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Multinodular Malignant Cutaneous Mast Cell Tumor in
a Horse With Generalized Pruritus and Reactive
Fibrosis: A Case Report
Daniel Combarros, Itziar Wilhelmi-Vilarrasa, Caroline Lacroux, Marie-Odile
Semin, Maxence Delverdier, Margot Grebert, Catherine Trumel, Marie
Christine Cadiergues, Elodie Lallemand
To cite this version:
Daniel Combarros, Itziar Wilhelmi-Vilarrasa, Caroline Lacroux, Marie-Odile Semin, Maxence Delverdier, et al.. Multinodular Malignant Cutaneous Mast Cell Tumor in a Horse With General-ized Pruritus and Reactive Fibrosis: A Case Report. Journal of Equine Veterinary Science, WB Saunders, 2020, 87, pp.102921. �10.1016/j.jevs.2020.102921�. �hal-02947155�
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Multinodular malignant cutaneous mast cell tumour in a horse with generalized pruritus
and reactive fibrosis: a case report
Daniel COMBARROS a,b Itziar WILHELMI-VILARRASA a Caroline LACROUX c
Marie-Odile SEMIN a Maxence DELVERDIER c,d Margot GREBERT a Catherine TRUMEL a,e
Marie Christine CADIERGUES a,b Elodie LALLEMAND a,f
a- Department of Clinical Sciences, Université de Toulouse, ENVT, Toulouse, France
d.combarros@envt.fr; itziar.wv9@gmail.com; semin_marie@hotmail.com; m.grebert@envt.fr; c.trumel@envt.fr; mc.cadiergues@envt.fr@envt.fr; e.lallemand@envt.fr
b- UDEAR, Université de Toulouse, INSERM, ENVT, Toulouse, France - d.combarros@envt.fr; mc.cadiergues@envt.fr@envt.fr
c- Department of Basic Sciences, Université de Toulouse, ENVT, Toulouse, France - c.lacroux@envt.fr
d- IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France - m.delverdier@envt.fr
e- CREFRE, Université de Toulouse, INSERM, UPS, ENVT, Toulouse, France – c.trumel@envt.fr
f– InTheRes, Université de Toulouse, INRA, ENVT, Toulouse, France – e.lallemand@envt.fr
Corresponding author: Marie Christine CADIERGUES
© 2020 published by Elsevier. This manuscript is made available under the Elsevier user license
https://www.elsevier.com/open-access/userlicense/1.0/
Version of Record: https://www.sciencedirect.com/science/article/pii/S0737080620300125
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Abstract
Mast cell tumour (MCT) has long been considered as an uncommon neoplasm in horses. Cytological and behavioural evidence of its malignancy is usually lacking and only a few reports have described MCT displaying malignant behaviour.
An 18-year-old Friesian stallion presented with a one-year history of intermittent and progressive skin lesions on the left forelimb associated with intense, generalized pruritus and apathy temporarily responsive to glucocorticoids and antibiotics. The horse was alert and responsive with poor body condition and marked generalized pruritus. The left forelimb was markedly enlarged and surrounded by numerous firm 2 to 20-cm masses that were ulcerated and focally necrotic. A 7-cm round firm mass was observed on the left dorsal neck. Dermatological examination revealed generalized moth-eaten alopecia and scaling with erosions and ulcers secondary to pruritus.
A direct skin smear from the affected leg showed severe eosinophilic inflammation and neutrophilic inflammation with pleomorphic bacteria. Histopathology of the skin and biopsies of the underlying tissues revealed an abundant population of atypical mast cells consistent with a malignant MCT.
The horse was euthanized and necropsy revealed a marked fibrous reaction on longitudinal sections of the affected limb and the tumour could be detected on only a few histological slides.
Diagnosis of equine MCT can be challenging due to the massive accompanying fibrous reaction. MCT should be suspected in the presence of eosinophilic infiltration of the affected tissue and in cases of generalized pruritus not attributable to other causes.
