A Peripheral Primitive Neuroectodermal Tumor with ... - SAGE Journals

Vet Pathol 41:4, 2004

Brief Communications and Case Reports

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Vet Pathol 41:437–441 (2004)

A Peripheral Primitive Neuroectodermal Tumor with Generalized Bone Metastases in a Puppy H. E. V. DE COCK, M. D. M. BUSCH, M. M. FRY, M. MEHL, A. W. BOLLEN,

AND

R. J. HIGGINS

Abstract. A peripheral primitive neuroectodermal tumor (pPNET), most consistent with a human Ewing’s sarcoma, is described in a 5-month-old male Australian Shepherd puppy. The first tumor site detected was in the left frontal bone of the skull with apparent subsequent rapid metastases to multiple sites in the axial and appendicular skeleton and bone marrow, kidneys, and perihyphophyseal meninges. Radiographically, all bone lesions were lytic and there was also a humeral bone fracture. Histologically, the tumor was diagnosed as a small round blue cell tumor. At this stage, the differential diagnosis included a lymphoma, rhabdomyosarcoma, and a PNET of the peripheral nervous system. However, the cells had positive expression of triple neurofilament antigens as detected immunocytochemically. The cells were negative for a broad panel of canine-specific leucocyte cell marker antigens for desmin, smooth muscle actin, synaptophysin, and CD99. Ultrastructurally, the cells contained occasional dense core neurosecretory granules and intermediate filaments with intercellular desmosomal-like junctions and abundant glycogen clusters. Based on the age of the dog, the clinical history, the distribution of gross lesions, histologic characteristics of a small round blue cell tumor, and immunocytochemical and ultrastructural evidence of neuroectodermal differentiation, a diagnosis of a pPNET similar to a human Ewing’s sarcoma was made. Key words: Bone; dogs; Ewing’s sarcoma; immunocytochemistry; primitive neuroectodermal tumor; small round blue cell tumor; ultrastructure. In people the Ewing’s sarcoma family of tumors (EFT) is a very rare, complex tumor group in children and young adults composed of primitive cells with a putative neuroectodermal phenotype and generally occurring outside the nervous system.3,4,11,13,15 These highly aggressive peripheral primitive neuroectodermal tumors (pPNETs) are histologically categorized as pediatric ‘‘small round blue cell tumors.’’3,4,11,13 As a group, pPNETs have been clearly separated from classical PNETs of the peripheral nervous system (PNS) (e.g., neuroblastoma, ganglioneuroblastoma, and ganglioneuroma) by differences in their clinical, pathologic, and molecular genetic profiles.1–4,8,11,13,15 Currently the EFT group includes the intraosseous pPNET (or Ewing’s sarcoma), the extraosseous pPNET and the thoracopulmonary Askin’s tumor.3,4,13,15 The primary site of Ewing’s sarcoma arises in the

long tubular bones and pelvis with subsequent hematogenous metastases most commonly to lung, other bones, and the bone marrow.3,4,11,13,15 Such tumors can also occur in the skull, spine, and brain as primary tumors or can be of metastatic origin.3,4,15 Microscopically, the sheets of tumor cells are relatively monomorphous with a round to angular nucleus, a thin cytoplasmic margin, and irregular border.2–4,11,13 Immunocytochemically, there is inconsistent expression of one or more neuronal/neuroendocrine cell-specific marker antigens (e.g., synaptophysin, chromogranin A, or triple neurofilaments).2–4,11,13 However, positive expression of the cell surface glycoprotein p30/32MIC2 (CD99) has been a consistent antigenic marker for human pPNETs (90% of cases) but is not expressed in PNETs of the PNS.2–4,11,14 Ultrastructurally, dense core neurosecretory gran-

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Fig. 1. Humerus; canine. Radiograph of the left humerus shows a lytic lesion in the distal part of the diaphysis. Bar ⫽ 5 mm. Fig. 2. Humerus; canine. Gross morphology of the lesion visualized in Fig. 1. A white friable mass with central hemorrhage expands the medullary cavity and perforates adjacent cortical bone of the distal humerus. Bar ⫽ 1 cm. Fig. 3. Frontal bone; canine. Impression smear of a lytic bone lesion shows many round cells with large immature nuclei and small amounts of basophilic cytoplasm. Nuclei vary in shape from round to indented or fissured. Note the mitotic figure in the upper right. Wright’s–Giemsa. Bar ⫽ 20 ␮m.



