Expression Of VEGF-C And VEGF-D And Their Impact On Lymphangiogenesis And Lymphatic Metastasis In Breast Carcinoma

Breast carcinoma is one of the most common female malignant tumors. The incidence of breast carcinoma shows a gradual upward trend in the world. It has ranked No.1 in China's big cities. Invasion and metastasis of tumor cells is a major cause of death in patients with carcinoma. The lymphatic system plays an important role in the process of invasion and metastasis. To develop an in-depth understanding of the metastasis of solid tumors, it is of great significance to study the mechanism of how tumor cells spread through the lymphatic system. Research on the mechanism of lymphatic metastasis has developed slowly due to the lack of clear lymphangiogenesis factors and markers, In recent years, as tge lymphatic endothelial growth factor and lymphatic endothelial-specific markers have been found, there comes the possibility to conduct the study of lymphangiogenesis and its relationship with the proliferation of lymphatic metastasis. D2-40, a marker specifically reflecting lymphatic instead of vascular, is a newly lymphatic endothelial cell-specific marker which can clearly distinguish of lymphatic and vascular of the tissue.Vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor D (VEGF-D) are the two new members of the VEGF family, which can be combined with VEGFR-2 and VEGFR-3 respectively to regulate the physiological functions of lymphatic and vascular. A few latest studies have indicated VEGF-C and VEGF-D can induce lymphangiogenesis and stimulate the proliferation and migration of lymphatic endothelial cell. VEGF-C and VEGF-D has aroused a growing attention among researchers. However, so far there has been little research on the relationship between VEGF-C and VEGF-D and their impact on lymphangiogenesis and lymphatic metastasis in breast carcinoma. Aimed to explore the mechanism of how VEGF-C and VEGF-D affecting the lymphangiogenesis and lymphatic metastasis of breast carcinoma, an immunohistochemical study was made of both breast carcinoma tissues and adjacent normal tissue to detect VEGF-C, VEGF-D and D2-40.OBJECTIVEThis research is aimed to observe the expression status of VEGF-C, VEGF-D and D2-40 in breast carcinoma tissue, and explore the clinical pathological characteristics of these factors and the underlying mechanism, as well as the relationship among the expression of VEGF-C,-D, lymphangiogenesis and lymph metastasis of breast carcinoma.METHEDSSixty-eight cases of breast carcinoma with complete clinical and pathological data were analyzed. A wide range of raw data has been adopted, including medical record number, name, sex, age, race, home address, telephone number, tumor's location, quadrant, size of tumor, pathology number, pathological type, histological grade, clinical stage, lymph node metastasis, expression status of Er, Pr and Her-2. All the patients in these cases have undergone curative surgery without preoperative chemotherapy and radiotherapy. In order to reduce the differences among race, only cases from Han ethnic group were selected. Cases such as recurrence of breast carcinoma were excluded.Based on the corresponding paraffin blocks acquired from the department of pathology, the immunohistochemical sections were made by a two-step way of shortcut immunochemistry. Immunohistochemical staining steps are as follows. First of all, deparaffinaged the paraffin section; secondly, repaired the antigen in citrate buffer solution by microwave 25 minutes; thirdly, used the solution of peroxidase to quench the activity of endogenous peroxidase about 3 minutes in microwave, then washed the sections three times separately by Double distilled water and PBS; fourthly, the solution of antibodies to VEGF-C, VEGF-D or D2-40 was applied respectively and the sections were incubated at 37℃in electric constant temperature incubator about 1 hours, then washed the sections three times by PBS; fifthly, the solution of Goat anti-rabbit HRP polymer LgG was applied and the sections were incubated at 37℃in Electric constant temperature incubator about about 30 minutes, then washed the sections three times by PBS; sixthly, used the solution of DAB to colourate about 5minutes; At last, mild Staininged with Hematoxylin after washed with water, Hydrochloride- Alcohol differentiation about 6 seconds, after dehydration by alcohol and transparent by xylene, mounted by neutral gum. In order to ensure the unity and comparability, the solution and reaction condition was kept to be identical. If the staining result is doubtful, the experiment would be carried out again. Antibody was replaced by PBS as negative control and positive control is the known positive reaction sections.For VEGF-C and VEGF-D expression analysis, immunohistochemical reactions were assessed using a semi-quantitation method combined with the quantity scores of tumor