From the Southwest Hospital, Southwest Eye Hospital, Third Military Medical University, Chongquing, China.
The authors have no financial or proprietary interest in the materials presented herein.
Address correspondence to Yuli Yang, PhD, Southwest Hospital, Southwest Eye Hospital, Third Military Medical University, No. 30 Gaotanyan Main Street, Shapingba District, Chongqing, 400038 China. E-mail: email@example.com
Primitive neuroectodermal tumor (PNET) is a term for a group of small round cell tumors that arise from primitive neuroectodermal progenitor cells. Once believed to be confined to the central nervous system, PNETs are now increasingly recognized to occur outside the central nervous system and are designated as peripheral PNETs (pPNETs).1 According to different degrees of neuroectodermal differentiation, pPNETs were further divided into pigmented neuroectodermal tumor, Askin tumor, and Ewing sarcoma/pPNET groups.2 Ewing sarcoma/pPNETs most commonly affects the long bones of the limbs, the ribs, and the pelvis.3–6 Ewing sarcoma/pPNET of the head and neck region is unusual, comprising 1% to 4% of all cases of Ewing sarcoma/pPNET.7,8 Although Ewing sarcoma/pPNETs occur more commonly in children, primary orbital involvement is extremely rare. Only 20 reports on Ewing sarcoma/pPNET of the orbit appear in the literature.9–25 We describe an additional patient with an orbital Ewing sarcoma/pPNET, features of which are compared with the previous cases in the literature.
A 6-year-old boy presented with the complaint of abruptly decreasing visual acuity for approximately 50 days in February 2009. His best-corrected visual acuity was 20/250 in his right eye and 20/20 in his left eye. External ocular examination showed localized swelling and erythema of the right lower eyelid. There was 6 mm of right proptosis with supranasal duction (Fig. 1). Examination of the extra-ocular movements revealed limitation of downward and lateral gaze of the right eye. Bilateral anterior segments and intraocular pressures were normal. Funduscopy of the right eye also showed normal findings.
Figure 1. A 6-year-old boy with a mass in the infratemporal portion of the right orbit.
Computed tomography scan revealed a mass in the region of the right inferotemporal orbit with extension into the infratemporal fossa and masseteric space, and there was erosion of the lateral orbital wall (Fig. 2). A complete blood cell count and serum laboratory values were normal. Bone marrow aspiration showed normal findings. Chest and long bone radiography were normal and abdominal ultrasonography revealed no organ involvement.
Figure 2. Computed tomography scan showing a mass in the region of the right inferotemporal orbit with extension into the infra-temporal fossa and masseteric space and there was erosion of the lateral orbital wall: (A) horizontal view and (B) coronal view.
An anterior orbitotomy eyelid crease was performed to remove the tumor. The mass adhered to the adjacent structures. After the loose adhesions were overcome by careful dissection, the mass was extracted without damage. Following the surgery, the patient was administered radiation therapy for the entire right orbit and chemotherapy for the residual tumors. The chemotherapeutic regimen consisted of multiple cycles of vincristine, actinomycin D, and cyclophosphamide. However, the tumor recurred, and the patient died approximately 14 months after surgery because the tumor had metastasized to the brain.
The excised tumor mass was fixed in 4% buffered formaldehyde and embedded in paraffin. Serial sections through the tumor were performed using routine histopathologic techniques and stained with hematoxylin–eosin and periodic acid-Schiff stains. Immunohistochemical reactions were performed using the streptavidin-biotin method. The following antibodies were applied: desmin, myogenin, leukocyte common antigen, synaptophysin, neurospecific enolase, and MIC2 (CD99). These antibodies were applied synchronously with appropriate positive control slides. For the negative control, buffered saline was used.
