Determination Of Lymphatic Vessel Density And VEGF-C Expression In Breast Cancer Patients Receiving Neoadjuvant Chemotherapy

BACKGROUND & OBJECTIVE: Recently, increasing attentions have been paid to the role of lymphangiogenesis in tumor metastasis via lymphatics, though its exact clinical relevance by far remains controversial. In the present study, we measured lymphatic vessel density (LVD) in breast cancer and compared the result with that in benign breast diseases to gain more insight into the role of lymphangiogenesis in tumor development and metastasis. Moreover, the changes of tumor apoptosis, VEGF-C expression, as well as peri- or intratumoral LVD in breast cancer patients before and after neoadjuvant chemotherapy were investigated with an attempt to illustrate the influence of cytotoxic agents on tumor cells, lymphangiogenesis and LVD. METHODS: A total of forty-five patients with primary breast cancer, proven by core needle biopsy, underwent three cycles of neoadjuvant DE (docetaxel and epirubicin, n=25), or CEF (cyclophosphamide, epirubicin, and 5-fluorouracil, n=20). Breast-conservative surgery or modified mastectomy was performed 14-21 days after the final cycle of chemotherapy. Lymphatic vessels in cut-off and surgically resected specimens were identified through immunohistochemical staining for the lymphatic endothelial specific marker podoplanin. The ratios of podoplanin-positive lymphatics and the peri- or intratumoral LVD were measured separately. VEGF-C expression in tumor tissues was analysed by immunohistochemistry. Apoptosis of tumor cells before and after neoadjuvant chemotherapy was examined using the terminal deoxynucleotidyl transferase-mediated dTUP nick end labeling (TUNEL) technique. The LVD and VEGF-C expression in 26 specimens of benign breast diseases was also studied with the same methods. RESULTS: Of the 86 tumor samples, podoplanin-stained peri- or intratumoral lymphatics were identified in 61 (70.9%) and 17 (19.8%) cases, respectively. The mean LVD at the periphery of tumor before chemotherapy was 9.56, significantly higher than that in perilobular tissues in patients with benign diseases (5.43, P = 0.012). The mean peritumoral LVD after chemotherapy was 3.29, significantly lower than that before therapy (P < 0.001). The mean intratumoral LVD before and after chemotherapy was 1.90 and 0.90, respectively; no significant difference was found between them (P = 0.055). The mean peritumoral LVD in lymph node-positive or node-negative patients was 11.82 and 6.95, respectively, implying a positive relationship between peritumoral LVD and axillary lymph node status before treatment (P = 0.0048). The ratio of positive VEGF-C expression in tumor and benign disease samples was 75.61% and 46.15%, respectively, with a significant difference (P=0.014). VEGF-C expression was positively correlated with lymph node status. No evident change in VEGF-C expression was detected before and after treatment (P =0.123). Apoptosis index before and after neoadjuvant chemotherapy was 2.81% and 19.87%, respectively, with a statistically significant difference (P < 0.001). CONCLUSION: Compared to benign disease, breast tumor exhibits sufficiently higher lymphatic vessel counts in the peripheral stromata. Lymphangiogenesis does exist inside the tumor bodies. Chemotherapy substantially reduces the number of lymphatic vessels at the tumor periphery, though the reduction is not distinct within tumor. The peritumoral LVD is associated with lymph node involvement. The degree of LVD reduction is positively correlated with the response of breast cancer to neoadjuvant chemotherapy. The expression of VEGF-C is much stronger in breast tumor, as compared with benign disease, and it reduced after chemotherapy, whereas the reduction is not evident. Apoptosis of tumor cell may be effectively induced by preoperative chemotherapy.