International Journal of Current Research and Review
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IJCRR - 8(20), October, 2016

Pages: 10-16

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CLINICOPATHOLOGICAL AND IMMUNOPHENOTYPIC CHARACTERISTICS OF EWINGS SARCOMA FAMILY OF TUMORS: SPECIAL EMPHASIS ON ROLE OF FRIEND LEUKEMIA INTEGRATION - 1(FLI-1) ANTIBODY AND OCCURRENCE OF TUMOR ON RARE SITES

Author: Alka Mittal, Jayanti Mehta, Kalpana Mangal, Alpana Jain, Neetu Agarwal, Madan Kumar Solanki

Category: Healthcare

Abstract:Objective: Ewings sarcoma /Primitive neuroectodermal tumor (ES/PNET) is one of the aggressive malignant round cell tumors affecting mainly children and adolescents. This study aimed to emphasize the role of FLI-1 antibody along with CD99 in diagnosing ES/PNET and to keep this tumor in differential diagnosis even at some rare sites.
Material and Method: This descriptive and retrospective study was conducted from January 2014 \? May 2016 and included 58 patients of ES/PNET. Histopathological and IHC findings were reviewed and the diagnosis of ES/PNET was confirmed. The tumors were further categorized into classic Ewings, PNET and atypical subtypes. Panel of IHC markers included vimentin, CD99, FLI-1, PanCK, LCA, Desmin, Myogenin, NSE, Synaptophysin, S100 and Chromogranin.
Results: Clinical Findings- We studied 58 patients of ES / PNET of whom 63% (37/58) were males, 37% (21/58) were females. The age ranged between 3- 65 years (mean 20 years).
Histological Findings: 57% of cases (33/58) exhibited classic morphology, 40% (23/58) were classified as PNET. There were only two cases of atypical Ewings sarcoma including large cell and Clear cell variant.
Immunohistochemical Findings: Strong membranous CD99 expression was seen in 100%, FLI -1 nuclear staining in 85% , NSE in 50%, S100 in 30%, Synaptophysin in 10% and EMA in 2% (1/58) cases. All other markers included in the panel were negative.
Tumor on Rare Sites: 10 Cases were of primary extradural spinal PNET, 1 intramedullary spinal, 2 brain parenchyma, 2 nose and nasopharynx , and one rare case of urinary bladder.
Conclusion: Clinical details, histomorphology and IHC includingCD99 and monoclonal FLI-1antibodies may help in supporting diagnosis of EFT if cytogenetic and FISH techniques are not available at any centre.

Keywords: Ewings sarcoma, Primitive neuroectodermal tumor, CD99, FLI-1v

Full Text:

 

Immunostaning for NSE was seen in 50% cases, S100 in 30% cases and Synaptophysin in 10% cases. EMA was focally positive in clear cell variant (Figure 7).

All other markers included in the panel were negative (table 2).

Discussion

This series comprised patients of Ewings sarcoma / PNET of all ages who were enrolled and treated in a tertiary care center, Jaipur, Rajasthan.

There was male preponderance and the mean age was 20 years. The most common sites of involvement were bone of both upper and lower extremities. The patients presented most often with swelling followed by pain, some have fever and limbs weakness which is in accordance with other studies10.

Histomorphogically the incidence of classic type was the most common followed by PNET and atypical variants, which is similar to a study done by Folpe et al11. In our study we had only 2 cases of atypical Ewing’s sarcoma which is quite less as compared to one study12and quite similar to other study done by Priya D et al. in which they found 4 cases of atypical Ewing’s sarcoma out of total 51 cases of EFT studied13.

These entire EFT show strong membranous CD99 Positivity. The differential diagnosis that could be considered for Ewings sarcoma with CD99 positivity included lymphoblastic lymphoma, poorly differentiated synovial sarcoma, small cell osteosarcoma, rhabdomyosacoma, desmoplastic small round cell tumor (DSRCT) small cell carcinoma and merkel cell carcinoma14. As CD-99 has not proven to be specific marker for EFT despite initial promise, we included other quite specific marker for EFT that is FLI-1. FLI-1 was positive in 85% cases including atypical Ewings sarcoma. Nuclear staining was considered positive with positive endothelial cell nuclei and a subset of lymphocytes serving as internal control.

Results are similar to study done by Llombart bosch et al in 2001, they found FLI-1 expression in 84% (16/19) cases of Ewings sarcoma family of tumors out of 48 studied cases of small round cell tumors15.

Our results are slightly higher to study done by Folpe AL in 200016.He studied 132 cases of CD99 positive mimics of Ewing’s  sarcoma/ primitive neuroectodermal tumors  including 41 cases of Ewings sarcoma. He found FLI-1 positivity in 71%  cases of EFT (29/41) and 88% cases of lymphoblastic lymphoma (7/8), rest all were found to be negative. 

