Paravertebral malignant peripheral nerve sheath tumour (MPNST) in a horse

Authors


Summary

A 10-year-old Lipizzaner gelding was presented with intermittent ataxia and hindlimb weakness. Ultrasonographic examination identified a mass cranial to the tuber sacrale. A provisional diagnosis of a soft tissue sarcoma was made based on a biopsy specimen. Owing to the extensive nature of the tumour and the associated poor prognosis, the horse was subjected to euthanasia on humane grounds. A post mortem examination revealed a locally infiltrative soft mass within the left lumbosacral epaxial musculature. Histologically, an infiltrative neoplasm predominantly composed of pleomorphic spindle or stellate-shaped cells was identified. Neoplastic cells exhibited strong S-100 protein and GFAP expression and variable vimentin, NSE, NGFR and myoglobin expression. They were uniformly negative for pan-cytokeratin, melan A, laminin and desmin. The ultrastructural examination revealed pleomorphic cells with long cytoplasmic processes and an absence of melanosomes. Based on these results, a diagnosis of malignant peripheral nerve sheath tumour was made. This report contributes further information to assist in the diagnosis of these poorly defined neoplasms in animals, especially when they occur in uncommon locations.

Introduction

Peripheral nerve sheath tumours (PNSTs) arise from various cell types of peripheral nerves. They are infrequent in domestic animals and are rarely malignant (Koestner and Higgins ; Maxie and Youssef 2007). In horses, PNSTs are mainly seen in the eyelids and skin but the malignant variant has occasionally been reported in other sites, namely the heart, oral cavity and intestine (Williamson and Farrell 1990; Andreasen et al. 1993; Kirchhof et al. 1996; Quinn et al. 2005; Snook and Wakamatsu 2011).

In man, PNSTs are subclassified as schwannomas or neurofibromas, based on the cell of origin or the arrangement of the cells forming Antoni A and B areas and Verocay bodies (Weiss and Goldblum 2001). In animals, a similar distinctive morphology is inconsistently described; therefore the use of the term PNST and malignant PNST (MPNST) has been suggested by the World Health Organisation to encompass a range of neoplasms originating from peripheral nerves (Koestner et al. 1999; Koestner and Higgins ).

This report describes a MPNST in a highly unusual location, the lumbosacral region of a horse, which effaced the epaxial musculature and was clinically associated with muscle weakness.

Case history

A 10-year-old Lipizzaner gelding was presented with an unusual hindlimb gait and a large swelling of the caudal lumbar region. The swelling had been present for many years but had recently increased in size. Prior to presentation, the horse had fallen over in the field and assumed a dog sitting position.

Clinical findings

On admission, vital parameters were within normal limits. No neurological deficits were detected but the horse showed profound bilateral hindlimb weakness. There was a marked reduction in the cranial phase of the stride of both hindlimbs at walk and trot. The horse showed reluctance to bear weight entirely on either hindlimb or to protract the limbs. Urination and defaecation was normal. The gelding was treated with phenylbutazone (Equipalazone)1 (2.2 mg/kg bwt per os b.i.d.) but no significant improvement was seen in his condition.

Radiographic evaluation of the lumbar swelling revealed a well demarcated, smooth outlined, soft tissue opacity cranial to the tuber sacrale, about 12–15 cm in size. There appeared to be complete lysis of the dorsal spinous process of the sixth lumbar vertebra, with partial lysis of the dorsal spinous process of the fifth lumbar vertebra, leaving only the cranial portion intact with a sclerotic margin to its caudal border (Fig1a). Ultrasonography revealed a space-occupying mass which seemed to be well demarcated from the surrounding musculature, with a homogenous echogenicity interspersed with focal hypoechoic and hyperechoic regions (Fig1b). Gamma scintigraphy revealed asymmetry of the sacroiliac joint with a focal area of moderately increased radiopharmaceutical uptake within the lumbar region, associated with the lumbar dorsal spinous processes.

Figure 1.

a) Latero-lateral radiograph of the lumbar region revealing a well demarcated, smooth outlined, soft tissue opacity cranial to the tuber sacrale. Note the almost complete lysis of the sixth lumbar vertebra, with partial lysis of the fifth lumbar vertebra, leaving only the cranial portion intact with a sclerotic margin to its caudal border (white arrow). b) Ultrasonogram of the mass (white asterisk) in longitudinal section (cranial to the left) revealing a well demarcated mass of homogenous echogenicity, interspersed with focal hypoechoic and hyperechoic regions which seemed to be well demarcated from the surrounding musculature.

