Lymphangiogenesis in breast carcinoma is present but insufficient for metastatic spread

Introduction: The lymphatic vasculature is an important route for the metastatic spread of human cancer. However, the extent to which this depends on lymphangiogenesis or on invasion of existing lymph vessels remains controversial. The goal of this study was to investigate the existence of lymphangiogenesis in invasive breast carcinoma: by measuring the lymphatic vessels density (LVD) and lymphatic endothelial cell proliferation (LECP) and their correlation with various prognostic parameters in breast cancer, including lymphovascular invasion (LVI).Methods: Lymphatic vessels density was investigated in 75 specimens of invasive breast carcinoma by immunostaining for D2-40 using the Chalkley counting method. Endothelial proliferation in lymphatic vessels was analyzed by dual-color immunohistochemistry with D2-40 and Ki-67.Results: Decrease of intra and peritumoral LVD in invasive breast carcinoma compared to fibrocystic breast disease was detected (p=0.002). Lymphatic endothelial cell proliferation was significantly higher in invasive breast cancer (p=0.008) than in the fibrocystic breast disease. LECP showed a correlation with histological grade of the tumor (p=0.05). Involvement of axillary lymph nodes with metastatic tissue was in strong correlation only with existence of lymphatic vascular invasion (p=0.0001).Conclusion: These results suggest that development of breast cancer promotes proliferation of lymphatic endothelial cells whose level correlates with histological grade of tumor, but in a scope that is insufficient to follow growth of tumor tissue that invades them and destruct them. This might explain the decrease of lymphatic vessels density.


INTRODUCTION
Th e major cause of death from breast cancer is dissemination of the primary tumor leading to forma-tion of metastases. Spread to axillary lymph nodes is often the fi rst step of generalization (1). Tumorassociated lymphatic vessels are considered to be the main route of tumor cells to axillary lymph nodes (2). Recently, lymphangiogenesis, the formation of new lymphatic vessels, has become a new research frontier in tumor metastasis since the discovery of the two major lymphatic vessel growth factors-C (VEGF-C) and -D (VEGF-D), as well as reliable lymphatic markers that have allowed observation and isolation of lymphatic endothelium (3). Specifi c lymphatic endothelial markers have become available, making analysis of lymphatics in cancer possible. D2-40 antibody was reported to detect a fi xation-resistant epitope on a 40 kDa O-linked sialoglycoprotein expressed in lymphatic endothelium but not blood vessels, and can be used to assess lymphangiogenesis specifi cally in conventionally processed formalin-fi xed and paraffi n-embedded tissue specimens (4,5). Although proliferating lymphatics, that promote nodal metastasis, have been demonstrated in experimental breast tumors, it has yet to be determined whether the same phenomena occurs in spontaneous human breast cancers. Th e existence of lymphangiogenesis in breast carcinoma is one of the most controversial areas in the breast cancer literature. Th e aim of this study was to investigate existence of lymphangiogenesis in invasive breast carcinoma: by measuring lymphatic vessels density (LVD) and lymphatic endothelial cell proliferation (LECP), and their correlation with various prognostic parameters in breast cancer, including lymphovascular invasion (LVI).

Clinico-pathological data
Seventy-fi ve cases of invasive carcinoma (IC) of the breast were identifi ed by searching the database of the Institute of Pathology, Medical faculty of Sarajevo, Bosnia and Herzegovina. All patients with IC of the breast underwent partial or total mastectomy with axillary lymph node dissection. No neoadjuvant chemotherapy or radiotherapy was administered before the surgical treatment. Ten samples fi brocystic breast tissue were used as a control group. Th e patient age at the time of surgery ranged from 37 to 87 years, (mean 59.43 years). Clinicopathological characteristics of the studied cases are shown in Table 1.

Lymphatic vessels quantifi cation
Sections stained with D2-40 were used for the evaluation of LVD using the Chalkley counting method. Each section was fi rst scanned at low-power magnifi cation (×40) to select the most vascularized areas; three hot spots were selected. Two authors fi rst examined 10% of specimens to agree on which fi elds to be used as hot spots. A 25-point Chalkley eyepiece graticule was applied to each hot spot and oriented to permit the maximum number of points to hit on, or within the areas of immunohistochemically highlighted microvessel using ×200 magnifi cation. A Chalkley count for an individual tumor was taken as the mean value of the three graticule counts (6).

