Pigmented Epidermal Plaques in Three Dogs
Papillomavirus was identified in pigmented epidermal plaques (PEP) from three dogs: a miniature schnauzer with hyperadrenocorticism and hypoglobulinemia, an American Staffordshire terrier with hypoglobulinemia, and a Pomeranian with unconfirmed hypothyroidism. Squamous cell carcinoma (SCC) arose within several plaques in the Pomeranian. Clinical improvement coincided in the first two cases with treatment of the concurrent disease and the administration of low-dose oral interferon-α. This is the first report of PEP in an American Staffordshire terrier and a Pomeranian. The potential for malignant transformation of PEP to SCC emphasizes the need for recognition and clinical management of PEP.
Introduction
Pigmented epidermal plaques (PEP) have been reported as a condition in the pug, miniature schnauzer, Chinese shar pei, and in individual cases of an English setter and an English pointer.1–5 Affected dogs have multiple cutaneous macules, papules, and plaques of heavily pigmented hyper-plastic epidermis. The dark pigmentation in the lesions results from an abnormal distribution of melanin within the epidermis and an accumulation of melanin in melanophages, rather than from a proliferation of melanocytes. Earlier designations that implied melanocytic hyperplasia, such as lentiginosis profusa, are no longer used.36 Ultrastructural studies of PEP have identified intranuclear inclusion bodies compatible with papillomavirus.3
Pigmented epidermal plaques in dogs may be analogous to epidermodysplasia verruciformis in humans, in which several closely related papillomaviruses cause a generalized distribution of pink to brown macules and verrucous papules and plaques.37–21 Although the viruses that cause epidermodysplasia verruciformis are widespread in the human population, active infections that result in the development of disease are rare.79 An X-linked or autosomal genetic predisposition associated with at least two genes on separate chromosomes leads to increased susceptibility to this group of viruses and the development of disease in predisposed individuals.22 In addition, renal transplant patients and other immunosuppressed individuals that are not members of genetically predisposed groups have a substantially higher incidence of epidermodysplasia verruciformis than the general population.23–26 Development of epidermodysplasia verruciformis in these latter individuals is attributed to defects in cell-mediated immunity and local mechanisms of immune surveillance secondary to their immuno-suppression.1923–27 The plaques in half of epidermodysplasia verruciformis-affected humans undergo malignant transformation to squamous cell carcinoma (SCC) or other nonmelanocytic skin cancers in sun-exposed sites.89131624252829 Although approximately 18 types of human papillomaviruses have been identified in epidermodysplasia verruciformis lesions, most neoplasms are associated only with human papillomavirus-5.912132123243031
This report presents cases of PEP from a miniature schnauzer with hyperadrenocorticism, an immunoglobulin-deficient American Staffordshire terrier, and a Pomeranian with unconfirmed hypothyroidism in which SCC arose by malignant transformation within multiple plaques. Papillomavirus was identified in plaques from all three animals.
Case Reports
Case No. 1
An 11.8-kg, 10-year-old, spayed female miniature schnauzer was presented to the University of Illinois Veterinary Teaching Hospital (UI-VTH) with a 2-year history of pigmented plaques on the flanks and dorsal thorax [Figure 1]. Both the number and size of the plaques had increased during the several months prior to presentation. Multiple, darkly pigmented macules and plaques involved the skin caudal to the dog’s right ear, the interscapular area, over the thoracolumbar spine, at the base of the tail, over both flanks, and on the ventral thorax, abdomen, and groin. The plaques ranged in diameter from 2 to 25 mm and in height from macular to 5 mm. They had a rough surface, a waxy texture, and were friable. Multiple superficial and deep skin scrapings were negative for mites and ova. Impression smears of the plaques and adjacent skin contained no bacteria or Malassezia spp. The abdomen was pendulous, and the dog had gradually become polyphagic and polyuric, but the owner could not state when those signs first appeared.