A large core biopsy specimen was obtained from the centre of the mass (5–6 cm deep) via ultrasonographic guidance. Histopathological examination of the tissue sample led to the provisional diagnosis of a malignant mesenchymal tumour. Due to the extensive nature of the neoplasm and the poor prognosis, with no possibility for surgical removal, the horse was subjected to euthanasia on humane grounds.

Post mortem findings

The carcase was subjected to a full post mortem examination. The main finding was a large (12 × 8 cm in cross section), firm, partially encapsulated, yellow-tan mass, located paravertebrally within the multifidi muscles of the left lumbosacral region and extending from the fifth lumbar (L5) to the first sacral (S1) vertebra. It was effacing the musculature, the spinous processes and the left and dorsal parts of the vertebral arches of L5 and L6, diverting the spinous process of the sacral bone to the right. The mass was extending into the spinal canal and compressed the spinal cord (Figs2a,b). No further significant gross changes were observed.

Figure 2.

Cross sections through the vertebral column and the neoplastic mass. a) Section through the body of L5. The mass is effacing the processus spinosus and the left and dorsal part of the vertebral arch (arrow: right part of the vertebral arch), compressing the spinal cord (SC). L5: vertebral body of L5; TP: transverse process. b) Section through the body of S1, showing the caudal portion of the mass within the multifidi muscles. In this area, the neoplasm appears well demarcated. S1: vertebral body of S1; TP: transverse process; VA: vertebral arch; TS: tuber sacrale.

Light microscopic, immunohistological and ultrastructural findings

The mass and surrounding tissues were fixed in formalin and representative areas trimmed and routinely paraffin wax embedded. Sections (3–5 μm) were prepared and stained with haematoxylin and eosin (H&E) and a range of special stains (Masson's trichrome, phosphotungstic acid haematoxylin, Masson Fontana, Periodic Acid Schiff [PAS]). Oil Red Orange (ORO) stained frozen sections were prepared to demonstrate fat.

Histologically, a neoplastic mass of variable cellularity, infiltrating the surrounding connective tissue was detected (Fig3a). The neoplastic cells formed haphazardly arranged bundles and whorls or exhibited a multifocal storiform pattern (Fig3b). They were embedded in a fine fibrovascular stroma, as confirmed by the Masson's trichrome stain. Cells were polymorphic, 20–50 μm long, spindle to stellate shaped, with a small amount of cytoplasm (Fig3c). Multifocally, multinucleated neoplastic cells were observed (Fig3d). Neoplastic cells did not display cytoplasmic striation, melanin (Masson Fontana stain) or fat (ORO stain) accumulation but occasionally exhibited PAS-positive cytoplasmic globules. Mitotic figures were rare. Multifocal acute and chronic haemorrhage was present throughout the neoplastic process.

Figure 3.

Histological features and immunohistological expression pattern of the neoplastic process. a) Neoplastic cells invade (arrowheads) the adjacent pre-existing fibrous connective tissue (arrows). H&E stain, x10. b) Storiform arrangement of neoplastic cells. H&E stain, x20. c) Closer view of spindle-shaped neoplastic cells. H&E stain, x40. d) Focal aggregates of multinucleated neoplastic cells. H&E stain, x40. e) Neoplastic cells exhibit strong cytoplasmic expression of S-100 protein. PAP method, Papanicolaou's haematoxylin counterstain, x40. f) Neoplastic cells show moderate to strong GFAP expression. PAP method, Papanicolaou's haematoxylin counterstain, x40. g) Neoplastic cells exhibit weak to moderate vimentin expression. PAP method, Papanicolaou's haematoxylin counterstain, x40. h) Variable expression of NSE in neoplastic cells, PAP method, Papanicolaou's haematoxylin counterstain, x40. i) Neoplastic cells exhibit variable NGFR expression. ABC method, Papanicolaou's haematoxylin counterstain, x40. j) Weak to moderate myoglobin expression in neoplastic cells. PAP method, Papanicolaou's haematoxylin counterstain, x40.

Immunohistology was performed to further characterise the neoplastic cells, detecting vimentin (mouse anti-swine vimentin, clone V9)2, laminin (rabbit-anti-laminin)3, cytokeratins (mouse anti-human pan-cytokeratin, clone AE1/AE3)2, desmin (mouse anti-swine desmin, clone D33)2, glial fibrillary acidic protein (GFAP; rabbit anti-GFAP)2, neuron specific enolase (mouse anti-human NSE, Clone NSE-1G4)4, myoglobin (rabbit anti-human myoglobin)1, melan-A (mouse anti-human melan A, clone A103)2, S-100 (rabbit anti-cow S-100 protein)2 and nerve growth factor receptor (mouse anti-human NGFR clone 7F10)5. Sections underwent antigen retrieval and the antibody-antigen reaction was visualised using the peroxidise-anti-peroxidase (PAP) or avidin-biotin complex peroxidase (ABC) method and diaminobenzidine as chromogen. The majority of neoplastic cells exhibited strong S-100 protein and GFAP expression and showed a variable expression of vimentin, myoglobin, NSE and NGFR (Figs3e-j). They were negative for laminin, cytokeratins, desmin and melan-A.