Table 1.

Immunocytochemical reactivity of the canine pPNET.

Antibody to (clone)

Synaptophysin (SY38) Neurofilament triplet Chromogranin A S100 NSE Melan A Pancytokeratin (Lu5) Skeletal myosin Desmin Vimentin (3B4) CD18 CD45 CD45r CD79a (HM57) CD3 (CD-12) CD99 (HO36-1.1)

439

Source

Dako Corporation, Carpinteria, California Zymed Laboratories, South San Francisco, California INCstar Corporation, Stillwater, Minnesota Novocastra Labs, Newcastle upon Tyne, UK Dako Corporation, Carpinteria, California Novocastra Labs, Newcastle upon Tyne, UK Biocare, Walnut Creek, California Biogenex Laboratories, San Ramon, California Biogenex Laboratories, San Ramon, California Dako Corporation, Carpinteria, California Gift of P.F. Moore, UC Davis, Davis, California Gift of P.F. Moore, UC Davis, Davis, California Gift of P.F. Moore, UC Davis, Davis, California Dako Corporation, Carpinteria, California Dako Corporation, Carpinteria, California Novocastra Labs, Newcastle upon Tyne, UK

ules and intercellular desmosomal-like junctions are the diagnostic features most supportive of neuronal differentiation.3,7,8,12 Focal aggregates of glycogen are also prominent.8 However, the human EFT is best distinguished from the PNETs of the PNS, and other tumors, by their consistent expression of chromosomal 22 translocations.1,3,11,15 These result in gene fusions between the EWSR1 gene and the closely related ETS protooncogenes FLI1 or EGR and the diagnostically useful expression of the chimeric fusion gene in 90% of the tumors.1,3,4,15 Such discriminating molecular genetic analysis is not yet available for canine tumors. One previous case of an intraosseus pPNET, in a young dog, fulfilling most of the human criteria for EFT has been described.12 In addition to the bone marrow, lesions were found in the skin, heart, and adrenal gland. One adult beagle has been described with gross and microscopic lesions consistent with an extraosseous pPNET.6 A pPNET in a 2-month-old Colobus monkey with brain lesions and widespread extraneural soft tissue organ and lumbar vertebral involvement has been described.7 Here we report a 5-month-old, intact male Australian Shepherd dog with a multicentric small round blue cell tumor whose location and morphologic and phenotypic features are most consistent with an intraosseous pPNET (Ewing’s sarcoma). Initial clinical signs noted were an acute lateral strabismus and mydriasis of the left eye with a swollen

Result

⫺ Strong ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

orbit. Soon after, the dog began limping on the left pelvic limb and several days later it experienced extreme pain in both thoracic limbs. Thirty days after the onset of these clinical signs, the dog was referred to the Veterinary Medical Teaching Hospital of the School of Veterinary Medicine at the University of California, Davis. On clinical evaluation the puppy was lame in both thoracic limbs, more prominent on the left than the right side and also in the left pelvic limb. Radiographs showed numerous lytic lesions in the spinous processes and vertebral bodies of some thoracic and lumbar vertebrae (segments T1, T2, L5, and L6), the mandible, and left frontal bone. There was an increased opacity in the left frontal sinus. Lytic lesions were found also bilaterally in the distal humeri (Figs. 1, 2), proximal, and distal radial metaphyses and in the distal femoral and tibial metaphyses. Single lytic lesions were in the left ulnar metaphysis and the right tibial crest and diaphysis. Mixed proliferative and lytic lesions were found also in the third and fourth distal metaphyses of metatarsal bones. A fracture with abundant callus formation was evident in the left distal humerus. Results of a complete blood count and buffy coat smear were unremarkable. Abnormalities in the neuromuscular chemistry panel included hypochloridemia (102 mM/liter; normal 105–116 mM/liter), hypercalcemia (12.2 mM/liter; normal 9.9–11.4 mM/liter), hyperphosphatemia (8.4 mM/li-

