Retroperitoneal extraosseous peripheral primitive neuroectodermal tumor in a Formosan serow: case report and literature review

Abstract

A 10-y-old female captive Formosan serow (Capricornis swinhoei) was inactive and was azotemic. An autopsy was performed following her death, and multiple irregularly shaped, white-to-gray masses of 0.5–2 cm diameter were noted on both ureters, the left adrenal gland, urinary bladder, and uterus. Microscopically, organs were effaced by a poorly demarcated, highly infiltrative neoplasm, composed of neoplastic round cells arranged in islands, sheets, or nests with occasional rosette formation. The neoplastic cells were small: ≤2 red blood cell (≤ 15 μm) diameter. The neoplastic cells were positive for CD56, CK, FLI-1, and NSE, but negative for desmin, GFAP, melan A, NF, PAX-8, S100, synaptophysin, and vimentin. Therefore, the diagnosis of retroperitoneal extraosseous peripheral primitive neuroectodermal tumor (pPNET) was made. pPNET with FLI-1 expression has not been reported previously in animals, to our knowledge.

Primitive neuroectodermal tumors (PNETs) are highly malignant tumors composed of small round cells, originating from multipotent progenitor cells of the neuroectoderm.^13,18 Based on anatomic location, PNETs are categorized as central nervous system PNETs (such as medulloblastomas) and peripheral PNETs (pPNETs).^12,13,26 pPNETs can arise from a variety of anatomic locations and can be subdivided into 2 types: extraosseous and osseous. In humans, the most common site of extraosseous pPNETs is the thoracopulmonary region (namely Askin tumors), but extraosseous pPNETs at other anatomic locations, such as the head, neck, kidney, and retroperitoneal or paraspinal regions, have been reported.¹³ PNETs usually have divergent phenotypic differentiation (neuronal, epithelial, and mesenchymal), and thus they usually have diverse immunohistochemical (IHC) characteristics.^11,13,18,47

In humans, ~90% of PNETs are associated with t(11;22)(q24;q12) translocation, causing fusion between the Ewing sarcoma breakpoint region 1 (EWSR-1) gene on chromosome 22 and the Friend leukemia integration 1 (FLI-1) gene on chromosome 11 (EWS-FLI fusion). This translocation results in juxtaposition of the amino-terminal domain of EWSR-1 to the carboxyl-terminus of FLI-1 and the expression of FLI-1 protein in PNETs.^1,11,13,29 Therefore, IHC staining of FLI-1 has been considered essential for the diagnosis of PNETs. However, no PNET cases in animals with IHC staining of FLI-1 have been reported (Table 1), to our knowledge. We report herein the gross, histologic, and IHC characteristics (especially FLI-1) of an extraosseous pPNET in a Formosan serow (syn. Taiwan serow; Capricornis swinhoei).

Table 1.

Primitive neuroectodermal tumors reported in animals.