A complete blood count (CBC), total thyroxine (T4), and thyroid-stimulating hormone (TSH) concentrations were within reference ranges. Elevations of serum alkaline phosphatase (SAP, 572 U/L; reference range, 12 to 110 U/L) and the corticosteroid-induced isoform of SAP (501 U/L; reference range, 0 to 40 U/L) were detected. Thoracic radiographs were unremarkable. On abdominal ultrasonography, neither adrenal gland was enlarged (left, 4.8-mm thickness on caudal pole; right, 5.2-mm thickness on caudal pole; reference range, 4 to 7 mm). Baseline serum cortisol was elevated at 221 nmol/L (reference range, 58 to 144 nmol/L). Two hours after administration of corticotropin (ACTH) gela (2.2 IU/kg intramuscularly), serum cortisol increased to 576 nmol/L (2-hour post-ACTH reference range, 186 to 566 nmol/L). Cortisol was suppressed 8 hours after administration of dexamethasone (0.01 mg/kg intravenously) to 27.6 nmol/L (8-hour post-dexamethasone reference range, 0 to 42 nmol/L). Serum immunoglobulin concentrations were measured, and all were below reference ranges. Immunoglobulin G (IgG) was 923 mg/dL (reference range, 1000 to 2000 mg/dL); immunoglobulin A (IgA) was 8 mg/dL (reference range, 20 to 150 mg/dL); and immunoglobulin M (IgM) was 15 mg/dL (reference range, 70 to 270 mg/dL).
Full-thickness, 6 mm-diameter punch biopsies of papules and plaques were obtained using local anesthesia. Tissues were fixed in 10% neutral-buffered formalin for light microscopy, and additional 2-mm samples were immersed in Karnovsky’s fixative (2.5% glutaraldehyde with 2% paraformaldehyde) buffered with 0.1 molar phosphate for transmission electron microscopy (TEM).3 Some sections from formalin-fixed blocks were assayed by immunohistochemistry for papillomavirus group-specific antigen. Papillomavirus controls were sections of normal canine skin (negative) and sections from canine oral papilloma (positive).
On histopathology, the plaques were hyperkeratotic and exophytic. The epidermal surface had numerous papilliferous folds [Figure 2], with an abrupt change from normal to affected epidermis. The epidermis was heavily pigmented, with melanin present in epidermocytes. Melanocytes did not appear increased in number. Pigment was present within keratinocytes in all cell layers of the epidermis, and dermal pigmentary incontinence was prominent. Although all epidermal layers were thickened, the most dramatic changes affected the stratum granulosum. Cells in the stratum granulosum contained large, keratohyalin-like granules that often obscured the nuclei. Rare cells of this layer contained smudged, intranuclear inclusion bodies. The superficial dermis contained prominent interstitial melanophages along with occasional scattered lymphocytes and plasma cells. Scattered nuclei within epidermal plaques exhibited immunoreactivity for papillomavirus group-specific antigen.
Transmission electron microscopy revealed intranuclear inclusion bodies within the stratum granulosum cells. Particles within the inclusion were arranged in a crystalline array [Figure 3]. At higher magnification, the particles were hexagonal and 25 to 30 nm in diameter. The organization, size, and shape of the particles were consistent with papillomavirus.
To control the animal’s hyperadrenocorticism, oral mitotaneb was initiated at a dose of 35 mg/kg q 12 hours for 5 days, after which baseline cortisol was 149 nmol/L and post-stimulation cortisol was 307 nmol/L. Polyphagia, polydipsia, and polyuria decreased. Mitotane was administered at the same dose, three times weekly for 4 weeks, at which time baseline cortisol was 76.9 nmol/L and post-stimulation cortisol was 219 nmol/L. No new plaques developed after initiation of mitotane therapy, and the texture of existing plaques became less waxy. The dog was then given a maintenance dosage of mitotane (35 mg/kg q 12 hours 2 days per week). Interferon-α 2Ac (IFN-α 2A) was also prescribed (1000 IU per os [PO] on a 21-day on, 7-day off schedule). Most plaques over the dog’s dorsal thorax resolved completely, and few new plaques developed over a 2-year period.
Case No. 2
A 22-kg, 3-year-old, spayed female, white and fawn American Staffordshire terrier was presented to the UI-VTH with multiple pigmented plaques ranging in diameter from 2 to 20 mm on the inner aspects of the apices of both ear pinnae. The first plaque was observed on the inner apex of the left ear 2 years prior to presentation. Additional papules and plaques developed on the inner aspects of the apices of both pinnae. Cytological examination of impression smears of interdigital skin from all four feet revealed Malassezia spp. Impression smears of skin from the left axilla contained cocci. The dog had a history of recurrent small intestinal bacterial overgrowth, inflammatory bowel disease, vaginitis, otitis, and pyoderma beginning at 4 months of age. Immunoglobulin levels measured when the dog was 7 months of age were below reference ranges (IgG, 801 mg/dL; IgA, 7 mg/dL; IgM, 60 mg/dL). Immunotherapy using a staphylococcal antigen-based immunomodulatord was initiated when the animal was 2 years of age; but after 1 year, this had not resulted in any clinical improvement.
