Sensitivity and specificity of cluster differentiation and friend leukemia integration1 for the diagnosis in a series of molecularly confirmed ewing sarcoma family of tumors

Sudha S Murthy; Daphne Fonseca; Sundaram Challa; Suseela Kodandapani; Sahithi Shilpa Arya; Sandhya Devi Gundimeda; B Vishal Rao; Faiq Ahmed; Manasi Mundada; Nambaru Lavanya; Senthil J Rajappa; Krishnam Raju Alluri; Veeraiah Koppula; T Subramanyeshwar Rao

doi:10.4103/oji.oji_8_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 5 | Issue : 2 | Page : 55-59

Sensitivity and specificity of cluster differentiation and friend leukemia integration1 for the diagnosis in a series of molecularly confirmed ewing sarcoma family of tumors

Sudha S Murthy¹, Daphne Fonseca², Sundaram Challa², Suseela Kodandapani², Sahithi Shilpa Arya², Sandhya Devi Gundimeda², B Vishal Rao², Faiq Ahmed², Manasi Mundada², Nambaru Lavanya², Senthil J Rajappa³, Krishnam Raju Alluri⁴, Veeraiah Koppula⁵, T Subramanyeshwar Rao⁶
¹ Datar Cancer Genetics, Nashik, Maharashtra; Department of Laboratory Medicine, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
² Department of Laboratory Medicine, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
³ Department of Medical Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
⁴ Department of Radiation Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
⁵ Department of Radiology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
⁶ Department of Surgical Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India

Date of Submission	10-Feb-2021
Date of Decision	27-May-2021
Date of Acceptance	06-Jun-2021
Date of Web Publication	21-Aug-2021

Correspondence Address:
Daphne Fonseca
Department of Laboratory Medicine, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad - 500 034, Telangana
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/oji.oji_8_21

Abstract

Background: Immunohistochemistry (IHC) is a cost-effective and routinely available ancillary technique for the diagnosis of Ewing sarcoma family of tumors (ESFT). However, molecular confirmation is needed for precise diagnosis. Aim: This study aimed to determine the sensitivity and specificity of the commonly used IHC markers cluster differentiation (CD99) and friend leukemia integration1 (FLI1) in a series of molecularly confirmed ESFT. Materials and Methods: Retrospective review of the ESFT confirmed by either fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) during a period of 10 years was done. The demographic, clinical, and radiologic data were noted from medical records. The histology was reviewed with CD99, FLI1, and additional markers, wherever performed. The sensitivity and specificity of CD99 and FLI1 for the diagnosis of ESFT were calculated. Results: There were 72 ESFT patients in the study period, confirmed by FISH (EWSR1 rearrangement) in 53 and RT-PCR (EWS-FLI1) in 19. The female-to-male ratio was 1.06. The median age at diagnosis was 21 years. The cases included 22 skeletal and 50 extraskeletal sites. The positivity of CD99 and FLI1 was 98.46% and 94.83%, respectively, and both were positive in 55/72 (76.39%) cases. The sensitivity and specificity of CD99 were 98.46% and 20%, and those of FLI1 were 94.83% and 28.57%, respectively. Conclusion: Although the sensitivity for CD99 and FLI1 was high, the specificity was low toward the diagnosis of ESFT. The combined use of CD99 and FLI1 could confirm only 76.39% of molecularly confirmed ESFT, emphasizing the need for a precise diagnosis by molecular technique.

Keywords: Ewing sarcoma family of tumors, Immunohistochemistry, Molecular diagnosis

How to cite this article:
Murthy SS, Fonseca D, Challa S, Kodandapani S, Arya SS, Gundimeda SD, Rao B V, Ahmed F, Mundada M, Lavanya N, Rajappa SJ, Alluri KR, Koppula V, Rao T S. Sensitivity and specificity of cluster differentiation and friend leukemia integration1 for the diagnosis in a series of molecularly confirmed ewing sarcoma family of tumors. Oncol J India 2021;5:55-9

How to cite this URL:
Murthy SS, Fonseca D, Challa S, Kodandapani S, Arya SS, Gundimeda SD, Rao B V, Ahmed F, Mundada M, Lavanya N, Rajappa SJ, Alluri KR, Koppula V, Rao T S. Sensitivity and specificity of cluster differentiation and friend leukemia integration1 for the diagnosis in a series of molecularly confirmed ewing sarcoma family of tumors. Oncol J India [serial online] 2021 [cited 2023 Jun 2];5:55-9. Available from: https://www.ojionline.org/text.asp?2021/5/2/55/324235

