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EQUINE VETERINARY EDUCATION / AE / AUGUST 2019


lateral cornea where the keratectomies were performed as an adjunctive therapy to help prevent recurrence of neoplasia. A radiation dose of 100 Gy was applied to each site.


Two grafts of porcine urinary bladder extracellular matrix


Fig 1: Photograph of the affected cornea at initial presentation. There are two irregular pale pink corneal masses rising minimally from the dorsal corneal surface. There are pinpoint areas of faint pigmentation present throughout the entire cornea.


vascularisation was present extending from the limbus with multifocal regions of anterior stromal haze and pinpoint areas of faint epithelial pigmentation. The remainder of the ophthalmic examination OS was within normal limits. Differential diagnoses for the corneal lesions included


neoplasia (squamous cell carcinoma, vascular tumour, mast cell tumour, lymphosarcoma, melanocytic tumour), granulation tissue from a previous injury, infectious keratitis and eosinophilic or other immune-mediated keratitis. Under standing sedation, topical anaesthetic (proparacaine 0.5% ophthalmic solution) was applied to the left cornea, and a shave biopsy of one of the corneal masses was performed using a microsurgical blade (No. 6400 Beaver Mini-Blade). Cytology of impression smears was highly cellular, containing binucleate and multinucleated cells with moderate-to- marked anisokaryosis, anisocytosis and anisonucleolosis. Neoplasia, and particularly squamous cell carcinoma, was suspected due to the numerous criteria for malignancy and lack of inflammatory cells present. Histopathology of the shave biopsy showed focal corneal fibroplasia with no neoplastic cells present. Excisional biopsy with a superficial keratectomy was recommended to obtain a definitive diagnosis. High-frequency ocular ultrasound to determine the depth of the lesions prior to surgery was considered, but was deemed unnecessary. The lesions appeared superficial on examination, and any visible pathological areas would be excised with surgery and followed by adjunctive therapy.


Surgical procedure One week following initial examination, the horse was anaesthetised and placed in right lateral recumbency. A superficial keratectomy, approximately 20 mm by 15 mm in size and 50% corneal depth, was performed to completely encompass both corneal masses and the surrounding areas of stromal opacity on the dorsal cornea. A second superficial keratectomy, approximately 8 mm in diameter, was performed adjacent to the lateral limbus where stromal haze and corneal surface irregularity were visible upon microscopic examination. This abnormal region was not appreciated on initial examination. Following the keratectomies, strontium-90 beta irradiation was applied in three separate locations to the dorsal and


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(ACell Vet Corneal Discs)2 were trimmed to size and sutured over the keratectomy sites using 8-0 polyglactin suture in a simple interrupted pattern. A ventral subpalpebral lavage line was placed for administration of post-operative topical medications. A lateral temporary tarsorrhaphy was placed for protection of the surgical site during initial healing. The horse recovered well from anaesthesia and was discharged the following day. Topical medications OS included neomycin/ polymixin B/gramicidin ophthalmic solution six times per day, serum six times per day and atropine 1% ophthalmic solution once daily. The horse was also treated orally with flunixin meglumine at a dose of 1.1 mg/kg twice daily, and sulfamethoxazole and trimethoprim at a dose of 25 mg/kg twice daily.


Histopathology Histopathological examination of the dorsal corneal biopsy revealed aggregates and clusters of large, round-to-oval neoplastic cells within the surface epithelium, along the stratum basale and within the stroma (Fig 2). The cells had large, round-to-oval nuclei, 1-2 nucleoli, an abundant amount of lightly basophilic cytoplasm and distinct cell borders. A few cells contained occasional brown granules interpreted to be melanin (Perl’s stain for iron was negative, ruling out the possibility that the pigment was haemosiderin). There was moderate-to-marked anisocytosis and anisokaryosis, and a high mitotic index (25 per 10 – 400 9 fields). Additionally, marked infiltrate of lymphocytes and plasma cells, occasional eosinophils, and fibrosis and neovascularisation were observed within the adjacent stroma. Surgical clearance of neoplastic cells appeared complete along the deep and outer margins. The lateral corneal biopsy did not contain neoplastic cells, but had a substantial number of melanophages and other inflammatory


cells along the epithelial–stromal interface. Based on these findings, consideration was given to a poorly melanised malignant melanoma or a poorly differentiated squamous cell carcinoma. Immunohistochemistry (IHC) was performed for four


markers: monoclonal mouse anti-vimentin, clone V9; polyclonal rabbit anti-S100 protein; monoclonal mouse anti-human melan-A, clone A103; and monoclonal mouse anti-human cytokeratin, clone MNF1163.These markers were chosen to try to differentiate squamous cell carcinoma, a tumour of epithelial origin that would likely express cytokeratin, from a malignant melanoma, a tumour of neuroectodermal origin that would likely express vimentin, S100 and potentially melan-A (Desnoyers et al. 1990; Ramos-Vara et al. 2000, 2014; de Wit et al. 2004). Neoplastic cells had strong cytoplasmic immunoreactivity for vimentin and S100, negative staining for melan-A and weak cytoplasmic immunoreactivity for cytokeratin (Fig 3). Poorly melanised malignant melanoma with chronic lymphoplasmacytic keratitis was diagnosed based on the results of IHC, the pattern of neoplastic cells within the cornea, and the presence of a modest amount of brown granular pigment within a few neoplastic cells.


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