Several pigmented plaques were removed by surgical excision with the dog under general anesthesia. Tissues were processed for light microscopy, immunohistochemistry, and TEM. Plaques were formed by localized, irregular thickening of the epidermis. All layers of the epidermis were thickened, and hyperkeratosis was marked. The epidermal cells of the stratum granulosum contained giant, irregular, keratohyalin-like granules that obscured nuclei. Hyperpigmentation was moderate, and mild pigmentary incontinence was present in the dermis. Multiple nuclei within epidermal plaques were immunoreactive for papillomavirus group-specific antigen. Stratum granulosum cells contained intranuclear inclusion bodies consistent with papillomavirus; they were similar in size, shape, and arrangement to those described for case no. 1.
The plaques were ablated using a carbon dioxide (CO2) laser, and IFN-α 2A was administered at the same dose used in case no. 1. No plaques had recurred at the laser ablation sites 18 months after the procedure. No new lesions had developed while the animal was on IFN therapy; however, small papules developed on the inner aspects of both ear pinnae when IFN was discontinued after 12 months because of financial constraints. Those plaques failed to resolve.
Case No. 3
A 4.9-kg, 8.5-year-old, spayed female Pomeranian was presented to the UI-VTH with a 4-year history of multiple, black, hyperkeratotic macules and plaques with friable, papillomatous projections. Erythematous, exudative, and ulcerative lesions developed within some plaques 7 months before presentation. Eighteen months prior to presentation, hypothyroidism was diagnosed by the referring veterinarian on the basis of low T4 values, although test results were unavailable. The owner reported a diminished rate of increase in plaque size and number coincident with administration of thyroid supplementation; however, existing plaques failed to regress. The referring veterinarian surgically removed plaques that had ulcerated and become invasive, and the microscopic diagnosis of several was SCC.
Physical examination at UI-VTH revealed multiple areas of hyperpigmentation and crusting that were most severe on the thorax, ventral abdomen, and groin. Abundant, friable, darkly pigmented, elevated, roughened, proliferative plaques ranging in diameter from 10 to 30 mm were present in the same area. A 10 mm-diameter, darkly pigmented, rough-surfaced macule lined the inner right pinna, and a hyperkeratotic pigmented plaque was on the inner left pinna. A 3 × 2-cm pigmented mass on the dog’s left upper lip adjacent to the nasal planum and a 5 × 2-cm mass extending from the dog’s right axilla to the medial aspect of the elbow were ulcerated. Results of a CBC, serum biochemical profile, and thoracic radiographs were unremarkable. Thyroid levels were not remeasured; thus, the diagnosis of hypothyroidism was not confirmed.
Biopsies of pigmented plaques from two sites on the caudal ventral abdomen, the left upper lip, and the right axilla were taken with the animal under general anesthesia. Tissues were processed for light microscopy and immunohistochemistry as described for case no. 1. Formalin-fixed tissues in paraffin-embedded blocks were deparaffinized using serial xylene washes, then they were post-fixed and processed for TEM.
The epidermis of a pigmented plaque from the skin over the abdomen was prominently folded, with significant retention of keratin over the skin surface. Histopathological features were similar to those described for case no. 1. No evidence of neoplasia was observed. Squamous cell carcinoma in situ was identified histopathologically in a second plaque in the skin over the abdomen. Arising within a pigmented plaque was a region of markedly thickened epidermis with mild disorganization and multifocal dyskeratosis. The integrity of the basement membrane remained intact. Plaque-like masses from the lip and right axilla contained poorly demarcated, highly infiltrative, nonencapsulated areas diagnosed as SCC [Figure 4]. The SCC arose within pigmented, hyperkeratotic plaques. The SCC consisted of numerous coalescing cords and nests of moderately to markedly atypical squamous epithelial cells that infiltrated the dermis and were associated with marked reactive fibrosis. Keratinization with the formation of keratin pearls was present centrally within nests. Lymphatic invasion was present.
Several keratinocytes in the peritumoral stratum granulosum of the hyperplastic epidermis stained positively with an antibody to papillomavirus group-specific antigen. Occasional neoplastic squamous epithelial cells within keratin pearls and dyskeratotic foci also exhibited faint cytoplasmic staining with an antibody to papillomavirus group-specific antigen. Intranuclear inclusion bodies consistent with papillomavirus were present in stratum granulosum cells, as in case nos. 1 and 2.
The owners elected topical retinoid therapy for the SCC because of its lower cost. The plaques within which SCC arose were treated topically using tretinoind (0.025%, approximately 25 mg, two to three times per week) for several months. Therapy was eventually discontinued by the owner because of financial constraints and lack of response. Thyroid supplementation was continued at the same dosage.
