Malignant Peripheral Nerve Sheath Tumour ...

ARTICLE IN PRESS J. Comp. Path. 2007,Vol. 137,137^141

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SHORT PAPER

Malignant Peripheral Nerve Sheath Tumour (Malignant Schwannoma) in the Diaphragm of a Goat ´ez, F. Rodrı´guez, A. Godhino, G. A. Ramı´rez, P. Herra M. Andrada and A. Espinosa de los Monteros Departamento de Morfolog|¤a, Facultad deVeterinaria, Universidad de Las Palmas de Gran Canaria, Trasmontana s/n, 35413 Arucas, Las Palmas, Spain

Summary This report describes a malignant schwannoma in the diaphragm of a 2-year-old goat.The immunohistochemical and histological features indicated a diagnosis of malignant schwannoma rather than neuro¢brosarcoma. The diaphragm represents an unusual location for neoplasia in both domestic animals and human beings. A possible anatomical origin from the phrenic nerve is discussed. r 2007 Elsevier Ltd. All rights reserved. Keywords: goat; peripheral nerve sheath tumour; schwannoma; tumour

Peripheral nerve sheath tumours (PNSTs) are a heterogeneous group of neoplasms arising from cells surrounding the axons of neurons in peripheral nerves. They include schwannoma (neurilemmoma), which originates from Schwann cells, and neuro¢broma, which originates from both Schwann cells and ¢broblast or perineurial cells (or a mixture of both). Schwannomas and neuro¢bromas are well characterized and, in human neuropathology, are considered to be distinct (Summers et al., 1995; Weiss and Goldblum, 2001). In domestic animals, however, distinction between the two forms by light microscopy is less clear cut, and for this reason both are classi¢ed as PNSTs by theWorld Health Organization (WHO) (Hendrick et al.,1998; Koestner et al.,1999). On the basis of histological appearance and biological behaviour, PNSTs are divided into benign PNST (BPNST) and malignant PNST (MPNST) forms, and both have been reported in cattle, sheep, goats, horses, dogs and cats (Theilen and Madewell, 1987; Veazey et al., 1993; Anderson et al., 1999; Koestner and Higgins, 2002; Chijiwa et al., 2004).

Correspondence to: G.A. Ram|¤ rez. (e-mail: [email protected]). 0021-9975/$ - see front matter

doi:10.1016/j.jcpa.2007.05.003

PNSTs may exhibit a wide range of histological patterns of sarcomatous growth, and ultrastructural or immunohistochemical examination (or both) may be necessary to establish a diagnosis. Primary neurogenic tumours of the diaphragm, particularly those showing malignant di¡erentiation, are uncommon in human patients (Moinuddeen et al., 2001; Kumbasar et al., 2004) and rare in animals (Anderson et al., 1999). Moreover, neoplasia in the goat is rare (Bastianello, 1983; Veazey et al., 1993). This report describes the histological and immunohistochemical diagnosis of a malignant schwannoma (MPNST) in the diaphragm of a goat. Tissue samples (including lung, liver, diaphragm, kidney, spleen and lymph nodes) from a slaughtered 2-year-old female Majorera goat were submitted to the Pathology Service of the Veterinary Faculty. Several coalescing nodules on the left thoracic surface of the diaphragm and one nodule in the left hepatic lobule had been detected at the abattoir. The diaphragmatic nodules, which were round to oval, measured from 0.5 to 2 cm in diameter; many coalesced to form a multilobulated, well-demarcated mass with a smooth surface originating from the pars costalis of the left thoracic diaphragm, in a linear plaque-like r 2007 Elsevier Ltd. All rights reserved.