Introduction

The Ewing sarcoma family of tumors (ESFT) encompass all the previous terminologies such as Ewing sarcoma (ES) of bone, extraskeletal ES, peripheral primitive neuroectodermal tumor, Askin's tumor of thoracopulmonary region, and atypical ES based on the presence of a unifying pathognomonic chromosomal translocation.^{[1],[2],[3],[4],[5],[6],[7],[8],[9]} These tumors involve the bones, soft tissues, and viscera. The age, site, and morphology of these tumors are mimicked by many other tumors such as lymphoblastic lymphoma, rhabdomyosarcoma, neuroblastoma, poorly differentiated synovial sarcoma, desmoplastic small round cell tumor (DSRCT), and others.^{[1],[4],[5],[6],[7],[9],[10],[11],[12]} The treatment and prognosis of these various tumors are different, necessitating accurate diagnosis. The diagnosis is challenging, especially in adults and in extraskeletal or visceral location. The diagnosis is based on clinical, morphologic, immunohistochemical, and molecular features.

About 60%–75% ESFTs have classical morphology,^[7],[13] and others may show variant morphology.^[8] Immunohistochemistry (IHC) is a cost-effective ancillary technique that can be easily applied for the routine diagnosis. Introduction of newer and more specific antibodies has widened the scope of IHC. ESFTs characteristically are positive for cluster differentiation (CD99), friend leukemia integration1 (FLI1), and caveolin1. Diffuse membranous expression of CD99 is characteristic for ES but not specific. Nuclear FLI1 expression is seen in most ES cases with the t (11;22)(q24; q12) translocation, whereas ERG immunoreactivity is seen in a small subset of cases harboring the t (21;22)(q22; q12) translocation.^[9] In addition, these tumors may express synaptophysin, chromogranin, S-100, pan cytokeratin (PCK), desmin, and other markers variably. Although IHC is useful and sensitive to identify the morphological subgroups and differentiate from mimics, the specificity is low and the immunophenotype is shared by many other tumors.^{[5],[7],[8],[11]} As the specificity is low, molecular confirmation of histologic diagnosis may become necessary for a definite diagnosis, which is clinically important.^[5],[8],[12] Molecular studies may involve the demonstration of chimeric fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) or gene rearrangement by fluorescence in situ hybridization (FISH).

CD99 and FLI1 are used routinely for the diagnosis of ESFT. The present study aims to evaluate the sensitivity and specificity of CD99 and FLI1 in a series of molecularly confirmed ESFT for their routine use in surgical pathology, highlighting the advantages and limitations.

Materials and Methods

All patients with molecularly confirmed ESFT either by FISH or RT-PCR, during the period September 2009 and August 2019 were included in the study. The demographic, clinical, and radiological findings were noted from the medical records. The biopsies and resected specimens were fixed in 10% buffered formalin and processed for paraffin sections. The sections were stained with hematoxylin and eosin. The morphology was reviewed, and architectural details, cellularity, atypia, and mitoses; areas of necrosis and differentiation, if any were noted [Figure 1]a. Depending on the age, site, and morphology, a panel of IHC markers was performed including CD99, FLI1, PCK, leucocyte common antigen (LCA), Tdt, desmin, B-cell lymphoma 2 (Bcl2), TLE1, WT1, CD56, chromogranin, synaptophysin, vimentin, and others as appropriate.

Figure 1: (a) H and E: Section shows fibrous tissue with infiltrating tumor arranged in sheets, inset H and E, ×400: Section shows sheets of round cells with scanty cytoplasm and hyperchromatic nucleus, (b) cluster differentiation, ×100: shows intense membrane positivity, (c) friend leukemia integration 1, ×100: shows strong nuclear positivity, (d) flourescence in situ hybridization image shows EWSR1 rearrangement in 82% of the cells

Click here to view

Ten cases that were evaluated with a clinical suspicion of ESFT but were proven to be other types of tumors by IHC, FISH, and/or RT-PCR were included as control. These included three each of lymphoma and synovial sarcoma and two each of DSRCT and angiomatoid fibrous histiocytoma. CD99 was performed in all and FLI1 in seven cases, in addition to other IHC markers.

Statistics

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CD99 and FLI1 in the diagnosis of ESFT were calculated.

