Journal of Cytology
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CASE REPORT  
Year : 2014  |  Volume : 31  |  Issue : 1  |  Page : 44-46
Cytodiagnosis of metastatic Ewing's sarcoma of orbital mass and its confirmation by demonstration of EWS/friend leukemia integration 1 fusion gene


1 Department of Pathology, S. C. B. Medical College, Cuttack, Odisha, India
2 Department of Oncopathology, Acharya Harihar Regional Cancer Center, Cuttack, Odisha, India

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Date of Web Publication15-Apr-2014
 

   Abstract 

Ewing's sarcoma (EWS) is an undifferentiated sarcoma of bone. Its morphologic appearance resembles many other malignant small round cell tumors. Due to the morphologic overlap, there is diagnostic difficulty and for accurate diagnosis, requires special studies such as immunohistochemistry, electron microscopy, cytogenetics, and molecular genetic analysis. We report a case of metastatic EWS from orbital mass in a 14-year-old female child diagnosed by cytology after clinicopathologic evaluation. She presented with low back ache of 1 year followed by proptosis of the right eye and swelling of the right side chest wall. Cytosmear and Tru-cut biopsy was taken from the orbital mass showed features of EWS. It was confirmed later by further studies including demonstration of EWS/friend leukemia integration-1 fusion gene by molecular genetic analysis.

Keywords: Cytology; Ewing′s sarcoma; Ewing′s sarcoma/friend leukaemia integration-1 fusion gene; molecular genetics

How to cite this article:
Kar A, Das U, Parija NC, Rout N. Cytodiagnosis of metastatic Ewing's sarcoma of orbital mass and its confirmation by demonstration of EWS/friend leukemia integration 1 fusion gene. J Cytol 2014;31:44-6

How to cite this URL:
Kar A, Das U, Parija NC, Rout N. Cytodiagnosis of metastatic Ewing's sarcoma of orbital mass and its confirmation by demonstration of EWS/friend leukemia integration 1 fusion gene. J Cytol [serial online] 2014 [cited 2020 Aug 7];31:44-6. Available from: http://www.jcytol.org/text.asp?2014/31/1/44/130700



   Introduction Top


Ewing's sarcoma (EWS) is an aggressive osteolytic tumor of bone and rapidly disseminates to other sites. It is the second most common malignant bone tumor in children and young adults. [1] It shares the histological features with other malignant small round cell tumors (MSRCT). Therefore the defining cytogenetic abnormality is a balanced translocation t(11;22) expressing the EWS/friend leukemia integration-1 (FLI-1) chimeric fusion protein. Metastatic EWS should be diagnosed early as it is difficult to control and treatment needs a multimodality approach constituting chemotherapy, surgery and radiotherapy.


   Case Report Top


The case we present here is a 14-year-old female child who presented with low backache since 1 year followed by right eye proptosis for 4 months and swelling over right side chest wall since 3 months [Figure 1]a. Her routine and biochemical investigations were within normal limits. Tests for HIV, hepatitis C virus and HBsAg were negative. Her chest radiograph was normal. Magnetic resonance imaging revealed involvement of posterior element of the sacrum with L5 vertebra and a presacral soft tissue mass measuring 11.8 cm × 7.5 cm. Large abdominal soft tissue mass in pancreatic region measuring 6.2 cm × 4.8 cm was seen indicating a possibility of nodal involvement. Venous invasion with tumor thrombus in left internal and common iliac veins and lower inferior vena cava was noticed. Bone scan revealed multiple skeletal deposits. Fine needle aspiration smears from orbital mass were highly cellular showing cells arranged both in clusters and singly dispersed [Figure 1]c. Cells were both light and dark types with a thin rim of pale cytoplasm and small round nuclei having small inconspicuous nucleoli. Rosette like structures, but no true rosettes were seen. Considering the clinical presentation, a provisional diagnosis of MSRCT possibly EWS was given. Two Tru-cut biopsy samples were taken from the same mass. From one sample, paraffin block was prepared.
Figure 1: (a) Pa.. ent with right orbital mass and chest wall mass. (b) Photograph of patient after chemotherapy. (c) Cytology of orbital mass (H and E ×100)

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Histologically the tumor cells were arranged in irregular nests separated by dense fibrous stroma [Figure 2]a. The cells were 1-2 times larger than small lymphocytes with scant eosinophilic or clear cytoplasm, centrally placed round nuclei with finely distributed chromatin and inconspicuous nucleoli [Figure 2]b. Mitotic figures were few; necrosis scanty and rosettes were not seen indicating the absence of neural differentiation. Periodic-acid Schiff (PAS) stain was positive demonstrating glycogen in cytoplasm. The paraffin block was subjected to CD99 immunohistochemical marker and was strongly and diffusely positive [Figure 2]c. Other sample was preserved in Trizol reagent for molecular genetic analysis to look for EWS-FLI-1 translocation. Total ribonucleic acid (RNA) was extracted from snap frozen tumor tissue using Trizol reagent. Reverse transcription-polymerase chain reaction (RT-PCR) was performed with ABL oligonucleotide primer to check for the quality of the RNA isolated, which amplified a 300 bp product. [2] The EWS-FLI-1 product was type 1 fusion (330 bp) [Figure 2]d.
Figure 2: (a) Photomicrograph showing small round cells in tissue section (H and E ×100). (b) Under higher magnification, (H and E ×400). (c) Diffuse and strong positivity with CD99 (IHC, x400). (d) Reverse transcrip�� onpolymerase chain reaction analysis of Ewing's sarcoma

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   Discussion Top


EWS is a primary nonosteogenic malignant tumor of bone with peak incidence in second decade of life. [3] It affects long bones and flat bones of pelvis, skull, and vertebrae. We report the above case in a 14-year-old girl with involvement of L5 vertebra and sacral bone with metastasis to multiple sites at the time of presentation.