cells in hotspots and staining intensity. LMD was assessed in the tumor slides after the immunohistochemical staining with anti-D2-40 antibody. After scanning the immunostained section at low magnification, the three areas of carcinoma with the greatest number of distinctly highlighted intratumoral lymphatic foci (hot spots) were selected by two observers at the same time. The two observers then independently evaluated the slides for LMD by counting, using X200 magnification without knowledge of patient status or stains used. Single immunoreactive endothelial cells, or endothelial cell clusters separate from other microvessels were counted as lymphatic vessels. The highest number of vessels counted was recorded and used in the statistical analysis.Statistical analysis was conducted with the adoption of the SPSS13.0. Mean differences in microvessel counts were compared with the use of paired chi-square test. Spearman correlation analysis was used to analyze the relationship between the two factors. The Fisher exact probability andχ2 tests were used to compare variables, and a value of P＜0.05 was considered to be statistically significant.RESULTS1. Both VEGF-C and VEGF-D expression in breast carcinoma were significantly higher than that in control breast tissues (P<0.001). VEGF-C staining in malignant tissues was positive in 44 of the 68 patients (64.7%). In the control group, VEGF-C was weakly stained in 6 of 68 patients (8.8%), other 60 were negative. VEGF-D staining in breast carcinoma was observed as positive in 40 of 68 patients (58.8%). In the control group, VEGF-D was evident only in 4 case (5.9%) as positive, other 64 were negative.2. VEGF-C expression was more frequently found in tumors with lymph node metastasis than in those without it (χ2=9.555, P=0.023). A significant relationship between VEGF-C expression and clinical stage (χ2=10.757, P=0.005) was also detected. There was no significant correlation between VEGF-C expression and tumor size (χ2=3.780, P=0.151), histological grade (χ2=2.332, P=0.312), hormone receptor status (χ2=2.839, P=0.417;χ2=0.614, P=0.893), Her-2 status (χ2=3.401, P=0.334). High expression of VEGF-D was significantly associated with the presence of axillary LN (χ2=9.719, P=0.021) and clinical stage (χ2=9.905, P=0.007). There was no significant correlation between VEGF-D expression and tumor size (χ2=2.294, P=0.318), histological grade (χ2=2.541, P=0.281), Her-2 status (χ2=1.622, P=0.654), hormone receptor status (χ2=0.849, P=0.838;χ2=3.283,P=0.350).3. The expression of D2-40 proteins was significantly higher in breast carcinomas than in control tissues (χ2=66.129, P=0.000). D2-40 staining in malignant tissues was positive in 62 of the 68 patients (91.2%). In the control group, D2-40 was only stained in 15 of 68 patients (22.1%).The LMD median for the 68 cases of breast carcinoma was 13.0, and the mean was 13.72±8.095. A significant correlation was found among LMD, tumor size (F=26.779, P=0.000), histological grade (F=40.246, P=0.000), lymph node metastasis (F=39.727, P=0.000), and TNM clinical stage (F=71.731, P=0.000). There was no significant correlation among LMD and ER (F=2.435, P=0.056), PR (F=1.362, P=0.355) and Her-2 (F=2.227, P=0.094).4. VEGF-C staining was negative when the mean LMD was 8.00±7229; when VEGF-C staining was "+","++" and "+++",LMD was 10.13±7.030,17.17±5.797 and 23.25±3.770, respectively. There was a significant correlation between VEGF-C expression and LMD (F=13.672, P=0.000), and the results were similar with VEGF-D. VEGF-D staining was negative when the mean LMD was 9.14±8.049; when VEGF-C staining was "+","++" and "+++", LMD was 9.09±5.563, 18.11±5.645 and 23.11±3.408, respectively. There was a significant correlation between VEGF-C expression and LMD (F=14.446, P=0.000)5. There was a significant positive correlation among VEGF-C expression, LMD (r=0.565, P<0.01), and lymph node metastasis (r=0.626, P<0.01), and the results were similar with VEGF-D. There was a significant positive correlation among VEGF-D expression, LMD (r=0.510, P<0.01), and lymph node metastasis (r=0.600,P<0.01).CONCLUSION1. Both VEGF-C and VEGF-D expression in breast carcinoma are significantly higher and are detected a significant relationship with clinical stage and lymph node metastasis. VEGF-C and VEGF-D play an important role in occurrence and development in breast carcinoma.2. Lymphangiogenesis is mainly distributed in the tumor marginal zone in breast carcinoma. A prominent correlation was found among LMD, tumor size, histological grade, lymph node metastasis, and clinical stage. Lymphangiogenesis may be related to breast cancer invasion and metastasis.3. By inducing peritumoral lymphangiogenesis, VEGF-C and VEGF-D may increase the risks of lymph node metastasis in breast carcinoma.4. VEGF-C, VEGF-D and LMD may serve as predictors of lymph node metastasis and prognostic factors in breast carcinoma.