Angiogenic Profile of Soft Tissue Sarcomas Based on Analysis of Circulating Factors and Microarray Gene Expression Sam S. Yoon, Neil H. Segal, Peter J. Park, Kara Y. Detwiller, Namali T. Fernando, Sandra W. Ryeom, Carlos Cordon-Cardo, Murray F. Brennan, Samuel Singer Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA (SSY, KYD, NTF) Sarcoma Disease Management Team, Departments of Surgery and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY (NHS, CC, MFB, SS) Harvard-Partners Center for Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA (PJP) Division of Vascular Biology, Boston Children’s Hospital, Harvard Medical School, Boston, MA (SWR)

Tumor Angiogenesis Tumors are comprised of a variety of cell types Angiogenesis (new blood vessel formation) is required to grow beyond 1-2 mm 3 Hypoxia in tumors leads to stabilization of HIF-1  and expression of proangiogenic genes Hanahan D et al. Cell 1996:86: Semenza GL. Nat Rev Cancer 2003:3:721-32

Hanahan D. Eur J Cancer 1996:32A: Hamano Y et al. BBRC 2005:333:292-8 Balance of Angiogenic Factors Leptin Angiopoietin 2

Circulating Angiogenic Factors - 1 Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) –Most widely studied growth factors for endothelial cell –Circulating levels elevated in a variety of cancers –Prognostic significance in certain cancers Endostatin –Endogenous inhibitor of tumor angiogenesis –C-terminal fragment of collagen XVIII –May inhibit growth of micro-metastases Poon RT et al. J Clin Oncol 2001:19:1207 O’Reilly MS et al. Cell 1997:88:277-85

Initial Study of Circulating Angiogenic Factors in Sarcoma Patients 76 patients with soft tissue sarcomas have widely variable levels of VEGF, bFGF, and endostatin VEGF and bFGF levels are fold higher in patients with soft tissue sarcoma Endostatin levels are generally high in patients without metastases and low in patients with metastases Low bFGF levels prior to resection predicted a higher risk of recurrence Yoon SS (Singer S) et al. Ann Oncol 2004:15:1261-6

Circulating Angiogenic Factors -2 Angiopoietin 2 (Ang2) –Competes with Ang1 for binding of Tie2 receptor on endothelial cells –May cause blood vessel destabilization followed by VEGF-induced new vessel formation –Circulating levels not measured in sarcoma patients Leptin –Circulating hormone produced by adipose tissue –Levels significantly higher in females than males –Found to promote angiogenesis Yancopoulos GD et al. Nature 2000:407:242-8 Sierra-Honigmann MR et al. Science 1998:281:1683-6

Microarray Analysis of Gene Expression Global snapshot of gene expression Microarray analysis of sarcomas –Differentiate between histologic subtypes –Better classify equivocal histologic subtypes –Determine prognosis and response to chemotherapy Microarray analysis not previously used to study angiogenesis-related gene expression in sarcomas Nielsen TO et al. Lancet 2002:359:1301-7; Segal NH et al. Am J Pathol 2003:21: Segal NH et al. J Clin Oncol 2003:21: ; Ohali A et al. Oncogene 2004: Lee YF et al. Cancer Res 2004:7201-4

Hanahan D. Eur J Cancer 1996:32A: Hamano Y et al. BBRC 2005:333:292-8 Balance of Angiogenic Factors 2 Leptin

Objectives To develop profiles of angiogenesis-related gene and protein expression for soft tissue sarcomas To determine differences in these profiles based on size and histologic subtype To identify targets for anti-angiogenic therapies

Methods Blood samples were collected from 108 patients with primary soft tissue sarcoma and 30 healthy controls –Plasma levels of VEGF, bFGF, Ang2 and leptin were determined by ELISA Tumor samples collected from 38 patients with soft tissue sarcoma along with 14 normal tissue samples –Affymetrix U133A oligonucleotide microarrays –List of 200 angiogenesis-related genes compiled –List of 107 hypoxia-related genes compiled –Hierarchical clustering analysis used to identify groups with similar gene expression patterns

Clinicopathologic Factors

Circulating VEGF and bFGF Levels n=108 n=30

Circulating Ang2 and Leptin Levels n=108 n=30n=51 n=14

VEGF (pg/ml) Ang2 (pg/ml) Circulating VEGF and Ang2 Levels by Tumor Size

bFGF (pg/ml) Leptin (pg/ml) Circulating bFGF and Leptin Levels by Tumor Histologic Subtype Fibrosarcoma n=11 Leiomyo- sarcoma n=9 Other sarcoma n=88 Well-diff liposarcoma n=25 Other liposarcoma n=16 Other sarcoma n=67

Hierarchical Clustering Analysis Based on All Genes Segal NH et al. Am J Pathol 2003:21:

Hierarchical Clustering Analysis Based on Angiogenesis-Related Genes

Top Up-regulated and Down-regulated Angiogenesis-Related Genes

Top Up-regulated Hypoxia-Related Genes Detwiller KY (Yoon SS) et al. Cancer Res 2005:65:5881-9

Summary Soft tissue sarcomas have varied levels of circulating angiogenic factors based on size and histologic subtype –VEGF, bFGF, Ang2 levels are elevated Soft tissue sarcomas have altered patterns of angiogenesis- related gene expression compared to normal tissues –Histologic subtypes often share similar patterns of angiogenesis-related gene expression –PDGFR-  and HIF-1  are highly upregulated in sarcomas Analysis of circulating angiogenic factors and angiogenesis-related gene expression may provide useful biologic information in designing or analyzing clinical trials of anti-angiogenic agents

A phase II study of neoadjuvant bevacizumab and radiation therapy for resectable soft tissue sarcomas Blood: analysis for circulating angiogenic factors Biopsy: analysis of microvessel density, VEGF and HIF1-1a protein expression; analysis of microarray gene expression Perfusion CT: analysis of blood volume and blood flow

Acknowledgments Massachusetts General Hospital –Thomas DeLaney –David Harmon –Francis Hornicek –David Kirsch –Andrew Rosenberg –Kevin Raskin –Dushyant Sahani –Herman Suit Yoon Laboratory –Kara Detwiller –Namali Fernando –Sung Hwan Kim –Lila Gollogly Memorial Sloan-Kettering –Samuel Singer –Neil Segal –Murray Brennan –Adam Olshen –Carlos Cordon-Cardo Harvard-Partners Center for Genetics and Genomics –Peter Park

Microarray Gene Expression Levels of VEGF, bFGF, Ang2, and Leptin

Hierarchical Clustering based on Expression of VEGF-Related Genes

bFGF (pg/ml) Circulating bFGF Levels by Tumor Location

Hierarchical Clustering Analysis Based on Hypoxia-Related Genes Detwiller KY (Yoon SS) et al. Cancer Res 2005:65:5881-9