Total RNA was extracted from paraffin-embedded tissues. The sections were deparaffinized in three changes of xylene and three washes with 100% ethanol. After drying, tissue pellets were re-suspended in 200 μL of lysis buffer (20 mmol/L Tris-HCL, PH 8.0, 20 mmol/L ethylenediaminetetraacetic acid, 2% sodium dodecyl sulfate) containing proteinase K. After incubation at 55°C overnight, 1.0 mL of Trizol (Invitrogen, Carlsbad, CA) was added to the sample and total RNA was extracted according to the manufacturer’s instructions. The RNA pellets were reconstituted in 20 μL of diethylpyrocarbonate-treated water and stored at −80°C. Thereafter, 1 μg of the extracted RNA was reverse-transcribed into cDNA using 50 units of SuperScript II (Invitrogen, Carlsbad, CA) reverse transcriptase, 100 ng of random primer, 0.5 mmol/L dNTP, and 40 units of RNase inhibitor, and was then incubated with 2 units of RNase H for 20 minutes at 37°C.
Polymerase chain reactions (PCR) were performed to detect the EWS-FLI-1 fusion transcripts. PCR was performed for EWS-FLI-1. PCR amplifications were performed in a total volume of 50 μL containing 5 μL of RT reaction product as template DNA, 1 × PCR buffer, 1.5 mmol/L of magnesium chloride, 0.1 mmol/L of each deoxynucleotide, 100 ng of each sense and antisense primer, and 1.25 U of Taq DNA polymerase. Programmable temperature cycling was performed with the following cycle profile: 95°C for 5 minutes, followed by 40 cycles of 94°C for 1 minutes, 65°C for 1 minutes, and 72°C for 2 minutes. After the last cycle, an extended 10 minutes at 72°C was followed by cooling to 4°C. Cultured cells of the human Ewing sarcoma cell line were used as known positive controls carrying the EWS exon 7-FLI-1 exon 6 translocation. A negative control in which the RT enzyme was omitted was used to exclude DNA contamination. Cases of unrelated tumors were used as negative controls. A reaction mixture of reagents devoid of template was included in each PCR procedure as a blank control.
On gross inspection, the excised tumor consisted of white, soft, and lobulated parts and measured 3.7 × 3.6 × 1.8 mm. It was partially encapsulated with moderate contexture. Microscopically, the tumor was composed of irregularly shaped islands of small round cells with basophilic cytoplasm and pleomorphic nuclei. There were frequent abnormal mitoses. Near the borders of the tumor, the cells infiltrated the surrounding fibrous tissue in cords. Homer–Wright pseudorosettes were not found (Fig. 3). Regarding immunostaining (Fig. 4), the tumor cells were strongly and diffusely positive in membranous pattern for CD99 immunostaining. All other reactions were negative. There were intervening fibrous septa among tumor cells that were stained positively with periodic acid-Schiff (Fig. 5). EWS-FLI-1 fusion genes were not detected by reverse-transcription PCR.
Figure 3. Histology of the Ewing sarcoma showing islands of monotonous small round cells with foamy cytoplasm and pleomorphic nuclei. There were no rosettes (hematoxylin–eosin, original magnification ×50).
Figure 4. The tumor cells immunostained with anti-MIC2 antibodies (original magnification ×200).
Figure 5. There were intervening fibrous septa among tumor cells that were stained positively with periodic acid-Schiff (original magnification ×50).
Ewing sarcoma/pPNET is one of the most malignant bone and soft tissue tumors in adolescents and young adults.26 Among the phenotypic spectrum of the Ewing family of tumors, Ewing sarcoma is at the most undifferentiated end of the spectrum, lacking most of the features of neural differentiation; the other end of the spectrum comprises the most differentiated pPNET.27 Recently, cytogenic findings revealed that Ewing sarcoma and pPNETs share the same specific reciprocal chromosome translocation t(11;22)(q24;q12).28,29 pPNET may demonstrate varying degrees of neural differentiation and the progressive process begins with neurospecific enolase expression, followed by Homer–Wright rosette formation, phenotypic ganglion cell differentiation, and neurofilament protein expression.30 At the light microscopy level, dense clumping of chromatin and mitotic figures are more common in pPNET. The rosette formation and fibrous background are necessary for the diagnosis of pPNET.
Regarding the immunophenotypes, Ewing sarcoma and pPNET may share some similar immunophenotypes, with the exception that pPNET should be positive for some neural markers, such as neurospecific enolase synaptophysin. According to the immunohistochemical and cytogenic findings, our case favors Ewing sarcoma rather than pPNET.
