Ilkyu Han

1. Gene Expression Signature from Tumor Initiating Population Predicts Clinical Outcome in Undifferentiated Pleomorphic Sarcoma Ilkyu Han Program in Developmental and Stem Cell Biology, Hospital for Sick Children University Musculoskeletal Oncology Unit, Mount Sinai Hospital, Toronto, Canada Department of Orthopaedic Surgery, Seoul National University Hospital, Seoul, Korea

2. Sarcoma • Group of malignant tumors arising from mesenchymal tissue • Classified by the histological resemblance to mesenchymal tissue differentiation osteosarcoma rhabdomyosarcoma liposarcoma

3. Undifferentiated Pleomorphic Sarcoma • Most common subtype, 25% of patients in sarcoma clinical trials • More undifferentiated of the sarcoma types → Possible role of Mesenchymal Stem Cells • Most death caused by cancer recurrence Mohseny et al. Stem Cells 2011

4. Tumor Initiating Cells (TICs) • Small population of cancer cells possess the ability to initiate and maintain tumors • Self-renewal & differentiation: ‘cancer stem cell’ • Identification of TICs • Fractionating primary tumors into populations that can form a xenograft • Transplanting tumors into secondary mice

5. TICs in Sarcoma • Side population (SP) assay: - Based on their ability to exclude Hoechest dye 333342 - SP fraction is enriched in TICs SP • Identified in UPS, osteosarcoma, synovial sarcoma Wu et al. Cancer Res 2007

6. Implications of TIC Concept • Distinct properties of TICs vs. non-TICs • TICs may be the drivers of cancer recurrence • Clinical parameters closely related to properties of TICs rather than non-TICs Ebben et al. Expert Opin 2010

7. Clinical Relevance of TICs • Correlation with single trait • Ability to be xenografted • TIC marker expression • Gene expression profiling • Universalproperties of TICs (vs. non-TICs) • Breast cancer and leukemia Liu et al. NEJM 2007 Eppertet al. Nature Med 2010

8. Hypothesis • If the TIC concept accurately reflects the biology of UPS, then properties of the TIC population, such as a gene expression signature would correlate with outcome Purpose • To establish a TIC gene expression signature and test its association with clinical outcome in UPS patients

9. Isolation of TICs • CD45 (-), Side population (SP) fraction : 0.13% (0.01~0.46%) • 15 primary UPS samples: SP and NSP fractions NSP SP CD45 Staining +Verapamil

10. UPS TIC Gene Signature UPS TIC Gene Expression Signature SP NSP • Global transcriptional profiling of SP and NSP fractions (Illumina Human HT-12) • Genes differentially expressed between SP and NSP cells • Fold difference≥1.5 and p<0.01

11. UPS TIC Gene Signature Genes in the Signature • S1PR1: Human ESC pluripotency • PARD6A : EMT, stem cell marker • PEBP1: oncogenesis • SERPINF1: oncogenesis, decrease Wnt signaling • MIF: oncogenesis • STAT2: oncogenesis • NODAL3: ESC pathway (NODAL) • SERPINE2: oncogenesis • ST13: oncogenesis

12. UPS TIC Gene Signature Clustering in SP Fraction • Unbiased analysis of SP fraction • → Clustered into two distinct clusters • Two clusters seemed to correlate with patient outcome

13. Clinical Outcome UPS TIC Gene Expression Signature Score • To compare the samples: relative expression of the genes in the UPS TIC signature • To correlate the UPS TIC signature with clinical outcome • Single number for each sample : Median of log2 expression values of 93 genes

14. Clinical Outcome Correlation with Clinical Outcome • SP fraction (15 patients) • High vs. Low score group • : median value of UPS TIC score • Clinical outcome: overall survival and disease-free survival • Overall Survival (p=0.031) • Disease-free Survival (p=0.007)

15. Clinical Outcome Validation in External Data • Bulk (unsorted) tumor samples • TIC gene expression persists in non-TICs • - Clinically feasible • Published dataset in soft tissue sarcoma • - Francis et al., BMC Genomics 2007

16. Clinical Outcome Validation in External Data • Soft tissue sarcoma (n=114), UPS (34%, n=48) • Standard treatment • 27k cDNA microarray • 53 of 93 genes in the UPS TIC signature identified Francis et al. BMC Genomics 2007

17. Clinical Outcome UPS (n=48): Univariate Analysis • Kaplan-Meier Analyses • High vs. Low score group: median value of UPS TIC score • Metastasis-free Survival (p=0.050) • Disease-free Survival (p=0.026)

18. Clinical Outcome UPS (n=48): Multivariate Analysis • Cox Multivariate Analyses • Histologic grade, tumor size, tumor depth • Metastasis-free Survival • Disease-free Survival

19. Clinical Outcome All Types (n=114): Univariate Analysis • Kaplan-Meier Analyses • High vs. Low score group: median value of UPS TIC score • Metastasis-free Survival (p=0.044) • Disease-free Survival (p=0.062)

20. Clinical Outcome All Types (n=114): Multivariate Analysis • Cox Multivariate Analyses • Histologic grade, tumor size, tumor depth • Metastasis-free Survival • Disease-free Survival

21. Summary • This is the first study to define a gene expression signature from sarcoma TICs and to relate this signature to expression profiles in bulk tumor samples, thereby allowing a correlation with the clinical outcome. • Patients with high TIC gene expression signature scores had worse clinical outcome in UPS, even when accounting for established prognostic variables.

22. Conclusion • The gene expression signature identified in this study suggests the clinical and biologic relevance of the TIC concept in UPS.

23. Acknowledgement • Alman Lab • Benjamin A. Alman • QingxiaWei • Shingo Sato • TCAG • Pingzhao Zhu • Department of Surgery • Jay S. Wunder • Peter Ferguson • Department of Oncology • Ana Carneiro • MefNilbert • Department of Tumor Biology • Ola Myklebost • Stine H. Kresse • Department of Orthopedic Surgery • Han-SooKim