Soluble VEGFR-2 Expression in Head and Neck Malignant Tumors
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Poster Design & Printing by Genigraphics ® - 800.790.4001 Natalie Silver MS MD University of Kentucky Department of Otolaryngology Email: [email protected] Objective 1) Define the spatial pattern of expression of soluble vascular endothelial growth factor receptor 2 (sVEGFR-2), a recently discovered receptor that has an important role in tumor lymphangiogenesis, in head and neck malignant tumors. 2) Examine the relationship between sVEGFR-2 expression and lymphatic vessel density in head and neck malignant tumors. Methods One hundred and ten paraffin-embedded tissue samples from patients with malignant tumors were stained using immunohistochemistry for sVEGFR-2 and podoplanin, a marker for lymphatic vessels. Tissues studied included: squamous cell carcinoma of the larynx (61), oral cavity (16), oropharynx (20), hypopharynx (5), nasopharynx (1). Papillary (4), medullary (2) and follicular (1) thyroid cancers were also examined. Results sVEGFR-2 expression was identified in all squamous cell carcinomas of the head and neck. Expression was specific to the endothelial cells in blood vessels of both malignant tissue as well as adjacent normal tissues. sVEGFR-2 was not expressed in lymphatic vessels. This secreted protein was also expressed in tissue stroma. Decreased expression of sVEGFR-2 was correlated with increased lymphatic vessel density in tumors, measured by podoplanin expression. sVEGFR-2 was minimally expressed in normal thyroid tissue but was abundant in the basement membrane of papillary thyroid cancer cells. Conclusion We have provided the first evidence of sVEGFR-2 expression in head and neck malignant tumors. Its expression correlates with lymphatic vessel density in head and neck malignant tumors and ongoing studies will reveal the precise function of sVEGFR-2 in nodal metastasis and disease progression. Soluble VEGFR-2 Expression in Head and Neck Malignant Tumors 1 Natalie Silver MS MD, 2 Judit Baffi MD PhD, 3 Yolanda Brill MD, 1 Thomas Gal MPH MD, 2 Romulo Albuquerque MD PhD, 2 Jayakrishna Ambati MD, 1 Joseph Valentino MD 1 University of Kentucky Department of Otolaryngology, 2 University of Kentucky Department of Ophthalmology, 3 University of Kentucky Department of Pathology sVEGFR-2 expression was identified in squamous cell carcinomas of the head and neck in all subsites examined. Expression was specific to the endothelial cells in blood vessels in both malignant tissue as well as adjacent normal tissues. This secreted protein was also expressed in tissue stroma as well as within malignant tumor cell nests. (Figure 2a and b) Decreased expression of sVEGFR-2 correlated with increased lymphatic vessel density in peri-tumoral stroma, measured by podoplanin expression. sVEGFR-2 was not expressed in lymphatic vessels, but was expressed in endothelial cells of blood vessels as measured by CD34 expression. (Figure 2c; Figure 3a and b) sVEGFR-2 was minimally expressed in normal thyroid tissue but was abundant in the basement membrane of papillary thyroid cancer cells. This correlated with peritumoral vascularity and was inversely related to lymphatic vessel density. (Figure 4a-c) Institutional IRB approval was obtained. 42 tissue samples from patients with head and neck squamous cell carcinoma who were previously enrolled in a Phase II clinical trial conducted at the University of Kentucky were obtained. Tissue microarrays were also obtained from US Biomax containing head and neck malignant tumors. Tumor subsites and pathology are illustrated in Table 1. Sections of formalin-fixed and paraffin embedded samples were deparaffinized. Epitope retrieval was performed by heating in citrate buffer. Endogenous peroxidase activity was blocked by incubating the sections in hydrogen peroxide in PBS. Primary polyclonal rabbit anti sVEGFR2 antibody was added to the slides