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1- Introduction
Mast cell tumour (MCT) has long been considered as an uncommon neoplasm in horses [1, 2]. However, some surveys indicate a prevalence of up to 6.9% of equine tumours [3], thus positioning MCT at the 6th position in terms of neoplasm frequency in the horse, behind sarcoid, squamous cell carcinoma, lymphoma, melanoma and gonadal tumour [4, 5]. Although MCT is typically a cutaneous neoplasia in horses [6, 7], many other different locations have been reported, such as the
subcutaneous tissue [8], cornea [9], bone [10] or tendons [11]. Although few studies have been done of the breeds of horses diagnosed with MCT, as compared with the general equine population, Arabian horses seem to be overrepresented [1, 2, 4, 8]. Mean age at presentation ranges between 9.5 and 12.7 years, depending on the studies [2, 12].
The usual lack of cytological and behavioural evidence of malignancy (neither metastasis nor
recurrence after excision) has led some authors to qualify MCTs as “cutaneous mastocytosis” [2, 3, 6, 13, 14] when the neoplasm is confined to the skin. However, a few reports have described them as being locally invasive [9], fast growing and invading local lymph nodes [15] or even multicentric with numerous cutaneous and subcutaneous nodules, and involvement of the pulmonary, hepatic and spleen parenchyma [16].
2- Case history
An 18-year-old Friesian stallion presented with a one-year history of intermittent and progressive skin lesions of the left forelimb associated with intense and generalized pruritus, several episodes of hyperthermia, reduced appetite, marked weight loss and apathy. A temporary improvement of this situation had been obtained by the combined administration of antibiotics, always based on bacterial cultures and antibiograms (benzylpenicillin- Duplocilline, Intervet, France – 40 mL intramuscularly
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every other day, five times, then cefquinome – Cobactan 2.5%, Intervet, France – 30 mL intramuscularly once daily for 7 days 3 times at 6 week-intervals)and glucocorticoids
(dexamethasone, Rapidexon, Eurovet, Netherlands – 20 mL intravenously for 5 days) which reduced lesion size and pruritus, and increased appetite. Limb biopsies had revealed superficial perivascular dermatitis with a heavy eosinophilic infiltrate and epidermal hyperplasia. The horse had been treated with repeated applications of topical phoxime solution (Sebacyl 50% solution, Bayer Healthcare, France - 1 mL of a 50% solution diluted in 1 L of water) without improvement. At the time of presentation, the horse was being treated with enrofloxacin (Baytril 10% oral solution, Bayer Healthcare, France – 35 mL/day orally since 28 days), dexamethasone (Dexadreson, Intervet, France – 20 mL intramuscularly every other day for 10 days) and topical antiseptics (Douxo Pyo shampooing, CEVA Santé Animale, France applied every day). The limb lesions were worsening, treatment was no longer relieving the pruritus and the horse remained apathetic. A new mass had recently appeared on the dorsal neck.
3- Clinical findings
Physical examination at hospital admission revealed an alert, responsive horse with poor body condition (Body Condition Score of 1/5; 479 kg, estimated ideal weight 600 kg) (Fig. 1a) and marked generalized pruritus. Temperature was normal. The left forelimb was markedly enlarged (twice the normal size) and was surrounded by numerous (approximately 20) firm 2 to 20-cm masses that were ulcerated, focally necrotic and covered with a purulent, haemorrhagic discharge (Fig. 1b,c). There was little flexion of the carpus and fetlock upon manipulation. A 7-cm round firm mass was present on the left dorsal neck (Fig. 1d). Dermatological examination revealed generalized moth-eaten alopecia and scaling with erosions and ulcers secondary to pruritus (Fig. 1e,f). Pruritus was permanently present during the consultation and was extremely pronounced.
Differential diagnosis concerning the limb included epitheliotropic lymphoma, sarcoid, fibroma/ fibrosarcoma, squamous cell carcinoma, equine eosinophilic granuloma/equine nodular
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collagenolytic granuloma, fungal granuloma, cutaneous habronemiasis, onchocerciasis, cutaneous pythiosis, multisystemic eosinophilic epitheliotropic disease, and exuberant granulation tissue. Regarding the body cutaneous lesions, differentials included generalized atypical sarcoptic, psoroptic, chorioptic or demodectic mange, multisystemic eosinophilic epitheliotropic disease, erythema multiforme, adverse drug reactions, sarcoidosis, epitheliotropic lymphoma, severe atopic dermatitis with secondary bacterial and/or yeast overgrowth. The body skin lesions might also be secondary to limb tumour metastasis or paraneoplastic itch.
