Feline Cutaneous Fibropapillomas: Clinicopathologic Findings and Association with Papillomavirus Infection

Vet Pathol 8:2 26 (200) Feline Cutaneous Fibropapillomas: Clinicopathologic Findings and Association with Papillomavirus Infection F. Y. SCHULMAN, A.E.KRAFFT, AND T. JANCZEWSKI Department of Veterinary Pathology (FYS) and Department of Cellular Pathology (AEK, TJ), Armed Forces Institute of Pathology, Washington, DC; and Veterinary Division, Marshfield Laboratories, Marshfield, WI (FYS) Abstract. Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopic findings supported the fibroblastic nature of the neoplastic cells. The 2 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. These tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 7 cats for which breed was reported were domestic shorthair cats. In /20 cases, there was confirmed exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 8 cats for which follow-up information was available. Metastasis was not documented in any of the cases. Two of the tumors tested by polymerase chain reaction (PCR) had no amplifiable D. The remaining 7 were positive for papillomavirus by PCR. papillomavirus D was detected in three other feline skin tumors (cutaneous mast cell tumor, malignant lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoids. Key words: Cat; feline; fibropapilloma; papillomavirus; polymerase chain reaction; sarcoid; skin tumor. In veterinary medicine, the term fibropapilloma is used to refer to a benign fibroblastic proliferation with overlying acanthosis, hyperkeratosis, and a downgrowth of rete ridges. 20,2 These tumors are generally attributed to papillomavirus infections and are most commonly seen in cattle and horses. 20 In cattle, at least three different bovine papillomaviruses (BPV, BPV2, and BPV) cause fibropapillomas. 20 Equine fibropapillomas are termed sarcoids and were first described in 6. 6 A bovine papillomavirus etiology for equine sarcoids was first proposed in. 2 Since then, a number of studies have provided evidence that bovine papillomaviruses play a role in the development of equine sarcoids.,, Bovine papillomaviruses and 2 have been implicated.,, To the best of our knowledge, fibropapillomas with papillomaviral associations have only been reported in cattle, horses, donkeys, mules, cervids, and a pronghorn antelope. 8,,7,20 Feline cutaneous fibropapillomas with histologic features similar to that of bovine fibropapilloma and equine sarcoid are occasionally seen in veterinary diagnostic services.,2 This report describes the microscopic and clinical features of these lesions as well as evidence for a papillomavirus etiology. Materials and Methods Tissue acquisition and preparation for histology Feline cutaneous fibropapillomas were collected from biopsy specimens submitted to Marshfield Laboratories, Veterinary Division, Marshfield, Wisconsin. Twenty-three tumors from 20 cats were collected from August through December. One author (FYS) collected of the 2 tumors, and other pathologists at the laboratory brought 4 others to her attention. The prevalence of this lesion cannot be determined since the number of cat lesions and cat tumors examined over the collection period are not known. Three other feline skin tumors, including a mast cell tumor, a high-grade malignant lymphoma, and a fibrosarcoma, served as controls. Tissue sections were processed routinely, paraffin embedded, sectioned at m, and stained with hematoxylin and eosin (HE). Electron microscopy For ultrastructural studies, tissue from two tumors (case s. 2 and 20) were deparaffinized, hydrated, and postfixed in % osmium tetroxide, then dehydrated, cleared, and embedded in epoxy resin. One-micron sections were cut and stained with toluidine blue for preliminary light microscopic examination. Thin sections (80 to 0 nm) were cut and stained with uranyl acetate and lead citrate and were examined with a Zeiss EM0 or a Zeiss EM00 transmission electron microscope. Polymerase chain reaction testing Polymerase chain reaction (PCR) testing was conducted on the tumors for which paraffin-embedded tissue was available and on all control tumors. To prepare D lysates, three 2-m sections were cut from formalin-fixed, paraffin- 2

22 Schulman, Krafft, and Janczewski Vet Pathol 8:, 200 embedded (FFPE) blocks. Sections were deparaffinized, and nucleic acid was released by proteinase K digestion in nonionic buffer (0 mm KCl, 0 mm Tris, ph 8, 0. mm EDTA, 0.% Tween 20) (60 g/00 l) overnight at C, followed by heating at C for 0 minutes. The digest was centrifuged at,000 rpm for minutes and the supernatant lysates were stored at 20 C. To design the consensus papillomavirus (PV) assay, the complete nucleotide sequences for bovine, canine oral, chimp, deer, European elk, hamster, ovine, rat, two rabbits, and one human papillomavirus were aligned pairwise using the D Star, Inc., software, searching for conserved regions. Two 2 base pair (bp) regions of the E gene were identified as the most highly conserved sequences among all PV species examined and were used to design degenerate primers to give a 76 bp PCR product. The consensus primers (with IUPAC mixed base codes) were as follows: PV--TATGTDT- CAAADTABYTCCAK- and PV--GGBCCTCCAAAY- ASWGGVAAD-. Positive controls included FFPE equine sarcoid tissue containing bovine papillomavirus (BPV) and plasmids containing cloned BPV, deer PV, cottontail rabbit PV (kindly provided by Carl Baker), canine oral PV (kindly provided by Bennett Jenson), and human PV type (ATCC 6446, Manassas, VA). A single-round hot start procedure with 2 P-dATP incorporation was used for PCR amplification. Radioactive nucleotide incorporation assay with product separation on denaturing polyacrylamide gels was used because it offers greatly enhanced sensitivity over ordinary ethidium bromide-stained gels for PCR assays that use highly degenerate primers. 