1. CXCR4 receptor is correlated to the development of metastases in leiomyosarcoma, pleomorphic sarcoma and liposarcoma S.C.D.U. Medical Oncology I.R.C.C. Candiolo Ordine Mauriziano S Aliberti, G Grignani, G Cavalloni, A Pisacane, P Allione, I Sarotto, Y Pignochino, M Motta, B Torchio, M Risio, M Aglietta.

2. “When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil.” S Paget The Lancet 1889

3. -3149 adult patients with soft tissue sarcoma were admitted and treated at Memorial Sloan-Kettering Cancer Center. -719 patients developed lung metastases (about 24%)

4. Tumor progression steps

6. Cancer cell Y Y chemokine receptor chemokine endothelium Non-target tissue Cancer cell Y Y endothelium different chemokine Target tissue

7. Pancreatic cancerKosshiba et al., Clin. Cancer Res. 2000 NeuroblastomaGeminder et al., J. Immunol. 2001 Breast cancerMuller et al., Nature 2001 RhabdomyosarcomaLibura et al., Blood 2002 Prostate cancerTaichman et al., Cancer Res. 2002 Colon cancerZeelemberg et al., Cancer Res. 2003 OsteosarcomaPerissinotto et al., Clin Cancer Res. 2005 Laverdiere et al., Clin Cancer Res. 2005 Non-small cell lung cancer cellsSu et al., Clin Cancer Res. 2005 Esophageal cancerKaifi et al., J Natl Cancer Inst. 2005 Carcinomatosis of Gastric CancerYasumoto et al., Cancer Res. 2006 GliomaEhtesham et al., Oncogene 2006 MelanomaBartolome et al., Cancer Res. 2006 Involvement of CXCR4/SDF-1 system in cancer progression

8. Purpose To analyze expression patterns of CXCR4, EGFR, angioinvasiveness (by standard immunohistochemical technique) along with other primary tumor features (T and grading). To assess if there is a correlation among the characteristics of primary tumor and the development of metastases.

9. Materials and methods We retrospectively evaluated expression patterns of CXCR4, EGFR and angioinvasiveness features on primary tumors in 67 pts. -Mean age = 57 yrs (24 - 85) -Male/female = 38/29 -Median follow-up = 57 mos (2 – 160) -Limbs/retroperitoneal = 47/20 -Histotype: leiomyosarcoma, pleomorphic sarcoma non-myxoid liposarcoma. -Grading: according to Coindre et al.

10. Materials and methods SiteN° Lung1340% Lung + other 1118% Liver618% Other39%

11. CXC4 immunohistochemistry CXCR4: Pharmagin 12G-5 monoclonal antibody Cytoplasmic positivityNuclear positivity

12. EGFR immunohistochemistry EGFR: pharmaDx monoclonal antibody DakoCytomation Positive = 3+ & 80% of cells Negative = 0/1+ & < 1% of cells

13. Angioinvasiveness features

14. Results Negative34/67 50.7% Positive33/67 49.3% Negative29/67 43.2% Low19/67 28.4% High19/67 28.4% Negative13/67 19.4% Low12/67 17.9% High42/67 62.7% CXCR4EGFR Angio- Invasiveness

15. Variable CXCR4 EGFR Angiogenesis Grading T Tumor burden 0.07 0.05 0.018 0.32 0.017 Univariate 0.067 0.03 0.06 0.02 Multivariate Correlation with development of metastases

16. Response according to metastasis

17. CXCR4 - stratified by grading CXCR4 pos p = 0.018 CXCR4 neg CXCR4 pos p = 0.05 Grade = 1 or 2Grade = 3

18. Where do we go from here?

19. CXCR4 expression on leiomyosarcoma cell lines Cell line% CXCR4+ cells SK-LMS-1 Leiomyosarcoma 20-70% SK-UT-1 Leiomyosarcoma 20-70% MES-SA Uterine leiomyosarcoma 10-12% 91266 Uterine leiomyosarcoma Negative

20. CXCR4 down-regulation by SDF-1 on CXCR4+ leiomyosarcoma cell lines

21. Regulation of CXCR4 expression by in vitro gene transduction in leiomyosarcoma cell line

22. AMD3465 selectively reduces CXCR4 transcript expression in lungs.

23. In this retrospective series we show how CXCR4 is associated with the development of lung metastases. We are currently verifying these results on larger series. We are developing an in vitro model in which we can modulate CXCR4 expression. The metastatic behavior of these histotypes needs further study but we believe CXCR4 may play a role in the recurrence of soft tissue sarcoma. Conclusions

24. Acknowledgments Medical Oncology: Surgical Pathology: Sandra Aliberti Paolo Allione Massimo Aglietta Alberto Pisacane Ivana Sarotto Mauro Risio Manuela Motta Bruno Torchio Molecular Oncology:Giuliana Cavalloni Ymera Pignochino