for 60 min at room temperature followed by incubation with a secondary HRP conjugated goat anti-rabbit antibody. Primary anti podoplanin (brown) and primary anti CD34 (red) were also added sequentially on a separate slide for double staining. After counterstaining of the sections with either hematoxylin or neutral red, they were embedded with mounting medium. Isotype of the primary antibodies were used for negative controls. Immunostaining was reviewed by a blinded independent pathologist. The expression of vascular endothelial growth factor (VEGF)-A ,VEGF-B, VEGF-C and VEGF-D in both experimental and clinical models of head and neck squamous cell carcinoma (HNSCC) has been determined and correlated with conventional clinicopathologic parameters, with particular reference to cervical nodal metastasis. Varying degree of expression of markers has been shown to correlate with pathologic staging and prognosis. 1 The vascular endothelial growth factor (VEGF) family of secreted proteins and their receptors are major regulators of blood vessel development (hemangiogenesis) and lymphatic vessel development (lymphangiogenesis). VEGF acts through a complex system of receptor tyrosine kinases, which can be membrane bound or soluble. New data concerning the receptor system are still emerging, thus contributing to the complexity of the system. Recently, a soluble form of VEGFR-2, termed sVEGFR-2, which is a result of alternative splicing, has been discovered. The newly discovered spliced variant of sVEGFR-2 binds the lymphangiogenic growth factor VEGF-C and thus inhibits VEGF-C- induced activation of VEGFR-3, consequently inhibiting lymphatic endothelial cell proliferation. 2 (Figure 1) Its inactivation in murine embryos permits hyperplasia of dermal lymphatics and invasion of lymphatics into the cornea. 3 Tumor lymphangiogenesis influences the metastatic behavior of malignant cells. A correlation has been found between the downregulation of sVEGFR-2 and the malignant progression of neuroblastoma, which is characterized by lymphogenic metastases in progressed stages. 4 These discoveries led to our investigation of the role of sVEGFR-2 in malignant tumors of the head and neck. INTRODUCTION MATERIALS AND METHODS 1. P Oc, Rhys-Evans P, Eccles SA. Expression of vascular endothelial growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis. Cancer 2001;92:556-68. 2. Pavlakovic H, Becker J, Albuquerque R, Wilting J, Ambati J. Soluble VEGFR-2: an antilymphangiogenic variant of VEGF receptors. Ann N Y Acad Sci 2010;1207 Suppl 1:E7-15. 3. Albuquerque RJ, Hayashi T, Cho WG, et al. Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth. Nat Med 2009;15:1023-30. 4. Becker J, Pavlakovic H, Ludewig F, et al. Neuroblastoma progression correlates with downregulation of the lymphangiogenesis inhibitor sVEGFR-2. Clin Cancer Res 2010;16:1431-41. 5. Bolzoni Villaret A, Barbieri D, Peretti G, et al. Angiogenesis and lymphangiogenesis in early-stage laryngeal carcinoma: Prognostic implications. Head Neck 2012. 6. Shibata MA, Ambati J, Shibata E, et al. The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model. BMC Med 2010;8:69. DISCUSSION/CONCLUSIONS RESULTS REFERENCES Figure 1. Schematic presentation of the VEGF ligands and their interaction with membrane-bound and soluble VEGF receptors. Inhibiory effects are exerted by sVEGFR-1 (anti- hemangiogenesis) and sVEGFR-2 (anti- lymphangiogenesis). 2 ABSTRACT CONTACT TABLES AND FIGURES Cancer Subsite/Pathology N=Number of Tissue Samples (Total 110) SCCA Oral Cavity Oropharynx Larynx Hypopharynx Nasopharynx 103 16 20 61 5 1 Thyroid Cancer Papillary Medullary Follicular 7 4 2 1 Table 1. Tissue samples examined by subsite/patholog y. Figure 2: a. H and E staining of a moderately differentiated SCCA of the larynx at 10X power. Arrow at invasive front of malignant tumor. b. IHC for sVEGFR-2 (blue) at 10X power with positive peritumoral expression and positive expression in vascular endothelial cells. c. IHC for podoplanin (brown)/CD34(red) at 10X power showing high vascular endothelial cell density correlating with expression of sVEGFR2, and very low density of lymphatic vessels. The histopathalogic assessment of lymphatic and blood vessel density has prognostic significance in head and neck cancers. Villaret et al recently demonstrated that the presence of lymphatic vessels in peritumoral fields had an impact on local and locoregional recurrence in early-stage laryngeal carcinoma. 