Download Abstract 427

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 mm3
• Hypoxia in tumors leads to
stabilization of HIF-1a and
expression of proangiogenic
genes
Hanahan D et al. Cell 1996:86:353-64
Semenza GL. Nat Rev Cancer 2003:3:721-32
Balance of Angiogenic Factors
Angiopoietin 2
Leptin
Hanahan D. Eur J Cancer 1996:32A:2386-9
Hamano Y et al. BBRC 2005:333:292-8
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 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 2000:407:242-8
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 2003:21:1775-81
Segal NH et al. J Clin Oncol 2003:21:1775-81; Ohali A et al. Oncogene 2004:8997-9006
Lee YF et al. Cancer Res 2004:7201-4
Balance of Angiogenic Factors
2
Leptin
Hanahan D. Eur J Cancer 1996:32A:2386-9
Hamano Y et al. BBRC 2005:333:292-8
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
Circulating VEGF and Ang2 Levels
by Tumor Size
10000
Ang2 (pg/ml)
VEGF (pg/ml)
400
300
200
100
0
<5cm (n=18)
>5cm,<10 cm
(n=30)
>10cm,<20cm >20cm (n=21)
(n=39)
8000
6000
4000
2000
0
<5cm (n=18)
>5cm,<10 cm >10cm,<20cm >20cm (n=21)
(n=30)
(n=39)
Circulating bFGF and Leptin Levels
by Tumor Histologic Subtype
25000
Leptin (pg/ml)
bFGF (pg/ml)
90
60
30
15000
10000
5000
0
0
Fibrosarcoma
n=11
20000
Leiomyosarcoma
n=9
Other
sarcoma
n=88
Well-diff
Other
Other
liposarcoma liposarcoma sarcoma
n=25
n=16
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
Gene name
Probe set Sarcoma samples (n=38) Normal tissues (n=14)
mean +/- sem
mean +/- sem
HIF-1a
200989 at
4,156.08 +/- 300.10
1,836.41 +/- 365.21
PLOD2
202620 s at
1,370.68 +/- 150.04
331.79 +/- 78.89
Annexin V
200782 at
6,549.06 +/- 462.77
3,108.48 +/- 361.10
BNIP3L
221478 at
1,771.35 +/- 122.75
743.54 +/- 132.63
Collagen 5 a1 212489 at
3,071.91 +/- 478.59
362.28 +/- 86.24
Vimentin
201426 s at
24,603.41 +/- 1,579.80
10,351.16 +/- 1,944.74
Collagen 5 a1 212488 at
5,286.67 +/- 925.23
737.84 +/- 188.65
PLOD2
202619 s 1t
1,407.84 +/- 203.68
393.69 +/- 88.29
p value
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
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 angiogenesisrelated gene expression compared to normal tissues
– Histologic subtypes often share similar patterns of
angiogenesis-related gene expression
– PDGFR-a and HIF-1a 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
Circulating bFGF Levels
by Tumor Location
bFGF (pg/ml)
8000
6000
4000
2000
0
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