Metastasizing extra-adrenal paraganglioma with neurological signs in four dogs

j. Comp. Path. 1993 Vol. 108, 283-290

Metastasizing Extra-adrenal Paraganglioma with Neurological Signs in Four Dogs M. E. Hines II, J. (3. Newton*, N. H. Altman, T. N. Hribernik~ and H. W. Casey* Division of Comparative Pathology, Department of Pathology, University of Miami School of Medicine, ]FIiami, FL, and *Department of Veterinary Pathology and ~Department of Veterinary Clinical Sciences, School oJ"Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA

Summary Extra-adrenal paragangllomas associated with vertebral pain and clinical neurological abnormalities as a result of' metastasis to the vertebral column were diagnosed in lbur dogs of different breeds by light microscopy. All were males (two intact and two neutered) aged 8 years. Metastatic neoplasms occurred as extradural masses with associated bone iysis at vertebrae C4 (9 cases), T I2-L1 (1 case) and L¢ (] case). The neoplastic cells exhibited similar morphology with little variation between cases. All neoplasms showed cytoplasmic granules after staining with the Churukian-Schenk modification of the Pascual argyrophil stain for neurosecretory granules or for membrane bound electron-dense granules (dense-core granules). On immunohistochemical examination the neoplastic cells gave positive results tbr neuron-specific enolase and negative results for chromogranin and epithelial membrane antigen. Multiple organ metastasis and metastasis to bone have been reported previously, but these cases were unusual due to the involvement of the spine as an apparent predilection site for metastasis, and the sex (male) and age of the animals affected.

Introduction Extra-adrenal paragangliomas include a diverse group of neuroendocrine neoplasms that may arise from multiple sites. The mammalian paraganglion system is composed of autonomic ganglia distributed symmetrically and segmentally in the para-axial regions throughout the thoracic and abdominal cavities. The system is subdivided into the branchometric, intravagal, aordcosympathetic and visceral-autonomic paraganglia on the basis of anatomical distribution (Glenner and Grimley, 1974). Paraganglia consist of discrete aggregates of histologically similar neurons along the central axis of the body which are connected to other similar neurons and function collectively as part of the autonomic nervous system (Glenner and Grimley, 1976). Neoplasms of various ganglia are collectively known as extra-adrenal paragangliomas or neuroendocrine carcinomas; however, they are often classified under other names that indicate the primary location of the neoplasm (Glenner and Grimley, 1974; Capen, 1985). 0021-9975/93/030283 + 08 $08,00/0

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T h i s r e p o r t describes four cases of e x t r a - a d r e n a l p a r a g a n g l i o m a that p r e s e n t e d clinically with neurological a b n o r m a l i t i e s due to metastasis to vertebral bone. Materials

and Methods

Dogs, Biopsy and Necropsy Biopsy specimens from the vertebral columns of four dogs presenting with clinical neurological abnormalities were submitted for histological examination to the surgical biopsy services at the Louisiana State University School of Veterinary Medicine. Details of the animals and pertinent clinical information are summarized in Table 1. Biopsy specimens were taken from vertebrae C4 in dogs 3 and 4, T1 l-L1 in dog 2, and L4 in dog 1. After euthanasia, all animals except dog 4 were necropsied. Histopathology and Immunohistochemist~y Biopsy specimens were fixed in 10 per cent neutral buffered formalin and embedded in paraffin wax and tissue sections (6 gm) were stained with haematoxylin and eosin (HE). The Churukian-Schenk modification of Pascual's argyrophil staining method (Churukian and Schenk, 1979) was used to demonstrate the presence of cytoplasmic neurosecretory granules (argyrophil granules) within tumour cells. Additional sections (3 gm) were stained for neuron specific enolase (NSE) (Anti-human Polyclonal Antibody, Dako Corp., Santa Barbara, USA), chromogranin (Anti-human Monoclonal Antibody, D a b Corp.) and epithelial membrane antigen (EMA) (Anti-human Monoclonal Antibody, Dako Corp.) by an avidin-biotin complex immunoperoxidase method (Nadji and Morales, 1986). Similarly treated sections of normal canine pancreas were used as positive control tissue. Sections of normal pancreas were also stained with synaptophysin (And-human Monoclonal Antibody, Dako Corp.). Electron Microscopy Specimens for transmission electron microscopy (TEM) were obtained from biopsy and necropsy tissues fixed in 10 per cent neutral buffered formalin for at least 48 h. Specimens were washed in 0' 1M sodium cacodylate buffer with sucrose 5 per cent, pH 7"4 (three changes, 10rain each). Specimens were post-fixed in 1"0 per cent osmium tetroxide in 0 . I M sodium cacodylate buffer (pH 7'4) for 1 h and then washed as before. The specimens were dehydrated in graded ethanol baths, washed in three 5-rain changes of propylene oxide and embedded in Spurr low viscosity epoxy resin (Weidmann et at., 1987). Thin sections (60-90 nm) were placed on copper grids,

