D2-40 is a commercially available mouse monoclonal antibody directed against human podoplanin, which is a mucin-type transmembrane protein present in lymphatic endothelial cells [21
]. It is a specific marker for lymphatic endothelium and has proven valuable in distinguishing lymph vessels from blood vessels and detecting lymphatic invasion in various malignant neoplasms [22
]. It has been reported that the basal epithelial cell layers of the epidermis and the myoepithelial cells of human breast tissue, prostate and salivary gland tissue can also be stained by D2-40; but the morphology of these cells are distinct from the characteristic morphology of lymphatic endothelium [25
In the present study, D2-40 staining was mainly located in the cytoplasm or cell membrane of the lymphatic endothelial cells, whilst the tumor cells and blood vessel endothelium had no D2-40 staining. The ductal cancer in situ (DCIS) foci displayed weak residual discontinuous myoepithelial staining. We found that the tumor lymphatic vessel invasion (LVI) was established when at least one tumor cell cluster ("tumor emboli") was clearly visible inside a D2-40 positive lymph vessel, according to criteria established by Hasebe et al. [27
]. Representative examples of the staining obtained in these studies are shown in Figure .
Figure 1 D2-40 labelled LMVD with IHC staining (200×). A-C Invasive ductal breast cancer: A. Stained lymphatic microvessels within a peripheral tumor (long arrow) with dilated tubes. Capillary vessel (short arrow) and tumor cell clusters were not stained; (more ...)
For LMVD assessment, we found that the mean number of visual microvessels was 12.95 ± 1.73 (range 1.04-42.10) lymphatic microvessels per 200× field (LMV per 200× field) in the 41 breast cancer specimens. Within the same subjects, the mean LMVD in normal breast tissue and lymph nodes were 2.24 ± 0.18 (range 1.00-5.03) and 5.49 ± 0.52 (range 0-32.01) LMV per 200× field, respectively. The differences observed in the LMVD between the three tissue types were statistically significant (Friedman test, P < 0.01). Dunnett's post-test showed that the mean LMVD in tumor tissues was higher than that observed in the normal breast tissues (P < 0.01) and lymph nodes (P < 0.01). However, LMVD differences between the normal tissue and the lymph nodes were not statistically significant (P > 0.05). The mean LMVD in the lymph nodes of the metastasis group was higher than that in the non-metastasis group (Mann-Whitney test, P = 0.003). Differences in the LMVD in the metastasis and non-metastasis groups was not found to be statistically significant in the cancer tissues, nor the normal breast epithelium (P = 0.409 and P = 0.377, respectively) (Table ).
Comparison of LMVD between the metastasis and non-metastasis groups
Our data show that LMVD in the breast cancer tissue was significantly higher than that in the normal tissues. We also found that LMVD in the lymph nodes with metastasis was higher than that without metastasis. Lymphatic vessels have discontinuous basement membranes and lack tight interendothelial junctions. Hence, it is possible that lymphatic vessels might be easier for tumor cells to enter than blood vessels [2
], and that LMVD enhancement could significantly increase the potential for tumor cells to invade the surface of lymphatic vessels [28
ECM1 and VEGF-C mRNA and protein expression
We used real-time RT-PCR to determine the mean relative expression levels of ECM1 mRNA (Table ) and VEGF-C mRNA (Table ) in breast cancer specimens, normal epithelia and lymph nodes from the patients. Differences in the ECM1 mRNA expression levels among these tissues were statistically significant (one-way ANOVA, P < 0.01) (Table ). Multiple comparison analysis (Tukey's test) showed that ECM1 mRNA expression levels in the breast cancer samples were significantly higher overall, compared to the normal tissue (P < 0.05) or to the lymph nodes (P < 0.05); however, no differences were found between normal tissues and lymph nodes (P > 0.05). In general, the results of VEGF-C mRNA expression among the three tissue types showed the same trends as those obtained for ECM1 (Table ).
