|Intratesticular benign peripheral nerve sheath tumour in a ferret (Mustela putorius furo) - case report
A domestic ferret was submitted for sterilisation due to right testis enlargement. Estradiol and cortisol concentrations were within normal physiological ranges, but testosterone was below and progesterone above normal. Microscopically, the right testis, with the exception of a small part of the epididymis, was replaced with neoplastic tissue. The tumour was composed of streams and bundles of closely packed spindle to ovoid cells forming whorls around collagen and capillaries, and separated by a collagenous matrix. In some areas, cells were loosely arranged and separated by a pale myxomatous matrix. The left testis showed atrophy. The majority of neoplastic cells expressed vimentin and S-100 protein while expression of collagen IV was moderate and there was no expression of glial fibrillary acid protein. On the basis of macroscopical and histopathological findings, and supported by immunohistochemical reactivity, the diagnosis of benign peripheral nerve sheath tumour (PNST) was made. This is the first report of benign PNST in ferret testis.
Key words: PNST, ferret, testis, immunohistochemistry, hormone
Peripheral nerve sheath tumours (PNST) are neoplasms arising from Schwann cells, perineurial cells, and intraneural fibroblasts. On the basis of morphological features and assessment of benignancy versus malignancy, they can be subdivided into schwannoma or benign PNST (previously called neurilemoma or neurinoma), neurofibroma or malignant PNST (Schulman and others 2009). Due to their uncertain histiogenesis, PNSTs in veterinary medicine are divided into benign and malignant variants in the most recent edition of the WHO International Histological Classification of Tumours of Domestic Animals (Koestner and others 1999). There are few studies of the incidence of neoplasia in ferrets (Brown 1997; Zeeland and others 2006). The most common tumours reported are insulinoma, lymphoma and adrenocortical neoplasms, followed by skin neoplasias and musculoeskeletal neoplasms (Dillberger and Altman 1989). Ovarian stromal tumours are the main neoplasia of the reproductive tract in female ferrets, while neoplasms of the male reproductive system are unusual (Beach and Greenwood 1993). A few studies have described findings of testicular neoplasia in ferrets: interstitial cell adenoma (Meschter 1989), Sertoli cell tumour in chryptorchid testis (Powers and others 2007) and mixed interstitial and Sertoli cell tumour (Batista-Arteaga and others 2011). In the available literature, there is only a single report of PNST in ferret, i.e. neurilemoma on the leg (Brown 1997). We now present the first case of benign PNST in a testicle of a ferret and discus in detail the reasons for this diagnosis.
A 2.2 kg, male, 6-year old, moderately obese domestic ferret from Zagreb Zoo, was submitted to the Surgery, Orthopaedics and Ophthalmology Clinic, Faculty of Veterinary Medicine, University of Zagreb in October 2010 for sterilisation due to right testis enlargement. During the operation, blood samples were taken for determination of estradiol, progesterone, testosterone and cortisol concentrations. Ultrasound evaluation of the abdomen was performed prior to the orchidectomy, which was performed through a scrotal incision with ligation of the spermatic cord the standard manner.
The testes were submitted to the Department of Veterinary Pathology for histopathological examination. The left testis was round and about 0.8cm in diameter (volume of cca. 0.27 cm3), while the right testis had a diameter of about 3.5 cm (volume of cca. 22.43 cm3). For comparison, the average testicular diameter in sexually mature ferrets is around 1.2cm (Sisk 1990), with an average volume of 0.94-1.38 cm3 (Schoemaker and others 2008). On the basis of these reported values, the left testis (0.8cm, 0.27 cm3) was slightly below the normal range, but the right considerably above (3.5cm, 22.43 cm3). The left testis was moderately firm and showed no macroscopic changes on the external or cut surface. The right testis was firm with a rugged, external surface and was whitish-grey and lobulated on the cut surface. The testes were fixed in 10% neutral buffered formalin, embedded in paraffin and processed routinely. Sections were stained by haematoxylin and eosin (HE).