← Fig. 4a. Humerus; canine. Lobules of neoplastic cells delineated by a thin fibrovascular stroma. Cells are demarcated from each other, have a round to angular nucleus with bluish cytoplasm and abundant mitotic figures. HE stain. Bar ⫽ 45 ␮m. Fig. 4b. Tumor cells have a sharp cytoplasmic border, and nuclei have marginated chromatin and an indistinct nucleolus. HE stain. Bar ⫽ 11 ␮m. Fig. 5. Bone marrow; canine. Strong and uniform cytoplasmic immunoreactivity of most tumor cells to triple neurofilament antibody. Indirect avidin–biotin–immunoperoxidase stain. Bar ⫽ 40 ␮m. Fig. 6. Humerus; canine. Positive immunoreactivity to vimentin antibody of the fibrovascular stroma but tumor cells are negative. Indirect avidin–biotin–immunoperoxidase stain. Bar ⫽ 50 ␮m. Fig. 7. Bone marrow; canine. Transmission electron photomicrograph illustrating the prominent intercellular desmosomal-like junctions between tumor cells (arrows) and clusters of glycogen. Bar ⫽ 0.5 ␮m.

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ter; normal 3.0–6.2 mM/liter), increased activities of alkaline phosphatase (163 IU/liter; normal 15–127 IU/liter), aspartate aminotransferase (51 IU/liter; normal 15–43 IU/liter), and creatine kinase (473 IU/liter; normal 43–320 IU/liter). Hypercalcemia and hyperphosphatemia were considered most likely due to the lytic bone lesions. Results of serum electrophoresis were unremarkable, and multiple bone marrow aspirates revealed a normally active bone marrow. Cytologic examination of a percutaneously obtained aspirate of the bone lesion near the left eye indicated a malignant round cell tumor (Fig. 3). Because of the severe pain and grave prognosis for the dog, the owners elected euthanasia. A complete necropsy revealed gross lesions restricted to the skeleton, kidneys, and the perihypophyseal meninges. The lesions in the long bones and the skull consisted of uniformly soft, white or tan, friable masses with often large areas of hemorrhage and necrosis noted on the cut surface. The masses expanded the medullary cavities and sometimes perforated adjacent cortical bone (Fig. 2). In the spinous processes and vertebral bodies of affected thoracic and lumbar vertebrae, multiple areas of osteolysis were noticed. The capsular surface of the cranial pole of the right and cranial and caudal pole of the left kidney had circular white depressions that extended in the underlying parenchyma. Representative tissue samples of lesions were immersion-fixed in 10% neutral buffered formalin, routinely processed for paraffin embedding, then 4 ␮m thick sections were stained with hematoxylin and eosin (HE). Selected tissue was also processed routinely for transmission electron microscopy (TEM). On histologic evaluation, neoplastic cell infiltrations were seen in the lesions in the long bones and skull, both kidneys, and the perihypophyseal meninges. The lesions consisted of variably sized, compact lobular nests or broad sheets of cells sometimes delineated by thin fibrous septae (Fig. 4a). The small round to elongate monomorphous cells had a thin rim of basophilic cytoplasm and had an oval, often twisted nucleus with marginated chromatin and indistinct nucleoli (Fig. 4b). The incidence of mitotic figures was up to 4 per 40⫻ high-power field (0.16 mm2), whereas the proliferative index, as determined by MIB-1 immunostaining, was about 40%. Prominent necrosis and hemorrhage were present within the central aspect of larger tumor masses. Within the bones, the tumor cells invaded and often perforated cortical bone. Histochemically, about 2% of tumor cells had positive periodic acid–Schiff granular cytoplasmic staining not resistant to diastase. Immunocytochemically, using an indirect avidin–biotin–immunoperoxidase technique and antibodies as previously described, the cells were strongly positive for triple neurofilament antigens but were otherwise negatively immunoreactive to the listed antigens including synaptophysin (Table 1, Fig. 5).5,9 Appropriate laboratory control canine tissues were run in parallel and were positively immunoreactive for all antibodies used. Endogenous positive control immunoreactivity in appropriate tissues from the affected dog was also demonstrated with all antibodies except to Melan A, which was not checked. A mouse monoclonal antibody NCL-CD99 (clone HO36-1.1) (Novocastra Labs, Newcastle upon Tyne, UK) was used at 1 : 250 dilution with overnight incubation at 4 C. From this dog, cells from both unaffected tissue (lymph node, spleen, pancreas) and tumor