Immunohistochemistry procedures

As per the protocol for automated immunostainer (Roche Ventana Benchmark XT), IHC was performed on formalin-fixed paraffin embedded (FFPE) tissues. Antibodies used were cluster of differentiation 99 (CD99) (12E7Dako; ready to use [RTU]), Friend leukemia integration (FLI1) (MRQ1; Cell marquee, Rocklin, CA, USA, 1:50). For CD99 and FLI1, weak/moderate/strong staining was considered positive staining and equivocal staining as negative staining. Diffuse membranous staining was considered positive for CD99 [Figure 1]b and nuclear positivity for FLI1 [Figure 1]c. Focal staining in the tumor cells was reported as focal positivity, no staining as negative; endothelial cells, and lymphocytes were used as internal control.

Fluorescence in situ hybridization procedures

FISH test was performed on FFPE sections, using locus-specific Vysis dual color break-apart probe for EWSRI gene located at 22q12 (Vysis, Inc., Downer s Grove, IL, USA), and the results for EWSR1 gene rearrangement were reviewed. Break in EWSR1 gene was considered to be positive for EWSR1 gene rearrangement [Figure 1]d with the established cutoff of 15% after viewing hybridization signals in 50 nonoverlapping nuclei. Typical break-apart signal pattern of one fusion and separated 3′ orange (centromeric) and 5′ green (telomeric) was noted in 36% and atypical signal pattern was seen in 20% each with deletion of 3′ centromeric orange signal (1 fusion and one 5′ green) and another clone showing deletion of 5′ (telomeric) green signal. Additional clone also demonstrates 3' and 5' orange and green signals connoting loss of normal copy of EWSR1 gene.

Reverse transcriptase polymerase chain reaction procedures

RT-PCR test was performed either in-house or outsourced. Total RNA from FFPE tissue, using Recover All Total Nucleic Acid Isolation kit (Invitrogen by Thermo Scientific, Baltics UAB, Lithuana) was extracted. As per manufacturer's protocol, 1 μg of RNA was reverse transcribed to cDNA using TRUPCR High Retro Transcriptase Starter kit (3B BlackBiotech India Ltd, Bhopal, India). 2% agarose gel was used to check the amplicons. EWS-FLI1 was subtyped as Type 1, EWS (exon 7) with FLI1 (exon6) of 330 bp (base pairs) and Type II, and EWS (exon7) and FLI1 (exon5) of 390 bp. Sequencing of the PCR products was performed at Bio-serve on 3500Dx Genetic Analyzer. Bio-edit software program (http://www.mbio.ncsu.edu/Bioedit/bioedit.html) was used to identify sequences.

Results

A total of 72 patients evaluated during the study period. The basic characteristics of the patient profile are mentioned in [Table 1]. Thirty-five patients were males and 37 were females with the age ranging from 7 months to 65 years. The median age of presentation was 21 years. Site of tumor origin was skeletal in 22 patients and extraskeletal in 50 patients inclusive of 15 in visceral organs. [Figure 1] shows the histopathological findings, CD99 and FLI1 marker positivity, and EWSR1 gene rearrangement in FISH technique. EWSR1 gene rearrangement was confirmed by FISH in 53 patients, and EWS-FLI1 fusion transcript was noted by RT-PCR in 19 patients. Bulk of the specimens was core biopsies, and the resection specimens included were hysterectomy, nephrectomy, and excision samples from soft tissues. All the tumors showed similar histology comprising of small round cells with foci of necrosis and mitoses [Figure 1]a. There were no rosettes or spindle cells or significant stromal changes.

Table 1: Basic characteristics of the patient profile

Click here to view

The different immunohistochemical markers performed in molecularly diagnosed ESFT patients are mentioned in [Table 2]. CD99 was performed on 65/72 cases and FLI1 on 58/72 cases. CD99 showed diffuse strong positivity [Figure 1]b in 62 with focal positivity in 2. FLI1 showed strong nuclear positivity [Figure 1]c in 47 with focal positivity in 8. One case was negative for CD99, and three cases were negative for FLI1 in molecularly confirmed cases. The positivity of CD99 and FLI1 was 98.46% and 94.83%, respectively, and both were positive in 55/72 (76.39%) cases. Both the markers were 100% positive in RT-PCR confirmed cases, whereas the positivity was 97.83% for CD99 and 92.31% for FLI1 in FISH positive cases. Vimentin was positive in all, and LCA was negative in all cases tested. PCK and Bcl2 both were positive in 16.67% of cases.