Immunohistochemistry was performed for exact tumour classification. Primary antibodies used were specific for the following antigens: vimentin (VIM) (Clone V9, Dakocytomation, No. M0725, diluted 1:200), S-100 protein (S-100) (DakoCytomation, No. Z0311, diluted 1:400), glial fibrillary acid protein (GFAP) (clone 6F2, DakoCytomation, No. M0761, diluted 1:300) and collagen IV (col IV) (clone CIV 22, DakoCytomation, No. M0785, diluted 1:25). Antigen retrieval for S-100 was not performed and for VIM was performed with microwave pretreatment with proteinase K (DakoCytomation, Code S3004) 5 min. Antigen retrieval for col IV was performed with ethylene diamine tetraacetic acid (EDTA) buffer, pH 9 (DakoCytomation, Code S2367) 3 x 5 min. and for GFAP with citrate buffer pH 6 (DakoCytomation, Code S2031) 20 min. in a Dako PT Link module. Endogenous peroxidase activity was blocked by 5-min. treatment with hydrogen peroxide (Dako No. S2023). Dako REALTM EnVisionTM Detection System, Peroxidase/DAB, Rabbit/Mouse (No. K5007) was used to detect the antibodies against VIM, S-100, col IV and GFAP.
Standard electro-luminiscence serum hormone testing showed an estradiol concentration of 5.00 ng/L, progesterone of 18.6 nmol/L, testosterone of 0.087 nmol/L and cortisol of 40.8 nmol/L. Ultrasound examination of all abdominal organs, prostate and both adrenal glands (left = 7.6 mm length, right = 7.8 mm length) appeared normal (reference values reported by Neuwirth and others 1997, are: left = 8.6±1.2 mm; right = 8.9 ±1.6 mm).
Microscopically, with the exception of a small part of epididymis, the remainder of the right testis was replaced with neoplastic tissue (Fig. 1). The tunica albuginea was not penetrated by neoplastic cells. Neoplastic tissue was composed of interlacing streams and bundles of closely packed spindle to ovoid neoplastic cells and separated by small amounts of collagenous matrix. These cells often formed whorls around the collagen and capillaries (Antoni A like areas) (Fig. 2). In some areas, cells were more loosely arranged and separated by a pale amphophilic, myxomatous matrix (Antoni B like areas) (Fig. 3). Borders of neoplastic cells were variably distinct and the cytoplasm was scant and eosinophilic. Nuclei varied from round to elongate with finely stippled, marginated or condensed chromatin, with one to two poorly distinct nucleoli. Mitotic figures were less than 1/10 HPF. Areas of coagulative necrosis and haemorrhage were scattered throughout the tumour. The left testis showed marked atrophy. All of the aforementioned microscopic features, except for necrosis and haemorrhage, suggested a benign neoplasm. Furthermore, the shape of the cells and their arrangement clearly indicated a neoplasm from the group of spindle cell tumours. The two different patterns with typical Antoni A and Antoni B like areas with the accompanying histological features are characteristic of benign PNST (Bergmann and others 2009; Chijiwa and others 2004). Although there were areas of coagulative necrosis and haemorrhage within the tumour, there were no other histological criteria of malignancy (infiltrative cell growth and aggressive behaviour, presence of atypical mononuclear or multinucleated cells, cellular pleomorphism, high mitotic index) to make a diagnosis of malignant PNST (Chijiwa and others 2004). Finally, even though histological features of the tumour were clearly suggestive of benign PNST, to exclude other spindle cell tumours, IHC stains for VIM, S-100 and col IV were applied.
IHC staining for VIM was strongly positive in cytoplasm (Fig. 4) and S-100 was moderately positive in the nuclei and cytoplasm of majority of neoplastic cells (Fig. 5). Col IV was moderately extracellularly positive (Fig. 6) whereas staining for GFAB was negative in the tumour.