The complete blood count and blood smear evaluation revealed extreme leukocytosis (42.75 10^3 cells/µL, RI 5.79-13.75 10^3/µL) with marked neutrophilia (32.49 10^3 cells/µL, RI 2.7-9.6 10^3/µL), monocytosis (1.07 10^3 cells/µL, RI 0.13-0.59 10^3/µL), eosinophilia (5.64 10^3 cells/µL, RI 0.06-0.58 10^3/µL) and basophilia (0.684 10^3cells/µL, RI 0.01-0.13 10^3/µL). As the automated basophil count has not been validated in horses [17], the differential and basophil counts were assessed from a 1000-cell manual count. One basophil population of cells with cytoplasm filled with round, small, dark purple granules was clearly identified because the nuclei were obviously lobulated, but some cells with similar granulations and a round nucleus were also observed, and distinguishing between basophils and mast cells was very difficult (Fig. 2a,b). The biochemistry panel was within normal limits.
Limb radiography showed severe soft tissue swelling but no signs of bone involvement.
Ultrasonography revealed a very thick diffuse hyper-echogenic subcutaneous tissue with some fluid- filled cavities on the dorsal part of the limb. The mass on the dorsal neck had a 2-cm thick wall and was filled with a small quantity of anechoic fluid which, on cytological examination, revealed eosinophilic inflammation and no infectious agents. Abdominal ultrasound showed no abnormality. Multiple skin scrapes and trichograms were negative for parasites. Cytological examination of the limb discharge from the ulcers and fistula showed a mainly eosinophilic and neutrophilic
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stained skin surface tape strips revealed moderate bacterial overgrowth on the trunk. Multiple deep biopsies of the affected limb were performed and submitted for bacterial and fungal culture and histopathology. Trunk body biopsies were also submitted for histopathology. All samples were fixed in 10% formalin solution.
While awaiting the results, the horse was treated with injectable penicillin G (Duplocilline, Intervet, France – 35 mL intramuscularly once daily for 9 days), gentamicin (G4, Virbac, France – 78 mL intraveinously once daily for 5 days) and flunixin meglumine (Finadine, Intervet, France – 10 mL intraveinously once daily for 2 days), oral hydroxyzine (Hydroxyzine 25 mg Mylan, Mylan Medical SAS, France – 600 mg orally thrice daily the last two days), topical 3% chlorhexidine shampoos (Douxo Pyo Shampooing, CEVA Santé animale, France – once daily), moisturizer spray (Ermidra spray, MP labo, France – once daily) and limb bandages. After a few days, granulation tissue developed over the ulcerated skin of the left forelimb and pruritus was slightly reduced.
Bacterial culture revealed a Streptococcus dysgalactiae strain resistant to gentamicin and fluorinated quinolones. Fungal culture was negative.
4- Histopathological examination and necropsy findings
Biopsies from the affected leg revealed a dense dermal infiltrate with newly-formed vessels and congestion, moderate oedema and focal haemorrhages. The dermal infiltrate was composed of pleomorphic round and fusiform cells with central hyperchromatic and often multinucleolated nuclei, a high nucleus to cytoplasmic ratio and marked anisokaryosis (Fig. 3a,b). Eosinophils and neutrophils were also abundant. Tissue sections were stained with Toluidine blue which revealed metachromatic granules within the cytoplasm of the neoplastic cells, consistent with mast cells (Fig. 3c). Marked cellular atypia was observed within the mast cell population thus including anisokaryosis,
anisocytosis, hyperchromatism, prominent nucleolation, bizarre nuclei and a high mitotic index [29 mitoses/10 high power fields (HPF, 400x) – range 15-40]. Approximately 70% of the mast cells
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(defined per 500 neoplastic cells) expressed the Ki-67 protein (Ki-67 Antigen, MIB-1, Agilent
Technologies, USA – 1/50 - Envision Detection Systems, Peroxidase/DAB, Rabbit/Mouse, Agilent) (Fig. 3d). The findings were consistent with a malignant MCT.
The fluid-filled cavities were confirmed as necrotic tissue on histopathology.
Trunk skin biopsies revealed perivascular neutrophilic superficial dermatitis associated with epidermal hyperplasia and neutrophilic serocellular crusts. No infectious agents were visualized either with hematoxylin-eosin stain or periodic acid-Schiff stain.
Based on the aggressive clinical and histological behaviour of the tumour, the prognosis for successful treatment was guarded and the owners elected to have the horse euthanized.