Species	Age	Sex	Location	IHC-positive	IHC-negative	Reference
Baboon (Papio papio)	NA	F	Cerebellum	NA	NA	5
Brahman crossbred calf	6 mo	NA	Spinal cord	NSE, S100, SYN, vimentin	Chro-A, GFAP	4
Colobus monkey (Colobus guereza kikuyuensis)	2 mo	M	Facial region, 4th ventricle, retroperitoneal cavity	GFAP, NSE, SYN	NF	30
Cynomolgus monkey (Macaca fascicularis)	3.5 y	M	Left cerebellar hemisphere and hindbrain	GFAP, NF, NSE, S100, SYN, vimentin	None	34
Dog
Australian Shepherd	9 y	CM	Intraocular	SYN	IRBPd, GFAP, opsin, S-Ag	39
	5 mo	M	Left frontal bone	NF	CD18, CD3, CD45, CD45r, CD79a, CD99, chro-A, CK, desmin, melan A, NSE, S100, skeletal myosin, SYN, vimentin	8
Beagle	10 mo	F	Intraocular	SYN	IRBPd, GFAP, opsin, S-Ag	39
Border Collie	4 y	SF	Cerebellum	GFAP, NSE, S100, vimentin	Nestin, NF, SYN,	43
Chow Chow	9 y	SF	Intraocular	None	IRBPd, GFAP, opsin, S-Ag, SYN,	39
German Shepherd	1 y	M	Thoracolumbar vertebral column	NSE, SYN	BS-1, CD3, CD45R, CD79a, chro-A, CK, desmin, GFAP, histiocyte antigen, IgG, IgM, melan A, MHC-II, NF, S100, SMA, vimentin	23
Golden Retriever	2.5 y	F	Left cerebral hemisphere	Vimentin	Chro-A, CK, GFAP, NF, NSE, S100, SYN	25
Labrador Retriever	15 wk	M	Cerebellum and brainstem	β-tubulin, nestin, NSE, S100, SYN, vimentin	GFAP	19
	4 y	CM	Left tympanic bulla, vertebral canal, cranial vault	NSE, vimentin	CD99, CK, GFAP, S100	16
Labrador Retriever mix	11 y	CM	Intraocular	SYN	IRBPd, GFAP, opsin, S-Ag	39
Malamute	1 y	F	Intraocular	IRBPd, GFAP, opsin, S-Ag, SYN	None	Regan, et al.39
Miniature Dachshund	9.6 y	SF	Cerebrum	β-tubulin, GFAP	Iba1, Oligo-2	35
Mixed	1 y	M	Intraocular	GFAP, SYN	IRBPd, opsin, S-Ag	39
Pointer	6 y	F	Cerebellar vermis region	NSE, S100, vimentin	CD3, CD45, CD79a, CK, GFAP, SYN	6
Polish Lowland Sheepdog	2.3 y	CM	Cerebellum	GFAP, S100, vimentin	CD3, CD79a	33
Rottweiler	2 y	M	Intraocular	SYN	IRBPd, GFAP, opsin, S-Ag	39
Terrier mix	7.4 y	CM	Intraocular	None	IRBPd, GFAP, opsin, S-Ag, SYN	39
Dromedary camel (Camelus dromedarius)	9 y	CM	3rd lumbar vertebra	GFAP, NSE, vimentin	CK, SYN, CD3, CD79a	46
Formosan serow	10 y	F	Retroperitoneal cavity	CD56, CK, FLI-1, NSE	Desmin, GFAP, NF, PAX8, S100, SYN, vimentin	Current study
Electric eel (Electrophorus electricus)	NA	NA	Facial region	NA	NA	24
Holstein heifer	18 mo	F	Cerebrum	NSE, vimentin	GFAP, NF, S100, SYN	31
Japanese Black cattle	7 y	F	Mandibulopharygeal region	NF, NSE	S100, vimentin	44
Jersey cow	2 y	F	Left lateral parietal and cranial vault	GFAP, S100, vimentin	Chro-A, NSE, SYN	37
Kowari (Dasyuroides byrnei)	4.5 y	F	Cerebellum	NA		2
Paint horse	2 y	CM	Scrotum, inguinal canals, retroperitoneal cavity	GFAP, NF, NSE, S100, SYN	CD99, desmin, muscle actin	12
Quarter Horse	Adult	M	Brainstem	NA	NA	20
Roe deer (Capreolus capreolus)	10 y	F	Mesencephalon	NF, NSE, SYN	β-tubulin, chro-A, CK, GFAP, NF, S100, vimentin	26
Squirrel monkey (Saimiri sciureus)	>20 y	M	Cerebrum	NF, NSE, vimentin	GFAP, SYN	22
Striped dolphin (Stenella coeruleoalba)	Juvenile	F	Cerebrum	S100, vimentin	CK, GFAP, NSE, SYN	3
Umbrella cockatoo (Cacatua alba)	NA	F	Cerebellum	SYN	GFAP, NSE, NF	10