5 Several growth factors in the VEGF family are important for peritumoral lymphatic and blood vessel growth in head and neck cancers. We suspect that sVEGFR-2 may play a key role in the regulation of peritumoral lymphatic invasion. Shibata et al demonstrated that gene therapy with alternative splicing variant esVEGFR-2 significantly suppresses tumor growth and lymph node metastasis in a mouse mammary cancer model. 6 • IHC demonstrated and inverse correlation of sVEGFR-2 expression and lymphatic vessel density in head and neck cancers. •Papillary thyroid cancer has dramatic expression of sVEGFR-2 unlike normal thyroid. •The expression pattern of this receptor supports a potential role in lymphatic spread of malignant tumors of the head and neck. •The precise function of sVEGFR-2 in metastasis and disease progression in these tumors should be evaluated further. Figure 3: a. Poorly differentiated nonkeratenizing squamous cell carcinoma of the tonsil at 10X stained for podoplanin (brown) and CD34 (red) showing abundant peritumoral lymphatics. b. Very weak expression of sVEGFR-2 (blue) correlating with increased lymphatic vessel density. Figure 4: a. Normal thyroid tissue at 10X power with minimal positive sVEGFR-2 staining. b. Intense sVEGFR-2 staining in papillary thyroid cancer. c. IHC for podoplanin/CD34 showing minimal expression of lymphatic vessels. a b c a b a b c Podoplanin \ CD34 \ sVEGFR-2 \ CD34 \ \ podoplanin Adapted from: http://www.rosenthallab.com/gallery/images/VEGF_VEGFR.jpeg.
Transcript of Soluble VEGFR-2 Expression in Head and Neck Malignant Tumors
Poster Design & Printing by Genigraphics® - 800.790.4001
Natalie Silver MS MDUniversity of Kentucky Department of OtolaryngologyEmail: [email protected]
Objective
1) Define the spatial pattern of expression of soluble vascular endothelial growth factor receptor 2 (sVEGFR-2), a recently discovered receptor that has an important role in tumor lymphangiogenesis, in head and neck malignant tumors. 2) Examine the relationship between sVEGFR-2 expression and lymphatic vessel density in head and neck malignant tumors.
Methods
One hundred and ten paraffin-embedded tissue samples from patients with malignant tumors were stained using immunohistochemistry for sVEGFR-2 and podoplanin, a marker for lymphatic vessels. Tissues studied included: squamous cell carcinoma of the larynx (61), oral cavity (16), oropharynx (20), hypopharynx (5), nasopharynx (1). Papillary (4), medullary (2) and follicular (1) thyroid cancers were also examined.
Results
sVEGFR-2 expression was identified in all squamous cell carcinomas of the head and neck. Expression was specific to the endothelial cells in blood vessels of both malignant tissue as well as adjacent normal tissues. sVEGFR-2 was not expressed in lymphatic vessels. This secreted protein was also expressed in tissue stroma. Decreased expression of sVEGFR-2 was correlated with increased lymphatic vessel density in tumors, measured by podoplanin expression. sVEGFR-2 was minimally expressed in normal thyroid tissue but was abundant in the basement membrane of papillary thyroid cancer cells.
Conclusion
We have provided the first evidence of sVEGFR-2 expression in head and neck malignant tumors. Its expression correlates with lymphatic vessel density in head and neck malignant tumors and ongoing studies will reveal the precise function of sVEGFR-2 in nodal metastasis and disease progression.