The dog was returned to the UI-VTH 1 year later. A 20 mm-diameter, ulcerative, hyperpigmented mass had developed rapidly on the upper left lip at the prior excision site of the SCC. A second, 20 mm-diameter, ulcerative plaque had also developed on the left upper lip caudal to the first one. A subcutaneous mass was palpated in the right ventral neck. The hyperkeratotic, hyperpigmented areas on the ventral abdomen persisted. Two, 10 mm-diameter, erythematous, fluctuant masses were found within the hyperpigmented area on the caudal ventral abdomen. Darkly pigmented nodules were present on the dog’s ears and the caudal aspects of both rear limbs. The masses on the lip, neck, and caudal ventral abdomen were surgically excised. Histopathological examination revealed numerous discrete and coalescing cords of moderately polymorphic squamous epithelial cells accompanied by focal areas of keratinization. Anaplasia was moderate to marked, and mitotic activity was increased. Epithelial cells had breached the basement membrane and invaded the subjacent dermal connective tissues. The microscopic diagnosis for all excised masses was SCC. Immunohistochemical labeling for papillomavirus group-specific antigen was negative.
Discussion
Plaques from the cases in this report had histopathological and ultrastructural features consistent with those of PEP.332 The ultrastructure of intranuclear inclusion bodies within epidermocytes was consistent with papillomavirus. Plaques described in this report were distinct from those of canine oral papillomavirus. Although canine oral papillomavirus can change its tissue tropism to infect skin, lesions are not hyperpigmented.3133–36 In the Pomeranian (case no. 3), the hyperkeratotic pigmented plaques on the lip developed subsequent to the cutaneous plaques, and they did not reflect a change of tropism from the oral cavity to the skin. Neither the clinical nor the histopathological appearance of the lesions was consistent with oral papillomatosis.3133–36 Likewise, lesions in this report were distinct from exophytic cutaneous papillomas, which often occur on the head, eyelid, and paws of primarily older male dogs. Exophytic papillomas are not usually pigmented and often regress spontaneously.435 Pigmented papules have been reported in a boxer with iatrogenic hyperadrenocorticism, but they differed from PEP in that they were endophytic, keratohyalin granules were rare, and both intracytoplasmic and intranuclear inclusion bodies were observed.37 The late 1 (L1) gene encodes the major capsid protein in papillomaviruses, a critical component of viral pathogenesis. Analysis of the nucleotide sequence of this gene is used to identify or compare papillomaviruses. The nucleotide sequence of the L1 open reading frame identified in the boxer’s papules was 70% homologous with that of canine oral papillomavirus, whereas PEP shares only 57% sequence homology with canine oral papillomavirus L1.3738
The study reported here documents PEP in an American Staffordshire terrier and a Pomeranian, two breeds in which PEP has not been described previously. The etiology of PEP has not yet been fully defined. Breed predisposition may reflect a genetically-determined increased susceptibility to infection by this virus or virus group, as in humans with epidermodysplasia verruciformis. Pugs and miniature schnauzers are over-represented in reports of PEP.34 The role of papillomavirus in lesions examined before the routine use of immunohistochemistry is unknown. Ideally, extant tissues from earlier cases will be reexamined.
Two cases in this report had PEP and a concurrent disease. The diagnosis of hypothyroidism in the Pomeranian was unconfirmed, and immunoglobulin levels were not measured. Impaired immune function associated with excessive cortisol production in the miniature schnauzer and low immunoglobulin levels in both the miniature schnauzer and the American Staffordshire terrier could have contributed to increased susceptibility to papillomavirus. However, direct causation of plaques by hyperadrenocorticism or immunoglobulin deficiency should not be inferred. Plaques in the miniature schnauzer failed to regress completely, despite the sharp reduction of cortisol levels induced by mitotane.
