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)
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Sam S. Yoon, Neil H. Segal, Peter J. Park, Kara Y. Detwiller,
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 - PowerPoint PPT Presentation
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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)
– 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
• 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