Conjunctival Inverted Papilloma Progressing to Carcinoma. First Report in Horse

veterinary sciences

Case Report Conjunctival Inverted Papilloma Progressing to Carcinoma. First Report in Horse

Vito Biondi † , Annamaria Passantino † , Michela Pugliese, Salvatore Monti, Alessandra Sfacteria * and Simona Di Pietro

Department of Veterinary Sciences, University of Messina, 98168 Messina, Italy; [email protected] (V.B.); [email protected] (A.P.); [email protected] (M.P.); [email protected] (S.M.); [email protected] (S.D.P.) * Correspondence: [email protected] or [email protected] † The authors labeled with † contributed equally to this paper.

Abstract: A five-year-old, entire female Arabian horse with a 6-month history of a non-painful nodule on the conjunctiva of the right eye was evaluated. Ophthalmological examination showed a firm, smooth and fleshy conjunctival mass that raised the suspicion of a conjunctival neoplasm. Histological evaluations showed that the mass was composed of an endophytic growth consisting of numerous long papillary projections of hyperplastic stratified squamous epithelium supported by thin fibrovascular stalks. Typical features of squamous cell carcinoma with disorganized cell growth and infiltration of surrounding tissues were detectable within the mass. Inverted papilloma progressing to carcinoma was diagnosed. Follow-up examination showed that no local recurrence was present during the 12-month follow-up period. To the authors’ knowledge, this is the first report describing the inverted papilloma in the horse and, due to its progression to squamous cell carcinoma, warns about the inclusion of the inverted papilloma in the differential diagnosis of conjunctival Citation: Biondi, V.; Passantino, A.; neoplasm and driven treatments. Pugliese, M.; Monti, S.; Sfacteria, A.; Di Pietro, S. Conjunctival Inverted Keywords: horse; eye; conjunctiva; neoplasia; inverted papilloma; squamous cell carcinoma Papilloma Progressing to Carcinoma. First Report in Horse. Vet. Sci. 2021, 8, 108. https://doi.org/10.3390/ vetsci8060108 1. Introduction Papillomas or warts are common cutaneous tumors of young horses ranging from Academic Editor: Jong-Hyuk Kim 12 to 24 months of age that recognize in their pathogenesis either equine papillomavirus

Received: 22 April 2021 or noninfectious irritants [1,2]. Only the variety caused by the viral action is referred to Accepted: 8 June 2021 as papillomatosis. Papillomas are slow-growing masses that frequently affect the muzzle, Published: 10 June 2021 eyelids, and ears. Papillomas of the ocular region represent 7% of equine ocular neoplasms [3]. Grossly, they present as elevated horny masses with fronds grouped in clusters

Publisher’s Note: MDPI stays neutral measuring 2 to 10 cm in diameter [2]. Equine papillomas are benign exophytic tumors, with regard to jurisdictional claims in usually spontaneously regressing in 1 to 6 months, eliminating the necessity for inter- published maps and institutional afﬁl- vention [4]. Histologically, papillomas are characterized by numerous thin ﬁbrovascular iations. stalks covered by hyperplastic epithelium. The growth pattern may be exophytic, with the projections extending above the surface of the skin, or endophytic (inverted), with the projections extending into the dermis and hypodermis [5]. Inverted papilloma is an uncommon, benign endophytic proliferation of the epidermis; it has only been reported

Copyright: © 2021 by the authors. in dogs and humans [6]. In dogs, endophytic lesions are most commonly found on the Licensee MDPI, Basel, Switzerland. forelimbs and abdomen as solitary lesions located within the dermis, yet have not been This article is an open access article described so far in the conjunctiva even though the classical, exophytic, and conjunctival distributed under the terms and papilloma can affect the canine eye. In humans, inverted papilloma is mainly found in the conditions of the Creative Commons nasal cavity and paranasal sinuses, while the conjunctival one is exceedingly rare, more Attribution (CC BY) license (https:// than ever if associated with squamous cell carcinoma (SCC) [7]. creativecommons.org/licenses/by/ 4.0/).