7,8 In this consensus PV assay, the upstream primers are -fold degenerate while the downstream primers are 20-fold degenerate. Therefore, with so many mismatched primers in the reaction, the primers complementary to the new feline PV sequences are present at very low effective concentrations and the PCR reaction is not very efficient. The 0-l reactions contained PCR Buffer II (Perkin Elmer),. mm MgCl 2, 00 M dgtp, dctp, dttp, 2M datp, 2. um of each primer (Integrated D Technologies, Inc., Coralville, IA),.6 unit AmpliTaq Gold D Polymerase (Perkin Elmer), and 2. Ci 2 P-dATP, specific activity,000 Ci/mmol (Amersham). Reactions were heated at C for 0 minutes, followed by 40 cycles of C for minute, 42 C for minute, and 72 C for minute, and a final 72 C extension step for 7 minutes in a Perkin Elmer 700 Thermal Cycler. Products were separated on a 6% denaturing polyacrylamide gel and exposed to Kodak XAR- film for 24 hours at 70 C for autoradiography. A single band of 76 bp was excised from the gel, electroeluted, ethanol precipitated, and cloned into the pcr 4-TOPO vector (Invitrogen, Carlsbad, CA) according to the manufacturer s instructions. 8 Selected colonies were sequenced using M primers and cycle sequencing on an ABI PRISM model 77. The nucleotide sequences obtained in forward and reverse orientations were compared to GenBank sequences by the National Library of Medicine BLAST sequence similarity search program at http://www.ncbi.nlm.nih.gov. Fig.. Skin; case. 6. Feline fibropapilloma that is composed of a dermal fibroblastic proliferation that surrounds and widely separates adnexa and is covered by hyperplastic epithelium with rete ridges. HE. Bar 20 m. Follow-up information The submitting clinicians and/or owners were contacted by telephone for follow-up information. In addition to trying to obtain missing demographic information, three basic questions were asked: ) Did the tumor recur (if so, how often and when)? 2) Was surgery the only treatment (if not, what other treatment)? ) Does the cat have known exposure to cattle? Results Histologically, the tumors were characterized by spindloid to stellate cells that had infiltrated and expanded the dermis and subcutis and surrounded and widely separated adnexa (Fig. ). Neoplastic cells were arranged haphazardly and in vague fascicles and were separated by variable amounts of collagenous matrix. The cellularity was often higher around adnexa and adjacent to the epidermis. The neoplastic cells had oval to elongate nuclei with finely stippled chromatin, usually single small nucleoli, small amounts of eosinophilic cytoplasm, and indistinct cell margins (Fig. 2). The mitotic rate varied in different areas of the tumor, being higher in the more cellular areas. The range of mitotic index was from one to five per 0 high power fields. The overlying epidermis was mildly to moderately hyperplastic with rete ridges (Fig. ). Multifocally, neoplastic cells were arranged perpendicularly to the dermal epidermal junction. Ten of the tumors were ulcerated. There were moderate numbers of mast cells

Vet Pathol 8:, 200 Feline Cutaneous Papillomas 2 Fig. 2. Skin; case. 6. Fibroblastic proliferation of feline fibropapilloma with mast cells (arrows) scattered throughout. HE. Bar 2 m. Fig.. Skin; case. 6. Superficial aspect of feline fibropapilloma with epidermal hyperplasia and long thin rete ridges. HE. Bar 2 m. Table. Papillomavirus PCR results for 2 feline cutaneous fibropapillomas. Tumor. Case. Papillomavirus PCR 2 4 6 7 8 0 2 4 6 7 8 20 2 22 2 2 4 6 7 8 0 0 2 4 4 6 7 7 8 20 * tissue not available. * Indeterminate, no amplifiable D Weak Weak Weak Indeterminate, no amplifiable D Weak scattered individually and in small clusters throughout all of the tumors. Ultrastructurally, neoplastic cells had elongate nuclei containing peripheralized heterochromatin and central euchromatin, few intracytoplasmic organelles comprised primarily of rough endoplasmic reticulum and mitochondria, and few cytoplasmic processes. Neoplastic cells were separated by many collagen fibrils. There were no external laminae or cellular junctions. Polymerase chain reaction (PCR) testing results are summarized in Table. Of the tumors examined, 2 had no amplifiable D. The papillomavirus PCR assay gave a product of 76 base pair (bp) in the other 7 feline cutaneous fibropapilloma samples that was identical in size to the products from the equine sarcoid tissue and from the plasmids containing BPV, deer PV, cottontail rabbit PV, canine oral PV, and human PV type (Fig. 4). The primers detected BPV, Fig. 4. Autoradiogram of 76 bp PCR product from P- 2 incorporation assay for papillomavirus with feline samples. Lanes and are weak positive; lanes 2, 4, and are positive.