Table 2: Positivity of immunohistochemistry markers in molecularly confirmed Ewing sarcoma family of tumors

Click here to view

Among other specific tumors (control group: n = 10), CD99 positivity was found in 8 out of 10 patients including focal positivity in 2. FLI1 was performed in seven, and it was positive in five including focal positivity in three.

The sensitivity, specificity, PPV, and NPV of CD99 were 98.46%, 20%, 88.89%, and 66.67%, respectively. The sensitivity, specificity, PPV, and NPV of FLI1 were 94.83%, 28.57%, 91.67%, and 40%, respectively. Diagnostic accuracy for CD99 was 88.00% and for FLI1 was 87.69% [Table 3].

Table 3: Efficacy of cluster differentiation 99 and friend leukemia integration 1 markers

Click here to view

Discussion

IHC is an economical and routine laboratory technique that finds application in cancer diagnostics including diagnosis of ESFT. Molecular studies allow a precise diagnosis and not only confirm the histologic diagnosis but also distinguish ESFT from the morphological mimics.^[5],[8],[12] However, FISH and RT-PCR may not be available or too expensive for routine use and more than 10% of cases may not yield good quality DNA or RNA.^[12],[14] In addition, there may be several limitations in technique and interpretation too.^[15],[16]

Soft-tissue sarcomas are a rare entity; hence, their diagnosis and management are a challenge. A comprehensive review with judicious use of IHC and molecular confirmation of diagnosis is needed for accurate management.

ESFT shows expression of CD99, FLI1, and markers of epithelial, mesenchymal, and neural differentiation. CD99 also known as MIC2 is a membrane-associated glycoprotein that is highly expressed in most cases of ESFT.^[10] It appears to prevent terminal neural differentiation and contributes to cell proliferation, migration, and metastasis of ESFT cells.^[17] CD99 shows high sensitivity for ESFT, and the sensitivity ranges from 90 to 100%.^[18] However, CD99 alone is unreliable to definitively diagnose ESFT as it also shows high expression in morphological mimics such as CIC- and BCOR-rearranged sarcomas, as well as in certain lymphoma subtypes and poorly differentiated synovial sarcoma.^{[12],[16],[19],[20],[21]}

FLI1 is a member of the ETS (erythroblastosis virus-associated transforming sequences) family of DNA-binding transcription factors, and it is involved in cellular proliferation and tumorigenesis.^[22] FLI1 was reported to be highly sensitive marker of ESFT and the sensitivity of FLI1 for ESFT ranges from 71% to 94%.^{[7],[18],[23]} However, FLI1 is also expressed in other tumors such as vascular tumors, lymphomas, synovial sarcomas, rhabdomyosarcomas, melanomas, Merkel cell carcinomas, and carcinomas of lung and breast.^{[7],[13],[18]} The combination of CD99 and FLI1 was suggested to improve the specificity of diagnosis for ESFT.^[23]

Rekhi et al. reported 92.3% and 94.4% diffuse positivity for CD99 and FLI1, respectively, in 58 molecular confirmed cases.^[18] The authors observed that a diagnosis of ESFT in a malignant round cell sarcoma with a classic histology shows diffuse membranous positivity for CD99, intranuclear FLI1 positivity, and LCA negativity.^[18] In the present study of 72 molecularly confirmed ESFT, the positivity of CD99 and FLI1 was 98.46% and 94.83%, respectively, along with negativity for LCA (wherever tested), similar to the earlier studies.^{[8],[18],[24]} In the present study, presence of both CD99 and FLI1 positivity was noted in 76.39% and these findings were similar to 76% and 73.08% by Gamberi et al. and Jambhekar et al., respectively.^[5],[24]

Gamberi et al. in a series of 156 ESFT observed that 76% of cases showed moderate to strong CD99, FLI1, and/or caveolin antibody positivity.^[5] These antibodies were negative in 24%, and the diagnosis was established by EWSR1 translocation. In the present study, CD99 was diffuse membranous positive in 62, focal positive in 2, and was negative in one. FLI1 was strong nuclear positive in 47, focal positive in 8, and was negative in three. CD99 negative ESFT, though small in number, was reported earlier.^[13],[24]