 

Introduction

Ewings sarcoma/Primitive neuroectodermal tumor (ES/PNET) is one of the aggressive malignant small round cell tumors that may arise virtually anywhere but is most common in bone, deep soft tissue of extremities and parenchyma.

This tumor was first reported in 1918 by Arthur Purdy stout as ulnar nerve tumor1. Three years later in 1921 James Ewing reported a round cell neoplasm calling it a “diffuse endothelioma of bone “in the radius2.The soft tissue Ewing’s sarcoma was first described in 1975 by Angervall and Enzinger3.Seemayer and colleagues described Peripheral neuroectodermal tumors (PNET) arising in soft tissues at the same time4.In 1979, Askin and coworkers described the malignant small cell tumor of the thoracopulmonary region (Askin tumor) similar to PNET5.

With the advent of IHC, cytogenetic and molecular  techniques it is universally accepted that these tumors shared identical features and these were designated as ES/PNET family of tumors(EFT) 6,7.70-80% of cases show morphology of classic Ewings sarcoma and up to 20% show atypical features including large cell, spindle cell, sclerosing, clear cell, vascular or admantinoma like patterns8. Diagnosis of PNET was considered when homer wright rosettes were seen or when any two different neural markers were positive according to criteria proposed by Schmidt et al9.

A panel of immunomarkers  such as vimentin, CD99, FLI-1, Desmin, Myogenin, Leucocyte common antigen (LCA), cytokeratin (CK) ,Neural markers like non specific enolase (NSE) ,S100,Chromogranin  and Synaptophysin are usually used to distinguish various small round cell tumors.CD99(MIC2) has not proven to be specific for Ewings sarcoma family of tumors despite initial promise. Approximately 90% of EFT have a specific t (11; 22) (q24; q12) that results in fusion of the EWS and FLI-1 protein; hence we included both CD99 and FLI-1 in the panel.

Although molecular testing is gold standard but many centers still do not have this facility, hence IHC plays an important role in differentiating these tumors. The aim of our study was to analyse the clinicopathological and immunophenotypic characteristics of 58 cases of Ewing’s sarcoma/ primitive neuroectodermal tumors from tertiary care centre in Jaipur, Rajasthan with special emphasis on role of FLI-1 protein, and occurrence of tumors at some rare sites.

Material and Method

This descriptive and retrospective study was conducted from January 2014 – May 2016 and included 58 patients of ES/PNET. Clinical details were collected from the case files. Clinical features such as age, sex, site of involvement, radiological findings, and soft tissue extension were evaluated.

The paraffin blocks of all cases were retrieved. Histopathological and immunohistochemical (IHC) findings were reviewed and the diagnosis of ES/PNET was confirmed. The tumors were further categorized into classic Ewings, PNET and atypical subtypes.

Diagnosis of PNET was considered when Homer Wright or Flexner Wintersteiner rosettes were seen or when any two different neural markers were positive. Atypical Ewings sarcoma was labeled when cells were large pleomorphic with coarse chromatin, prominent nucleoli and absent rosettes.

 

Panel of IHC markers included vimentin, CD99, FLI-1, CK, LCA, Desmin, Myogenin, NSE, Synoptophysin, S100, and Chromogranin. Additional IHC markers like glial fibrillary acidic protein (GFAP), INI-1, terminal deoxyneotidyl transferase (Tdt) etc. were also included according to site of lesion and differential diagnosis.

IHC analyses for all above markers were done on 4µ thick section taken on tissue bond coated slides by immunoperoxidase method with respective antibodies as per manufactures instructions. Antigen retrieval was done by the heat induced epitope retrieval method in diva decloaker chamber by using citrate buffer (PH 6.0) after tris buffer wash. Endogenous peroxidase was blocked by hydrogen peroxide solution. Sections were incubated with primary antibody for 1 h 30 min. Biocare’s HRP polymer is applied as secondary antibody and 3’3 diaminobenzidine as chromogen substrate.

Appropriate positive and negative controls were included.

Results

Clinical details                                                                                                          

This retrospective and descriptive study included 58 patients of ES / PNET of whom 63% (37/58) were male, 37 %( 21/58) were female. The age ranged between 3- 65 years (mean age 20 years).

 

Most common site of involvement was skeletal in 61% (35/58), followed by soft tissue in 29% (17/58) and parenchyma of rare sites in 10% (6/58) cases. Rare sites included 10 cases of primary extradural spinal PNET, 1 case of intramedullary spinal, 2 cases of brain parenchyma, one case of nose, nasopharynx and urinary bladder each (Table 1).

The most common bones involved were of upper and lower extremities followed by iliosacral region. The patients presented with local swelling as most common complaint followed by pain, fever, weakness of limbs and pathological fracture.