At necropsy, longitudinal section of the left forelimb revealed infiltration of the subcutaneous tissue with an abundant whitish homogenous and firm tissue sparing the tendons, ligaments, joints and bones. The regional lymph nodes were normal in size and appearance. Diffuse moderate
hepatomegaly was observed and associated histologically with steatosis. Multiple small ulcers were mainly observed on the gastric mucosa at the margo plicatus, sometimes coalescing to form large 5 to 6 mm ulcers with raised edges and necrotic centre and graded IV according to the European College of Equine Internal Medicine Consensus Statement [18]. Other abdominal or thoracic organs were macroscopically normal. Tissue samples from normal and alopecic skin, the dorsal neck mass, left axillary and left prescapular lymph nodes, liver, spleen and left forelimb (multiple sites) were collected at necropsy. Samples were fixed in 10% formalin solution and processed for histopathology. The histological examination did not reveal any signs of internal organ or lymph nodes metastasis. A heavy fibrous reaction was widely found on the affected forelimb samples along with eosinophilic infiltration and neovascularization/angiogenesis (Fig.3e,f). Neoplastic mast cells infiltrating the dorsal neck mass were detected but the numbers of cells and cellular atypia were less abundant than in the limb tumour.
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5- Discussion
Equine MCTs are considered to exhibit benign biological behaviour, to seldom recur after surgical excision and may even spontaneously regress [2]. However, up to one third of the equine MCTs analysed in the pathology laboratory of one university appeared to be poorly differentiated and displayed infiltrative growth, moderate to marked anisokaryosis, a high degree of proliferation and aberrant KIT expression [19]. However, in another study, thesame observations were not associated with clinical outcome [20], suggesting that histological appearance might not be indicative or
predictive of biological behaviour. Prospective studies are needed to determine the malignant nature of equine MCTs [21], however malignant MCTs exhibit a high proliferation rate and cellular atypia, in combination with aberrant KIT protein expression. There is evidence that cytoplasmic c-KIT
expression is a useful diagnostic marker for malignant equine MCTs and membranous c-KIT
expression for benign equine MCTs [19,21]. However, atypical cells with morphological features and immunophenotype of mast cells can be discussed as histiocytic-like neoplastic mast cells in equine species [22].
In the current case, several clinical signs were observed that have been described in dogs as suggestive of MCTs exhibiting aggressive behaviour [23, 24]. These included rapid growth, local irritation/inflammation, local infiltration/poor demarcation from adjacent tissues, ulceration and satellite nodules. In our case, the MCT was associated with a very strong fibrous reaction, which has already been observed in other cases described in the literature [2, 16, 25, 26]. Reactive fibrosis could be secondary to massive eosinophil degeneration and necrosis. This type of reaction seems to appear more frequently in horses than in other species [2]. In the present case, the intense pruritus led to chronic self-trauma of the affected limb which exacerbated the fibrous tissue formation. This reactive fibrosis can make the diagnosis of a MCT challenging when performing biopsies as sampling could potentially concern only the fibrous tissue and miss any neoplastic cells.
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In addition, the suspected malignant behaviour was supported histologically by marked anisokaryosis and anisocytosis and by the high mitotic rate and Ki-67 expression. In the present case the mitotic index was very high (29/10 HPF x400). However, a high or low mitotic rate in MCT does not seem to always correlate with a poor or good prognosis in the horse [15]. Probably, no specific histological variable can independently indicate prognosis but all such variables should be taken together with the clinical picture, to determine the most likely behaviour [16]. Combining Ki-67 expression with toluidine blue staining is useful in differentiating MCTs from other lesions such as eosinophilic granuloma or Multisystemic Eosinophilic Epitheliotropic Disease (MEED) in horses.
Ki-67 protein is a marker of proliferation expressed during the cell cycle that can be detected by immunohistochemistry and is significantly associated, in small animal practice, with MCT tumour prognosis, independently of the tumour grade (increased mortality, recurrence and metastasis) [27]. In the present case, approximately 70% of the mast cells stained positive for Ki-67 suggesting a high degree of malignancy. However, due to the high variability of the histological appearance of MCTs in large animals, Ki-67 represents a less useful prognostic marker than in small animals [28].