Transmission electron microscopy was performed on formalin-fixed neoplastic tissue after post fixation in 4% paraformaldehyde/2.5% glutaraldehyde in a sodium cacodylate buffer and secondary fixation in osmium tetroxide. The neoplastic cells exhibited long cytoplasmic processes and electron-lucent nuclei as well as a moderate amount of rough endoplasmic reticulum. Melanosomes or fragments of basement membranes were not detected (Fig4).

Figure 4.

Transmission electron micrographs. a) Neoplastic cells exhibit variably dilated endoplasmic reticulum, filled with mildly electron dense granular material (arrows). b) Neoplastic cells in proximity to collagen fibres (C), showing long cytoplasmic processes (arrows). x13,500.

The histological examination of other organs and tissues including the brain and spinal cord did not show any significant pathological changes apart from the presence of diffuse meningeal oedema and occasional axonal swelling of the spinal cord at the level of L5/6.

Diagnosis

Based on the morphological features and the local infiltrative growth, the neoplasm was diagnosed as a soft tissue sarcoma. Malignant peripheral nerve sheath tumour (MPNST), giant cell sarcoma, rhabdomyosarcoma, liposarcoma and amelanotic melanoma were considered as differential diagnoses. The lack of striation, melanin or fat in neoplastic cells, together with the expression of S-100, GFAP, NGFR and NSE and the ultrastructural features (i.e. long cytoplasmic processes, lack of melanosomes) excluded a rhabdomyosarcoma, melanoma, giant cell sarcoma and liposarcoma and allowed for a final diagnosis of MPNST.

Discussion

This report describes a MPNST in a horse in a previously unreported location, the epaxial musculature. The clinical data suggest that the neoplastic process had been present for several years, but had only recently started to grow, which would suggest an initially benign process. The nerve of origin of the neoplasm was not identified, but due to its location it is likely that it had developed in the dorsal branch of the left spinal nerve of L5. This presentation shares several characteristics with the so-called giant sacral schwannomas described in man (Pongsthorn et al. 2010). Like the process in the present case, these generally grow slowly and are only diagnosed when they reach a substantial size and induce osteolysis (Pongsthorn et al. 2010). In our case, osteolysis of the spinous processes and of the left and dorsal parts of the vertebral arches of L5 and L6 was identified but not of the sacral bone as it is seen in human cases.

Peripheral nerve sheath tumours rely on immunohistology for a definite diagnosis which was in the present case supported also by the ultrastructural features, to rule out the large number of potential differential diagnoses. Both in man and in animals, PNSTs have been reported to exhibit focal to multifocal S-100 protein and variable GFAP, NSE, NGFR and myoglobin expression (Chijiwa et al. 2004; Kar et al. 2006; Maxie and Youssef 2007; Ramírez et al. 2007; Snook and Wakamatsu 2011). The expression of the above markers as well as vimentin, and in particular the strong reaction for S-100 in combination with a variable expression of NGFR is characteristic for the malignant form, MPNST (Chijiwa et al. 2004; Maxie and Youssef 2007; Ramírez et al. 2007; Schöniger et al. 2011; Snook and Wakamatsu 2011).

This present report on a MPNST in a horse in an unusual location, the epaxial musculature, further highlights the need for a varied methodological approach to diagnose these rare neoplasms, especially when they occur in uncommon locations.

Acknowledgements

The authors wish to thank the technicians in the Histology Laboratory and Electron Microscopy Unit, Veterinary Laboratory Services, School of Veterinary Science, University of Liverpool, for excellent technical assistance. G. Nikolaou's senior clinical scholarship is supported by the Animal Health and Veterinary Laboratories Agency, UK.

Authors' declaration of interests

No conflicts of interest have been declared.

Manufacturers' addresses

  1. 1

    Dechra Veterinary Products, Shrewsbury, Shropshire, UK.

  2. 2

    Dako, Glostrup, Denmark.

  3. 3

    Sigma Aldrich Ltd, Gillingham, Dorset, UK.

  4. 4

    Zymed Laboratories, Carlsbad, California, USA.

  5. 5

    Novocastra Laboratories Ltd, Newcastle upon Tyne, UK.

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