To our knowledge, this is the second report of a benign PNST in a domestic ferret and first report of this tumour in the testis (Brown 1997). The diagnosis of benign PNST was based on macroscopic and histopathological findings which were identical to those described in the literature (Bergmann and others 2009; Chan and others 2007; Chijiwa and others 2004; Koestner and Higgins, 2002; Rothwell and others 1986). Since there is no single specific IHC marker that is able to define PNST, a panel of reagents is generally employed. PNSTs are mesenchymal spindle cell tumours and are in 100% of cases positive for vimentin (Bergmann and others 2009; Chijiwa and others 2004). S-100 protein identifies Schwann cells hence, most PNSTs express this molecule (Bergmann and others 2009; Chijiwa and others 2004). Since Col IV is a basement membrane component of Schwann cells, PNSTs can also be positive to this antibody (Gaitero and others 2008). Finally, GFAB - which is a neuronal marker, may be variably expressed (around 67%) in PNSTs (Bergmann and others 2009; Chijiwa and others 2004). Spindle cell tumours which can have similar IHC reactivity are malignant PNST, rabdomyosarcoma and other tumours of vascular origin (Bergmann and others 2009; Chijiwa and others 2004). In our case, typical histological findings excluded potential diagnosis of these tumours. Morover, IHC reactivity to VIM, S-100 protein and col IV confirmed the diagnosis of benign PNST (Chan and others 2007; Gaitero and others 2008; Koestner and Higgins 2002; Schulman and others 2009).
Estradiol (5.00 ng/L) and cortisol (40.8 nmol/L) levels were within the reference ranges of 8±3 ng/L (Carroll and Baum 1989) and 53±42 nmol/L (Rosenthal and Peterson 1996), respectively. However, the progesterone level (18.6 nmol/L= 5.85ng/mL) was approximately twice the normal value (3.1±0.42 ng/mL) (Carlson and Rust 1969) and the testosterone level (0.087 nmol/L) was more than 100 times lower than the normal value (9-73 nmol/L) (Schoemaker and others 2008).
Since PNSTs are not hormone-secreting neoplasms, this raises some questions concerning the altered plasma levels of progesterone and testosterone. One explanation could be that progesterone is produced, in male animals, mainly by the adrenal glands and is then converted to testosterone in testes (Eckstein and others 1987; Maitra and Abbas 2005). Elevated progesterone and decreased testosterone level could be result of replacement of the testicular tissue of the right testis with neoplastic tissue and left testis atrophy. Since a very small amount of functional testicular tissue remained, a very low conversion rate of progesterone to testosterone could be assumed. As a result, testosterone levels would drop and progesterone concentration be increased in plasma.
Considering that surgical castration of male ferrets is a common practice to prevent reproduction and to reduce interspecies aggression (Schoemaker and others 2008) it may be difficult to define the prevalence of testicular tumours in ferrets (Batista-Arteaga and others 2011). According to this, our finding of intratesticular benign PNST can contribute in a better understanding of the incidence and clinic significance of testicular neoplasm in ferrets.
This work was supported by Republic of Croatia, Ministry of Science, Education and Sports, project no. 053-053-2264-2260.
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Fig. 1. Right testis, ferret. With the exception of a small part of the epididimis, the rest of the right testis was replaced with neoplastic tissue (H&E, x10).
Fig. 2. Right testis, ferret. Antoni A like areas, neoplastic cells form whorls around collagen and capillaries (H&E, x10).
Fig.3. Right testis, ferret. Antoni B like areas, neoplastic cells loosely arranged and separated by a pale amphophilic, myxomatous matrix (H&E, x10).
Fig.4. Right testis, ferret. Neoplastic cells with strong cytoplasmic staining for VIM (IHC, x40).
Fig.5. Right testis, ferret. Neoplastic cells are moderately positive in nuclei and cytoplasm for S-100 (IHC, x40).
Fig.6. Right testis, ferret. A moderate extracellular col IV positive reaction (IHC, x20).