Sebaceous cell differentiation in a canine oral papilloma

Abstract

Papillomas caused by viral infection are well-known tumors in animals. Microscopic features typically include neoplastic epithelium with hyperkeratosis and koilocytes. An 8-y-old castrated male Shih Tzu dog was presented with a small exophytic mass on the external upper lip. The mass was diagnosed as a viral papilloma based on microscopic and immunohistochemical examination. Sebaceous cell differentiation was found in the neoplastic epithelium of the tumor, which is a rare finding in humans or animals.

Viral papilloma caused by canine papillomavirus is a common and well-recognized lesion in dogs. Canine oral papilloma caused by canine oral papillomavirus is commonly referred to as warts on the lip, buccal mucosa, tongue, palate, pharynx, or eyelid.⁸ Papilloma can be diagnosed easily by typical morphologic and histopathologic characteristics. Morphologically, the tumor shows pedunculated or “cauliflower-like” growth, and solitary or multiple tumors may be present. The major histologic features include multiple papillary projections consisting of thick neoplastic epithelium with hyperkeratosis supported by a fibrovascular stalk. The cytoplasm of neoplastic cells may be highly vacuolated, may contain enlarged keratohyalin granules, and nuclei may contain eosinophilic inclusions.^3,12,13

Papillomaviruses infect the stratum basale through injured stratified epithelium of mucosae or skin, and the virus replicates in the nuclei of basal cells. Basal cells are immature keratinocytes that divide and differentiate into cells of the suprabasal layers, cells of which no longer divide. However, production of infective virions occurs in suprabasal cells. At this point, the virus interferes with the cell cycle by blocking G1/S and G2/M arrest, especially in the cells of the strata spinosum and granulosum.^2,13 Consequently, these virus-infected cells continue to divide, leading to papillary growth of the epithelium. Sebaceous cell differentiation is not included in the usual histologic features of viral papilloma. Only one case report of sebaceous gland differentiation in squamous papilloma (non-viral papilloma) from the gingiva of a cat has been reported, to our knowledge.⁷ We describe herein the histologic features of a case of sebaceous cell differentiation in a canine viral papilloma.

An 8-y-old castrated male Shih Tzu dog was presented to a veterinary clinic with a 3–4 mm diameter exophytic black mass on the external aspect of the upper lip. The mass was removed surgically, fixed in 10% neutral-buffered formalin, processed routinely, sectioned at 4 µm, and stained with hematoxylin and eosin. For immunohistochemistry (IHC), monoclonal anti-human papillomavirus antibody (ab2417; Abcam, Cambridge, UK) was used because this antibody has been shown previously to react with canine cutaneous viral papillomas.¹ Monoclonal anti-adipophilin antibody (sc-377429; Santa Cruz Biotechnology, Dallas, TX) was used for detection of sebaceous cells. Adipophilin is an adipocyte differentiation–related protein expressed in intracytoplasmic lipids of sebocytes,¹⁰ and has been used for diagnosis of sebaceous cell tumors in humans. Hydrogen peroxide (3%) solution was used to inhibit endogenous peroxidase activity. A microwave antigen-retrieval process was performed using citrate buffer. The antigen–antibody complex was labeled with an avidin–biotin peroxidase complex solution (ABC kit, Vector Laboratories, Burlingame, CA) and a DAB (3,3’-diaminobenzidine) substrate kit (Invitrogen, Carlsbad, CA). Slides were counterstained with Mayer hematoxylin.