ESFT expresses epithelial, mesenchymal, and neural differentiation. In the present study, vimentin was positive in all and LCA was negative in all cases tested. PCK and Bcl2 both were positive in 16.67% of cases. Desmin was negative in the cases tested. These results were in agreement with earlier studies.^{[18],[25],[26],[27]}

The present study highlights the usefulness of IHC in the routine diagnosis of ESFT. Although the sensitivity for both markers was high (98.46% for CD99 and 94.83% for FLI1), the specificity was low (20% and 28.57%, respectively). The combined use of CD99 and FLI1 could confirm only 76.39% of molecularly confirmed ESFT, emphasizing the need for further confirmation by other orthogonal tests.^[28] Equivocal IHC results and uncommon sites should prompt molecular testing for diagnosis.^[29] Other IHC markers with good sensitivity and specificity include caveolin, NKX2.2, and protein kinase PKC-ß.^[30],[31] However, these were not performed in the present study due to nonavailability in the study period and the retrospective nature of the study. Newer IHC markers ATP1A1, BCL11B, and GLG1 constitute potential specific markers for ES with 96% specificity and extremely effective at discriminating ES from Ewing-like sarcomas.^[16]

Conclusion

The present study emphasizes the sensitivity and specificity of the commonly used IHC markers CD99 and FLI1 for the diagnosis of ESFT which had confirmation by FISH or RT-PCR. It highlights the importance of use of both IHC and molecular characterization in genetically and biologically diverse patients from the Southern part of India.

Acknowledgment

If any. We would like to thank the technical team of Histopathology, IHC, FISH and Molecular pathology, including U Ravinder, D. Vijaya, D Ramanarasamma, Khaja Akther Hussain, Ms. M. Padma Mr. K. Ramachander Reddy, and Mrs K M Swarnalata, for rendering technical support in all areas of services.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

