| Objective: The carcinoma of larynx is very common malignant neoplasm in otolaryngology and head and neck surgery. Today surgery assisted by chemotherapy and biological threatment is still the best way to cure the cancer. Despite the continuous improvement of comprehensive treatment method, but still 5-year survival rate of laryngeal cancer was not significantly increased, the main reason for treatment failure is local recurrence and lymph node metastasis. Therefore, understanding the mechanism of lymphatic metastasis has important clinical significance in diagnosis and treatment of laryngeal cancer.Vascular endothelial growth factor (VEGF) is a kind of heparin binding growth factor. VEGF-C, a new VEGF homolog, is the most important known lymphangiogenesis inducing factor, can selectively promote the lymphatic control proliferation and expansion, enhance lymphatic permeability, imporve the growth of related lymphatic vessels around the tumor, and promote is metastasis and spread of tumor cells via lymphatic vessels.VEGFR-3 belonged to the family of receptor tyrosine kinase, was the first molecular marker cloned in lymphatic endothelial cells, and was specific VEGF receptor. Through a variety of pathological and physiological approach, VEGF-C reacts to its receptor VEGFR- 2 and VEGFR-3 reaction, stimulating hyperplasia of blood vessels and lymphatic. D2-40 was a newly discovered antigen-specific monoclonal anti-M2A antibody, and was considered the most specific marker of new lymphatic endothelial cells.In this study, immunohistochemical staining and flow cytometry were used to detect VEGF-C, VEGFR-3 and D2-40 from the qualitative and quantitative aspects, to explore the expression of three types of proteins in laryngeal carcinoma, providing new ideas for understanding the mechanism of lymphatic metastasis of laryngeal cancer and the its prognosisMethods:Materials were collected from patients with laryngeal cancer admitted in Department of Otolaryngology, Head and Neck Surgery from January 2008 to December 2009 at the Fourth Hospital of Hebei Medical University. There were 40 cases of laryngeal carcinoma, and 40 cases of the corresponding para-carcinoma tissues (at least 0.5cm from the tumor margin), and over the same period 19 cases were taken, in which normal laryngeal mucosa of laryngeal diseases with non-surgical treatment served as controls. SPSS 13.0 was applied to analyze the results of experiment.P≤0.05 indicating significant difference.P≤0.01 indicating very significant difference.Results:1 The expression of VEGF-C protein1.1 The results detected by IHC:The expression of VEGF-C protein in laryngocarcinoma,para-carcinoma and normal laryngeal mucosa tissues by S-P immunohistochemical methods.The expression of VEGF-C protein descent from laryngocarcinoma to para-carcinoma and normal laryngeal mucosa tissues , there is statistic significance(P<0.01). The expression of VEGF-C protein in laryngocarcinoma is obviously higher than those in para-carcinoma(P<0.01)and in normal laryngeal mucosa(P<0.01)tissues respectively.No significant difference was found between the expression of para-carcinoma and normal laryngeal mucosa tissues(P>0.05). Expression of VEGF-C protein was correlated with lymph node metastasis(P<0.01), clinical stage(P<0.01) and patients'clinical classification(P<0.01),but that wasn't correlated with pathological grading of tumor, tumor size, smoking history, patients'age and sex(P>0.05).1.2 The results detected by FCM: The quantitative expression of VEGF-C protein in laryngeal carcinoma tissues ,para- carcinoma and normal laryngeal mucosa tissues is statistic significance(P<0.01). The quantitative expression of VEGF-C protein in laryngeal carcinoma tissues is obviously higher than those in para-carcinoma(P<0.01)and in normal laryngeal mucosa(P<0.01)tissues respectively. No significant difference was found between the expression of para-carcinoma and normal laryngeal mucosa tissues(P>0.05). Expression of VEGF-C protein was correlated with lymph node metastasis (P <0.01),and clinical stage (P<0.01)and patients'clinical classification, ,but that wasn't correlated with pathological grading of tumor, tumor size, smoking history, patients'age and sex(P>0.05).2 The expression of VEGFR-3 protein2.1 The results detected by IHC: The expression of VEGFR-3 protein in laryngocarcinoma ,para-carcinoma, normal laryngeal mucosa tissues by S-P immunohistochemical