Assessment of lymphatic endothelial cell proliferation
Th e fractions of proliferating lymphatic endothelial cells (LECP%) were calculated in each hotspot as the number of lymphatic endothelial cells with Ki-67stained nuclei per 100 lymphatic endothelial cells. Cases in which the average number of lymphatic endothelial cells in three hotspots was <10 were excluded for statistical analysis.

STATISTICAL ANALYSIS
Statistical analysis was performed using SPSS for Windows (version 13; SPSS, Chicago, Il, USA). Two-tailed unpaired t test was performed to identify the diff erences between two groups. One-way ANOVA test was performed for comparing the groups that were used for the t test. Th e correlations between the variables were assessed by the Spearman rank sum test. P values <0.05 were considered as statistically signifi cant.

Benign breast samples
In these 10 cases, the lymphatic vessels were dispersed around the lobules in the interlobular stroma, adipose tissue, and adjacent to blood vessels. Th ese vessels were elongated and linear in most areas and tortuous focally. Lymphatic vessels were not identifi ed within the intralobular stroma ( Figure 1A). Lymphatic endothelial cell proliferation was observed in 3 of 10 cases.

Invasive carcinoma
Lymphatic vessels were identifi ed within invasive tumors except in areas adjacent to preexisting ducts and lobules; the latter were interpreted as preexist-ing vessels 'entrapped' within the tumor ( Figure  1B). Blood vessels within the tumor mass were not associated with perivascular lymphatic vessels. Th e density of intratumoral and peritumoral lymphatic vessels in invasive carcinoma was signifi cantly lower (p=0.002) in comparison to fi brocystic disease (Figure 2). Conversely, density of peritumoral lymphatic vessels was signifi cantly higher in comparison to intratumoral lymphatic vessel density (p=0.0001).

A B
Lymphatic endothelial cell proliferation was observed in the fi brocystic breast disease (in 3 of 10 cases) but was signifi cantly higher (p=0.008) in invasive breast cancer. LECP% showed correlation (p=0.05) with histological grade of the tumor (Figure 3). Signifi cant correlation was not found between lymphatic vascular density and lymphatic endothelial cell proliferation. Involvement of axillary lymph nodes with metastatic tissue showed strong correlation (p=0.0001) only with existence of lymphatic vascular invasion (Figure 4).

