological features of these literature cases were homogeneous with pseudoepitheliomatous hyperplasia and dyskeratotic cells and some authors described particular cells with a clear and “glassy” cytoplasm [4, 9], suggestive of viral infection [14]. In addition, pregnancy seems to be a favorable factor, as a regression of STIP has been detected during gestation in two literature cases, even if spontaneous regressions were reported in six patients. Indeed our patient reported a moderate regression of STIP symptoms in pregnancy.

The presence of HPV-15 in STIP in our case may open a new scenario in the pathogenesis of such lesions. HPV-15 is a cutaneous HPV of beta genus [17] that induces skin infections and tumors in immunosuppressed as well as in immunocompetent patients [18]. This virus was also found in pre-neoplastic lesions like actinic keratoses [19]. Moreover, the presence of viral transcripts for the E7 gene strongly suggests that HPV-15 is not a simple passenger in this lesion but it may act as a driver of these subungual tumors.

To the best of our knowledge, this is the first report of HPV in STIP. No previous publications have reported an attempt to identify HPV by sensitive methods like PCR in subungual tumours; only Simmons [11] attempted to identify HPV virus by an immunoperoxidase technique, but he failed to detect any papillomaviruses. The technique used, that may not have been sensitive enough for the detection of HPV in these lesions, may account for this negative result.

HPV infections could be the cause of these tumors. The cells in which the mutant X chromosome is active are deficient for the NEMO protein and, after an initial hyper-proliferation, are then gradually eliminated through apoptosis, leading to the classic progressive resolution of the skin lesions. This scenario could be dramatically modified in the presence of cutaneous HPV because these viruses exert an anti-apoptotic activity. The late appearance of STIP is consistent with the long latency of the papillomavirus infection. Moreover in the literature most of the STIP cases are reported as dyskeratosis, or described as “glassy appearing” [4] or “clear aspect with pseudo-vacuolated cytoplasm and pyknotic nuclei” [9]. These histological features are also conceivable with a pathological role of papillomaviruses in these tumors. Nevertheless the possibility that the virus is present as the result of a coincidental infection of the lesions cannot be excluded. Studies on a large number of STIP patients are needed to address this issue.

Although STIP may resolve spontaneously, surgical or medical approaches have been undertaken. Abimelec et al. tried a therapy with topical fluorouracil, that was effective in relieving and reducing the keratotic mass [15]. Mascaro et al. [9] used etretinate 1 mg/kg for three months with clinical resolution of the tumors. It has been reported that vitamin A and its analogues inhibit the proliferation of cells associated with HPV infection by restoring apoptosis processes [20] and by inhibiting viral transcription [21], suggesting promising effects of retinoid therapy in inhibiting the progression of early cervical lesions to cancer. Moreover successful therapy was achieved in HPV 2 positive multiple verrucae vulgaris by retinoids and interferon alfa [22].

On the basis of these previous results, we tried topical 0.05% retinoic acid cream, twice a day, achieving a cure of the STIP after six months. The retinoic acid effect could be related to its action on ungual keratinocyte differentiation (classical action of retinoids) and/or on its experimentally-proved antiviral activity [20-22]. Although the hypothesis of a spontaneous resolution has to be taken in account (table 2), in our case these subungual tumors lasted for three years without spontaneous resolution, and only after the topical therapy with retinoic acid did the tumors resolve.

In conclusion we made a diagnosis of IP starting from the late event of this disease consisting of subungual tumors; and therefore, in our opinion, all subungual tumors should be considered as a possible late manifestation of this pathology. Further, the presence and, in one of them, the expression of HPV-15 in the lesions suggest a possible role of these viruses in the genesis of these tumors, representing the “rationale” for the use of different therapeutic approaches. Retinoids, with their synergic effect on the differentiation pathway and on the viral activity, or immunomodulators like imiquimod which has been shown to have an indirect effect on HPV infection, are promising candidates for a topical treatment of these lesions.
Table 1 Differential diagnosis

	STIP	Subungual keratoacanthoma	Viral Warts	Squamous cell carcinoma	Epidermoid Cysts
Age	12-15 years	30-70 years	No predictable	60-70 years	No predictable
Sex	Women	Men > Women 3.5:1	No predictable	Men >> Women	No predictable
Number of lesions	Multiple	Single/Multiple	Single/Multiple	Single	Single/Multiple
Localization	Under nail	Under nail	Beside and Under nail	Beside and Under nail	Under nail
Pain	Yes	Yes	No	Yes ±	Yes (late onset)
Associated nail changes	Onycodystrophy	No	No	No	No
Evolution	Sometimes Spontaneous regression	Sometimes Spontaneous regression	Sometimes Spontaneous regression	No Spontaneous regression	No Spontaneous regression
Histology	Hyperkeratosis Parakeratosis Dyskeratotic cell	Hyperkeratosis, vertically oriented parakeratosis, some dyskeratotic cells	Acanthosis Hypergranular koilocytes	Cystic Space	Atypia Mitosis

Table 2 STIP literature data

Patient (ref)	Age/sex	Symptoms	Spontaneous/Pregnancy regression	Histology	Dental lesions	Ocular lesions	Therapy
1 [3]	20/F	Pain, trunk pigmentary lesions	Yes/Yes	PEH^*, dyskeratosis	Yes	No	Surgery
2 [8]	16/F Twin of 3	Pain, alopecia	Yes/No	Acanthosis, papillomatosis, parakeratosis, dyskeratotic and clear cells in granular layer	Yes	No	Surgery
3 [8]	16/F Twin of 2	Pain	Yes/No	See above	Yes	No	Surgery
4 [9]	28/F	Pain, leg achromic lesions	No/No	PEH^*, hyperkeratosis, hypergranulosis, dyskeratotic cells	Yes	No	Etretinate
5 [11]	22/F	Pain, leg achromic lesions	No/No	PEH^*, dyskeratotic cells	Yes	No	Surgery
6 [12]	15/F	Tenderness, leg achromic lesions	No/No	See above	Yes	No	Surgery
7 [12]	23/F	Tenderness, alopecia, leg achromic lesions	Yes/No	Not performed	Yes	No	Spontaneous resolution
8 [13]	24/F	Pain, leg whorled pigmented lesions	No/No	Acantosis,hyperkeratosis, parakeratosis (diagnosis of subungual keratoacanthoma)	Yes	No	Surgery
9 [13]	31/F	See above	No/Yes	See above	No	No	Surgery
10 [14]	25/F	Pain, leg achromic lesions	No/No	Acanthosis, hyperkeratosis, hypergranulosis (viral-wart-like histology)	Yes	Yes	Surgery
11 [15]	10/F	Pain, alopecia, leg achromic lesions	No/No	Papillomatosis, hyperkeratosis, focal dyskeratosis	Yes	Yes	Fluoro- uracyl
12 [4]	25/F	See above	Yes/No	Acanthosis, papillomatosis, glassy and dyskeratotic cells	Yes	Yes	Surgery
13 [4]	18/F	Pain, leg achromic lesions	Yes/No	Acanthosis, hyperkeratosis, dyskeratotic cells	No	Yes	Surgery
14 [16]	65/F	See above	No/No	PEH^*, dyskeratotic cells, presence of cystic structures	No	No	Acitretin

^*PEH: pseudo epitheliomatous hyperplasia.

Acknowledgements

Conflict of interest: None declared. Financial support: Work partially supported by Italian Ministry of Health grant.

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