ANTICANCER RESEARCH 25: 3591-3596 (2005)
Expression of Vascular Endothelial Growth Factor and its Receptor, KDR/Flk-1, in Soft Tissue Sarcomas EMILIOS E. PAKOS1, ANN C. GOUSSIA2, PERICLES G. TSEKERIS1, DIONYSIOS J. PAPACHRISTOU2, DIMITRIOS STEFANOU2 and NIKI J. AGNANTIS2 1Department
of Radiation Therapy and 2Department of Pathology, University Hospital of Ioannina, Medical School, University of Ioannina, 45110 Ioannina, Greece
Abstract. The aim of the present study was to evaluate the association of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, with tumor grade, microvessel density (MVD) and clinical outcome in patients with soft tissue sarcomas (STSs). Tissue specimens of 28 patients with STSs were analyzed immunohistochemically using specific monoclonal antibodies. Tissue samples were obtained prior to any treatment. Half of the STSs exhibited strong expression of VEGF that was associated statistically significantly with high tumor grade. Strong expression of KDR/Flk-1 was detected in only 2 sarcomas. No association was demonstrated between VEGF and KDR/Flk-1 expression. MVD was significantly associated with tumor grade and was higher in sarcomas with strong VEGF expression. Limited data on clinical outcome precluded solid analyses for an association with disease progression. This study provides further evidence on the role of VEGF and MVD in tumor aggressiveness in STSs. The formation of new blood vessels within the tumor stroma is an essential requirement for the growth of neoplasms (1, 2). The angiogenetic drive of a tumor results from the balance between promoters and inhibitors in the local tumor environment. Among promoters, vascular endothelial growth factor (VEGF) is one of the most important angiogenetic factors in vivo. VEGF is a dimeric glycoprotein, that acts as a mitogen for endothelial cells and induces angiogenesis and changes in vascular permeability, both involved in tumor vascularization and growth (3-5). It also functions as a potent anti-apoptotic factor for endothelial cells in newly-formed
Correspondence to: N.J. Agnantis, MD, Professor of Pathology, Department of Pathology, P.O.B: 1186, University Hospital of Ioannina, Medical School, University of Ioannina, 45110, Ioannina Greece. Tel: +30 26510-97795, Fax: +30 26510-97898/97858, email:
[email protected] Key Words: Soft tissue sarcoma, VEGF, Flk-1, MVD, angiogenesis.
0250-7005/2005 $2.00+.40
vessels (6, 7). This key regulator of angiogenesis exerts its effects by binding with high affinity to two tyrosine kinase receptors, which are expressed on the surface of endothelial cells; VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) (8, 9). Tumor angiogenesis is assessed morphologically by measuring intratumoral microvessel density (MVD) with various endothelial markers such as CD34, CD31 and Factor VIII. VEGF expression has been found to correlate with MVD (10). VEGF is overexpressed in many tumor types, and it has been proposed as a prognostic indicator correlated with decreased survival in several cancers (11-14). Soft tissue sarcomas (STSs) are tumors with high vascularization (15). Angiogenesis may be involved in the progression of STS since sarcoma cells produce large amounts of VEGF and VEGFRs in vitro (16). However, only a few studies have focused on the association of VEGF and its receptors with tumor grade and MVD of STSs, as well as their prognostic value, with contradictory results. In that respect, a retrospective study was conducted in order to evaluate the expression of VEGF and its receptor, KDR/Flk-1, in patients with STSs. We also tried to evaluate the association of VEGF and KDR/Flk-1 expressions with tumor grade, MVD and disease progression.
Patients and Methods Patients. The medical records of 28 patients with the diagnosis of primary, non-metastatic STS were retrospectively reviewed. We accepted all patients regardless of the type of STS. All patients were treated surgically with radical tumor excision at the Ioannina University Hospital, Greece, and had available histological tissue from the surgical specimens. Patients with incomplete tumor resection were excluded from the study. None of the patients had received any adjuvant therapy before surgery. For each patient, data on demographic characteristics, the type and location of the STS, the grade of tumor, the surgical margins and the maximal size on MRI were recorded. Also data on additional therapy, such as chemotherapy and radiotherapy, were recorded. The histological diagnosis and the grade of tumor were confirmed for each sample
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ANTICANCER RESEARCH 25: 3591-3596 (2005) Table I. Characteristics of 28 patients with STS included in the study.