24 Schulman, Krafft, and Janczewski Vet Pathol 8:, 200 Fig.. Comparison of 76 bp feline sequence to E gene sequences of bovine papillomaviruses type (BPV, GenBank X0246.) and type 2 (BPV2, GenBank M202). A dash indicates an identical nucleotide in BPV aligned with the feline sequence. The numbering at the ends refers to the nucleotide position within the complete PV genome. Underlined sequences are the binding sites for PV consensus PCR primers. deer PV, and cottontail rabbit PV reproducibly and canine oral PV and human PV type inconsistently, reflecting the variable PCR efficiency on different runs. The band intensity from the FFPE equine sarcoid sample was similar to that obtained with many of the cat samples. The weak band found in one feline case (case. 4) was cloned, sequenced, and compared with known PV sequences. BLAST analysis showed that the 76 bp feline sequence was most closely related to the E gene sequences of BPV and BPV2, with 7 and 64% homology, respectively (Fig. ). The feline sequence had very limited homology with other known papillomavirus sequences, including deer PV, European elk PV, ovine PV, BPV4, and canine oral PV. papillomavirus D was identified in the three control tumors, all of which had amplifiable D. Follow-up information was available on 8 of the 20 cats and was obtained months to years after histologic diagnosis. Most of the clinical information is presented in Table 2. Additionally, all but one case was submitted from veterinary clinics in Wisconsin. The other came from Minnesota. Discussion Feline fibropapillomas have many similarities to equine sarcoids. The histology of feline fibropapillomas and equine sarcoids is virtually identical, characterized by a fibroblastic proliferation with overlying epithelial hyperplasia and rete ridges. 4,20,2 Electron microscopy confirmed the fibroblastic nature of the neoplastic cells. In horses, sarcoids are usually first observed in young animals between and 6 years of age. 0 In this study, 2 of the 8 cats of known age were less than years old. In horses, sarcoids most Table 2. Summary of clinical information for 20 cats with cutaneous fibropapillomas.* Case. 2 4 6 7 8 0 2 4 6 7 8 20 Age at Initial Tumor Sex Breed Location Recurrence months SF SF F 4 6 F 2 M 4 M 2 SF 2 0 months SF F months F 4 4 M DLH Face LR digit Upper lip L nostril Tail RF digit Eyelid Neck Vulva Him Digit Eyelid Lip L digit RR digit Lip Lip L ear L nostril Digit Vent abd Twice in years Euthanized Inyear In 6 months Deceased times in years Lost to follow-up Lost to follow-up In months Inyear In 28 months Known Exposure to Cattle Other Clinical Information BB pellet wound 2 months prior to tumor FIV Treated for lymphoma treatment of recurrence; scabbed over and fell off several times; reduced in size Had radiation treatment after fifth surgery Deceased * not available; castrated male; SF spayed female; F female; M male; domestic shorthair; Him Himalayan; LR left rear; L left; RF right front; RR right rear; Vent abd ventral abdomen.