Table 1 Clinical signs in four dogs with metastatic vertebral extra-adrenal paraganglioma

Dog no.

Breed

Age(years)

Sex

Clinicalsigns and radiographiealfindings Back pain, hind limb ataxia and paresis, urinary incontinence,lysisof L4. Back pain, hind limb ataxia and paresis, cord deviation TI0-T12, heart base mass. Cervical pain, muscle fasciculation,lysisof C4. Cervical pain, lysis of C4

1

GermanShepherd

8'3

M

2

Boxer

7'8

M

3

GoldenRetriever

8.8

M*

4

AmericanStaffordshireTerrier

8"5

M*

* = Neutered.

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stained with uranyl acetate and Reynold's lead citrate, and examined in a Zeiss model 10 transmission electron microscope at 80 keV.

Results

Macroscopical Findings At necropsy, dog 1 had a lobulated, dark, mottled, reddish-tan mass (9 x 6 x 5 cm) at the base of the heart. The mass was adherent to the right atrium and aorta and completely surrounded the pulmonary trunk. Multifocal centrally located areas of necrosis were present within the mass. Multiple reddish nodules, 1-2 mm in diameter, were scattered throughout the lung lobes. A hemi-laminectomy site was noted where the original biopsy was taken from vertebra L4. At the base of the heart dog 2 had a lobulated, dark coloured mass, 5-6 cm in diameter, which wrapped around but did not impinge on the great vessels. A similar, extradural, spinal cord mass, 2-3 c m in diameter, was found at vertebrae T10-T12. The right kidney had a solitary mass, 3 cm in diameter, at the cranial pole, with multiple nodules, < 1.0 c m in diameter throughout the cortex. The lungs had multiple masses, < 0 . 5 c m in diameter, throughout all lobes. The tracheobronchial lymph nodes were greatly enlarged (5-6 cm long) and necrotic. Dog 3 had two, dark, firm, lobular masses (4 x 3 x 3 crn and 3 x 2 x 2 cm) within the cranial mediastinum. A similar mass (4 x 3 x 3 cm) was noted in the paralumbar area just caudal to the right kidney; a cut section appeared orange in colour. An osteolytic extradural mass (2 x l x 2cm) was found in vertebra C4 and a smaller (0"5 x 0"75 x 0"5 cm) but similar extradural mass was noted in the craniodorsolateral portion of vertebra C2. Radiographically, dog 4 showed an osteolytic extradural mass involving vertebra C4, but necropsy was not performed and there was no follow-up after biopsy.

Histological Findings Biopsy and necropsy specimens from the metastatic vertebral masses as well as from the primary thoracic sites (three cases) were histologically similar in all cases on HE-stained sections (Fig. 1). The masses were generally composed of sheets and islands of round to polygonal neoplastic cells arranged in nests, and lobules interspersed within a delicate fibrovascular stroma. The cells had round, often hyperchromatic or vesicular, nuclei with moderate anisokaryosis, multiple small nucleoli and abundant eosinophilic granular cytoplasm. Some sections demonstrated fine, basophilic, dust-like, cytoplasmic granules. Mitotic figures were rare ( < 1 per high power field). Most sections contained multifocal areas of necrosis. Invasion of tumour cells into bony tissues with areas of ischaemic necrosis was present in vertebral sections.

Findings made by Special Techniques T u m o u r sections stained with Churukian-Schenk argyrophil stain d e m o n strated large numbers of tumour cells containing dense brown to black

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Fig. 1.