Localized disease at presentation was seen in 29% (17/58) cases whereas locally advanced disease was seen in 15% (9/58) cases. Soft tissue extension was noted in 74 % ( 20/27) of osseous cases.

Follow up related to recurrence, metastasis and treatment history was available in very few patients hence not included in this study.

Histological Characteristics

57% of cases (33/58) exhibited classic morphology (Figure1). Microscopically tumor shows solidly packed, lobular pattern of strikingly uniform round cells with high N/C ratio. The individual cells have a round to ovoid nucleus with a distinct nuclear membrane, fine chromatin and 2-3 inconspicious nucleoli. The cytoplasm was ill defined scanty, pale, clear and sometime vacuolated. Neoplastic cells were positive for PAS and digested with D-PAS indicating accumulation of glycogen.

Some of the cases showed darker crushed spindled cells. The number of mitotic figures varied from 1 per 10 HPF to 7 per 10 HPF. Most of the cases showed necrosis.

40% (23/58) cases were classified as PNET characterized by presence of rosettes (figure 2). Most of the rosettes contain a central solid core of neurofibrillary material (Homer Wright rosettes) rarely rosettes contain a central lumen or vesicle (Flexner Wintersteiner rosettes).

 There were only two cases of atypical Ewings sarcoma in the present study showing large cell and clear cell variant. Large cell variant was seen in nasopharynx and clear cell variant was noted in spinal cord. In large cell variant, cells were large, pleomorphic with coarse chromatin, prominent nucleoli and absent rosettes (figure 3).Large areas of necrosis were also seen. In clear cell variant, tumor cells had abundant clear cytoplasm with small round cell morphology (figure 4).

Immunohistochemical Findings

All of the cases (58/58) studied showed strong membranous CD99 expression (figure 5) and cytoplasmic Vimentin.

FLI -1 nuclear staining was noticed in 85% cases (49/58) (Figure 6).

 

Previous study done on FLI-1 was by using polyclonal antibody. Rossi et al17used monoclonal antibody and found 100% positivity for FLI-1 in EFT cases(15/15).We also used polyclonal  antibody that is why positivity rates were lower in our study.

NSE was expressed in50% and S100 in 30% which was similar to previous study done by Kavalar et al18, where NSE and S-100 were expressed in 66.6% and 25.4% cases respectively.

We had 10 cases of primary intraspinal PNET at our centre .All of our cases were extradural. A case study done by Sundar Venkataraman found that only 19 cases of primary intraspinal PNETs have been reported to date19.To best of our knowledge we had half of this data at our centre within such a short time duration.

One interesting case at this location was atypical Ewings sarcoma with ciear cell type showing focal EMA positivity.EMA positivity highlights the epithelial differentiation in the EFT. Previously many studies were done on expression of broad spectrum of AE1/AE3 antibody showing positivity rates ranging from 20% to 40% 20,21. Machado et al in 2011 studied 415 genetically confirmed ESFT, for expression of CK, EMA and CEA. They found 19.2 %, 6.6% and 20.8% positive cases for these antibodies respectively22.

We had 2 cases of primary intracranial peripheral PNET (pPNET). The IHC panel included GFAP, EMA, CD-99, FLI-1, CD-34, synaptophysin, INI-1, NSE and MIB-1 score. The cases were positive for CD-99, FL-1and NSE. MIB-1 labeling index was 6% and 45% respectively (Figure 8). PNET at any site show variable but generally high MIB-1 score varying from 0-85%.23 These cases are different from CNS PNET in the clinical behavior, treatment, prognosis, IHC and molecular genetic study.24

We had one case of primary intramedullary spinal PNET in 12 year old male child at D11-L2 label. Bone scan and MRI brain were done to know any other site of primary tumor. Both investigations did not reveal any evidence of tumor elsewhere and a diagnosis of primary intramedullary spinal PNET was made. The diagnosis of such a tumor is very crucial as management strategies are relatively unclear and have a poorer outcome as compared to other primary intramedullary spinal tumors25.

We had one another interesting case of primary PNET of urinary bladder in 31 year old female patient. These are extremely rare and to our knowledge only 12 cases have been reported so far in the English literature26.

We had other two interesting cases of nasal cavity and nasopharynx in40 and 10 year old male patients respectively. It is very rare in head and neck region and accounts for only 2-3% of all the cases27.

Conclusion

Clinical details, histomorphology and IHC including CD99 and monoclonal FLI-1antibodies may help in supporting the diagnosis of EFT even in absence of cytogenetic and FISH techniques. EFT should be kept in differential diagnosis of all malignant small round cell tumours even at rare sites.  

Acknowledgment

Authors wish to express and convey their sincere thanks and gratitude to all those who helped for completion of this research article.

Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to author’s /editors/ publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

Authors acknowledge that we did not get any grants for this study; required funds were generated by ourselves.

The Authors declare and acknowledges that we have no conflict of interest.

 

 

 

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A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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