ECM1 expression in breast cancer specimens, normal epithelium and lymph nodes
VEGF-C expression in breast cancer specimens, normal epithelium and lymph nodes
We found that ECM1 was mainly located in the cytoplasm of the cells, with scant staining noted on the cell membrane or the stroma; no nuclear staining was seen. Within the draining lymph nodes, ECM1 staining was specific for the metastatic cancer cells and occurred primarily in their cytoplasm. Notably, in the normal breast epithelium, there was little or no staining at all. Representative examples of the ECM1 staining patterns are shown in Figure . The ECM1 positive staining rates among tumor tissue (31, 75.6%), normal breast tissue (4, 9.8%) and lymph nodes (13, 31.7%) were significantly different (χ2 = 39.08, P < 0.01). Multiple comparison (χ2 division, α = 0.0125) analysis showed the ECM1 positive staining rate in cancer tissue was higher than in normal tissue and the lymph nodes (P < 0.001). Differences in the ECM1 positive staining rate between normal tissues and lymph nodes were not significant, however (P > 0.0125, Table ).
Figure 2 Representative IHC staining of ECM1. A-D: ECM1 was detected primarily in the cytoplasm of breast cancer cells (200×): A. ECM1 negative; B. + ECM1 staining; C. ++ ECM1 staining; D. +++ ECM1 staining. E-F normal breast epithelium (200×): (more ...)
In breast cancer cells, VEGF-C staining was observed in the cytoplasm; such staining was often more intense at the invasive edge or in the intraductal component (Figure ). In contrast, very little or no staining was observed in normal ductal epithelium. According to the criteria used to evaluate the immunostaining, VEGF-C expression in the cancer specimens (33/41, 80.5%) was higher than that in the normal tissues and lymph nodes (19/41, 46.3% and 15/41, 36.6%, respectively; P < 0.01). However, the difference in the values obtained from the normal epithelium and the matched lymph nodes was not significant (P > 0.0125, Table ).
Figure 3 Representative IHC staining of VEGF-C (200×). A-D: VEGF-C staining was mainly located in the cytoplasm of breast cancer cells: A. VEGF-C negative; B. + VEGF-C staining; C. ++ VEGF-C staining; D. +++ VEGF-C staining. E-F normal breast epithelium: (more ...)
Differences in ECM1 and VEGF-C expression between the metastatic and non-metastatic groups
Statistically significant differences in ECM1 mRNA expression in tumor tissues between the metastasis group and the non-metastasis group were not found (Mann-Whitney test, P = 0.314); no statistically significant differences were found for the normal breast epithelium tissues or the lymph nodes, between the metastasis and non- metastasis groups (P = 0.754 and P = 0.178, respectively; Table ). The results of VEGF-C mRNA expression between the metastasis group and the non-metastasis group showed similar trends as those observed for ECM1 (Table ).
Comparison of ECM1 expression levels between the metastasis- and non-metastasis groups
Comparison of VEGF-C expression levels between the metastasis- and non-metastasis groups
Differences in the ECM1 positive staining rates between metastatic (15/19, 78.9%) and non-metastatic (16/22, 72.7%) tumors were not significant (P > 0.05). Similar results were obtained for the normal tissue groups (metastatic: 3/19, 15.8%; non-metastatic:1/22, 4.55%) (Fisher's exact test, P > 0.05, Table ). The ECM1 positive rate in the lymph node metastases was 68.4% (13/19). Likewise, no differences were apparent in VEGF-C expression in the three tissues (i.e., cancer tissue, normal tissue and lymph nodes) between the metastasis and non-metastasis groups (Fisher's exact test, P > 0.05, Table ).
In the metastatic group, the difference in the ECM1 positive staining rate between the primary tumor (15/19, 78.9%) and the metastatic focus (13/19, 68.4%) was not statistically significant (P > 0.05, Table ). In two of the cases, we found that the primary tumor was ECM1 negative, whilst ECM1 was expressed in the corresponding lymph node metastases. Similarly, the difference in the VEGF-C positive staining rate between the primary tumor and the metastatic focus was not significant (P > 0.05, Table ). In addition, the VEGF-C staining rates in the two tissue types (i.e. cancer tissues and lymph nodes) were both 68.4% (13/19), although the cases that had positive staining did not all coincide with each other.