methods. The expression of VEGFR-3 protein descent from laryngocarcinoma to para-carcinoma and normal laryngeal mucosa tissues , there is statistic significance(P<0.01). The expression of VEGFR-3 protein in laryngocarcinoma is obviously higher than those in para-carcinoma(P<0.01)and in normal laryngeal mucosa(P<0.01)tissues respectively.No significant difference was found between the expression of para-carcinoma and normal laryngeal mucosa tissues(P>0.05). Expression of VEGFR-3 protein was correlated with lymph node metastasis (P<0.01),clinical stage(P<0.01) and patients'clinical classification of tumor (P<0.05),but that wasn't correlated with pathological grading of tumor, tumor size, smoking history, patients'age and sex(P>0.05).2.2 The results detected by FCM: The quantitative expression of VEGFR-3 protein in laryngeal carcinoma tissues ,para- carcinoma and normal laryngeal mucosa tissues is statistic significance(P<0.01). The quantitative expression of VEGFR-3 protein in laryngeal carcinoma tissues is obviously higher than those in para-carcinoma(P<0.01)and in normal laryngeal mucosa(P<0.01)tissues respectively. No significant difference was found between the expression of para-carcinoma and normal laryngeal mucosa tissues(P>0.05). Expression of VEGFR-3 protein was correlated with lymph node metastasis(P<0.05),clinical stage(P<0.05),and patients'clinical classification(P<0.01) of tumor,but that wasn't correlated with pathological grading of tumor, tumor size, smoking history, patients'age and sex(P>0.05).3 The expression of D2-40 protein3.1 The results detected by IHC: The expression of D2-40 protein in laryngeal carcinoma,para-carcinoma, normal laryngeal mucosa tissues by S-P immunohistochemical methods. The expression of D2-40 descent from laryngocarcinoma to para-carcinoma and normal laryngeal mucosa tissues, there is statistic significance(P<0.01). The expression of D2-40 protein in laryngocarcinoma is obviously higher than those in para-carcinoma(P<0.01)and in normal laryngeal mucosa(P<0.01)tissues respectively.No significant difference was found between the expression of para-carcinoma and normal laryngeal mucosa tissues(P>0.05). Expression of D2-40 was correlated with lymph node metastasis (P<0.05),clinical stage(P<0.05) and patients'clinical classification of tumor (P<0.05),but that wasn't correlated with pathological grading, tumor size, smoking history, patients'age and sex(P>0.05).3.2 The results detected by FCM: The quantitative expression of D2-40 protein in laryngeal carcinoma tissues ,para- carcinoma and normal laryngeal mucosa tissues respectively , there is statistic significance(P<0.01). The quantitative expression of D2-40 protein in laryngeal carcinoma tissues is obviously higher than those in para-carcinoma(P<0.01)and in normal laryngeal mucosa(P<0.01)tissues respectively. No significant difference was found between the expression of para-carcinoma and normal laryngeal mucosa tissues(P>0.05). Expression of D2-40 was correlated with lymph node metastasis(P<0.01),clinical stage(P<0.01)and patients'clinical classification of tumor (P<0.01),but that wasn't correlated with pathological grading of tumor, tumor size, smoking history, patients'age and sex(P>0.05).4 There is multiple linear regression relationship in the expression of VEGF-C,VEGFR-3 and D2-40 in laryngeal carcinoma, and it is a positive correlation. The FI of D2-40 increase along with that of VEGF-C and VEGFR-3 increases, The multiple linear regression equation is YD=0.753XC+0.466XR-0.430(F=9.973,P=0.001).Conclusions:1 VEGF-C, VEGFR-3 and D2-40 protein were expressed in laryngeal squamous cell carcinoma, laryngeal cancer tissue and normal mucosa tissue.2 The quantitative and qualitative expression of VEGF-C, VEGFR-3 and D2-40 protein in laryngeal squamous cell carcinoma was significantly higher than that of para-carcinoma tissue and normal laryngeal mucosa, which may be related to the development of laryngeal cancer.3 VEGF-C, VEGFR-3 and D2-40 protein expression in laryngeal carcinoma was relevant to lymph node metastasis clinical stage, and clinical type while irrelevant to pathological grade, tumor size, smoking, age and sex . Suggest These three may possible be relevant to laryngeal cancer development and metastasis.4 Laryngeal carcinoma D2-40 protein and VEGF-C, VEGFR-3 expression was positively correlated.
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