Primitive neuroectodermal tumor of the ulnar nerve was initially described in 1918 by Stout.31 Until the late 1970s, Ewing sarcoma/pPNET was not recognized as an entity. Ewing sarcoma/pPNET most commonly affects the long bones and it accounts for 8% to 10% of all primary malignant bone tumors.32 Primary orbital Ewing sarcoma/pPNETs are extremely rare. Harbert and Tabor reported the first case in a 19-year-old boy presenting with a proptosis due to a mass in the lateral portion of the right orbit.9 To our knowledge, only 20 cases of primary orbital Ewing sarcoma/pPNET have been reported. Orbital Ewing sarcoma/pPNETs are heterogeneous from a clinical and histopathologic standpoint (Table). Among reported cases there was a male-to-female ratio of 12 to 8 and the mean age at presentation was 17 years (range: < 1 to 61 years). One common feature of these tumors seems to be their proclivity to arise in the lateral orbit. Extraorbital extension has been noted in 12 cases. There are 4 patients with systemic metastases.21,22,24
Table: Reported Cases of Primary Ewing’s Sarcoma Involving the Orbit
The differential diagnosis of small round cell tumors of childhood and adolescence34 may be difficult. Small round cell tumors of childhood and adolescence are a diverse group of tumors of neural, mesenchymal, and lymphoid derivation that may share a similar histologic appearance of small, poorly differentiated cells, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma, and malignant lymphoma. However, accurate diagnosis and staging of these tumors is essential because therapy and prognosis are tumor specific. Currently, CD99-O13 immunostaining and molecular studies using PCR are definitive for Ewing sarcoma to detect characteristic chromosomal translocations. Approximately 90% of tumors exhibit a fusion of the EWS and FLI-1 genes resulting from a characteristic t(11;22). This results in the juxtaposition of the 5′ end of the EWS gene to the 3′ end of the FLI-1 gene.
The EWS gene has been poorly characterized, but is known to encode an RNA-binding protein. The human FLI-1 gene is a member of the ETS family of DNA transcription factors. The translocation results in a novel fusion protein that has been shown to have transforming activity in NIH 3T3 cells.34 In approximately 5% of the Ewing sarcoma family of tumors, a fusion of the EWS and ERG genes resulting from a t(11;22) can be detected. In these tumors, the ERG gene is believed to function similarly to the FLI-1 gene present in the classic translocation because both ERG and FLI-1 are members of the ETS family of DNA-binding factors and share significant homology.35 The presence of the fusion proteins resulting from these translocation events can be detected in tissues using reverse-transcription PCR.
The Ewing sarcoma family of tumors is characterized by the presence of specific translocations resulting in the formation of the MIC2 surface protein36 and this could be detected by the specific antibody CD99-O13 in immunostaining. The absence of MIC2 protein or t(11;22) translocation in the neoplasms would suggest that they are not Ewing sarcoma. CD99 immunostaining was positive in our case, but EWS exon 7-FLI-1 exon 6 translocation was not detected. Ramzi et al.37 reported on a series of 58 patients with Ewing sarcoma; EWS exon 7-FLI-1 exon 6 translocation was only detected in 45 patients (78%) and some other types of gene translocations existed in Ewing sarcoma. That may explain why we did not detect EWS exon 7-FLI-1 exon 6 translocation in our case.
There is no consensus for the best treatment strategy for Ewing sarcoma/pPNETs; they progress rapidly with poor prognosis and often have metastasized at the time of diagnosis.38 Curative treatment now exists for most patients with localized tumors and consists of surgery and systemic chemotherapy or radiation therapy for control of the primary tumor.39 There is no doubt that the addition of adjuvant chemotherapy has been of considerable benefit to these patients. The drugs used, including cyclophosphamide, doxorubicin, vincristine, etoposide, and ifosfamide, produced good results.40 The small number of reported cases makes evaluation of the different therapeutic modalities difficult. Therefore, no comparable data exist for orbital Ewing sarcoma/pPNETs until now.
Ewing sarcoma/pPNET rarely affects the orbit. It is prevalent among the children with no gender proclivity. It progresses rapidly with poor prognosis, therefore accurate diagnosis must be made as soon as possible according to immunohistochemical analysis and cytogenic findings. Appropriate treatment consists of local resection, radiotherapy, and adjunctive chemotherapy.