C = castrated male; Chro-A = chromogranin A; CK = cytokeratin; CM = castrated male; F = intact female; FLI-1 = friend leukemia integration 1; GFAP = glial fibrillary acidic protein; IHC = immunohistochemical; IRBPd = interphotoreceptor retinoid-binding protein; M = intact male; mo = month; MHC-II = major histocompatibility complex class II; NA = not available; NF = neurofilament; NSE = neuron-specific enolase; PAX8 = paired-box gene 8; S = spayed female; S-Ag = S-antigen; SF = spayed female SYN = synaptophysin; y = year.

A 10-y-old female captive Formosan serow of the Taipei Zoo was inactive, and blood examination revealed increased serum creatinine (592 µmol/L; reference interval [RI]: 62–141 µmol/L), urea (25.4 mmol/L; RI: 5.0–8.9 mmol/L), and potassium (7.1 mmol/L; RI: 3.5–5.1 mmol/L). The RIs of serum biochemistry were based on previous studies of selected Caprinae species (Chinese goral, Naemorhedus griseus).⁴⁵ The animal did not improve with supportive treatment and was found dead the next day; an autopsy was conducted. Multiple irregularly shaped, white-to-gray masses of 0.5–2 cm diameter were noted on both ureters and extended to peripheral soft tissues and organs in the retroperitoneal space, including the left adrenal gland and sublumbar lymph node (LN), and adjacent organs, including urinary bladder and uterus. The growths infiltrated the lumen of the left ureter and thereby obstructed urinary flow, leading to marked dilation of the left ureter and inducing hydronephrosis of the left kidney. Cut sections of the masses were lobulated and homogeneous beige with a firm texture. The wall of the urinary bladder was hardened with some raised masses of 0.5–1 cm diameter, and the dorsal aspect of the urinary bladder adhered to the uterus. In addition, there was a 0.5 × 0.5 × 0.5 cm raised white-to-yellow nodule on the surface of the liver.

Representative tissue samples were collected from the brain, spinal cord, eyes, tongue, thyroid glands, trachea, tracheobronchial LN, lungs, heart, diaphragm, gastrointestinal tract, spleen, liver, both adrenal glands, whole urogenital tract with sublumbar LN, sciatic nerves, and skeletal muscle. The samples were fixed in 10% neutral-buffered formalin, processed routinely, and sections stained with hematoxylin and eosin. Histologic examination revealed that the masses on the ureters were poorly demarcated and highly infiltrative neoplasms with areas of necrosis; neoplastic round cells were arranged in islands or sheets separated by various amounts of fibrovascular stroma. Peripheral palisading of the neoplastic cells with rosette formation was observed. The neoplastic cells were highly pleomorphic with scant eosinophilic cytoplasm, and their nuclei were hyperchromatic and round-to-ovoid with stippled chromatin and conspicuous nucleoli (Fig. 1). The diameter of the neoplastic cells was ≤ 2 red blood cells (≤ 15 μm diameter). Lymphovascular invasion was noted. Neoplastic cells extensively infiltrated the cortex and medulla of the left adrenal gland, urinary bladder, and uterus. The hepatic nodule was composed of the same neoplastic cell population, indicative of hepatic metastasis. The cell morphology and aggressive behavior of the neoplasm were compatible with malignant small round cell tumor (MSRCT) in humans, a term used for a group of neoplasms characterized by small, round, relatively undifferentiated cells.^36,41

Figure 1.

Retroperitoneal extraosseous peripheral primitive neuroectodermal tumor in a Formosan serow. Peripheral palisading of the neoplastic cells with rosette formation is observed (*). The neoplastic cells have scant eosinophilic cytoplasm and contain a hyperchromatic and round-to-ovoid nucleus with stippled chromatin and conspicuous nucleoli. H&E. Bar = 50 μm.