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growth. On cut section, the mass consisted of bulging white to pale grey tissue with a ¢brous consistency (Fig. 1a). There was no adhesion to adjacent organs. The presence of a single white nodule, 1.5 cm in diameter, was con¢rmed in the liver (Fig. 1b). Tissue samples were ¢xed in 10% neutral bu¡ered formalin, processed by routine methods and embedded in para⁄n wax for light microscopical and immunohistochemical examination. Sections (4^5 mm) were stained with haematoxylin and eosin (HE), periodic acid-Schi¡ (PAS), or Masson’s trichrome. For immunohistochemistry (IHC) the avidin biotin peroxidase complex (ABC) method (Vector Corporation, Burlingame, CA, USA) was used. Sections (3 mm) were labelled with commercially available monoclonal antibodies against vimentin (Dako, Glostrup, Denmark; diluted 1 in 100), nerve growth factor receptor (NGFr; Dako; 1 in 50), neuron-speci¢c enolase (NSE; Dako;1in1000), neuro¢laments (NF; EuroDiagnostica, Arnhem, The Netherlands; 1 in 20), a and g-smooth muscle actin (SMA; Enzo, Farmingdale, NY, USA; 1 in 200), calponin (Dako; 1 in 400), and cytokeratin AE1/AE3 (Dako; 1 in 100). Polyclonal (rabbit) antisera against S-100 protein (Dako; 1 in 100), glial ¢brillary acidic protein (GFAP; EuroDiagnostica;1 in 100), myoglobin (Dako; 1 in 2000), and desmin (EuroDiagnostica; 1 in 100) were also employed. For ‘‘visualization’’, 3-amino-9-ethylcarbazole (AEC; Sigma-Aldrich Co., St Louis, MO, USA) was used. Tissues were counterstained with Mayer’s haematoxylin.

Histological examination revealed a neoplastic process with a common cell type and growth pattern. The tumours were relatively well-demarcated but not completely circumscribed by connective tissue. Neoplastic cells in¢ltrated from the diaphragmatic surface, extending deeply into the connective tissue and among the muscular bundles. Some of the muscular ¢bres appeared degenerate and atrophied. The mass was mainly composed of densely packed, pleomorphic spindle cells, arranged in interwoven fascicles and bundles with a sparse ¢brillar collagenous stroma (Fig.2).These cells occasionally formed palisading areas and whorllike aggregates. They exhibited moderate amounts of lightly acidophilic, vacuolated cytoplasm and indistinct cell borders. The nuclei were large and elongated to fusiform, with a vesicular-chromatin pattern and one to three small but prominent nucleoli. Other nuclei showed marked anisokaryosis and were di¡usely hyperchromatic. Some areas of the tumour showed anaplastic spindle-shaped or plump cells, with epithelioid characteristics, arranged in streaming or interlacing bundles. Other atypical cells consisted of mononucleated and multinucleated bizarre giant cells with prominent cytoplasmic projections. Intracytoplasmic PAS-positive globules were occasionally seen within atypical giant cells (Fig. 2, inset). Mitotic ¢gures were frequently observed (43 per high-power ¢eld). The walls of small arterioles and the nerves within the tumour were in¢ltrated by neoplastic cells. Di¡use mononuclear in¢ltrate, consisting of mature

Fig. 1a,b. (a) Several sections of the diaphragmatic tumoral mass, showing relatively well-demarcated nodules, displacing and in¢ltrating the muscle tissue. (b) Cut surface of the hepatic nodule.

ARTICLE IN PRESS Malignant Schwannoma in a Goat

lymphocytes and plasma cells, was frequently observed. Multifocal lymphocytic in¢ltrates were also commonly seen, especially in peripheral areas and associated with muscle in¢ltration by tumour cells. Histological features of the hepatic nodule di¡ered slightly from those of the diaphragmatic mass. Although the nodule was relatively discrete, neoplastic

Fig. 2. Diaphragmatic tumour, composed of pleomorphic, large spindle cells exhibiting lightly acidophilic cytoplasm, indistinct cell borders, and large fusiform nuclei with prominent nucleoli.The neoplastic cells are arranged in interwoven fascicles and bundles with sparse collagenous stroma. HE. 400. Inset: Mononucleated, bizarre giant cell showing intracytoplasmic PAS-positive globules. PAS. 1000.