cells (from bone marrow and kidney) were uniformly negative for CD99 reactivity compared with strongly positive expression in similar normal human control tissue (lymph node, pancreatic islet cells, and tumor cells) when immunostained in parallel.14 Normal canine lymph node, spleen, and pancreatic islet cell tissue from our laboratory control tissue bank was also immunocytochemically negative for CD99. Positive immunoreactivity to vimentin of the fibrovascular stroma was in sharp contrast to the negative tumor cells (Fig. 6). With TEM, the tumor cell cytoplasm contained small numbers of various organelles, mainly mitochondria, occasional small dense core secretory granules, dense focal clusters of glycogen rosettes, and numerous rudimentary desmosome-like intercellular junctions (Fig. 7). Based on the results of the gross and microscopic evaluation of HE sections, a tentative morphologic diagnosis of a small round blue cell tumor was made. However the differential diagnosis of this complex tumor group in young dogs includes at least a rhabdomyosarcoma, neuroblastoma of the PNS, or lymphoma.2–4,10,11,13 A rhabdomyosarcoma was excluded on the basis of the lack of any cytoplasmic striations histologically, further supported both by negative immunocytochemical staining for skeletal myosin and desmin, and by the absence ultrastructurally of myofilaments with Zbands. A neuroblastoma was ruled out based mainly on the distribution of gross lesions, together with histologic absence of Homer–Wright rosettes, and lack of neuritic processes by TEM.3,4,8,11 In children, correct differentiation between a primary osseous lymphoma and EFT is a major diagnostic challenge that additionally requires immunophenotyping, molecular genetic analysis, and ultrastructural evaluation.2,3,8,10,11,15 In this dog, the consistent negative expression by tumor cells for all canine leukocyte cell–specific markers (see Table 1), together with the ultrastructural findings of intercellular desmosome-like junctions and some cytoplasmic dense core secretory vesicles excluded a diagnosis of an osseous lymphoma.8,9–11,13 Thus, based on the age, clinical history of orbital swelling with subsequent axial and appendicular skeletal involvement, the histologic and ultrastructural findings, and immunocytochemical profile, the diagnosis of this canine tumor best fits the clinicopathologic criteria of an intraosseous human pPNET (Ewing’s sarcoma).2–4,8,10,11 Admittedly, because CD99 expression could not be detected in relevant normal dog tissue (lymph node, spleen, and pancreatic islet cells) and the lack of molecular genetic analysis, the diagnosis still depends somewhat on circumstantial evidence. Our study suggests that either CD99 is not expressed in the dog or more likely that the HO36-1.1 antibody, reliably immunoreactive in human tissue, does not recognize the canine epitope of this conserved cytoplasmic membrane–bound glycoprotein.2,14,15 Whether the dog or other animal pPNETs can present profiles similar to the other clinicopathologic subtypes occurring in people is unknown. One previously documented intraosseous Ewing’s sarcoma in an 8-month-old dog had no skull or spinal vertebral lesions.12 Another instance in an adult dog seems to best fit the criteria for an extraosseous Ewing’s sarcoma.6 One histologically and immunocytochemically documented pPNET has been reported in a 2-month-old Co-



lobus monkey in which the primary site appeared to be an abdominal retroperitoneal mass with local spread to adjacent lumbar vertebrae, as well as to brain, lymph nodes, skeletal and cardiac muscle, lung, and liver.7 Whereas the lesions in this monkey pPNET do not neatly fit into one of the human EFT subtypes, it does support the hypothesis that the neural crest progenitor cell of EFT can differentiate into various neuroectodermal lineages. More importantly, because human Ewing’s sarcoma is overall the fourth most common bone tumor, and the second most common malignant bone tumor in children, it is surprising that this tumor has been so rarely identified in animals.3,4,6,7,11,12 Based on our experience, we suspect it has been most likely overlooked and misdiagnosed as a lymphoma with osseous metastases.