The most probable causes of pruritus in this case were excluded. The animal had received an appropriate acaricidal treatment and there was no evidence of parasites in the multiple diagnostic tests. The animal had no previous history of any pruritic disease that might suggest an allergic background and the histopathological analyses of the skin biopsies from the trunk were not
consistent with a drug reaction, skin tumour or eosinophilic epitheliotropic disease. Even if bacterial overgrowth was diagnosed, treatment of this syndrome with systemic antibiotics and daily topical antiseptic shampoos only mildly alleviated the pruritus. The limb lesions and pruritus seemed linked because there was a response to different treatments that were associated with a reduction in tumour size and pruritus. It is possible that the very severe skin pruritus was secondary to the systemic release of histamine [29] or pruritogenic cytokines like IL-31 [30] by the tumour cells. Biopsies of alopecic trunk skin did not show any infiltration of the dermis or epidermis by mast cells
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that could explain a local release of cytokines, as in systemic mastocytosis or in skin mast cell
metastasis. Paraneoplastic itch (PI) has been defined, by a Special Interest Group of the International Forum on the Study of Itch, as follows: sensation of itch as a systemic (not local) reaction to the presence of a tumour or a haematological malignancy neither induced by the local presence of cancer cells nor by tumour therapy. Thus PI does not describe drug-induced itch in patients with cancer or itch secondary to infections or specific dermatosis in cancer patients. In human medicine, PI is more frequently associated with haematological malignancies but has also been reported with solid tumours [31]. It usually disappears with remission of the tumour and can return with its relapse [32]. In the present case, unfortunately, as the horse was euthanized, it was not possible to observe if the pruritus disappeared after tumour removal. In consequence, PI cannot be proven and remains purely hypothetical.
The horse in this case also developed several episodes of hyperthermia. The association between MCT and fever has already been described in veterinary medicine [33]. The mechanisms involved in paraneoplastic fever are not fully understood but could represent the release of pyrogenic cytokines by cancer cells such as interleukin (IL)-1, IL-6, tumour necrosis factor and interferon [34]. In the present case, fever could also be related to the heavy and deep bacterial infection or to tumour necrosis and the consequent release of TNF and other pyrogens from necrotic tissue [35]. Equine Squamous Gastric Disease (ESGD) has been unclearly associated with various types of clinical signs in adult horses, such as poor appetite, poor body condition, weight loss, chronic diarrhea, poor coat condition, bruxism, behavioral changes, acute or recurrent colic, and poor performance [18]. In the present case, it seems difficult to determine the exact link between ESGD and the set of symptoms which was observed.
Large numbers of eosinophils were found on direct smears of the leg lesions, on histology and in the peripheral blood count. Eosinophilia and basophilia are uncommon in horse and are mainly
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was not observed or not reported, probably because not investigated [2, 9, 11, 15, 25, 28]. However, in one horse with multicentric mastocytoma, marked eosinophils infiltration of the largest cutaneous tumour and of different organs was observed. Mast cells are known to release chemotactic factors and cytokines, like CCL5 and IL-16, that attract eosinophils [37, 38] and MCTs are therefore
consistently associated with large numbers of eosinophils on histology in both large and small animals [15, 39, 40].
Basophilia is seldom reported in horses but since haematology analysers are unable to detect these cells, a manual differential leukocyte count is needed to determine any increase in the number of basophils and is rarely performed. It is therefore likely that basophilia is underestimated. One case of marked basophilia reported in a horse was associated with basophilic enterocolitis suspected to be a variant of eosinophilic enteritis secondary to immediate Type I hypersensitivity [41]. Four cases of basophilia have also been reported in a series of 23 cases of horses with Daboia (Vipera) palaestinae snake envenomation [42]. In this series, basophilia was a poor prognosis factor. If we consider the differential of basophilia in other species, it has been reported in dogs with dirofilariosis and other parasites or infectious agents, in various hypersensitivity and inflammatory conditions, in neoplasia including mast cell tumor with or without mastocytemia, lymphoma and basophilic leukemia [43].
