Study Of The Correlation Between Angiogenesis/lymphangiogenesis And HER-2 Gene And Their Prognostic Significance In Human Breast Cancer

Objective: In order to demonstrate the molecular mechanism of poor prognosis in HER-2 overexpressing breast carcinoma, the study of the correlation between angiogenesis/lymphangiogenesis and HER-2, the effect of them in the progression and prognosis was carried out. The aim of the study is to provide novel molecular markers for lymph node metastasis and prognosis in breast carcinoma. Meanwhile, to provide new clue and theory for clinic treatment of breast carcinoma patients with HER-2 overexpression.Methods: (1) By using TaqMan real-time quantitative reverse transcription polymerase chain reaction(Real-time qRT-PCR) technique and immunohistochemistry (IHC), the mRNA and protein expression of VEGF-A,-C,-D were detected in two breast cancer cell lines (SKBR-3 with HER-2 overexpression, MDA-MB-231 with HER-2 low expression) prior and post-interfered with Herceptin. (2) Real-time qRT-PCR was performed to detect mRNA expression of VEGF-A,-C,-D genes in 61 cases of primary breast infiltrating ductal carcinoma and 29 cases of benign mammary lesions. Statistical analysis were used to investigate the relationship between the expression of VEGF-A,-C,-D gene and clinicopathological variables of breast carcinoma and their effect on prognosis. (3) 160 formalin-fixed paraffin-embedded infiltrating ductal carcinomas were selected. The protein expression of VEGF-A,-C,-D, microvessel density (MVD), lymphatic vessel density (LVD), microvessel invasion (MVI) and lymphatic vessel invasion (LVI) were detect by IHC. Statistical analysis were performed to investigate the relationship between these markers and others clinicopathological parameters of breast carcinomas. (4) In these samples, the gene amplification status and protein expression of HER-2 were detected by Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC). The correlation between HER-2 gene and angiogenesis/lymphangiogenesis factors was studied by statistical analysis. Logistic regression model was used to investigate the relationship between all markers and lymph node metastasis. Univariate and Cox's multivariate regression analysis were performed to evaluate the effect of all markers on prognosis.Result: (1) The mRNA expression of VEGF-A,-C,-D in SKBR-3 cell interfered with Herceptin was 0.247, 0.519, 0.930 of that without Herceptin treatment. VEGF-A,-C protein expression of SKBR-3 cells detected by IHC was strongly positive, VEGF-D was moderately positive without Herceptin treatment. After interfered with Herceptin, VEGF-A,-C protein expression Of SKBR-3 cells reduced to moderately positive , while VEGF-D expression had no obvious change. After interfered with Herceptin, the mRNA and protein expression of VEGF-A,-C,-D in MDA-MB-231 cell had no obvious change. (2) In human primay breast cancer, the mRNA expression of VEGF-A,-C was significantly higher than that in benign mammary lesions ((4.19 vs 2.24, 4.29 vs 3.69) (P<0.01). The percentage of VEGF-D mRNA expression in breast cacinoma was 73.77%, while it was 51.72% in benign mammary lesions(P<0.05). The overexpression percentage of VEGF-A,-C,-D was 54.4%, 56.3%, 47.5%, respectively. But in most of prara-tumor benign mammary tissue, VEGF-A,-C,-D was negative or weakly positive. The mRNA and protein expression of VEGF-A,-C,-D was not associated with other clinicopathological parameters of breast carcinoma, such as the age of patients, size of tumors, clinical stage and histological grade of infiltrating ductal carcinoma(P>0.05). (3) In primary breast cancer, There was positive correlation between VEGF-A protein expression and MVD(r=0.268, P<0.01), between VEGF-C protein expression and LVD(r=0.248,P<0.01). MVD was associated with tumor size(P<0.05). LVD was associated with lymph node metastasis and TNM stages(P<0.05). (4) The gene amplification and protein expression of HER-2 were positively correlated to the protein expression of VEGF-A,-C (r=0.205, r=0.253, r=0.203, r=0.215, P<0.05) . HER-2 protein expression was positively correlated with VEGF-D protein expression(r=0.184, P<0.05), but there was no correlation between HER-2 gene amplification and VEGF-D protein expression(r=0.132, P>0.05). HER-2 gene had no significant correlations with MVD, MVI, LVD, LVI(P>0.05). (5) Univariate analysis indicated that gene amplification and protein expression of HER-2 were associated with lymph node metastasis (P_uni<0.05). Uni- and Multivariate analysis all indicated that LVD and LVI were associated with lymph node metasasis (P_uni<0.05 , P_mult<0.05). The ratio of VEGF-D/VEGF-C mRNA expression was associated with lymph node metastasis, it was significantly lower in lymph node metastasis positive cases than that in negative ones(0.93 vs 1.58)(P_uni<0.05, P_mult<0.1). (6) Univariate analysis indicated that gene amplification and protein overexpression of HER-2, protein overexpression ofVEGF-A,-C, high MVD, MVI, high LVD, and LVI was associated with patient's worse desease-free survival(P_uni<0.05). Multivariate analysis indicated that besides TNM stages and lymph node metasasis, LVI is the only independent prognostic factor of breast cancer (P_mult<0.05).Conclusion: (1) VEGF-A,-C,-D overexpression is a common phenomenon in human primary breast carcinomas. They all play roles in breast cancer's carcinogenesis. VEGF-A is an important angiogenic factor. While, VEGF-C is an important lymphangiogenic factor. (2) The ratio of VEGF-D/VEGF-C mRNA, LVD and LVI can predict lymph node metastasis of breast cancer. There may be antagonistic interaction between VEGF-C and VEGF-D. (3) The markers of angiogenesis/ lymphangiogenesis(the expression of VEGF-A,-C, MVD, LVD, MVI, LVI) all have effect on pateint's prognosis. But only LVI is an independent factor of prognosis. It may better predict prognosis in breast cancer patients than other markers. (4) In SKBR-3 cells, HER-2 gene regulates VEGF-A,-C expression, but not VEGF-D expression. Through up-regulation of VEGF-A,-C expression, HER-2 induces angiogenesis/lymphangiogenesis, promotes prolification and metastasis of primary breast cancer. Angiogenesis/lymphangiogenesis mediated by HER-2 gene is an important molecular mechanism of poor prognosis in breast cancer patients.