References 1 Arvand A, Denny CT: Biology of EWS/ETS fusions in Ewing’s family tumors. Oncogene 20:5747–5754, 2001 2 Devoe K, Weidner N: Immunohistochemistry of small round-cell tumors. Semin Diagn Pathol 17:216–224, 2000 3 Ginsberg JP, Woo SY, Johnson ME, Hicks MJ, Horowitz ME: Ewing’s sarcoma family of tumors: Ewing’s sarcoma of bone and soft tissue and the peripheral neuroectodermal tumors. In: Principals and Practices of Pediatric Oncology, eds. Pizzo PA, and Poplack DG, 4th ed., pp. 973–1083. Lippincott Williams and Wilkins, Philadelphia, PA, 2002 4 Hadfield MG, Quezado MM, Williams RL, Luo VY: Ewing’s family of tumors involving structures related to the central nervous system: a review. Pediatr Dev Pathol 3: 203–210, 2000 5 Higgins RJ, LeCouteur RA, Vernau KM, Sturges BK, Obradovich JE, Bollen AW: Granular cell tumor of the canine central nervous system: two cases. Vet Pathol 38: 620–627, 2001 6 Hosokawa S, Suzuki S, Hibino N, Fukuta T, Imai T, Hayakawa K, Nakanowatari J, Sagami F: Peripheral primitive neuroectodermal tumor (peripheral neuroepithelioma) in a dog. Contemp Top Lab Anima Sci 37:66–69, 1998

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7 Long PH, Schulman FY, Koestner A, Fix AS, Campbell MK, Cameron KN: Primitive neuroectodermal tumor in a two month-old black and white Colobus monkey. Vet Pathol 35:64–67, 1998 8 Mierau GW, Weeks DA, Hicks MJ: Role of electron microscopy and other special techniques in the diagnosis of childhood round cell tumors. Hum Pathol 29:1347– 1355, 1998 9 Moore PF, Affolter VK, Olivry T, Schrenzel MD: The use of immunological reagents in defining the pathogenesis of canine skin diseases involving proliferation of leukocytes. In: Advances in Veterinary Dermatology, eds. Kwochka KW, Willemse T, and von Tscharner C, vol. 3, pp. 77–94. Butterworth Heinemann, Oxford, UK, 1998 10 Ozdemirli M, Fanburg-Smith JC, Hartman D-P, Azumi N, Miettinen M: Differentiating lymphoblastic lymphoma and Ewing’s sarcoma: lymphocyte markers and gene rearrangement. Mod Pathol 14:1175–1182, 2001 11 Parham DM: Neuroectodermal and neuroendocrine tumors principally seen in children. Am J Clin Pathol 115(Suppl 1):S113–S128, 2001 12 Rudolph R, Weiss E, Biel M: Multiples Ewing-Sarkom bei einem Hund. Zentralbl Veterinarmed A 16:426–437, 1969 13 Shimada H, Brodeur GM: Tumors of peripheral neuroblasts and ganglion cells. In: Russell and Rubenstein’s Pathology of Tumors of the Nervous System, eds. Bigner DD, McLendon RE, and Bruner JM, 6th ed., vol. 2, pp. 512–517. Arnold, London, UK, 1998 14 Weidner N, Tjoe J: Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC–2 and is useful in diagnosing Ewing’s sarcoma and peripheral neuroepithelioma. Am J Surg Pathol 18:486–494, 1994 15 West DC: Ewing sarcoma family of tumors. Curr Opin Oncol 12:323–329, 2000 Request reprints from Dr. H. E. V. De Cock, Department of Pathology, Microbiology and Immunology, One Shields Avenue, Davis, CA 95616 (USA). E-mail: hedecock@ ucdavis.edu.