Mean age (±SD) Type of STS Liposarcoma MFH Fibrosarcoma Leiomyosarcoma Synovial sarcoma Rhabdomyosarcoma
Overall
VEGF +
VEGF–
47.5 (14.8)
45.9 (14.7)
49.1 (15.3)
9 7 4 4 2 2
2 4 3 2 1 2
7 3 1 2 1 0
Sex Male Female
17 11
8 6
9 5
Grade Low Intermediate High
12 3 13
2 3 9
10 0 4
KDR/Flk-1 Positive Negative
2 26
1 13
1 13
Mean MVD (±SD)
48.9 (25.1)
56.6 (6.7)
41.2 (23.3)
Postoperative therapy Radiotherapy Chemotherapy Both None
6 13 5 4
4 5 2 3
2 8 3 1
STS: soft tissue sarcoma, SD: standard deviation, MFH: malignant fibrous histiocytoma, MVD: microvessel density. VEGF and KDR/Flk1 positivity defined as expression in >10% of tumor cells.
by reviewing hematoxylin-eosin-stained sections. All patients were followed up in an outpatient basis at the Radiation Therapy Department. Their evaluation included assessment of treatmentrelated toxicity, local recurrences, distant metastases and death. Immunohistochemistry. Immunostainings were performed on formalin-fixed, paraffin-embedded tissue sections (3Ìm) by the labelled streptavidin avidin biotin (LSAB) method. The sections were deparaffinized in xylene and dehydrated. Subsequently, all sections were immersed in citrate buffer solution (10mM, pH 6.0) in plastic Coplin jars and subjected to microwave irradiation twice for 10 min. After quenching the endogeneous peroxidase activity with 0.3% hydrogen peroxide in methanol, the sections were incubated overnight at 4ÆC with primary monoclonal antibodies directed against VEGF (dilution 1:50, clone JH121, Neomarkers, CA, USA), KDR/Flk-1 (1:2000, DBS, CA, USA) and CD34 (1:50, Biogenex, San Ramon, CA, USA). Non-specific binding was blocked by incubating the sections for 30 min with Blocking Solution (Dako, Denmark). Detection was carried out using the Envision System kit (Dako) with diaminobenzidine as chromogen. Finally, the sections were counterstained with Harris hematoxylin,
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dehydrated and mounted. To assess the specificity of the reaction, negative controls were included in each staining run and consisted of tumor sections that were subjected to the whole procedure with omission of the primary antibody. Two observers (A.G. and D.P.), who were blinded to the clinical outcome, examined all sections independently and reached consensus on all items. Assessment of vascularity. Any single brown staining of endothelial cells or clusters of endothelial cells, with or without a lumen, were considered as individual countable microvessels. Vessels with thick muscular walls were excluded. The density of tumor microvessels was determined as described in previous studies (17). Using light microscopy, the three most intense regions of neovascularization ("hot spot areas") were selected and counted at X200 magnification (0.739 mm2 per field). Final MVD was the mean of the values obtained for the three fields. Assessment of VEGF and KDR/Flk-1 expressions. The staining was evaluated only in the areas with well-preserved tissue morphology and away from necrosis or artifacts. The immunoreactivity for VEGF was found in the cytoplasm of tumor cells, and the staining intensity varied among tumors. The expression of KDR/Flk-1 was observed in the cell membrane and the cytoplasm of cells. The immunostaining for VEGF and KDR/Flk-1 was semiquantitatively estimated based on a scale from – to ++: – , when no staining of tumor cells was observed; +, when 0%) were also performed with similar results regarding VEGF, but with varying results when the KDR/Flk-1 receptor was analyzed. Finally, the present study was not adjusted for other angiogenesis-related parameters that may interact with VEGF or KDR/Flk-1, or may be independent prognostic factors in STSs (29, 30). The present study provides further evidence for the relationship of VEGF and the degree of malignancy and tumor vascularization in STSs. In the future, large, prospective and homogenous studies should examine the role of such angiogenesis-related factors in tumor aggressiveness and clinical outcome. The possible implication of such factors in the development and progression of STSs may offer an additional therapeutic option with improved anti-angiogenic agents previously used in experimental models, such as antisense oligonucleotides, monoclonal antibodies and soluble VEGFRs.
Acknowledgements The authors thank Mrs A. Christodoulou for her technical assistance and Mr E. Evangelou for the statistical appraisal of this paper.
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Received March 31, 2005 Accepted June 10, 2005
