1. Comparison of genetic, antigenic and clinical features of extra- osseous Ewing Sarcoma (EO- EWS) and osseous Ewing sarcoma (O-EWS) Amanda Rivera-Begeman; Carrye Cost; Stephen Lessnick; Richard Smith; Charles Timmons; Patrick Leavey Annual CTOS Meeting; Seattle, Washington, November 2007

2. Background Ewing's sarcoma family of tumors (EFT) Osseous EWS (O-EWS), Extraosseous ES (EO-EWS), Peripheral primitive neuroectodermal tumor (pPNET) and Askin's tumor of chest wall (Carvajal,R. et al; Hematol Oncol Clin North Am 2005) Cell surface protein MIC-2 (CD99) expressed on both O-EWS and EO-EWS (Ambros, IM; Cancer; 1991) FLI-1 nuclear immunostain + ve in 70% of EWS and PNET cases (Folpe et al; Am J Surg Pathol; 2000)

3. Background EWS/FLI-1 is seen in patients with EFT (O’Sullivan,MJ et al; Hum Pathol; 2001) Prior reports of treatment for EO- EWS demonstrated no advantage to the addition of Doxorubicin (Raney RB et al.; J Clin Oncol; 1997) EO-EWS should be treated with strategies used for O-EWS vs. malignant mesenchymal tumors (Castex,M.P.; J Clin Oncol; 2007)

4. Objective To describe genetic, antigenic, and clinical features of patients with EO- EWS, primarily those of intra- abdominal origin To compare genetic and antigenic features of EO-EWS to those of randomly chosen patients with O- EWS

5. Patients Eligibility criteria EFT treated at Children’s Medical Center Dallas (1995 – 2005; n=52) Availability of archival diagnostic material and clinical data

6. Patients Clinical Characteristic EO-EWS (n=11) O-EWS (n=11) Primary site Abdominal: n=9Pelvic: n=5 Brain: n=1Long Bone: n=5 Nasopharynx: n=1Rib: n=1 Mean tumor volume 1035 cm 3 494 cm 3 Metastases at diagnosis 25 Median age at diagnosis 14.911.4 Radiation 57 Surgery 83 Died 25

7. Methods AE1/AE3 Cytokeratin CAM5.2 CK8 CK7 CEACarcino-embryonic antigen VimentinMesenchymal FLI-1Nuclear stain CD99MIC2 CD56Neuro-ectodermal Synaptophysin Neuronal NSE Chromogranin Myogenin Muscle Desmin All immunostains were independently reviewed by 2 pathologists (CT, ARB) Interpretation was subjective +ve vs. –ve No grading was attempted

8. Fli-1 weak positive Fli-1 strong positive

9. CD99 (O13) Weak positive CD99 (O13) strong positive

10. Results – positive staining ImmunohistochemistryEO-EWSO-EWS CK AE1/AE311 CAM5.210 CK700 CK801 CEA22 Vimentin88 FLI-199 CD9911 Leu-7/CD5648 Synaptophysin44 NSE1110 Chromogranin00 Myogenin00 Desmin00

11. Results – positive staining ImmunohistochemistryEO-EWSO-EWS CK AE1/AE311 CAM5.210 CK700 CK801 CEA22 Vimentin88 FLI-199 CD9911 Leu-7/CD5648 Synaptophysin44 NSE1110 Chromogranin00 Myogenin00 Desmin00

12. Cytokeratin Cam 5.2 positive Carcinoembryonic antigen (CEA) positive

13. Results – positive staining ImmunohistochemistryEO-EWSO-EWS CK AE1/AE311 CAM5.210 CK700 CK801 CEA22 Vimentin88 FLI-199 CD9911 Leu-7/CD5648 Synaptophysin44 NSE1110 Chromogranin00 Myogenin00 Desmin00

14. Results – positive staining ImmunohistochemistryEO-EWSO-EWS CK AE1/AE311 CAM5.210 CK700 CK801 CEA22 Vimentin88 FLI-199 CD9911 Leu-7/CD5648 Synaptophysin44 NSE1110 Chromogranin00 Myogenin00 Desmin00

15. Results – positive staining ImmunohistochemistryEO-EWSO-EWS CK AE1/AE311 CAM5.210 CK700 CK801 CEA22 Vimentin88 FLI-199 CD9911 Leu-7/CD5648 Synaptophysin44 NSE1110 Chromogranin00 Myogenin00 Desmin00

16. Methods RT-PCR RNA extracted from formalin fixed, paraffin embedded tumor (Roche high pure RNA paraffin kit) PCR was performed for EWS/FLI-1 EWS/FLI-1 fusion type identified by melt curve analysis EWS-FLI-1 fusion type confirmed by agarose gel electrophoresis Alternate partners were examined as necessary ( ews/fev, etv1, etv4 and erg)

17. Results – EWS-FLI-1 type EO- EWS O- EWS EWS-FLI-110 Type 1710 Type 230 Non Type 1 or 2 11 Not type 1 or 2 EO-EWS: confirmed t(11:22)(q24;q12) but no translocation products amplified O-EWS: PCR product melting curve between types 1 and 2 but failed sequencing

18. Summary Negative FLI-1 nuclear staining does not exclude EWS-FLI-1 translocation positive EFT While CEA +ve staining can be seen in EO-EWS it does not differentiate this from O-EWS More type 2 fusions noted in patients with EO-EWS

19. Conclusion Variability may occur in immunostaining and genotype analysis of patients with extra-osseous Ewing sarcoma vs. osseous Ewing sarcoma.