To identify the cell origin of the neoplasm, an IHC panel was performed. Briefly, deparaffinized tissue sections were heated in retrieval solution (Trilogy; Cell Marque, Rocklin, CA) at 121°C in an autoclave for 15 min. Subsequently, tissue sections were incubated in 2.5% normal goat serum in Tris-buffered saline for 30 min at room temperature and then incubated with antibodies against neuron-specific enolase (NSE; diluted 1:800; clone BBS/NC/VI-H14; Dako, Glostrup, Denmark) at room temperature for 1 h. The sections were then treated with 3% hydrogen peroxide in methanol for 10 min followed by peroxidase-conjugated secondary antibodies for 60 min at room temperature. After exposure to 2% 3,3’-diaminobenzidine (Real DAB⁺ chromogen; Dako) for 3 min, the slides were counterstained with hematoxylin for 30 s. IHC staining for CD56 (diluted 1:50; clone RCD56; Zytomed Systems, Berlin, Germany), CD99 (diluted 1:200; clone PCB1; Novocastra Laboratories, Newcastle upon Tyne, UK), chromogranin A (diluted 1:300; clone Poly; Genemed Biotechnologies, South San Francisco, CA), cytokeratin (CK; diluted 1:400; clone AE1/AE3; Novocastra), desmin (diluted 1:100; clone DE-R-11; Leica Microsystems, Richmond, IL), FLI-1 (diluted 1:100; clone G146-222; BD Biosciences), glial fibrillary acidic protein (GFAP; diluted 1:250; clone GA5; Novocastra), melan A (diluted 1:100; clone A103; Dako), neurofilament (NF; diluted 1:200; clone N52.1.7; Novocastra), paired-box gene 8 (PAX8; diluted 1:100; clone BC12; Biocare Medical, Pacheco, CA), S100 (diluted 1:1000; clone 16/f5; Zytomed Systems), synaptophysin (diluted 1:800; clone 27G12; Novocastra), and vimentin (diluted 1:400; clone V9; Novocastra) was performed in an automated system (BondMax; Leica Microsystems). Normal tissues from this serow were used as internal controls for the IHC staining, including endothelial cells (FLI-1 and CD99), muscular arteries (desmin), adrenal gland (CD56, chromogranin A, synaptophysin), cerebellum (GFAP, NSE, NF, S100), kidney (PAX8), and skin (CK, vimentin). Most of the internal controls from the normal tissue of this serow for the above antibodies, except chromogranin A and CD99, were positive, indicative of good cross-reactivity; we excluded chromogranin A and CD99 from our investigation.

The neoplastic cells had membranous positivity for CD56, cytoplasmic positivity for CK/NSE, and cytoplasmic and/or nuclear positivity for FLI-1 (> 50%; Fig. 2). However, the cells were negative for desmin, GFAP, melan A, NF, PAX8, S100, synaptophysin, and vimentin (Supplementary Table 1). Based on the results of IHC staining patterns and the anatomic location of the neoplasm, we diagnosed PNET with hepatic metastasis and involvement of the left adrenal gland, urinary bladder, and uterus.

Figure 2.

Retroperitoneal extraosseous peripheral primitive neuroectodermal tumor in a Formosan serow. A. Neoplastic cells with membranous positivity for CD56. IHC. Bar = 100 μm. B. Neoplastic cells with cytoplasmic positivity for cytokeratin. IHC. Bar = 100 μm. C. Neoplastic cells with nuclear and/or cytoplasmic positivity for FLI-1 (>50% of cells). IHC. Bar = 100 μm. D. Neoplastic cells with cytoplasmic positivity for neuron-specific enolase. IHC. Bar = 100 μm.