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cells in¢ltrated the immediately adjacent hepatic tissue. The neoplastic tissue showed a disorganized pattern of atypical bundles and variably sized islands of pleomorphic cells.The bundles were sometimes further subdivided into smaller indistinct fascicles of neoplastic cells. As in the diaphragm, neoplastic cells in¢ltrated the nerves and blood vessel walls. Mitotic ¢gures and PAS-positive multinucleated giant cells demonstrating cell cannibalism (cytophagocytosis) were more prominent in the hepatic tumour than in the diaphragmatic tumour. The adjacent hepatic parenchyma showed marked hyperplasia of bile ducts, congestion, and fatty degeneration. Immunohistochemically, the tumour cells were strongly positive for vimentin, NSE and S100 protein (Fig. 3), moderately positive for AE1/AE3 (Fig. 4) and calponin, and weakly positive for NGFr. Moreover, neoplastic cells in the diaphragm showed a stronger intensity of immunoreaction than did those in the liver. Immunolabelling for NF, SMA, GFAP, myoglobin and desmin was negative. On the basis of gross morphology, histological features and immunophenotype, a diagnosis of MPNST was made. PNSTs have been classi¢ed, somewhat confusingly, in the veterinary literature as neurinomas, neurilemmomas, schwannomas, neuro¢bromas or neuro¢brosarcomas, depending on their presumed cell origin. However, this arbitrary classi¢cation is based largely on extrapolation from human tumour studies (Theilen

Fig. 3. Diaphragmatic tumour. (a) Anti-S100 protein antibody produced di¡use, intense immunolabelling of the neoplastic cells. (b) AntiNSE antibody also gave strong and di¡use immunolabelling of tumour cells, including the multinucleated, pleomorphic giant cells (arrows). ABC method, Mayer’s haematoxylin counterstain. 200.


G.A. Ramı´rez et al.

Fig. 4. Diaphragmatic tumour. Immunolabelling of neoplastic cells with anti-cytokeratin (AE1/AE3) antibody. Note the perinuclear dot-like pattern of immunolabelling in some cells. ABC method, Mayer’s haematoxylin counterstain. 400.

and Madewell,1987; Summers et al.,1995; Koestner and Higgins, 2002).The di⁄culty in assessing the cell origin lies in the fact that most PNSTs are relatively anaplastic (Summers et al., 1995; Anderson et al., 1999; Koestner and Higgins, 2002; Chijiwa et al., 2004). Immunophenotyping has been reported as helpful in distinguishing between neuro¢bromas and schwannomas (Veazey et al., 1993; Summers et al., 1995; Weiss and Goldblum, 2001; Chijiwa et al., 2004). In the case described here, the tumour cells with cytoplasmic PAS-positive granules appeared similar to granular cells. It is now generally accepted that granular cell tumours are derived from peripheral nerve components (Weiss and Goldblum, 2001; Chijiwa et al., 2004). Similar cells have also been recognized in PNSTs with eosinophilic cytoplasmic globules. Immunohistochemically, these cells are commonly positive for S-100 (Chijiwa et al., 2004), as in the present case. Uniformly intense immunolabelling for NSE and S100 protein is common in schwannomas, whereas neuro¢bromas are variable or negative (Veazey et al., 1993; Weiss and Goldblum, 2001; Chijiwa et al., 2004). In addition, neuro¢bromas contain interlacing bundles of elongated cells intimately associated with wire-like strands of collagen, and small to moderate amounts of mucoid material separating cells and ¢bres (Weiss and Goldblum, 2001). In the present study, Masson’s trichrome did not demonstrate signi¢cant amounts of ¢brous tissue, and PAS stain did not demonstrate mucoid material. These observations suggest that the tumour was a schwannoma rather than a neuro¢broma. The distinction between benign and malignant schwannoma was based on cytological criteria (pleomorphism, anaplasia, epithelioid cells, multinucleated