Microscopically, the mass had typical papillomatous exophytic growth supported by a fibrovascular stalk (Fig. 1A). The neoplastic epithelial cells in the suprabasal layers had marked cytoplasmic and nuclear vacuolation. Numerous koilocytes (keratinocytes with eccentric pyknotic nuclei and perinuclear halos) were present in suprabasal layers of the epithelium. Basophilic keratohyalin granules in cells of the stratum granulosum were larger and much more abundant than in normal cells (Fig. 1B). We did not identify intranuclear viral inclusions in the sections examined. These tumor cells showed no cellular pleomorphism. Melanin pigmentation was abundant in the stratum basale. Neoplastic cells with foamy cytoplasm similar to well-differentiated sebaceous cells were found within lower layers of the epithelium. These cells usually formed nests toward a fibrovascular stalk and were not associated with hair follicles (Fig. 1C, D). IHC for papillomavirus strongly labeled nuclei of keratinocytes, especially in the stratum basale (Fig. 2A). Staining of the cytoplasm in some keratinocytes in the papilloma was interpreted as nonspecific background because this staining was also present in controls. Adipophilin-stained lipid vacuoles in the cytoplasm of sebaceous cells were found in the papilloma (Fig. 2B). We used slides of a canine cutaneous sebaceous gland adenoma as positive control for adipophilin, and found that adipophilin was stained appropriately in sebaceous cells.

Figure 1.

Histology of an oral papilloma in a dog. A. Hyperplastic and hyperkeratotic epithelium showing exophytic growth supported by a fibrovascular stalk. B. Nuclei of epithelial cells are eccentric and pyknotic with cytoplasmic vacuolation (koilocytes, arrows). Note enlarged basophilic keratohyalin granules (arrowheads) in stratum granulosum. Inset: higher magnification of koilocytes with keratohyalin granules (arrowheads). C. Well-differentiated sebaceous cells (arrows) are seen along the lower layer of the epithelium, budding into a fibrovascular stalk not associated with hair follicles. D. Note foamy cytoplasm of sebocytes next to vacuolated keratinocytes. H&E.

Figure 2.

Immunohistochemistry of oral papilloma in a dog. A. Strong positive reaction against papillomavirus is seen in the nuclei of stratum basale cells. B. Adipophilin is expressed in the intracytoplasmic lipid of sebaceous cells.

Most cases of viral papilloma in dogs can be diagnosed easily based on typical histopathologic features. Sebaceous cell differentiation in papillomas not associated with hair follicles has been rarely reported in humans and animals.^7,9 In animals, there has been only one case report of sebaceous gland differentiation in a squamous papilloma of a cat, to our knowledge.⁷ According to that report, the mass was found on the gingiva of the maxilla; histologic features of sebaceous nests toward a fibrovascular stalk in that papilloma were similar to the features observed in our case. However, the squamous papilloma of the cat was a non-viral tumor that was distinguished from viral papilloma by histologic features, such as normal-sized keratohyalin granules, the absence of koilocytes, and IHC using anti-papillomavirus antibody.³

Sebaceous glands are normal follicular adnexa in the dermis, composed of basal cells peripherally and mature sebocytes centrally. Basal cells originate from bulge stem cells located at the outer root sheath of hair follicles, and differentiate into mature sebocytes.⁶ Excretory ducts open into the hair canal or directly onto the cutaneous surface. Sebaceous glands that arise independently from hair follicles are termed Fordyce spots, and are found as specialized glands in the lip, penis, and mammary nipple in humans.¹¹ Fordyce spots have not been reported in dogs, to our knowledge. Dogs only have serous or mucoserous cells, known as labial glands, in their lips. In humans, Bowen disease is squamous cell carcinoma in situ characterized by broad hyperkeratosis and variable numbers of neoplastic keratinocytes. Bowen disease is thought to have multiple etiologies, including human papillomavirus infection.⁴ Bowen disease rarely has basaloid, pilar, pilosebaceous, or sebaceous differentiation from keratinocytes.⁵ There is a case report of sebaceous differentiation in Bowen disease associated with infection of human papillomavirus 16 in a human.⁹ Considering the paucity of reports of papillomavirus leading to sebaceous differentiation of human papilloma, sebocyte differentiation in our case may not be viral related.^4,9

Based on microscopic findings, sebocytes in our case appear to have arisen from keratinocytes. We speculate that sebaceous differentiation can originate from keratinocytes as in Bowen disease of humans, with or without viral infection.⁹ However, correlation with papillomavirus infection remains unclear.