Representative section of extra-adrenal paraganglioma metastatic to the vertebral column in the four dogs. Note the lobular pattern and delicate fibrovascular stroma. HE. Bar= 50 I-tm.

Fig. 2.

Churukian-Schenk argyrophil staining Dr neurosecretory granules. Note the numerous black staining intracytoplasmic granules. Bar= 25 p.m.

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Fig. 3,

Immunohistochemical staining for neuron-speclfic enolase, Note the scattered cells containing clark cytoplasmic granules. Bal'= 50 p_m.

Fig. 4.,

Dense-core granules (ncuvosecretory granules). TEM. Bar =500 nm.

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cytoplasmic granules from dogs 1-3 (Fig. 2). Similar stained sections from dog 4 were repeatedly non-diagnostic. Immunohistochemical staining of additional sections of all four cases were positive for NSE (Fig. 3) and negative for chromogranin and EMA. Sections of normal canine pancreas were also negative for chromogranin and synaptophysin. Specimens from dogs 1, 3 and 4 were examined by T E M after fixation with neutral buffered formalin 10 per cent. Numerous small uniform electron-dense granules were seen in the cytoplasm (Fig. 4). Many of the cytoplasmic granules were membrane bound and morphologically typical of dense-core granules.

Discussion

The widely dispersed collections of specialized cells in the paraganglion system are related both functionally and structurally to the adrenal medulla. Both systems contain two separate populations of cells which are derived from primitive neural precursor cells. Chief (type I) cells are derived from neuroectoderm, contain neurosecretory granules and may secrete catecholamines in both normal and abnormal states. Sustentacular (type II) cells closely surround clusters of chief cells, are closely related to satellite cells of autonomic ganglia, lack neurosecretory granules and do not contain or secrete catecholamines. Chief cells contain NSE, chromogranin, and leu-enkephalin, whereas sustentacular cells contain S-100 protein (Kliewer et al., 1989). Neoplasms of various ganglia are collectively known as extra-adrenal paragangliomas or neuroendocrine carcinomas but are often :found under other names, including chemodectoma, aortic body tumour, carotid body tumour, heart,base tumour, chemoreceptor tumour and ganglioneuroma (Glenner and Grimley, 1974; Capen, 1985). The primary location is often more valuable than the histological features in assessing prognosis. The usual histological criteria that can be used to distinguish malignant from benign tumours, such as nuclear atypia and nuclear to cytoplasmic ratios, do not apply in most neuroendocrine tumours. Most paragangliomas are benign; however, features compatible with malignant neoplasms are often seen in benign paragangliomas (Kliewer et al., 1989). The large number of sites of origin of human extra-adrenal paragangliomas have not been observed in the dog. Canine paragangliomas are biologically similar to human tumours and they occur as multiple turnouts in 16 per cent of cases and at a rate of metastasis of 22 per cent (Patnaik el al., 1975). An association with endocrine tumours (pheochromocytomas and thyroid neoplasms) has been noted in man and in dogs. Aortic body tumours are the most common in dogs and occur most often in males. Brachiocephalic breeds have a significantly higher frequency of chemodectomas, possibly because of chronic hypoxia due to partly obstructed air passages (Patnaik el al., 1975). In this study, the neoplasms were morphologically compatible with extraadrenal paraganglioma (neurocndocrine carcinoma}. All cases were morphologically similar and on ultrastructural examination showed dense-core granules typical of the neurosecretory granules usually present in neuroendocrine tumours. Three out of four cases stained positively for neurosecretory