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.
2.	Khoury JD. Ewing sarcoma family of tumors. Adv Anat Pathol 2005;12:212-20.
3.	Sand LG, Szuhai K, Hogendoorn PC. Sequencing overview of Ewing sarcoma: A journey across genomic, epigenomic and transcriptomic landscapes. Int J Mol Sci 2015;16:16176-215.
4.	De Alava E, Lessnick SL, Sorensen PH. In: Fletcher CD, Bridge JA, Hogendoorn PC, Mertens F, editors. Ewing sarcoma in WHO Classification of Tumours of Soft tissue and Bone. 4^th ed. Lyon, France: IARC; 2013. p. 306-9.
5.	Gamberi G, Cocchi S, Benini S, Magagnoli G, Morandi L, Kreshak J, et al. Molecular diagnosis in Ewing family tumors: The Rizzoli experience – 222 consecutive cases in four years. J Mol Diagn 2011;13:313-24.
6.	O'Sullivan MJ, Perlman EJ, Furman J, Humphrey PA, Dehner LP, Pfeifer JD. Visceral primitive peripheral neuroectodermal tumors: A clinicopathologic and molecular study. Hum Pathol 2001;32:1109-15.
7.	Folpe AL, Hill CE, Parham DM, O'Shea PA, Weiss SW. Immunohistochemical detection of FLI-1 protein expression: A study of 132 round cell tumors with emphasis on CD99-positive mimics of Ewing's sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol 2000;24:1657-62.
8.	Pinto A, Dickman P, Parham D. Pathobiologic markers of the ewing sarcoma family of tumors: State of the art and prediction of behaviour. Sarcoma 2011;2011:856190.
9.	Wei S, Siegal GP. Round cell tumors of bone: An update on recent molecular genetic advances. Adv Anat Pathol 2014;21:359-72.
10.	Kovar H, Dworzak M, Strehl S, Schnell E, Ambros IM, Ambros PF, et al. Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. Oncogene 1990;5:1067-70.
11.	Noujaim J, Jones RL, Swansbury J, Gonzalez D, Benson C, Judson I, et al. The spectrum of EWSR1-rearranged neoplasms at a tertiary sarcoma centre; assessing 772 tumour specimens and the value of current ancillary molecular diagnostic modalities. Br J Cancer 2017;116:669-78.
12.	Machado I, Navarro L, Pellin A, Navarro S, Agaimy A, Tardío JC, et al. Defining Ewing and Ewing-like small round cell tumors (SRCT): The need for molecular techniques in their categorization and differential diagnosis. A study of 200 cases. Ann Diagn Pathol 2016;22:25-32.
13.	Llombart-Bosch A, Machado I, Navarro S, Bertoni F, Bacchini P, Alberghini M, et al. Histological heterogeneity of Ewing's sarcoma/PNET: An immunohistochemical analysis of 415 genetically confirmed cases with clinical support. Virchows Arch 2009;455:397-411.
14.	Machado I, Noguera R, Pellin A, Lopez-Guerrero JA, Piqueras M, Navarro S, et al. Molecular diagnosis of Ewing sarcoma family of tumors: A comparative analysis of 560 cases with FISH and RT-PCR. Diagn Mol Pathol 2009;18:189-99.
15.	Papp G, Mihály D, Sápi Z. Unusual signal patterns of break-apart FISH probes used in the diagnosis of soft tissue sarcomas. Pathol Oncol Res 2017;23:863-71.
16.	Baldauf MC, Orth MF, Dallmayer M, Marchetto A, Gerke JS, Rubio RA, et al. Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets. Oncotarget 2018;9:1587-601.
17.	Rocchi A, Manara MC, Sciandra M, Zambelli D, Nardi F, Nicoletti G, et al. CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. J Clin Invest 2010;120:668-80.
18.	Rekhi B, Vogel U, Basak R, Desai SB, Jambhekar NA. Clinicopathological and molecular spectrum of ewing sarcomas/PNETs, including validation of EWSR1 rearrangement by conventional and array FISH technique in certain cases. Pathol Oncol Res 2014;20:503-16.
19.	Doyle LA. Sarcoma classification: An update based on the 2013 World Health Organization Classification of Tumors of Soft Tissue and Bone. Cancer 2014;120:1763-74.
20.	Peter M, Gilbert E, Delattre O. A multiplex real-time pcr assay for the detection of gene fusions observed in solid tumors. Lab Invest 2001;81:905-12.
21.	Olsen SH, Thomas DG, Lucas DR. Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol 2006;19:659-68.
22.	Sharrocks AD, Brown AL, Ling Y, Yates PR. The ETS-domain transcription factor family. Int J Biochem Cell Biol 1997;29:1371-87.
23.	Llombart-Bosch A, Navarro S. Immunohistochemical detection of EWS and FLI-1 proteinss in Ewing sarcoma and primitive neuroectodermal tumors: Comparative analysis with CD99 (MIC-2) expression. Appl Immunohistochem Mol Morphol 2001;9:255-60.
24.	Jambhekar NA, Bagwan IN, Ghule P, Shet TM, Chinoy RF, Agarwal S, et al. Comparative analysis of routine histology, immunohistochemistry, reverse transcriptase polymerase chain reaction, and fluorescence in situ hybridization in diagnosis of Ewing family of tumors. Arch Pathol Lab Med 2006;130:1813-8.
25.	Schuetz AN, Rubin BP, Goldblum JR, Shehata B, Weiss SW, Liu W, et al. Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: Additional evidence of epithelial differentiation. Mod Pathol 2005;18:1403-10.
26.	Kavalar R, Pohar Marinsek Z, Jereb B, Cagran B, Golouh R. Prognostic value of immunohistochemistry in the Ewing's sarcoma family of tumors. Med Sci Monit 2009;15:R442-52.
27.	Rossi S, Orvieto E, Furlanetto A, Laurino L, Ninfo V, Dei Tos AP. Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody. Mod Pathol 2004;17:547-52.
28.	Murthy SS, Gundimeda SD, Challa S, Manjula V, Fonseca D, Rao VB, et al. FISH for EWSR1 in Ewing's sarcoma family of tumors: Experience from a tertiary care cancer center. Indian J Pathol Microbiol 2021;64:96-101. [PUBMED] [Full text]
29.	Murthy SS, Challa S, Raju K, Rajappa SJ, Fonseca D, Gundimeda SD, et al. Ewing sarcoma with emphasis on extra-skeletal Ewing sarcoma: A decade's experience from a single centre in India. Clin Pathol 2020;13:1-10.
30.	Yoshida A, Sekine S, Tsuta K, Fukayama M, Furuta K, Tsuda H. NKX2.2 is a useful immunohistochemical marker for Ewing sarcoma. Am J Surg Pathol 2012;36:993-9.
31.	Surdez D, Benetkiewicz M, Perrin V, Han ZY, Pierron G, Ballet S, et al. Targeting the EWSR1-FLI1 oncogene-induced protein kinase PKC-β abolishes ewing sarcoma growth. Cancer Res 2012;72:4494-503.