ECM1 and VEGF-C expression profiles and clinical characteristics
We evaluated whether correlations exist between expression of ECM1 or VEGF-C and the clinicopathological characteristics of the disease, (i.e. age; histological type or grade; tumor size; lymph node, ER, and PR status and Her-2/neu score). Tables and , summarize the ECM1 and VEGF-C data, respectively. ECM1 mRNA expression was not associated with any of the clinicopathological characteristics tested (Unpaired t test with Welch's correction, P > 0.05). The ECM1 protein positive rate was found to be associated with the status of the ER (χ2, P = 0.045). The ER status of patients with ECM1-positive tumors were more likely to be positive than those without ECM1 staining. However, ECM1 staining was not correlated with tumor size, lymph node status, PR status or the Her-2/neu score (Fisher's exact test, P > 0.05). We found that VEGF-C mRNA and protein expression were not associated with any of the clinicopathological characteristics tested (P > 0.05, respectively; Table ).
Correlations between ECM1 expression and clinicopathological characteristics
Correlations between VEGF-C expression and clinicopathological characteristics
Correlations between LMVD and ECM1 or VEGF-C
We found that histological sections that were ECM1-positive had higher LMVDs (Figure ). As shown in Table , differences in LMVD between the ECM1-positive cases and ECM1-negative cases were statistically significant for both the tumor tissues (Mann-Whitney test, P = 0.045), and the lymph nodes (Mann-Whitney test, P < 0.001). A positive correlation was further established between the LMVD and ECM1 staining intensity in both the breast cancer sections, and the lymph node sections (Spearman's correlation coefficient 0.347 and 0.604, respectively; P < 0.05, Table ). The correlation was not linear, however.
Figure 4 Representative examples of D2-40 and ECM1 staining in the matched sections (100×). Sections with ECM1-positive staining had higher lymphatic microvessel densities. A-D: Lymphatic microvessels labelled with D2-40. E-H: ECM1 IHC staining: Panels (more ...)
Correlation between ECM1 staining and LMVD (LMV per 200× field of vision)
Correlation between ECM1 staining intensity and LMVD (LMV per 200× field of vision)
We noted that the LMVD in the VEGF-C-positive lymph nodes was higher than that in the VEGF-C-negative ones (Mann-Whitney test, P < 0.001); LMVD in the lymph nodes was correlated with VEGF-C staining (Spearman's correlation coefficient: 0.566, P < 0.001). However, LMVD in the breast cancer specimens was not associated with VEGF-C staining (P > 0.05, Table ).
Correlation between VEGF-C staining and LMVD (LMV per 200× field of vision)
We further analysed the LMVD in breast cancer tissue and lymph nodes for both ECM1 and VEGF-C staining (Table ). The LMVD in both ECM1- and VEGF-C-positive (E+V+) tumor specimens was higher than that in both the ECM1- and VEGF-C-negative (E-V-) ones (Mann-Whitney test, P = 0.029; Figure ). Additionally, the LMVD in both E+V- and E-V+ tumor specimens was higher than in the E-V- specimens (Mann-Whitney test, P > 0.05). We also found that LMVD in E+V+ tumor specimens was higher than in the E-V+ and E+V- ones, although the differences did not reach statistical significance (Mann-Whitney test, P > 0.05). However, LMVD in the lymph nodes of the different assemblies for both ECM1 and VEGF-C staining (i.e. E-V-, E-V+, E+V- and E+V+) showed a statistically significant difference (one way ANOVA, P = 0.025), but did not show a significant tendency (Figure ).
LMVD with ECM1 and VEGF-C staining (LMV per 200× field of vision)
Figure 5 LVMD in breast cancer specimens and lymph nodes with ECM1 and VEGF-C staining. A: LMVD in both ECM1- and VEGF-C-positive (E+V+) tumor specimens was higher than in both ECM1- and VEGF-C-negative (E-V-) ones (P < 0.05). B: The LMVD within the 41 (more ...)