Background
Breast cancer is one of the most common malignant tumors in females. Prognostic factors are helpful in clinical management and have the potential to improve the disease-free survival (DFS) and overall survival (OS) in breast cancer [
1]. Several independent risk factors for survival have been identified, including tumor size, histological grade, nodal status, hormone receptor status, and HER-2 status [
2,
3]. However, these risk factors are insufficient to fully determine an individual’s prognosis. More risk factors are needed to be explored.
Lymphatic vessel was formerly considered as a passive participant in tumor metastasis and regarded mainly as a transportation channel for tumor cells. Now, it appears that lymphatic vessel provides a safe route for tumor cells dissemination, because of the discontinuous structure of the lymphatic basement membrane, an ultraminiature shear stress, and a high concentration of hyaluronic acid [
4]. Even so, it is still uncertain that whether the high lymphatic vessel density is a necessary condition for tumor metastasis. Many studies have demonstrated the unfavorable prognostic value of lymphatic vessel density in primary breast cancer [
5,
6]. However, Zhang et al. [
7] showed that lymphovascular invasion, but not lymphangiogenesis, was correlated with lymph node metastasis and poor prognosis in young breast cancer patients. Other studies found that the lymphatic vessel density in the lymph node metastasis negative group even was higher than that of the positive group in primary breast cancer [
8,
9]. Therefore, a meta-analysis study is needed to pool the results to clarify the prognostic value of lymphatic vessel density in breast cancer.
Lymphatic metastasis contains a series of sequential processes, such as tumor associated lymphangiogenesis, lymphovascular invasion, implantation of cancer cells in regional lymph nodes, and proliferation of micrometastasis in distant organs [
10]. Lymphovascular invasion, infiltration of tumor cells into lymphatic vessels, represents a high invasion feature of breast tumor cells. Determined by hematoxylin and eosin (H&E) staining in past time, lymphovascular invasion was widely investigated and showed a correlation with the clinicopathological outcomes of breast cancer [
11,
12]. At present, lymphatic vessels can be distinguished from blood vessels or retraction artifacts. Thus, using immunohistochemical staining, many studies have updated the investigation of the prognostic value of lymphovascular invasion [
13,
14].
With the identification of specific markers of lymphatic vessels, such as podoplanin/D2–40, LYVE-1, Prox-1 and VEGFR-3, many studies have demonstrated the importance of lymphatic system in tumor metastasis [
9]. Therefore, we conducted a meta-analysis study not only to estimate the effect of lymphatic microvessel density on patients’ survival, but also to update and re-estimate the prognostic value of lymphovascular invasion in breast cancer.
Discussion
The current meta-analysis study indicates that both lymphatic vessel density and lymphovascular invasion presence can predict poor prognosis in females with breast cancer. Compared with the high lymphatic vessel density, the presence of lymphovascular invasion in breast cancer appears to have weaker impacts on DFS and OS; but it is also significantly associated with poor survival. Furthermore, lymphovascular invasion was present in 29.56% of breast cancer patients, who would have poorer prognosis.
The metastasis routes of breast cancer consist of local invasion, hematogenous metastasis, and lymphatic metastasis. New blood and lymphatic vessels formed through physiological or pathological processes are called angiogenesis and lymphangiogenesis, respectively. It is well known that tumor angiogenesis, and its indicator blood vessel density are closely associated with the clinicopathological outcomes of breast cancer [
32]. A meta-analysis study performed by Uzzan et al. has shown that the high blood vessel density can predict poor survival in breast cancer (risk ratio = 1.54 for DFS and OS with the same 95% CI 1.29–1.84) [
9]. However, the prognostic value of lymphatic vessel density is still uncertain [
33]. With the development of lymphatic vessel biology, lymphatic vascular system has been considered as an active player involved in breast cancer [
34]. Our meta-analysis result shows that high lymphatic vessel density has unfavorable impacts on DFS (HR 2.02, 95% CI 1.69 to 2.40) and OS (HR 2.88, 95% CI 2.07 to 4.01). Compare with blood vessel density, lymphatic vessel density even displays a stronger predictive value in breast cancer.
The result that lymphatic vessel density is a risk factor of poor survival is supported by all included studies; however, the values of lymphatic vessel density were differentiated notably in these studies [
17,
21,
24]. The variation might be caused by patient sources, staining techniques, antibody categories and antibody dilutions. In addition, different counting methods of lymphatic vessel density, by using different hotspots (three [
19], four [
18], and five [
24]), magnification field (100× [
22], 200× [
24], 400× [
19]), and measuring unit (vessels/mm2 [
24], vessels/area [
22]), are also accounted for the variation of results. Furthermore, the cutoff value to divide lymphatic vessel density as low and high is a crucial factor that cannot be ignored. Because the asset value of lymphatic vessel density is not a normal distribution, seven in ten studies chose the median value as the cutoff value, other three studies took the mean or actual value as the cutoff value. Therefore, studies with more standardized and stricter design are required in the assessment of lymphatic vessel density.
Due to lack of the specific markers of lymphatic endothelium cells, most of the previous studies have detected lymphovascular invasion using H&E staining method [
11,
12]. One major challenge of this method is to distinguish lymphovascular invasion from retraction artifacts caused by tissue handling and fixation on H&E stained sections. Another challenge is that lymphovascular invasion may be missed if tumor cells are packed in a small vessel [
35]. With the help of specific markers, such as D2–40/podoplanin, LYVE-1, VEGFR-3, and Prox-1, lymphatic vessels can be effectively distinguished from blood vessels or retraction artifacts. A previous study has compared the reliability of immunohistochemical staining with that of H&E staining [
36]. The results showed that the detection rate of lymphovascular invasion widely ranged from 10 to 49% for H&E staining; however, the range was narrower using immunohistochemical staining (ranged from 21 to 42%) [
36]. It indicates that immunohistochemical staining should be more reliable for identifying lymphovascular invasion. Therefore, we conducted a meta-analysis to study the prognostic value of lymphovascular invasion, which was assessed by immunohistochemical staining instead of H&E staining [
35].
With the accumulating evidence, we conducted an update meta-analysis study to re-evaluate the prognostic value of lymphovascular invasion. The result shows that lymphovascular invasion, detected by immunohistochemical staining, has an unfavorable impact on survival, in line with the previous study [
36]. However, the result should be analysed more thoroughly. Mohammed et al. [
14,
31] has demonstrated that the impact of lymphovascular invasion is mainly found in breast cancer patients with negative lymph node metastasis and with a single positive lymph node metastasis. Moreover, the frequency of lymphovascular invasion per tumor lesion has no effects on prognosis in lymph node negative and lymph node positive patients [
14,
31]. Besides, the location of lymphovascular invasion [
23,
30] and the patients’ age [
25] also have influence on the survival of breast cancer patients.
The current meta-analysis study has some strengths. The results show that both lymphatic vessel density and lymphovascular invasion are unfavorable predictors on DFS and OS in breast cancer. The included 19 studies and 4215 participants enhanced the statistical power and provided more reliable results. However, some limitations should be considered. All included studies were observational studies with relatively small sample sizes. Selection bias and recall bias were inevitable. Besides, the values of lymphatic vessel density varied notably due to unmeasured or inadequately measured factors. It resulted that different cutoff values were used to define high and low lymphatic vessel density subgroups in different studies. Although there are no heterogeneities show in each subgroup, the deviations caused by different cutoff values cannot be ignored. Nevertheless, the conclusion that higher lymphatic vessel density is associated with poor survival is reasonable even with different cutoff values. Therefore, strictly controlled studies with larger sample sizes are needed.