Vet. Sci. 2021, 8, 108. https://doi.org/10.3390/vetsci8060108 https://www.mdpi.com/journal/vetsci Vet. Sci. 2021, 8, 108 2 of 6

2. Materials and Methods A five-year-old, entire female Arabian horse (400 kg bwt) presented a conjunctival mass of approximately 2 cm located on the lower eyelid conjunctiva of the right eye (RE). The owner reported a watery discharge and the presence of an enlarging mass over the last six months. The animal was active, and had been treated with tobramycin eye drops for a month without significant improvement. Ophthalmological examination of both eyes with a transilluminator (Heine, Gilching, Germany), a portable handheld biomicroscope (Portable Slit Lamp SL-14, Kowa, Osaka, Japan), and a direct ophthalmoscope (Heine BETA 200®, Heine, Gilching, Germany) was performed. Tear production (Schirmer’s tear test—Tear Strips, Biovision Limited, Dunsta- ble, UK) and intraocular pressure (IOP) (TonoPen® XL, Reichert Technologies, Depew, NY, USA) were also measured. Differential diagnoses of the mass included primary tumor and non-neoplastic lesions such as granuloma. The owner agreed to remove the growth surgically and signed the written consent. Before surgery, the animal underwent a complete general physical examination, which was normal, and laboratory tests, including complete blood counts (CBC) and serum biochemical profile (total protein, albumin, urea, creatinine, total bilirubin, aspartate aminotransferase, and aspartate aminotransferase) that were within the physiological ranges. The animal was pre-medicated with acepromazine (Prequillan®, Fatro Industria Farmaceutica Veterinaria S.p.A., Bologna, Italy) at a dose of 0.03 mg/kg intravenously. Sedation was induced using detomidine (Demosedan®,Vétoquinol Italia S.r.l., Bertinoro, Italy) 0.03 mg kg−1 and butorphanol (Dolorex®, MSD Animal Health S.r.l., Milan, Italy) 0.01 mg kg−1 intravenous administration. The eye and associated structures instilling 0.4% oxybuprocaine hydrochloride (Benox- inato Cl®, Alfa Intes S.r.l., Casoria, Italy) ophthalmic drops were desensitized. A retrobulbar nerve block using 5 mL of 2.0% lidocaine (Lidocaine 2%, Ecuphar Italia S.r.l., Milan, Italy) injected into the retrobulbar space was obtained. The mass was successfully excised, and the animal was standing calm without any signs of pain. The mass was sent to the pathology service of the Veterinary Teaching Hospital of the University of Messina; formalin-fixed and paraffin-embedded (FFPE) specimens were routinely cut and stained with H&E for histopathological evaluation. The slides, prepared as above, were observed under an optical microscope (DMI6000, Leica Microsystems, Wetzlar, Germany) connected to a camera and image analysis software (Leica Application Suite X, Leica Microsystems, Wetzlar, Germany). A numerical count of mitoses was determined and reported for 2.37 mm2 area at high power field (400×).

3. Results 3.1. Ophthalmological Finding Slit lamp biomicroscopy showed in RE third eyelid and conjunctiva moderately hyperemic and a greyish white mass smooth and regular, and raised above the surrounding tissue. The conjunctival mass was approximately 2 cm in size and localized close to the medial (nasal) canthus of the lower eyelid conjunctiva (Figure1). Vet. Sci. 2021, 8, x FOR PEER REVIEW 3 of 6 Vet.Vet. Sci. Sci. 20212021, ,88, ,x 108 FOR PEER REVIEW 3 of 6 3 of 6

Figure 1. Mass on the right eye, of approximately 2 cm, located on the lower eyelid conjunctiva close FiguretoFigure the 1.medial 1.Mass Mass oncanthus. on the the right right eye, eye, of approximately of approximately 2 cm, located2 cm, located on the loweron the eyelid lower conjunctiva eyelid conjunctiva close close toto the the medial medial canthus. canthus. The growth was also firm and moderately friable. The anterior chamber, the iris, the TheThe growth growth was was also also ﬁrm firm and and moderately moderately friable. friable. The anterior The anterior chamber, chamber, the iris, thethe iris, the vitreous, and the fundus were normal. vitreous,vitreous, and and the the fundus fundus were were normal. normal. Schirmer tear test were 18 and 27 mm/min RE and left eye (LE), respectively. IOP was SchirmerSchirmer tear tear test test were were 18 and 18 and 27 mm/min 27 mm/min RE and RE left and eye left (LE), eye respectively. (LE), respectively. IOP was IOP was foundfound to to be be 25 25 and and 23 23 mmHg mmHg RE andRE and LE, respectively.LE, respectively. found to be 25 and 23 mmHg RE and LE, respectively. UnderUnder general general anaesthesia, anaesthesia, the nodule the wasnodule excised. was As excised. soon as theAs mass soon was as clamped, the mass was Under general anaesthesia, the nodule was excised. As soon as the mass was itclamped, went completely it went completely away, showing away, an inner showing surface an coveredinner surface by ﬁliform, covered acuminate, by filiform, and acumin- clamped, it went completely away, showing an inner surface covered by filiform, acumin- hornyate, and fronds horny (Figure fronds2). (Figure 2). ate, and horny fronds (Figure 2).