Soluble VEGFR-2 Expression in Head and Neck Malignant Tumors1Natalie Silver MS MD, 2Judit Baffi MD PhD, 3Yolanda Brill MD, 1Thomas Gal MPH MD, 2Romulo Albuquerque MD PhD, 2Jayakrishna Ambati MD, 1Joseph Valentino MD
1University of Kentucky Department of Otolaryngology, 2University of Kentucky Department of Ophthalmology, 3University of Kentucky Department of Pathology
sVEGFR-2 expression was identified in squamous cell carcinomas of the head and neck in all subsites examined. Expression was specific to the endothelial cells in blood vessels in both malignant tissue as well as adjacent normal tissues. This secreted protein was also expressed in tissue stroma as well as within malignant tumor cell nests. (Figure 2a and b)
Decreased expression of sVEGFR-2 correlated with increased lymphatic vessel density in peri-tumoral stroma, measured by podoplanin expression. sVEGFR-2 was not expressed in lymphatic vessels, but was expressed in endothelial cells of blood vessels as measured by CD34 expression. (Figure 2c; Figure 3a and b)
sVEGFR-2 was minimally expressed in normal thyroid tissue but was abundant in the basement membrane of papillary thyroid cancer cells. This correlated with peritumoral vascularity and was inversely related to lymphatic vessel density. (Figure 4a-c)
Institutional IRB approval was obtained. 42 tissue samples from patients with head and neck squamous cell carcinoma who were previously enrolled in a Phase II clinical trial conducted at the University of Kentucky were obtained. Tissue microarrays were also obtained from US Biomax containing head and neck malignant tumors. Tumor subsites and pathology are illustrated in Table 1.
Sections of formalin-fixed and paraffin embedded samples were deparaffinized. Epitope retrieval was performed by heating in citrate buffer. Endogenous peroxidase activity was blocked by incubating the sections in hydrogen peroxide in PBS. Primary polyclonal rabbit anti sVEGFR2 antibody was added to the slides for 60 min at room temperature followed by incubation with a secondary HRP conjugated goat anti-rabbit antibody. Primary anti podoplanin (brown) and primary anti CD34 (red) were also added sequentially on a separate slide for double staining. After counterstaining of the sections with either hematoxylin or neutral red, they were embedded with mounting medium. Isotype of the primary antibodies were used for negative controls. Immunostaining was reviewed by a blinded independent pathologist.
The expression of vascular endothelial growth factor (VEGF)-A ,VEGF-B, VEGF-C and VEGF-D in both experimental and clinical models of head and neck squamous cell carcinoma (HNSCC) has been determined and correlated with conventional clinicopathologicparameters, with particular reference to cervical nodal metastasis. Varying degree of expression of markers has been shown to correlate with pathologic staging and prognosis.1 The vascular endothelial growth factor (VEGF) family of secreted proteins and their receptors are major regulators of blood vessel development (hemangiogenesis) and lymphatic vessel development (lymphangiogenesis).
VEGF acts through a complex system of receptor tyrosine kinases, which can be membrane bound or soluble. New data concerning the receptor system are still emerging, thus contributing to the complexity of the system. Recently, a soluble form of VEGFR-2, termed sVEGFR-2, which is a result of alternative splicing, has been discovered. The newly discovered spliced variant of sVEGFR-2 binds the lymphangiogenic growth factor VEGF-C and thus inhibits VEGF-C-induced activation of VEGFR-3, consequently inhibiting lymphatic endothelial cell proliferation. 2 (Figure 1) Its inactivation in murineembryos permits hyperplasia of dermal lymphatics and invasion of lymphatics into the cornea. 3 Tumor lymphangiogenesis influences the metastatic behavior of malignant cells. A correlation has been found between the downregulation of sVEGFR-2 and the malignant progression of neuroblastoma, which is characterized by lymphogenicmetastases in progressed stages. 4 These discoveries led to our investigation of the role of sVEGFR-2 in malignant tumors of the head and neck.