Neoplasms in the MSRCT group are characterized by poorly differentiated small round cells and can be epithelial, mesenchymal, neuroendocrine, melanocytic, or hematopoietic.^36,41 Differential diagnosis of MSRCTs includes desmoplastic small round cell tumor (DSRCT), dysgerminoma, melanoma, nephroblastoma, neuroblastoma, neuroendocrine carcinoma, PNET, round cell sarcomas such as lymphoma, small cell rhabdomyosarcoma, small cell osteosarcoma, and synovial sarcoma. Definitive diagnosis is difficult because neoplasms in the MSRCT group share a similar cell morphology.^28,36,41 Therefore, IHC staining is necessary to make the final diagnosis (Supplementary Table 1). In our case, the cross-reactivity of CD99 and chromogranin A was poor in control tissue; therefore, these stains were excluded. Previous studies in humans have demonstrated that > 20% of PNETs are positive for cytokeratin,^7,11,15,17 suggestive of epithelial differentiation. Therefore, the diagnosis of PNETs cannot be ruled out by the expression of epithelial markers. CD56, also known as neural cell adhesion molecule (NCAM), is a marker for neuroendocrine carcinomas and certain types of lymphomas, and PNET can also be positive for CD56 in some human cases.²⁸ Therefore, the differential diagnosis of our neoplasm was narrowed to neuroblastoma, neuroendocrine carcinoma, and PNET by the IHC markers tested (positive for CD56, NSE, and CK; negative for desmin, PAX8, and vimentin [Supplementary Table 1]).

According to studies of human PNETs, FLI-1 is considered crucial for distinguishing PNETs from other MSCRT.^1,11,27-29 In human studies, FLI-1 is a transcription factor and is generally localized within the nucleus, but it has been detected in the cytoplasm of some cells.³⁸ The cytoplasmic positivity may be associated with either cross-reactivity with a cytoplasmic protein or the presence of FLI-1 protein in the cytoplasm of these cells.³⁸ For the IHC staining of FLI-1, the neoplasm is defined as positive only if > 10% of neoplastic cells have nuclear positivity.¹⁴ Generally, neuroblastoma and neuroendocrine carcinoma are negative for FLI-1, and PNET is positive for FLI-1.^28,29 Therefore, our neoplasm is most likely PNET.

We hypothesize that the ureters and adjacent soft tissue were the primary site of our PNET because they were the most severely affected tissues. The other possible origin was from the spinal nerve roots in the lumbar region. However, considering the infiltrative behavior of our PNET, neoplastic cells would have had to directly invade from the spinal nerve roots to the spinal cord, which we did not observe in our case. Therefore, the spinal nerve roots from the lumbar region were less likely to be the primary location.

Several prognostic factors of PNETs have been reported in humans, such as stage, primary tumor site, size, age, and response to therapy.⁹ Some human studies have suggested that extraosseous pPNETs are highly malignant and have a worse prognosis than osseous pPNETs, and that extraosseous pPNETs of the urinary tract may be more aggressive than those in other anatomic locations.^21,32 Given the aggressive behavior of extraosseous pPNETs, early diagnosis with appropriate medical treatment may be the only way to achieve a relatively good clinical outcome. However, such an outcome is difficult to achieve in captive or free-ranging wild animals.

FLI-1 protein is involved in cell proliferation and tumorigenesis, and it is normally expressed in endothelial cells and hematopoietic cells.^29,40,42 In patients with PNETs, EWS-FLI fusion is present only in the neoplastic cells of PNETs and does not exist in normal cells.⁹ The presence of EWS-FLI fusion plays an important role in the transformation, growth, and metastasis of PNETs.^1,9 Therefore, it may be a useful biomarker for both diagnosis and targeted therapy. PNETs with FLI-1 expression have not been reported in any animal species, to our knowledge, and thus the tumorigenesis and the use of FLI-1 as a biomarker for diagnosis or therapy is poorly studied. Further investigations of cytogenetics (such as the chromosomal translocation of t(11; 22)(q24;q12) in humans) by fluorescence in situ hybridization and/or reverse-transcription PCR in animals with PNET or other common neoplasms are necessary to determine the role of FLI-1 in tumorigenesis and the use of FLI-1 as a marker in animals.