- Dehner LP. Peripheral and central primitive neuroectodermal tumors: a nosologic concept seeking a consensus. Arch Pathol Lab Med. 1986;110:997–1005.
- Dehner LP. Neuroepithelioma (primitive neuroectodermal tumor) and Ewing’s sarcoma: at least a partial consensus. Arch Pathol Lab Med. 1994;118:606–607.
- Ewing J. Diffuse endothelioma of bone. Proc NY Pathol Soc. 1921;21:17–24.
- Jereb B, Ong RL, Mohan M, et al. Redefined role of radiation in combined treatment of Ewing’s sarcoma. Pediatr Hematol Oncol. 1986;3:111–118. doi:10.3109/08880018609031206 [CrossRef]
- Senac MO Jr, Isaacs H, Gwinn JL. Primary lesions of bone in the 1st decade of life: retrospective survey of biopsy results. Radiology. 1986;160:491–495.
- Wilkins RM, Pritchard DJ, Burgert EO, et al. Ewing’s sarcoma of bone: experience with 140 patients. Cancer. 1986;58:2551–2555. doi:10.1002/1097-0142(19861201)58:11<2551::AID-CNCR2820581132>3.0.CO;2-Y [CrossRef]
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- Watanabe H, Tsubokawa T, Katayama Y, et al. Primary Ewing’s sarcoma of the temporal bone. Surg Neurol. 1992;37:54–58. doi:10.1016/0090-3019(92)90067-W [CrossRef]
- Harbert F, Tabor GL Jr, . Ewing’s tumor of the orbit: report of two cases. Am J Ophthalmol. 1950;33:1219–1225.
- Yamada S, Takahashi H. A dissected case of Ewing’s tumor (reticulum cell sarcoma) of the orbit. Hirosaki Med J. 1957;8:202–207.
- Howard GM. Neuroepithelioma of the orbit. Am J Ophthalmol. 1965;59:934–937.
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- Shuangshoti S, Menakanit W, Changwaivit W, et al. Primary intraorbital extraocular primitive neuroectodermal (neuroepithelial) tumor. Br J Ophthalmol. 1986;70:543–548. doi:10.1136/bjo.70.7.543 [CrossRef]
- Woodruff G, Thorner P, Skarf B. Primary Ewing’s sarcoma of the orbit presenting with visual loss. Br J Ophthalmol. 1988;72:786–792. doi:10.1136/bjo.72.10.786 [CrossRef]
- Wilson WB, Roloff J, Wilson HL. Primary peripheral neuroepithelioma of the orbit with intracranial extension. Cancer. 1988;62:2595–2601. doi:10.1002/1097-0142(19881215)62:12<2595::AID-CNCR2820621224>3.0.CO;2-Y [CrossRef]
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- Singh AD, Husson M, Shields CL, et al. Primitive neuroectodermal tumor of the Orbit. Arch Ophthalmol. 1994;112:217–221.
- Lam DS, Li CK, Cheng LL, et al. Primary orbital Ewing’s sarcoma: report of a case and review of the literature. Eye. 1999;13:38–42. doi:10.1038/eye.1999.8 [CrossRef]
- Choi RY, Lucarelli MJ, Imesch PD, et al. Primary orbit Ewing’s sarcoma in a middle-aged woman. Arch Ophthalmol. 1999;117:535–537.
- Kiratli H, Bilgic S, Gedikoglu G, et al. Primitive neuroectodermal tumor of the orbit in an adult: a case report and literature review. Ophthalmology. 1999;106:98–102. doi:10.1016/S0161-6420(99)90020-9 [CrossRef]
- Dutton JJ, Rose JG, DeBacker CM, et al. Orbital Ewing’s sarcoma of the orbit. Ophthalmic Plast Reconstr Surg. 2000;16:292–300. doi:10.1097/00002341-200007000-00008 [CrossRef]
- Alyahya GA, Heegaard S, Fledelius HC, et al. Primitive neuroectodermal tumor of the orbit in a 5-year-old girl with microphthalmia. Graefes Arch Clin Exp Ophthalmol. 2000;238:801–806. doi:10.1007/s004170000178 [CrossRef]
- David JW, Roger AD, Michael TG, et al. Primary Ewing’s sarcoma of the orbit. Ophthalmic Plast Reconstr Surg. 2001;17:300–303. doi:10.1097/00002341-200107000-00011 [CrossRef]
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- Weiss SW, Goldblum JR. Primitive neuroectodermal tumors and related lesions. In: Weiss SW, Goldblum JR, eds. Enzinger and Weiss’s Soft Tissue Tumors, 4th ed. St. Louis, MO: Mosby-Harcourt Brace; 2001:1265–1321.