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granules with Churukian-Schenk argyrophil stain. However, positive argyro. philic staining of dense-core granules seen by T E M in dog 4 was not apparent. O n immunohistochemical staining all cases gave a positive result for NSE. This substance is found particularly in neurons and neuroendocrine cells, and positive reactions are given by most neuroectodermal and neuroendocrine neoplasms, including carcinoid tumours and malignant melanoma (Rosai, 1989). Immunohistochemical staining for chromogranin was negative in all cases. Nearly all h u m a n types ofneuro-endocrine tumours react positively with this stain and it has become the most widely used pan-endocrine marker (Patnaik et al., 1975); however, in our laboratory canine and feline tumours morphologically and ultrastructurally resembling neuroendocrine neoplasms have been uniformly negative (unpublished data). Immunohistochemical staining for chromogranin and synaptophysin in normal canine pancreatic islet cells was also negative. These findings suggest that there is no cross-reactivity between h u m a n anti-chromogranin or synaptophysin monoclonal antibodies and canine neuroendocrine tumours or normal pancreas. Synaptophysin is usually expressed in normal, reactive and neoplastic cells of neuroectodermal and neuroendocrine types including those of pheochromocytoma, thyroid medullary carcinoma, endocrine pancreatic tumours and carcinoid tumours (Rosai, 1989). Immunohistochemical staining for EMA was negative in all cases. EMA is an excellent non-specific marker for most normal and neoplastic epithelia. It can also be expressed by mesotheliomas, meningiomas, a variety of mesenchymal neoplasms, normal and neoplastic perineural cells and some malignant lymphomas (Rosai, 1989). Extra-adrenal paragangliomas are not unusual neoplasms and have been reported to metastasize to bone (Szczech el al., 1973; Montgomery et al., 1980). The apparent tendency to metastasize to the vertebral column, with resulting neurological deficits, was an unusual finding; however, there was the possibility of a multicentric origin. The apparent age and sex incidence demonstrated by these tumours was also unusual. This study demonstrated the need for multiple diagnostic methods to confirm the diagnosis, since only ultrastructural identification of neurosecretory granules appeared to give consistent results. Acknowledgments This study was partly supported by NIH grant 1P40RR04326-01. We thank Drs C. Hedlund, V. Fucci, T. Vance, R. W. Kornegay, T. G. Snider III and Doo-Youn Cho (Louisiana State University) for help in various ways. The assistance of Mac Lopez and Cheryl Crowder (Louisiana State University) with the electron microscopy and Churukian-Schenk stains and Dr M. Nadji (University of Miami) for performing the immunoperoxidase staining is also greatly appreciated. References Capen, C. C. (1985). The endocrine glands. In: Pathology of Domestic Animals, 3rd edn, vol. 3. K. V. F. Jubb, P. C. Kennedy and N. Palmer, Eds, Academic Press, Orlando, FL, pp. 292-297. Churukian, C. J. and Schenk, E. A. (1979). A modification of Pascual's argyrophil method. Journal of Hislotechnology, 2, 102-103.

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Glenner, G. G. and Grimley, P. M. (1974). Tumors of the extra-adrenal paraganglion system (including chemoreceptors). In: Atlas of Tumor Pathology, Armed Forces Institute of Pathology, Washington DC, 2nd series, fascicle 9. Kliewer, K. E., Wen, D. R., Cancilla, P. A. and Cochran, A. J. (1989). Paragangliomas: assessment of prognosis by histologic, immunohistochemical and ultrastructural techniques. Human Pathology, 20, 29-39. Montgomery, D. L., Bendele, R. and Storts, R. W. (1980). Malignant aortic body tumor with metastasis to bone in a dog. Veterinary Pathology, 17, 214-244. Nadji, M. and Morales, A. R. (1986). Immunoperoxidase Techniques: A Practical Approach to Tumor Diagnosis, American Society of Clinical Pathologists Press, Chicago, IL. Patnaik, A. K., Liu, S. K., Hurvitz, A. I. and McClelland, A. J. (1975). Canine chemodectoma (extra-adrenal paragangliomas)--a comparative study. Journal of Small Animal Practice, 16, 785-801. Rosai, J. (1989). Special techniques in surgical pathology. In: Ackerman's Surgical Pathology, 7th edit., vol. 1, J. Rosai, Ed. C. V. Mosby Co., St Louis, MO, pp. 31-51. Szczech, G. M., Blevins, W. E., Carlton, W. W. and Cutlan, G. R. (1973). Chemodectoma with metastasis to bone in a dog. Journal of the American Veterinary Medical Association, 162, 376-378. Weidmann, J., Freund, M. and McGeever-Rubin, B. (1987). Comparative light and electron microscopy of paraffin-embedded tissue: an assessment of three methods. Journal of Histotechnology, 10, 163-166.

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Received, October 30th, 1992-] Accepled, January 7th, 1993_J