Vet Pathol 8:, 200 Feline Cutaneous Papillomas 2 commonly occur on the head, neck, ventral abdomen, and limbs. 6 All but two of these cat tumors were on the head, neck, ventral abdomen, and limbs. Equine sarcoids often recur following surgical excision, 4 and recurrence was reported in 7 of the 8 cats for which follow-up information was available. Equine sarcoids do not metastasize, and metastasis was not documented in any of the feline cases. Equine sarcoids are known to have breed predispositions and have been associated with certain major histocompatibility antigens, suggesting a genetic basis for sarcoid susceptibility. 4,, In this study, of the 7 cats for which breed was reported were domestic shorthair cats, but this is more likely a reflection of the demographics of the cat population sampled than a true breed predisposition. Both tumor types have a strong association with papillomaviruses. Equine sarcoids have been associated with bovine papillomaviruses and 2. In all of the feline tumors for which paraffin-embedded material was available and that had amplifiable D, papillomavirus D was identified by PCR. The nucleotide sequence from the feline tumor was distinct from, but most similar to, bovine papillomavirus type. Eleven of the 20 cats had known exposure to cattle and all but were submitted from veterinary clinics in Wisconsin, a state known for its dairy farms. The remaining nine cats did not have confirmed exposure to cattle but may have been exposed unbeknownst to the clinician or owner, i.e., prior to living with the present owner. While sarcoids are widely recognized and there is a plethora of information concerning their clinical behavior and association with papillomaviruses, there are no peer-reviewed articles on fibropapillomas in cats. There is one published letter reporting solitary dermal fibropapillomas in two cats, one in a 2-year-old cat on the lip and one in a -month-old cat on a hind foot. The lip fibropapilloma recurred once in months, but repeat surgery was reportedly curative. The other tumor did not recur. In their textbook, Yager and Wilcock also reported 4 cases of fibropapilloma in dogs and cats collected over years, but the number of feline cases and other specifics are not given. 2 While papillomavirus etiology was suspected in both reports, no direct evidence of a viral infection was provided. The reasons for the limited information on fibropapillomas in cats are not known. These tumors may not always be recognized as fibropapillomas. If only a portion of the tumor is submitted, especially if there is no epidermis included in the biopsy, the differential diagnosis may include fibrosarcoma, fibroma, amelanotic melanocytoma, and granulation tissue. In general, fibrosarcomas are more pleomorphic than fibropapillomas and have cytological evidence of malignancy. Fibromas are well circumscribed and less cellular than fibropapillomas. Both fibrosarcomas and fibromas lack epidermal participation. The clinical history, immunohistochemistry, and electron microscopy may be used to differentiate fibropapilloma from amelanotic melanocytoma. Granulation tissue is characterized by fibrous tissue oriented perpendicular to capillaries and not by haphazardly arranged fibroblasts, as are seen in fibropapillomas. If definitive diagnosis cannot be made from an incisional biopsy, excisional biopsy is recommended. In addition to misdiagnosis, there are other possible reasons for the apparent low incidence of feline cutaneous fibropapillomas. Cats may have a low susceptibility to papillomavirus infection and/or cellular transformation. If the papillomavirus is of bovine origin, cats may have less exposure to cattle than horses except in areas of concentrated dairy farming, such as Wisconsin. Cats that have greater contact with cattle, i.e., farm cats, may not be biopsied often. In addition, the molecular techniques for identifying viral D in these tumors have only recently become available. Any or all of these factors may contribute to the paucity of reports of feline fibropapillomas. A few cases of papillomavirus-associated cutaneous squamous papillomas in cats have been reported. 