FigureFigure 2. 2.Acuminate, Acuminate, horny horny fronds fronds at the at inner the inner surface surface of the conjunctivalof the conjunctival mass after mass surgery. after surgery. Figure 2. Acuminate, horny fronds at the inner surface of the conjunctival mass after surgery. TheThe nodule nodule did did not not invade invade the corneathe cornea and wasand approximately was approximately 2 cm in 2 diameter. cm in diameter. After After The nodule did not invade the cornea and was approximately 2 cm in diameter. After surgery,surgery, a topicala topical medication medication was was performed performed using us aning eye an ointment eye ointment 10 mg/g 10 chlortetracy- mg/g chlortetracy- clinesurgery, hydrochloride a topical medication (Ophtocycline, was Dechra performed Veterinary using Products an eye S.r.l.,ointment Torino, 10 Italy),mg/g fourchlortetracy- cline hydrochloride (Ophtocycline, Dechra Veterinary Products S.r.l., Torino, Italy), four timescline ahydrochloride day for 15 days. (Ophtocycline, After the treatment, Dechra the animalVeterinary was followedProducts up S.r.l., every Torino, 2 months Italy), four times a day for 15 days. After the treatment, the animal was followed up every 2 months duringtimes thea day first for year 15 to days. rule out After recurrences. the treatment, Any sign the of animal ocular recurrence was followed was not up revealed. every 2 months during the first year to rule out recurrences. Any sign of ocular recurrence was not re- during the first year to rule out recurrences. Any sign of ocular recurrence was not re- 3.2.vealed. Histological Finding vealed. Histologically, conjunctival epithelium invaginated downward the underlying submu- cosa3.2. givingHistological rise to Finding numerous long papillary projections of hyperplastic stratified squamous 3.2. Histological Finding epithelium,Histologically, supported conjunctival by thin fibrovascular epithelium stalks. invaginated Foci of a mixed downward chronic inflammatory the underlying sub- cell, mainlyHistologically, composed conjunctival of lymphocytes epithelium and plasma invaginated cells, were downward located at the the periphery underlying submucosa giving rise to numerous long papillary projections of hyperplastic stratified squa- ofmucosa the lesion giving and rise underneath to numerous the surface long papillar epithelium.y projections The stratum of hyperplastic basale laying stratified to the squamous epithelium, supported by thin fibrovascular stalks. Foci of a mixed chronic inflam- fibrovascularmous epithelium, stalks wassupported hyperplastic, by thin with fibrovascu a moderatelylar stalks. high mitoticFoci of counta mixed (2–5/high chronic inflammatory cell, mainly composed of lymphocytes and plasma cells, were located at the pe- powermatory field). cell, Amainly prominent composed hyperkeratosis of lymphocytes and keratohyalin and plasma granules cells, characterized were located the at the pe- proliferation.riphery of the There lesion was and a regular underneath epithelial the maturation surface epithelium. sequence even The though stratum many basale cells laying to riphery of the lesion and underneath the surface epithelium. The stratum basale laying to inthe the fibrovascular stratum granulosum stalks was were hyperplastic, swollen with centrallywith a moderately placed nuclei high often mitotic containing count not (2–5/high the fibrovascular stalks was hyperplastic, with a moderately high mitotic count (2–5/high well-defined,power field). circular, A prominent basophilic hyperkeratosis structures surrounded and keratohyalin by a clear halo granules and marginated characterized the power field). A prominent hyperkeratosis and keratohyalin granules characterized the chromatinproliferation. being There suggestive was a of regular koilocytes. epithelial Acanthocytes maturation were alsosequence admixed even within though cells ofmany cells proliferation. There was a regular epithelial maturation sequence even though many cells normalin the stratum appearance. granulosum These patterns were wereswollen compatible with centrally with the placed cytopathic nuclei effects often and containing the not in the stratum granulosum were swollen with centrally placed nuclei often containing not well-defined, circular, basophilic structures surrounded by a clear halo and marginated well-defined, circular, basophilic structures surrounded by a clear halo and marginated chromatin being suggestive of koilocytes. Acanthocytes were also admixed within cells of chromatin being suggestive of koilocytes. Acanthocytes were also admixed within cells of normal appearance. These patterns were compatible with the cytopathic effects and the normal appearance. These patterns were compatible with the cytopathic effects and the inclusions induced by papillomaviruses as already described in other equine papilloma- inclusions induced by papillomaviruses as already described in other equine papillomatous lesions (Figures 3 and 4). tous lesions (Figures 3 and 4).

