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

Microarray Gene ExpressionSam 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 mm3

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

Hanahan D et al. Cell 1996:86:353-64Semenza GL. Nat Rev Cancer 2003:3:721-32

Hanahan D. Eur J Cancer 1996:32A:2386-9Hamano 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 2001:19:1207O’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 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

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 2000:407:242-8Sierra-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 2003:21:1775-81Segal NH et al. J Clin Oncol 2003:21:1775-81; Ohali A et al. Oncogene 2004:8997-9006Lee YF et al. Cancer Res 2004:7201-4

Hanahan D. Eur J Cancer 1996:32A:2386-9Hamano 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 n=108 n=30

Circulating Ang2 and Leptin Levels

n=108 n=30 n=51 n=14

0

100

200

300

400

<5cm (n=18) >5cm,<10 cm(n=30)

>10cm,<20cm(n=39)

>20cm (n=21)

VE

GF

(p

g/m

l)

An

g2 (

pg/

ml)

Circulating VEGF and Ang2 Levels by Tumor Size

0

2000

4000

6000

8000

10000

<5cm (n=18) >5cm,<10 cm(n=30)

>10cm,<20cm(n=39)

>20cm (n=21)

bF

GF

(p

g/m

l)

Lep

tin

(p

g/m

l)

Circulating bFGF and Leptin Levels by Tumor Histologic Subtype

0

30

60

90

0

5000

10000

15000

20000

25000

Fibrosarcoman=11

Leiomyo-sarcoma

n=9

Othersarcoma

n=88

Well-diffliposarcoma

n=25

Otherliposarcoma

n=16

Othersarcoma

n=67

Hierarchical Clustering Analysis Based on All Genes

Segal NH et al. Am J Pathol 2003:21:1775-81

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

Gene name Probe set Sarcoma samples (n=38) Normal tissues (n=14) p valuemean +/- sem mean +/- sem

HIF-1 200989 at 4,156.08 +/- 300.10 1,836.41 +/- 365.21 <0.0001PLOD2 202620 s at 1,370.68 +/- 150.04 331.79 +/- 78.89 <0.0001

Annexin V 200782 at 6,549.06 +/- 462.77 3,108.48 +/- 361.10 <0.0001BNIP3L 221478 at 1,771.35 +/- 122.75 743.54 +/- 132.63 <0.0001

Collagen 5 1 212489 at 3,071.91 +/- 478.59 362.28 +/- 86.24 <0.0001Vimentin 201426 s at 24,603.41 +/- 1,579.80 10,351.16 +/- 1,944.74 <0.0001

Collagen 5 1 212488 at 5,286.67 +/- 925.23 737.84 +/- 188.65 <0.0001PLOD2 202619 s 1t 1,407.84 +/- 203.68 393.69 +/- 88.29 <0.0001

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 factorsBiopsy: analysis of microvessel density, VEGF and HIF1-1a protein expression; analysis of microarray gene expressionPerfusion 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

bF

GF

(p

g/m

l)

Circulating bFGF Levels by Tumor Location

0

2000

4000

6000

8000

Abdomen (n=42) Extremity (n=36) Trunk (n=30)

Hierarchical Clustering Analysis Based on Hypoxia-Related Genes

Detwiller KY (Yoon SS) et al. Cancer Res 2005:65:5881-9