2,,6 Unlike the fibropapillomas, feline squamous papillomas are plaquelike and are characterized histologically by epidermal and follicular hyperplasia with hypergranulosis and swollen cells containing clear cytoplasm and vesicular nuclei (koilocytes). Based on immunohistochemistry, electron microscopy, and molecular tests, the papillomavirus in feline cutaneous squamous papillomas has been considered to represent Feline domesticus papillomavirus-type ; however, results of D sequencing have not been reported. Squamous papillomas appear to affect immunodeficient cats. Two of the older cats with fibropapillomas had concomitant disease (lymphoma in one and feline immunodeficiency virus infection in another) that may well have caused immunosuppression. It seems logical that immunosuppression might play a role in viral oncogenesis and apply to both manifestations of cutaneous papillomavirus infection, i.e., squamous papilloma and fibropapilloma. In summary, these feline tumors share many clinicopathologic features with equine sarcoids and have a strong association with papillomavirus, suggesting a causal relationship. Additional studies of these tumors may help in the understanding of viral oncogenesis. Acknowledgements The authors thank Ms. V. Ferris, Mr. B. Jones, and Mr. J. Jenkins of the Armed Forces Institute of Pathology and Mr. D. Stoiber of Marshfield Laboratories for technical assis-

26 Schulman, Krafft, and Janczewski Vet Pathol 8:, 200 tance. We also thank all the clinicians and owners for their willingness to contribute to this study. Ms. S. Molter, Ms. M. Gregorich, and Ms. L. Zimmermann of Marshfield Laboratories provided invaluable logistic support. Drs. J. L. Carpenter, T. G. Taylor, M. J. Tomlinson, and D. A. Belote helped with the collection and characterization of the tumors. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or Department of Defense. References Angelos JA, Marti E, Lazary S, Carmichael LE: Characterization of BPV-like D in equine sarcoids. Arch Virol : 0, 2 Carney HC, England JJ, Hodgin EC, Whiteley HE, Adkison DL, Sundberg J: Papillomavirus infection of aged Persian cats. J Vet Diagn Invest 2: 24 2, 0 Egberink HF, Berrocal A, Bax HA, van den Ingh TS, Walter JH, Horzinek MC: Papillomavirus associated skin lesions in a cat seropositive for immunodeficiency virus. Vet Microbiol :7 2, 2 4 Goodrich L, Gerber H, Marti E, Antczak DF: Equine sarcoids. Vet Clin rth Am Equine Pract 4:607 62, 8 Gumbrell RC, Rest JR, Bredelius K, Batchelor DJ, Williamson J: Dermal fibropapillomas in cats. Vet Rec 42: 76, 8 6 Jackson C: The incidence and pathology of domesticated animals in South Africa. Ondersteport J Vet Sci Anim Indust 6:78 8, 6 7 Krafft AE, Taubenberger JK, Sheng Z-M, Bijwaard KE, Abbondanzo SL, Ives Aguilera NS, Lichy JH: Enhanced sensitivity with a novel TCR PCR assay for clonality studies in 6 formalin-fixed, paraffin-embedded (FFPE) cases. Mol Diagn 4:, 8 Lancaster WD, Sundberg JP: Characterization of papillomaviruses isolated from cutaneous fibromas of whitetailed deer and mule deer. Virology 2:22 26, 82 Lazary S, Gerber H, Glatt PA, Straub R: Equine leukocyte antigens in sarcoid-affected horses. Equine Vet J 7: 28 26, 8 0 Marti E, Lazary S, Antczak DF: Report of the first international workshop on equine sarcoid. Equine Vet J 2: 7, Mohammed HO, Rebhun WC, Antczak DF: Factors associated with the risk of developing sarcoid tumours in horses. Equine Vet J 24:6 68, 2 2 Olson C, Cook RH: Cutaneous sarcoma-like lesions of the horse caused by the agent of bovine papilloma. Proc Soc Exp Biol Med 77:28 284, Otten N, von Tschaner C, Lazary S, Antczak DF, Gerber H: D of bovine papillomavirus type and 2 in equine sarcoids: PCR detection and direct sequencing. Arch Virol 2:2, 4 Ragland WL, Spencer GR: Attempts to relate bovine papillomavirus to the cause of equine sarcoid: Equidae inoculated intradermally with bovine papilloma virus. Am J Vet Res 0:74 72, 6 Reid SW, Smith KT, Jarrett WFH: Detection, cloning and characterization of papillomaviral D present in sarcoid tumors of Equus asinus. Vet Rec :40 42, 4 6 Sundberg JP, Van Ranst M, Montali R, Homer BL, Miller WH, Rowland PH, Scott DW, England JJ, Dunstan RW, Mikaelian I, Jenson AB: Feline papillomas and papillomaviruses. Vet Pathol 7: 0, 2000 7 Sundberg JP, Williams E, Thorne ET, Lancaster WD: Cutaneous fibropapilloma in a pronghorn antelope. J Am Vet Med Assoc 8: 4, 8 8 Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG: Initial genetic characterization of the 8 Spanish influenza virus. Science 27:7 76, 7 Trenfield K, Spradbrow PB, Vanselow B: Sequences of papillomavirus D in equine sarcoids. Equine Vet J 7:44 42, 8 20 Yager JA, Scott DW, Wilcock BP: The skin and appendages. In: Pathology of Domestic Animals, ed. Jubb KVF, Kennedy PC, and Palmer N, 4th ed., vol., pp. 708 70. Academic Press, San Diego, CA, 2 2 Yager JA, Wilcock BP: Color Atlas of Surgical Pathology of the Dog and Cat. Wolfe, London, UK, p. 22, 4 Request reprints from Dr. F. Yvonne Schulman, Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC 2006-6000 (USA). E-mail: schulman@afip.osd.mil.