INTRODUCTION
MATERIALS AND METHODS
1. P Oc, Rhys-Evans P, Eccles SA. Expression of vascular endothelial growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis. Cancer 2001;92:556-68.2. Pavlakovic H, Becker J, Albuquerque R, Wilting J, Ambati J. Soluble VEGFR-2: an antilymphangiogenic variant of VEGF receptors. Ann N Y Acad Sci 2010;1207 Suppl 1:E7-15.3. Albuquerque RJ, Hayashi T, Cho WG, et al. Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth. Nat Med 2009;15:1023-30.4. Becker J, Pavlakovic H, Ludewig F, et al. Neuroblastoma progression correlates with downregulation of the lymphangiogenesis inhibitor sVEGFR-2. Clin Cancer Res 2010;16:1431-41.5. Bolzoni Villaret A, Barbieri D, Peretti G, et al. Angiogenesis and lymphangiogenesis in early-stage laryngeal carcinoma: Prognostic implications. Head Neck 2012.6. Shibata MA, Ambati J, Shibata E, et al. The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model. BMC Med 2010;8:69.
DISCUSSION/CONCLUSIONS
RESULTS
REFERENCES
Figure 1. Schematic presentation of the VEGF ligands and their interaction with membrane-bound and soluble VEGF receptors. Inhibiory effects are exerted by sVEGFR-1 (anti-hemangiogenesis) and sVEGFR-2 (anti-lymphangiogenesis). 2
ABSTRACT
CONTACT
TABLES AND FIGURES Cancer Subsite/Pathology
N=Number of TissueSamples
(Total 110)SCCA
Oral CavityOropharynxLarynxHypopharynxNasopharynx
10316206151
Thyroid CancerPapillaryMedullaryFollicular
7421
Table 1. Tissue samples examined by subsite/pathology.
Figure 2: a. H and E staining of a moderately differentiated SCCA of the larynx at 10X power. Arrow at invasive front of malignant tumor.b. IHC for sVEGFR-2 (blue) at 10X power with positive peritumoralexpression and positive expression in vascular endothelial cells. c. IHC for podoplanin(brown)/CD34(red) at 10X power showing high vascular endothelial cell density correlating with expression of sVEGFR2, and very low density of lymphatic vessels.
The histopathalogic assessment of lymphatic and blood vessel density has prognostic significance in head and neck cancers. Villaret et al recently demonstrated that the presence of lymphatic vessels in peritumoral fields had an impact on local and locoregional recurrence in early-stage laryngeal carcinoma. 5
Several growth factors in the VEGF family are important for peritumoral lymphatic and blood vessel growth in head and neck cancers. We suspect that sVEGFR-2 may play a key role in the regulation of peritumoral lymphatic invasion. Shibata et al demonstrated that gene therapy with alternative splicing variant esVEGFR-2 significantly suppresses tumor growth and lymph node metastasis in a mouse mammary cancer model.6
• IHC demonstrated and inverse correlation of sVEGFR-2 expression and lymphatic vessel density in head and neck cancers.
•Papillary thyroid cancer has dramatic expression of sVEGFR-2 unlike normal thyroid.
•The expression pattern of this receptor supports a potential role in lymphatic spread of malignant tumors of the head and neck.
•The precise function of sVEGFR-2 in metastasis and disease progression in these tumors should be evaluated further.
Figure 3: a. Poorly differentiated nonkeratenizing squamous cell carcinoma of the tonsil at 10X stained for podoplanin (brown) and CD34 (red) showing abundant peritumoral lymphatics. b. Very weak expression of sVEGFR-2 (blue) correlating with increased lymphatic vessel density.
Figure 4: a. Normal thyroid tissue at 10X power with minimal positive sVEGFR-2 staining.b. Intense sVEGFR-2 staining in papillary thyroid cancer. c. IHC for podoplanin/CD34 showing minimal expression of lymphatic vessels.
a b c
a b
a b c
Podoplanin\
CD34\
sVEGFR-2\
CD34\
\podoplanin
Adapted from: http://www.rosenthallab.com/gallery/images/VEGF_VEGFR.jpeg.