Early and correct diagnosis of EWS is essential for clinical management since it belongs to the group of primary MSRCT. This group includes neuroblastoma, lymphoma, desmoplastic small round cell tumor and rhabdomyosarcoma besides EWS and peripheral neuroectodermal tumor where neural differentiation is seen in addition to all the features of EWS. [4] Since the morphologic features in all these tumors are more or less similar, one takes the help of special stains in addition to other ancillary techniques for differentiation of one from the other. PAS stain detects the presence of large amounts of intracellular glycogen, but it is a nonspecific finding since many childhood tumors contain it and up to 35% of EWS may not contain detectable glycogen. CD99 is a useful marker in the differential diagnosis of these tumors as 90% of EWS/peripheral neuroectodermal tumors stain positive. Though neuroblastoma, the main tumor in the differential diagnosis is not positive for CD99, it is seen to be positive in other malignant small cell tumors such as small cell osteosarcoma, lymphoblastic lymphoma, mesenchymal chondrosarcoma, and alveolar rhabdomyosarcoma. Therefore one has to go for chromosomal analysis for final diagnosis. The balanced t(11:22)(q24:q12) translocation is a genotypic marker. The chromosome 22q12 breakpoints are clustered within a single gene designated EWS and chromosome 11q24 are within a gene called FLI-1. This translocation results in the expression of an aberrant hybrid protein in which the N-terminal part of EWS is linked to deoxyribonucleic acid binding domain (Ets domain) of the FLI-1 transcription factor. The EWS-FLI-1 protein is thought to be responsible for the origin of EWS. [5] Kelleher and Thomas [6] have suggested some promising therapeutics like EWS-FLI-1 gene silencing, growth factor-I receptor antagonists, mammalian target of rapamycin inhibition, KIT oncoprotein targeting, etc.Although cytogenetic analysis can detect a wide variety of chromosomal translocations, it is time consuming, technically difficult and success rate is variable. Hence, we used the reverse transcription followed by PCR assay for detecting the presence and type of EWS/FLI-1 fusion transcript. We got a 330 bp indicating type 1 fusion in the present case. The specific genetic alterations among other small round cell tumors can establish the genotypic diagnosis of these tumors. The most common site to which EWS spreads, or metastasizes, is the lungs. Metastatic Ewing's sarcoma is typically difficult to control, though patients with lung metastases have a better prognosis than patients with other distant metastases. Following diagnosis by Tru-cut biopsy and CD99 immunopositivity (which was confirmed by RT-PCR analysis), our patient received three cycles of chemotherapy and no radiotherapy. After one cycle of chemotherapy, the right eye proptosis and swelling over the chest wall disappeared [Figure 1]b. However after receiving next two cycles, she became bedridden and before receiving the 4 th course of chemotherapy, the patient expired (within 1 year of diagnosis). This case exemplifies the idea that every new lesion in a patient with EWS should be considered as a possible metastasis.

 
   References Top

1.Delattre O, Zucman J, Melot T, Garau XS, Zucker JM, Lenoir GM, et al. The Ewing family of tumors - A subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med 1994;331:294-9.  Back to cited text no. 1
    
2.Parija T, Shirley S, Uma S, Rajalekshmy KR, Ayyappan S, Rajkumar T. Type 1 (11;22)(q24:q12) translocation is common in Ewing′s sarcoma/peripheral neuroectodermal tumour in South Indian patients. J Biosci 2005;30:371-6.  Back to cited text no. 2
    
3.de Alava E, Kawai A, Healey JH, Fligman I, Meyers PA, Huvos AG, et al. EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing′s sarcoma. J Clin Oncol 1998;16:1248-55.  Back to cited text no. 3
    
4.Asif N, Khan AQ, Siddiqui YS, Mustafa H. Metastasis from scapular Ewing′s sarcoma presenting as sutural diastasis: An unusual presentation. Int J Shoulder Surg 2010;4:18-21.  Back to cited text no. 4
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5.Ross KA, Smyth NA, Murawski CD, Kennedy JG. The biology of Ewing sarcoma. ISRN Oncol 2013;2013:759725.  Back to cited text no. 5
    
6.Kelleher FC, Thomas DM. Molecular pathogenesis and targeted therapeutics in Ewing sarcoma/primitive neuroectodermal tumours. Clin Sarcoma Res 2012;2:6.  Back to cited text no. 6
    

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Correspondence Address:
Asaranti Kar
Qrs. No. JO - 1, S. C. B. Medical Campus, Cuttack - 753 007, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9371.130700

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