Regression and prevention of recurrence of the plaques occurred after concomitant therapy with low-dose oral human recombinant IFN-α. In the American Staffordshire terrier, plaques recurred when IFN therapy was discontinued. Low-dose oral IFN-α has been used to prevent or treat viral diseases in several species.2839–46 High doses of IFN inhibit the immune response, whereas low-dose IFN therapy stimulates immunity.4146 Orally administered IFN-α exerts both local and systemic immunostimulatory effects by binding to cell receptors associated with mucosal lymphoid tissue and acting in an autocrine and paracrine fashion to up-regulate the production of IFN-γ and additional IFN-α, ultimately leading to a systemic immune response characterized by a subset of thymic helper (Th) lymphocytes, Th1.154146 These cells produce cytokines such as inter-leukin (IL)-1, tumor necrosis factor (TNF)-β, and IFN-γ, which enhance cell-mediated immunity.154146
An alternative to oral IFN-α is topical imiquimod.g This immune modulator is an imidazoquinoline amine recently approved for topical use in humans. The drug’s antiviral and antitumor effects are caused by an induction of several cytokines that stimulate both innate and cell-mediated immunity, including IFN-α.4748 Remission has been achieved in Bowen’s disease, papillomavirus infections, and SCC in humans after topical application of imiquimod at a concentration of 5%.49–51 Imiquimod is an alternative to oral IFN-α and can be used in dermatopathies where IFN-α is effective, although its safety and efficacy in dogs have not yet been determined.5253
The association between papillomavirus infection and the development of neoplasia has been documented in several species.353642434854–59 In cats, papillomavirus infection can lead to multicentric SCC in situ (i.e., Bowen’s disease).3554–59 Mechanisms of neoplastic transformation by papillomaviruses include interference with the functions of p53 gene products, leading to decreased tumor suppression, and viral binding with retinoblastoma protein to increase cellular proliferation.2960–64
Surgical excision of SCC lesions is often curative, providing that the cancer has not metastasized.4 However, pigmented plaques are typically too numerous and widely distributed for surgical excision to be practical. Laser ablation may be a useful alternative to surgical excision, as it allows rapid removal of multiple masses and causes less tissue destruction than surgical excision.4 Laser ablation has been used palliatively in the management of multicentric SCC in cats.59 Oral and topical retinoid therapy has produced mixed results in cases of SCC in dogs.432 No improvement was observed when topical retinoids were administered in case no. 3.
Papillomavirus-associated SCC in dogs may be under-reported. Papillomavirus was not recognized at the time biopsies were initially submitted from the Pomeranian, because koilocytes were not observed, and the lesion was not evaluated by immunohistochemistry. Had the association between PEP and papillomavirus been known at the time, plaque progression might have been slowed. The drug, IFN-α, has both antiviral and antitumor properties, so its administration would have been appropriate before or after the development of SCC.3965 Identification of SCC arising within a PEP should prompt a histopathological and immunohistochemical search for papillomavirus. If an association with papillomavirus is demonstrated, the possibility of a concurrent disease state should also be investigated, and IFN-α therapy may be considered.
Conclusion
In this study, pigmented epidermal plaques were documented in an American Staffordshire terrier and a Pomeranian, two breeds in which PEP has not been described previously. Two of the dogs had concurrent disease, although the relationship between those disorders and plaque development was unclear. Plaques regressed in the two animals treated with IFN-α. Squamous cell carcinoma arose by malignant transformation from multiple plaques within the third dog. This risk of malignant transformation emphasizes the need to recognize PEP and to control plaque progression. Antiviral and antineoplastic therapy, such as low-dose oral IFN-α, may be an effective component of the therapeutic management of both PEP and papillomavirus-associated cutaneous neoplasia.
ACTHAR gel; Questcor, Union City, NJ 94587
Lysodren; Bristol-Myers Squibb, Princeton, NJ 08543
Roferon A; Hoffman-LaRoche, Nutley, NJ 07110
Staphage Lysate; Delmont Laboratories, Swarthmore, PA 19081
Retin-A; Ortho Dermatological, Raritan NJ 08869
Aldara; 3M Pharmaceuticals, Saint Paul, MN 55144
Acknowledgments
The authors thank Drs. J. Zacchary, A. Vitsky, and M. Wallig for assistance with histopathology; Drs. C. Ulbricht, S. Albright, and A. Hasty for assistance with the clinical cases; the Center for Microscopic Imaging for assistance with TEM; and the Veterinary Diagnostic Laboratory histology section for preparing histology and immunohistochemistry slides.
Citation: Journal of the American Animal Hospital Association 40, 5; 10.5326/0400411
Citation: Journal of the American Animal Hospital Association 40, 5; 10.5326/0400411
Citation: Journal of the American Animal Hospital Association 40, 5; 10.5326/0400411
Citation: Journal of the American Animal Hospital Association 40, 5; 10.5326/0400411
Multiple pigmented plaques on the trunk of a miniature schnauzer with hyperadrenocorticoidism (case no. 1).
Histopathological section (from case no. 1) of an exophytic plaque with papillomatous projections (Hematoxylin and eosin stain; 50×).
Transmission electron microscope image of hexagonal viral particles within an intranuclear inclusion body from case no. 1 (33,000×).
Histopathological section through an area of squamous cell carcinoma from case no. 3, showing keratin pearls and neoplastic epidermocytes within the dermis (Hematoxylin and eosin stain; 100×).