Circulating mast cells have not been described in the reported cases of MCTs in horses, but it was not always indicated whether a blood smear evaluation and manual differential leukocyte count were performed. Basophils and mast cells in horses contain round, small, dark purple granules that fill the cytoplasm and sometimes hinder visualization of the nucleus and make it difficult to distinguish between these two types of cell. Atypical mast cells with evident morphological features of malignancy, such as hypogranularity, would be easily identified in blood. In our case, some highly granulated cells with an obvious round nucleus were occasionally observed and could be neoplastic mast cells as the neoplastic cells from the lesions were granulated according to histopathology. Further examinations would have been necessary to confirm this hypothesis. Previous studies
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reported a strong positive staining of equine MCT with toluidine blue and expression of CD117 (cKIT) but these stains cannot efficiently differentiate basophils from mast cells as they stain both cell-types [19, 20, 25].
6- Conclusion
In conclusion, equine MCTs are uncommon neoplasms which can present a high degree of malignancy. Diagnosis can be challenging when the tumour is accompanied by a massive fibrous reaction, which is apparently a common finding. These neoplasms can be accompanied by other signs such as fever, gastric ulcers or local or diffuse pruritus. Prompt diagnosis is required for any
attempted treatment to be successful.
Declarations
Ethics approval and consent to participate
All diagnostic and therapeutic procedures were performed by licensed veterinarians in the course of routine veterinary health management.
Consent for publication
The owner of the horse gave her consent for publication.
Availability of data and materials
The data generated, and/or used during the work-up of this case cannot be made publicly available in the interests of retaining patient confidentiality, but are available from the corresponding author on reasonable request.
13 Competing interests
The authors declare that they have no competing interests.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors
Authors' contributions
Daniel Combarros – Investigation (clinical prior to euthanasia); original draft; review & editing. Itziar Wilhelmi-Vilarrasa – Investigation (clinical prior to euthanasia); review & editing.
Caroline Lacroux – Investigation (post-mortem examination and histopathology); review & editing. Marie-Odile Semin – Investigation (post-mortem examination and histopathology); review & editing. Maxence Delverdier – Investigation (post-mortem examination and histopathology); review & editing.
Margot Grebert – Investigation (clinical pathology); review & editing. Catherine Trumel – Investigation (clinical pathology); review & editing.
Marie Christine Cadiergues – Investigation (clinical prior to euthanasia); original draft; review & editing.
Elodie Lallemand – Investigation (clinical prior to euthanasia); review & editing.
Acknowledgements
The authors would like to acknowledge Lilly Elanco for supporting the position of D. Combarros as an ECVD resident during his first year of residency. They thank Diana Warwick for English editing and François Schelcher for his help with necropsy findings.
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Fig. 1: Initial physical examination. Poor body condition, markedly enlarged left forelimb and
generalized moth-eaten alopecia (a); close view of the left forelimb with ulcerated, focally necrotic multiple firm 2 to 20-cm masses covered with a purulent, haemorrhagic discharge on the lateral (b) and medial (c) aspects; left dorsal neck with a 7-cm round firm mass (d); alopecia and scaling with erosions and ulcers secondary to pruritus on the tail base (e) and the neck and face (f).
Fig. 2: Cytology from an 18-year-old horse with mast cell tumour. Blood smear showing basophil
with a lobulated nucleus (a). Basophilic granulated cell with a round nucleus suggestive of a mast cell or a basophil between an eosinophil and a neutrophil (b) (May-Grünwald Giemsa staining,
magnification X1000). Impression smear from the purulent discharge of the limb showing mainly eosinophilic and neutrophilic inflammation with variable numbers of pleomorphic bacteria (c, d) (RAL® staining, magnification X1000).
Fig. 3: Histopathological examination of biopsies taken from the left forelimb. Dense dermal
infiltrate composed of mast cells (blue arrow) with a central hyperchromatic and often
multinucleolated and/or vacuolated nuclei (yellow arrow), a high nucleus to cytoplasmic ratio and marked anisokaryosis and mitoses (green arrow, 15-40 per 10 HPF), accompanied by abundant
18
eosinophils (red arrows) and neutrophils (a, b); (H&E staining, magnification ×400, and × 1000, bars = 50 µm, and 10 µm). Toluidine Blue staining showing intracytoplasmic metachromatic granules (blue arrow) (c) (magnification x1000, bar = 10 µm). Immunohistochemical staining for Ki-67:
approximately 70% of mast cells expressed the Ki-67 protein (d) (magnification x1000, bar = 10 µm). Intense reactive fibrosis (e) and collagenolysis (*) (f) (H&E staining, magnification ×200, bars = 100 µm).