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Vet. Sci. 2021, 8, x FOR PEER REVIEWinclusions induced by papillomaviruses as already described in other equine papillomatous4 of 6 lesions (Figures3 and4).

Figure 3. (a) Conjunctival epithelium (double-edged arrow) invaginating downward the underlying submucosa (asterisk, HE 5×); to form (b) numerous long papillary projections of hyperplastic stratified squamous epithelium (arrow, HE 5×); (c) cross section of papillary fronds showing a fibrovascular core surrounded by hyperplastic stratified squamous epithelium with conserved normal ar- chitecture (arrow, HE 10×). Many koilocytes were identifiable (arrowhead) (d) a mixed chronic inflammatory cell infiltrate, mainly composed by lymphocytes and plasma cells, was interposed between the boundaries of the lesion and the surface epithelium (HE 2.5×. Inset. 40×).

In focal areas of the mass, the growth appeared disorganized, with loss of polarity of the keratinocytes and loss of normal keratinocyte maturation invading and infiltrating the FigureFiguresubmucosal 3.3. (a) Conjunctival Conjunctival tissues. This epithelium epithelium neoplastic (double-edged (double-edged growth arro was arrow)w) ofteninvaginating invaginating arranged downward downward in trabecular, the theunderlying underlying papillar, or submucosa (asterisk, HE 5×); to form (b) numerous long papillary projections of hyperplastic strat- submucosa (asterisk, HE 5×); to form (b) numerous long papillary projections of hyperplastic ifiednest squamouspatterns epitheliumwith cells (arrow, showing HE 5×);mild (c) tocross mo sectionderate of pleomorphism, papillary fronds showing multiple a fibrovas- nucleoli, dys- × stratifiedcularkeratinization, core squamous surrounded increased epithelium by hyperpla mitotic (arrow,stic stratified count HE 5 (4 squamous); up (c) to cross 9/high epithelium section power of with papillary field) conserved and fronds normal atypical showing ar- mitotic a fibrovascularchitecturefigures. Some(arrow, core “keratinic surroundedHE 10×). Many bypearls” hyperplastickoilocytes were were stratifiedidentifi identifiableable squamous as (arrowhead) well epithelium as foci (d) a withof mixed neutrophilic conserved chronic normalin- inflam- architectureflammatorymation surrounding cell (arrow, infiltrate, HE 10 themainly×). carcinomatous Many composed koilocytes by lymphocytes weregrowth identifiable. Acantholytic and plasma (arrowhead) cells, cells, was ( d koilocytesinterposed) a mixed chronicbe- and cells inflammatorytweenbearing the intranuclearboundaries cell infiltrate, of theeosinophilic lesion mainly and composed th inclusionse surface by epithelium lymphocytes were also (HE visible 2.5×. and Inset. plasma at the 40×). cells, boundaries was interposed of the ma- betweenlignant theneoplastic boundaries growth of the lesionor intermingled and the surface with epithelium the cancerous (HE 2.5× cells. Inset. (Figure 40×). 4). In focal areas of the mass, the growth appeared disorganized, with loss of polarity of the keratinocytes and loss of normal keratinocyte maturation invading and infiltrating the submucosal tissues. This neoplastic growth was often arranged in trabecular, papillar, or nest patterns with cells showing mild to moderate pleomorphism, multiple nucleoli, dyskeratinization, increased mitotic count (4 up to 9/high power field) and atypical mitotic figures. Some “keratinic pearls” were identifiable as well as foci of neutrophilic inflam- mation surrounding the carcinomatous growth. Acantholytic cells, koilocytes and cells bearing intranuclear eosinophilic inclusions were also visible at the boundaries of the malignant neoplastic growth or intermingled with the cancerous cells (Figure 4).