DISCUSSION
Th e invasion and metastasis of tumor cells are important biological features of neoplasm and the main cause for poor prognosis and death (7). Axillary lymph node status at time of diagnosis is the most signifi cant and durable prognostic factor in breast cancer patients (1). However, the extent to which this depends on lymphangiogenesis or on invasion of existing lymph vessels remains controversial. Lymphangiogenesis may be assessed either by LVD or lymphatic endothelial proliferation. In the cur-rent study, lymphangiogenesis was assessed by both these methods. We did not fi nd an increased LVD in breast cancer. Detection of dividing lymphatic endothelial cells became central to the current controversy on whether lymphangiogenesis occurs in breast cancer and whether lymphatic metastasis occurs through pre-existing or newly-formed lymphatic vessels (13)(14)(15). Th is controversy exists because neither the mere presence of pro-lymphangiogenic factors nor frequent incidence of lymph node metastasis constitutes a proof of tumor-induced, actively ongoing formation of new lymphatic vessels. Resolving this question has been sought by double staining using antibodies to specifi c lymphatic markers (LYVE-1 or D2-40) combined with antibodies to proliferative markers such Ki-67 (MIB-1) or PCNA. As a result, interpretation of overlapped lymphatic marker/ Ki-67 positivity might depend on whether the posi- tive cells are seen to be proliferating LEC, or dividing tumor cells that had invaded lymphatic vessels. Additional challenges in detection of proliferating LEC are: (a) a relatively low rate of vessel formation in well-established tumors; (b) a lower density and heterogeneity of tumor lymphatics compared with tumor blood vessels; and (c) variability in sprouting of new vessels at diff erent points along the parental lymphatic vessel (16), with the latter being undetectable in two-dimensional evaluation. Moreover, the formation of new lymphatic vessels might not require endothelial mitotic division if they originate from circulating progenitors or non-endothelial cells via trans-diff erentiation (17). Given these technical and biological limitations, it is not surprising that several studies failed to detect Ki-67 or PCNA markers on LYVE-1 or D2-40-labeled structures (13)(14)(15). However, evidence from several research groups also supports tumor-induced lymphangiogenesis and shows its clinical relevance to lymphatic metastasis. For instance, double Ki-67/ podoplanin staining of a large panel (N= 177) of invasive breast carcinomas determined that 29% of specimens displayed Ki-67 positive nuclei in 2.2% of intratumoral, peritumoral and peripheral lymphatics (18). Frequency of positive nuclei was strongly associated with a high lymphatic density (p = 0.001), LN metastasis and survival (18). An independent study detected a similar fraction of proliferating LEC (LECP%) in peritumoral lymphatics also identifi ed LECP% as an independent prognostic factor for LN metastasis (19). Studies that compared LECP% in infl ammatory and noninfl ammatory breast cancers found that the former have both a higher incidence of Ki-67 positive lymphatics (80% vs. 50%) and an increased median LECP% (20,21). Active lymphangiogenesis was also detected in positive sentinel LN (22,23) that displayed a signifi cantly higher median LECP% (p < 0.001) than uninvolved LN (23). Moreover, high frequency of Ki-67-labeled lymphatics in positive sLN was strongly associated (p = 0.01) with axillary metastasis (22), supporting the contention that tumor-induced lymphangiogenesis promotes dissemination from both the primary tumor and secondary metastatic sites. Nevertheless, with the exception of very active lymphangiogenesis in infl ammatory breast cancer (20,21), a relatively low fraction of di-viding lymphatic endothelial cells (2-6%) and some discrepancy between high %LVI and low %LECP in other breast cancer types suggest that both new and existing lymphatic vessels partake in lymphatic metastasis (24). In contrast to challenges mentioned earlier in the detection of dividing LEC, enumerating lymphatic vessels seemed initially a straightforward measure of lymphangiogenesis. To assess tumor vascularity, there are several methods including counting the number of immunohistochemically stained microvessels in vascular hot spots, grading of vascular, using image analysis systems (25) and applying the Chalkley grid. Th e Chalkley count technique was recommended in an international consensus report because it is considered to be a simple and acceptable procedure for daily clinical use and produced lower inter-observer variability compared to the more frequently used conventional microvessel density method (26). LVD has been studied in a number of human cancers, including melanoma (27) and prostate (28). As a result, fi ndings and interpretations from the studies that focused on infrequently occurring intratumoral lymphatic vessels (14), or those that compared a heterogeneous LVD pattern to more orderly tumor blood vessel distribution (13), fueled the debate whether lymphangiogenesis exists in breast cancer (13)(14)(15). Additional complexity arises from the fact that, in contrast to blood vessels, lymphatic vessels support spread of metastatic cells, but not tumor cell proliferation and expansion of the tumor mass. Kanngurn et al. (29) showed that microvessel density (MVD) Chalkley but not the LVD Chalkley count can be a predictive factor for axillary lymph node metastasis in breast carcinoma. Th erefore, subtle increases in LVD might be missed in tumor sections set aside for immunohistochemical analysis, although they might suffi ce for tumor dissemination in a patient. All methods used in study of Niemiec et al. (30) for assessment of lymphangiogenesis (LVD, DLV, LVD/MVD) were correlated to each other and to parameters indicating aggressive tumor behavior (high grade, TNP, HER2 subtype, basal marker expression), hence they might be used equivalently. Th e main evidence supporting the claim that lymphangiogenesis does not exist in tumors is detection of decreased LVD or absence of intratumoral lym-phatic vessels (LV) compared with normal breast tissue (13)(14)(15). Th e same studies, however, reported a signifi cant increase (p=0.0001) in peritumoral LVD (13,15) with some lymphatic vessels containing tumor emboli (13). Th ere is a wide range of opinions with regard to a prognostic value of intratumoral LVD. However, a consensus seems to exist with regard to increased density of peritumoral lymphatic vessels that might be suffi cient for tumor cell transit to lymph node even in the absence of intratumoral lymphatics.

CONCLUSION
Th e fi ndings from this study show that lymphangiogenesis in breast cancers, measured by lymphatic endothelial cell proliferation is present, but measured by lymphatic vascular density is absent. Th ese results suggest that development of cancer tissue in breast promotes proliferation of lymphatic endothelial cells whose level correlates with histological grade of tumor, but in a scope that is insuffi cient to follow growth of tumor tissue that invades them and destruct them. Th is might explain decrease of lymphatic vessels density.