atypical giant cells), biological behaviour (invasion of the surrounding soft tissues, metastasis, cytophagocytosis) and immunohistochemical features. These characteristics have been interpreted as malignant changes by others (Veazey et al., 1993; Summers et al., 1995; Chijiwa et al., 2004). The neoplastic cells were GFAP-negative, a feature more common in MPNSTs than in BPNSTs (Chijiwa et al., 2004). It has also been demonstrated that the expression pattern of intermediate ¢laments may change in anaplastic neoplastic cells (Hellmen and Lindgren, 1989). Anaplastic cells in the primary mass demonstrated cytokeratin immunolabelling. It is known that malignant schwannomas occasionally express cytokeratins, especially those demonstrated by the pancytokeratin AE1/AE3 ‘‘cocktail’’ (Anderson and Salter, 2005). In addition, the tumour described in this report was positive for calponin, a smooth muscle marker previously described for some MPNSTs and other sarcomas (Fisher et al., 2003). On the basis of morphological characteristics alone, MPNSTs are often confused with other soft tissue tumours (Veazey et al.,1993; Anderson et al.,1999; Fisher et al., 2003; Chijiwa et al., 2004). Di¡erential diagnosis of anaplastic PNSTs includes leiomyosarcoma, rhabdomyosarcoma and haemangiopericytoma. Most rhabdomyosarcomas are positive for myoglobin and negative for NGFr. In addition, 80% of haemangiopericytomas exhibit focal a-SMA expression, whereas PNSTs are negative (Chijiwa et al., 2004). In all sections of tumour tissue a positive immunoreaction for NGFr was observed, but immunolabelling for actin, desmin and myoglobin was negative, and Masson’s trichrome stain showed no longitudinal striations; pericyte or muscle cell origin was therefore unlikely. Only one previous case of malignant schwannoma, originating from a thoracic spinal nerve, has been identi¢ed in the goat (Veazey et al.,1993); in that case there were multiple nodular tumours in several organs, including liver, lung and spleen.The origin of the tumour in the present case remains unknown. PNSTs typically arise from the spinal roots and peripheral nerves of the limbs and occasionally from the cranial nerves (Theilen and Madewell,1987; Summers et al.,1995; Koestner and Higgins, 2002). The diagnosis of PNSTs in unusual locations may depend on immunolabelling (Anderson et al., 1999; Weiss and Goldblum, 2001). Tumour cells in the hepatic mass were more pleomorphic than those in the diaphragmatic mass. In addition, NSE, S100, and vimentin immunoreactivity was more intense in the diaphragmatic lesion than in the hepatic nodule. Labelling of neoplastic cells by NGFr and AE1/AE3 antibodies was observed only in the diaphragm; metastatic cells in blood vessels or cells growing in the liver were negative. These ¢ndings indicate a minor di¡erentiation of cells in the hepatic nodule, suggesting that


Malignant Schwannoma in a Goat

the diaphragmatic mass was the primary tumour. Primary neurogenic tumours of the diaphragm, particularly those showing aggressive changes, represent only about 10% of diaphragmatic neoplasms in man (Moinuddeen et al., 2001; Kumbasar et al., 2004). In animals, the only reported case of primary diaphragmatic PNST ocurred in a 1-year-old dog (Anderson et al., 1999). In the case reported here, the anatomical location of the tumour, the apparently linear growth, and the extension into surrounding diaphragmatic muscle tissue suggested that the phrenic nerve may have been the site of origin.This might have caused dysfunction of the phrenic nerve, but dysfunction would not necessarily result in obvious clinical signs (Greene et al.,1988). PNSTs in cattle often cause no clinical signs, being encountered in old animals at slaughter (Summers et al., 1995). In human beings, ca 20% of such neoplasms are discovered accidentally (Kumbasar et al., 2004). The real prevalence of neoplasia in the goat, although not well documented, would appear to be low. This may be due to innate resistance, incomplete reporting of tumours, or poor detection (Bastianello, 1983; Veazey et al.,1993). There is only one previous description of a caprine schwannoma showing malignant behaviour (Veazey et al., 1993). MPNSTs may have a high metastatic potential in this species, but information is lacking.

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Received, May 25th, 2006 Accepted, May 9th, 2007