FigureFigure 4. 4. ((aa)) AreasAreas ofof the tumor showing a disorganized disorganized ap appearancepearance and trabecular, papillar, or nest nestgrowth growth patterns patterns (arrowhead). (arrowhead). Papillary Papillary projection projectionss and and nests nests of of SCC SCC invading invading the submucosasubmucosa (ar- (arrow)row) (HE, (HE, 2.5×); 2.5× ();b (–bd–)d SCC) SCC cells cells showing showing mild mild to to moderate moderate pleomorphism, pleomorphism, dyskeratinization, dyskeratinization, and and keratin pearl formation ((b), arrow. 20×), acantholytic cells ((b), arrowhead), increased mitotic count ((c), arrow. HE 40×), koilocytes ((d), arrow), intranuclear inclusions ((d), arrowhead), and foci of neutrophilic inﬂammation ((d), asterisk. HE, 40×). Figure 4. (a) Areas of the tumor showing a disorganized appearance and trabecular, papillar, or nest growth patterns (arrowhead). Papillary projections and nests of SCC invading the submucosa (arrow) (HE, 2.5×); (b–d) SCC cells showing mild to moderate pleomorphism, dyskeratinization, and

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In focal areas of the mass, the growth appeared disorganized, with loss of polarity of the keratinocytes and loss of normal keratinocyte maturation invading and infiltrating the submucosal tissues. This neoplastic growth was often arranged in trabecular, papillar, or nest patterns with cells showing mild to moderate pleomorphism, multiple nucleoli, dyskeratinization, increased mitotic count (4 up to 9/high power field) and atypical mitotic figures. Some “keratinic pearls” were identifiable as well as foci of neutrophilic inflam- mation surrounding the carcinomatous growth. Acantholytic cells, koilocytes and cells bearing intranuclear eosinophilic inclusions were also visible at the boundaries of the malignant neoplastic growth or intermingled with the cancerous cells (Figure4).

4. Discussion In the horse, the eye can be the site of primary and metastatic neoplasms; squamous carcinoma in the limbic bulbar conjunctiva, the third eyelid, and the other eyelids being these sites directly exposed to ultraviolet radiation, are the most frequently occurring and concerning [3]. Precancerous epithelial changes as squamous plaque, squamous papilloma, squamous carcinoma in situ can temporally precede the onset of the neoplasm [8]. Squa- mous cell carcinoma progression from equine penile and preputial papilloma is frequent [9]. As for the classical pattern of papilloma, inverted papilloma is a benign neoplasm that can be associated with squamous cell carcinoma. In human patients, the occurrence of malignancy in all patients with inverted papilloma accounts for 9.1% [10]. Although this association is likely to occur, the exact relationship is unclear. Squamous cell carcinoma may present in the setting of inverted papilloma in three different circumstances. It has been reported that small foci of squamous cell carcinoma within inverted papilloma may differentiate; the malignancy may present as a separate synchronous lesion and not within inverted papilloma; and ﬁnally, metachronous carcinoma in areas of prior resection of benign inverted papilloma may rise [11–13]. The association between papillomavirus and the onset of epithelial neoplasms is well known; the papilloma resulting from the viral action is described based on the frond and cytopathic patterns other than the viral particles recognition by the mean of ancillary techniques [14,15]. In the present case, the macroscopic and microscopic appearance was suspicious of a viral etiology, although lacking molecular conﬁrmation. The progression from papilloma to squamous cell carcinoma has been recently ad- dressed as an emerging concern in canine oral papilloma [16]; the association between inverted papilloma and squamous cell carcinoma has not yet been reported. In the current paper, the association with malignancy, along with the malicious growth of inverted papilloma, recommends seeking it even in other species prone to develop papilloma. This case report, describing such evidence in a horse, warns about the need to include the inverted papilloma in the differential diagnosis of conjunctival neoplasm and drives the treatment paradigm for papilloma.

Author Contributions: Methodology, A.P., S.D.P., and A.S.; investigation, A.S. (histopathology), V.B. and S.M.; resources, A.S. and A.P.; writing—original draft preparation, A.S. and M.P.; writing— review and editing, A.S., A.P., and S.D.P.; funding, A.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by FFABR Unime grant. Institutional Review Board Statement: The horse underwent clinical and surgical procedures on call by the owner and under the relevant Italian guidelines on veterinary medicine good practice and European legislation on animal welfare. Informed Consent Statement: The informed consent statement was obtained from the horse owner. Acknowledgments: The authors would like to thank Cornelia Mannarino for the helpful technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. Vet. Sci. 2021, 8, 108 6 of 6

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