1. Maureen O’Sullivan 1,2 *, Tony Ng 1 *, Torsten Nielsen 1, Malcolm Hayes 1,3, Paul Clarkson 4, Catherine Pallen 5, Poul Sorensen 1,6, Doug Horsman 1,3 1 Department of Pathology and Laboratory Medicine, University of British Columbia, 2 Division of Anatomic Pathology, British Columbia’s Children’s Hospital, 3 Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, 4 Department of Surgery, UBC 5 Department of Pediatrics, Child and Family Research Institute, UBC, 6 Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, Canada *These authors contributed equally Novel translocation t(2;16)(q35;p11) producing an in-frame fusion of FUS and FEV in a Ewing sarcoma lacking EWS rearrangement

2. Molecular Pathology of Ewing Family Tumours Ancillary diagnostic and potentially prognostic value Typically, EWS (member of TET family) rearranged with any one of a number of Ets family members: Fli1, Erg, ETV1, ETV4, FEV. Recent report of additional TET family member FUS rearranged with Erg in 4 cases of EFT.

3. Case History 33 year-old man sustains pathologic fracture while snowboarding Plain film images show moth-eaten distal clavicle Incisional biopsy inadequate, but with suspicious population of small blue cells Excisional biopsy = EFT Patient treated with 11 courses of chemo, irradiation and then surgical excision = negative margins and 100% tumour kill.

5. Pathology

6. Cytogenetics

7. t(2;16)(q35;p11)

8. FISH with FUS Break-apart Probe

9. FISH with BAC RP11-207M4

11. RT-PCR, Cloning and Sequencing Primers : Forward - FUS exon 1; Reverse - FEV exon 3 Product of 1.4Kb on electrophoresis Cloned and sequenced

12. RT-PCR, cloning and sequencing FEV Exon 2 FUS Exon 10

13. In-frame FUS exon 10 to FEV exon 2 fusion Novel Translocation Another example of a translocation in EFT involving FUS New FUS breakpoint

14. FUS, FEV Breakpoints – modified from Janknecht; Gene 2005

15. ?Further Evidence of FUS or FEV rearrangement in EFTs? – TMAs being evaluated

16. Conclusion Novel Chromosomal Translocation t(2;16)(q35;p11) in Ewing sarcoma; Underscores the interchangeability of fusion partners in EFT translocations Consider FISH with FUS break-apart if EWS break- apart negative

18. 3 weekly cycles, 11 in total, alternating 6 cycles of ifosfamide 9g/m2 etoposide 500mg/m2 given over 5 days with 5 cycles of vincristine 2mg, doxorubicin 75mg/m2, cyclophosphamide 1g/m2 given on 1 day local control commenced after 5 cycles with radiation, 45 Gy in 25 fractions (chemotherapy continued concurrently) then 8 weeks post radiation surgery with resection of distal clavicle and surrounding tissues