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- Huvos AG. Bone Tumors: Diagnosis, Treatment and Prognosis. Philadelphia: WB Saunders Company; 1979:322–344.
- Harbert F, Tabor GL Jr, . Ewing’s tumor of the orbit: report of two cases. Am J Ophthalmol. 1950;33:1219–1225.
- Yunis EJ. Ewing’s sarcoma and related small round cell neoplasms in children. Am J Surg Pathol. 1986;10:54–62.
- May WA, Gishizky ML, Lessnick SL, et al. Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A. 1993;90:5752–5756. doi:10.1073/pnas.90.12.5752 [CrossRef]
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Reported Cases of Primary Ewing’s Sarcoma Involving the Orbit
|Year Published||Gender/Age (Y)||Orbital Location||Bone Invasion||Extension of Tumor||Treatment||Immuno-histochemistry||EM||Follow-up|
|1950||M/19||Right lateral||–||–||Excision+RT||NA||NA||10 mo (died)|
|1957||M/61||Right||–||–||Enucleation+RT||NA||NA||17 d (died)|
|1965||M/< 1||Right inferior||+||–||Excision+RT||NA||NA||14 mo (died)|
|1985||M/14||Right medial||+||Sinuses (OU); cribriform plate (OU)||Excision+RT+CT||NA||Glycogen||12 mo (alive)|
|1986||M/52||Right lateral||–||–||Excision+RT+CT||GFAP||NA||6 mo (alive)|
|1988||F/7||Left medial, posterior, superior||+||Intracranial space||Excision+RT+CT||NSE||Glycogen||45 mo (alive)|
|1988||M/6||Right roof, medial, lateral||+||Sinuses (OU); cranial fossa (OU)||Biopsy+RT+CT||Vimentin||Glycogen||9 mo (alive)|
|1993||F/10||Left superior, lateral||+||Cranial fossa (OS); apex of temporal fossa (OS)||Excision+RT+CT||NA||NA||18 mo (alive)|
|1994||F/10||Lateral||+||–||Exicision+RT+CT||CK, EMA, NSE||+||9 mo (alive)|
|1999||M/2||Bilateral medial||+||Sinuses (OU)||Biopsy+CT+RT||PAS, NSE||NA||24 mo (alive)|
|1999||F/43||Left supranasal||+||Ethmoid sinuses (OS)||Excision+RT+CT||NSE||NA||14 mo (alive)|
|1999||M/28||Right lateral||–||–||Excision||NSE, vimentin||NA||15 mo (alive)|
|2000||M/2||Right supratemporal||+||L1 vertebra||Excision+RT+CT||–||Neurosecretory granules||17 mo (died)|
|2000||M/7||Left superior||+||Lung; cranial fossa (OS)||Excision+RT+CT||–||NA||12 mo (died)|
|2000||F/5||Right lacrimal gland intraconal||–||–||Excision+CT||Synaptophysin||Neurosecretory granules||4 y (alive)|
|2001||F/17||Right roof||+||Anterior cranial fossa (OD); temporal fossa||Excision+CT||PAS||NA||1 y (alive)|
|2002||F/17||Right intraconal||–||Liver||Excision+RT+CT||NSE, EMA||Neurosecretory granules||5 y (alive)|
|2003||F/12||Right superotemporal||+||–||Excision+RT||Negative||Negative||6 mo (alive)|
|2009||M/22||Left lateral||+||Left temporal muscle||Excision+RT+CT||MIC-2||Neurosecretory granules||2 y (died)|
|Current case (2010)||M/6||Right inferotemporal||+||Infratemporal fossa (OD) masseteric space||Excision+RT+CT||PAS||NA||14 mo (died)|