Tumor Angiogenesis Tumors are comprised of a variety of cell types

Angiogenic Profile of Soft Tissue Sarcomas Based on Analysis of Circulating Factors and Microarray Gene Expression

Tumor Angiogenesis

• Tumors are comprised of a variety of cell types

• Angiogenesis (new blood vessel formation) is required to grow beyond 1-2 mm3

• Hypoxia in tumors leads to stabilization of HIF-1 and expression of proangiogenic genes

Balance of Angiogenic Factors

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

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 12-14 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

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

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

Balance of Angiogenic Factors

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

Circulating VEGF and Ang2 Levels by Tumor Size

Circulating bFGF and Leptin Levels by Tumor Histologic Subtype

Hierarchical Clustering Analysis Based on All Genes

Hierarchical Clustering Analysis Based on Angiogenesis-Related Genes

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

Top Up-regulated Hypoxia-Related Genes

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

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

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

Hierarchical Clustering based on Expression of VEGF-Related Genes

Circulating bFGF Levels by Tumor Location

Hierarchical Clustering Analysis Based on Hypoxia-Related Genes