Clinical Study On The Relation Of Lymphatic Microvessel Marked By Monoclonal Antibody D2-40 And The Expressions Of VEGF-A/VRGF-C In The Tissues Of Human Breast Cancer

Background and Objective:The spread and metastasis of tumor cell were critical cases involved in the decease of breast cancer, which was a malignant disease with complicated and systemic features. The tumor growth and metastasis mostly depended on the angiogenesis (blood vessel growth) and lymphangiogenesis (lymph vessel growth), which were continual and uncontrolled. Studies suggested that when the size of tumor tissue was over 0.2cm or 0.3cm, new vessel would be generated to provide essential oxygen and nutrition under the control effect of many positive or negative factors. Vascular endothelial growth factor A (VEGF-A) appeared to be one of the most critical factor for blood vessel development in breast cancer. VEGF-C was an importance factor for lymphatic vessel development, correlating with lymphangiogenesis in tumor tissues. The new blood capillary was similar to the new lymphatic vessels in many factors. In the past, lymphangiogenesis studied have been limited because that the lacking of specific lymphatic endothelial markers maked it difficult to distinguish the blood vessel and the lymphatic vessel on morphology, only by HE staining technique. These years, studies have found some molecules marking lymphatic endothelial, such as VEGFR-3 and LIVE-1, providing possible method for the research on lymph vessel. But several studies have conformed that these makers did not only express on the lymphatic vessels, but also expressed on some endothelial cell of blood vessels, that lowered the value of distinguishing the tumor blood vessels and lymphatic vessels. It was reported by Marks in 1999 that D2-40 was a monoclonal antibody that reacts with a 40-kd oncofetal antigen, better known as M2A antigen, that is expressed in fetal testis and germ cell.The new studies in 2006 reported that D2-40 was usually reported to detect lymphatic vessels selectively in the tissues of gastric cancer, prostate cancer and malignant melanoma by reacting selectively with thefixed antigen on lymphatic endothelium.,without reaction with mature lymphatic vessel and capillary vessel endothelium. But it was seldom studied in breast cancer.This study aimed to observing the morphologic characteristics of micro-lymphatic vessels marked by D2-40 in breast cancer by contrasting the capillary vessel marked with CD31; analyze the expression of VEGF-A, VEGF-C protein and VEGF-C mRNA and the correlation with clinical pathology factors by semiquantitative method; probe relationship of the dose of lymphatic micovessels (LMVD) marked by D2-40 with the the expression of VEGF-A /VEGF-C and the situation of lymphatic nodes metastasis. And this may provide theory basement and experiment evidence assisting the study on prognosis of breast cancer and the therapy method to prevent the angiogenesis and lymphangiogenesis.Method: 102 cases of breast cancer and 25cases of breast fibroadenoma were collected, which were all embedded in paraffin after surgery from Jan 2003 to Oct 2006. The expression of VEGF-A , VEGF-C and VEGF-C mRNA were assessed by methods of SP immunohistochemistry and hybridization in situ. The results of staining were assessed and analyzed by the semiquantitative method. The expressions of the positive micovessels marked by D2-40 and CD31 in 102 cases with breast cancer were evaluated by single-labeling or double-labeling immunohistochemistry (positive color is amethyst in BCIP/NBT coloration system and bright red in AEC coloration system ). The dose of lymphatic micovessels (LMVD) marked by D2-40 was counted.Al statistical calculations were carried out using the statistical software named Statistical Product and Services Solutions(SPSS) 14.0. The Student t-test and Chi-square test were used to analyze data.Result:In 102 breast cancer cases, D2-40 only marked the lymphatic vessels which had thin wall, irregular lumens and shapes, but without CD31 expressing. The positive rate of D2-40 was 76.5%.The mean LMVD was 21.6 lymphatic microvessels per×100 field visually. And the LVD at peripheral region of breast cancer was significant higher thanM LVD at intratumor region(p=0.000). The Layered analysis according to the number of metastasis nodes suggested that the number of lymphatic nodes infiltrated by tumor cells correlated with the dose of d2-40 positive lymphatic micovessels at peripheral region of breast cancer, but did not correlated with the LMVD at intratumor region(p>0.05).VEGF-A and VEGF-C antigen were mostly observed in the cytoplasm or membrane of cancer cells, and seldom in the cytoplasm of endothelium cell in thecancer stroma by the semiquantitative method of immunohistochemistry. Respectively, the positive rate were 64.7% and 55.9%, which were obviously higher than that of control group(P<0.05). VEGF-C mRNA mostly expressed in the in the cytoplasm of cancer cells, and positive rate was 59.8%, equal to the protein translational level(P<0.05). The expressions of VEGF-A and VEGF-C correlated with tumor size, status of lymph node,clinical stage (P < 0.05),but not with the age and and histology stage (P> 0.05). The expressions of VEGF-C mRNA correlated with status of lymph node and clinical stage (P < 0.05). And the expressions of VEGF-A , VEGF-C by ICH and VEGF-C mRNA by HIS had a positive correlation with the expressions of C-erbB-2 and 53(P<0.05).VEGF-C protein and VEGF-C mRNA both correlated with the dose of positive lymphatic vessels marked with d2-40((P<0.05). Along with the increasing of VEGF-C/VEGF-C mRNA expression, the dose of positive lymphatic vessels increased gradually, and the scale of functional lymphatic vessels at peripheral region of breast cancer also increased(P<0.05).Conclusions: In human breast cancer, new monoclonal antibody D2-40 had affinity with the lymphatic endothelium cell at peripheral region of breast cancer , and the number of lymphatic nodes infiltrated by tumor cells correlated with the dose of d2-40 positive lymphatic micovessels. So,the monoclonal antibody D2-40 may become a selective marker of lymphatic endothelium in human breast cancer. The positive lymphatic vessels, especially at the peripheral region, may provide a lymphatic metastasis road of breast cancer.We found that the expression of VEGF-C mRNA was equal to the protein translational level by the semiquantitative method, and the expressions of VEGF-A and VEGF-C correlated with tumor size, status of lymph node , clinical stage and the expression of P53, C-erbB-2.This suggested that oncogene C-erbB-2 and anti-oncogene P53 may up-regulation the expressions of VEGF-A and VEGF-C, playing an important role in the development of breast cancer. So VEGF-A and VEGF-C may become a biology marker to judge the prognosis of breast cancer.This study also found that the dose of d2-40 positive lymphatic micovessels was correlated with the overpression of VEGF-C in the VEGF-C protein and VEGF-C mRNA levelas.