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We read with interest the recent article by Vleugel et al, in which the presence of lymph vessels was investigated during breast cancer progression.1 The authors claim that the observed absence of intratumorous lymph vessels in invasive breast cancer can be explained by the absence of lymphangiogenesis. In our opinion, this is a rather bold conclusion because only lymph vessel density was assessed and not lymph endothelial cell proliferation. By analogy with angiogenesis, the mere presence of a number of vessels at a certain time point—for example, at surgery—provides no information concerning the degree of ongoing angiogenesis.2 Lymph vessel density is the net result of lymphangiogenesis and vessel regression or remodelling.
A second remark is that the authors made no distinction between different growth patterns of breast cancer, as described earlier by Colpaert et al.3 However, the recognition of growth patterns of malignant tumours is of crucial importance when evaluating (lymph)angiogenesis: breast carcinomas with an expansive growth pattern form a well circumscribed nodule consisting of carcinoma cells and desmoplastic connective tissue with ongoing angiogenesis, whereas breast carcinomas with an infiltrating growth pattern grow between pre-existing structures and co-opt blood vessels without disturbing the tissue architecture.3 In addition, liver metastases of breast cancer have a very high density of blood vessels, but this is a result of the co-option of pre-existing liver sinusoids and not the result of angiogenesis.4
We recently studied the presence of lymphangiogenesis in 31 breast carcinomas by double immunostaining for the proliferation marker Ki-67 and the lymph endothelial cell marker LYVE-1, taking into account the growth pattern.5 Intratumorous lymph vessels were present in eight of 10 breast carcinomas with an infiltrating growth pattern. In breast carcinomas with an expansive growth pattern, intratumorous lymph vessels were present in only seven of 18 cases (p = 0.04). Proliferating lymph endothelial cells were seen in 15 of 31 breast tumour specimens in the tumour parenchyma and at the tumour–breast tissue interface. Fractions of proliferating lymph endothelial cells ranged from 0% to 11.4%. Thus, lymphangiogenesis is ongoing during breast cancer growth and its extent depends on the tumour growth pattern. In the same study, higher mRNA expression of lymphangiogenesis related factors (vascular endothelial growth factor C (VEGF-C), VEGF-D, VEGF receptor 3; assessed by quantitative real time reverse transcription polymerase chain reaction) in inflammatory breast cancer versus non-inflammatory breast cancer correlated with higher fractions of proliferating lymph endothelial cells. The presence of this positive association between the expression of lymphangiogenic factors and the fraction of proliferating lymph endothelial cells is at variance with the hypothesis stated by Vleugel et al that inhibitors completely counter the lymphangiogenic signal in breast cancer.1
In addition, a methodological issue that needs further investigation is whether the expression of LYVE-1 is downregulated by the presence of breast cancer cells, as suggested by Stessels et al and by Williams et al.4,6 If this is true, slight variations in immunostaining protocols might be responsible for false negative results of intratumorous lymph vessel assessment.
In conclusion, our results are in favour of ongoing lymphangiogenesis during breast cancer progression, the extent of this process being modulated by the growth pattern, and advocate the assessment of the lymph endothelial cell proliferation fraction as a measure of lymphangiogenesis.