1. BIOLOGICAL MARKERS OF PROGNOSIS IN EWING'S SARCOMA: THE ROLE OF GAL3BP CTOS meeting, Seattle 2007 Katia Scotlandi, Diana Zambelli, Monia Zuntini, and Piero Picci Istituti Ortopedici Rizzoli Bologna, Italy

2. From experimental models to clinics TC-71MDR transfectantsDrug resistant cells 4 different variants, characterized by: MDR phenotype Loss of malignancy

3. Gene expression profile of these cells COMMON UP-REGULATED GENES

4. gene functional Networks operating in the regulation of mets capabilities

5. Primary lesions (n = 42) Metastasis (n = 7) Recurrences (n = 7) Sex Male Female 26 16 5252 3434 Age ≤ 14 years > 14 years 15 27 2525 1616 Tumor Site Extremity Pelvis Other 25 8 9 511511 5-25-2 Metastasis at Diagnosis No Yes 36 6 ---- ---- Type of Translocation EWS/Fli1 Type 1 EWS/Fli1 Type 2 EWS/Fli1 Type 3 Other 23 10 1 8 51015101 52005200 Validation by TaqMAN Real Time PCR array Genes: CDC42EP1, CTGF, FVT1, IRS1 LGALS3BP HINT IFITM2, STOML2 RPS4Y1 CANX CTGF KPNA2 RNP24

6. Comparison of the mean expression values of primary lesions with recurrences or metastases for each gene did not provide significant results. However remarkable reduction in the recurrences and metastasis groups was observed for some genes

7. p = 0.03 H L p = 0.04 H L Prognostic Value of 6 out of 12 genes IFITM2 EFS OVS RPS4Y1 p = 0.04 H L

8. p = 0.03 H L Prognostic Value HINT1 EFS OVS p = 0.06 H L H L p = 0.03 H L STOML2

9. Prognostic Value None of these 6 prognostic genes displayed correlation with clinical pathological features. However, GALS3BP status showed a statistically significant, positive correlation with the fusion type translocation (p= 0.0007, Fisher’s exact test), EWS/Fli-1 type 1 fusion occurring more frequently in LGALS3BP high- expressing cases (17/20, 85% vs 7/22, 32%) LGALS3BP EFS OVS p = 0.008 H L p = 0.005 H L

10. Prognostic Value As expected, EWS/Fli-1 type 1 expression also showed significant correlation with survival

11. Prognostic Value: Multivariate Analysis Event-Free SurvivalOverall Survival

12. Evaluation of Protein Expression by Tissue Array Analysis PRIMARY LESIONS (N = 60) Sex Male Female 33 27 Age ≤ 14 years > 14 years 28 32 Tumor Site Extremity Pelvis Other 33 11 16 Metastasis at Diagnosis No Yes 51 9 In collaboration with A.Llombart-Bosch, University of Valencia, Spain

13. Prognostic Value: LGALS3BP Protein Expression 39 LOW21 HIGH (Monia Zuntini1*, Diana Zambelli1*, Filippo Nardi1, Maria Cristina Manara1, Massimo Serra1, Stefano Ferrari2, Patrizia Bacchini3, Antonio Llombart-Bosch4, Stefano Iacobelli5, Piero Picci1, and Katia Scotlandi1 Biological indicators of prognosis in Ewing’s sarcoma: an emerging role for lectin galactoside-binding soluble 3 binding protein (LGALS3BP) (Submitted to JCO)) H L p = 0.03 H L OVS EFS

14. GAL3-BP LGALS3BP (also known as 90K or Mac-2 binding protein) was originally identified as a tumor- associated antigen in breast cancer It binds galectins and appears to be have a role in cell-cell adhesion A dualistic role depending on the cellular context In EWS, high expression is associated with better prognosis Molecular mechanisms?? Studies are in progress

15. LGALS3BP/90K in EWS cell lines Protein mRNA

16. TC/90K CLONES Screening of 90 clones by ELISA 20 CLONES

17. Future plans  Analyze the presence of 90K in the serum of EWS patients  Growth ability, both in monolayer and soft-agar  Adhesion to different substrates  Migration and invasion processes  Chemo sensitivity to drugs commonly applied in the treatment of EWS patients  Evaluate the biological behavior of TC/90K clones

18. Thanks to: EU project EUROBONET AIRC Regione Emilia Romagna and Italian Ministry of Health