First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours

Abstract

The lymphatic system is the primary pathway of metastasis for most human cancers. Recent research efforts in studying lymphangiogenesis have suggested the existence of a relationship between lymphatic vessel density and patient survival. However, current methodology of lymphangiogenesis quantification is still characterised by high intra-and interobserver variability. For the amount of lymphatic vessels in a tumour to be a clinically useful parameter, a reliable quantification technique needs to be developed. With this consensus report, we therefore would like to initiate discussion on the standardisation of the immunohistochemical method for lymphangiogenesis assessment. Metastasis is the leading cause of cancer mortality. Metastatic cancer cells can escape from their site of origin and spread to distant organs through invasion of the vascular system and/or the lymphatic system. Tumour vascularisation is widely accepted as a bona fide indicator of tumour growth, metastases and patient survival. In 1996, Peter Vermeulen et al (1996) published a first international consensus on the methodology and criteria of the evaluation of angiogenesis quantification in solid tumours and 5 years later, a second consensus report, in which new concepts and mechanisms of tumour vascularisation were integrated, appeared (Vermeulen et al, 2002). Both reports were aimed at improving the standardisation of angiogenesis quantification in order to allow intratumourous microvessel density to be applied as a prognostic indicator and, moreover, as a reliable predictor of the risk of malignant transformation of premalignant lesions and of response to cancer treatment. Contrary to angiogenesis, the de novo formation of lymphatic vessels or lymphangiogenesis and its role in promoting the metastatic spread of tumour cells has only recently become a focal point of cancer research with an increasing number of studies showing a relationship between patient survival and lymphatic density in different tumour types. In order to confirm the potential prognostic value of lymphangiogenesis in patients with cancer, a quantification method that is characterised by a low intra-and interobserver variability needs to be developed. In this first consensus report, we would like to provide an overview of current concepts of the lymphatic vasculature and its regulating factors and propose guidelines for the estimation of the ongoing lymphangiogenesis in solid human tumour sections.

Lymphatic vessel density is associated with CD8+ T cell infiltration and immunosuppressive factors in human melanoma

“…45 The majority of studies on LVD and clinical outcome of melanoma patients have used staining quantification methods based on visual inspection and selection of “hot spots” (e.g. Weidner and Chalkley method).…”

“…However, depending on their tissue location and state of development, LEC expression of these markers can be variable, and furthermore, other cell types within the tumor microenvironment can also express these markers. 45 , 71 Some tumor-associated macrophages express Lyve-1, and cancer-associated fibroblasts found in the stroma can express podoplanin. As expected, in our tissue sections, LECs expressed podoplanin and nuclear Prox-1 in all tissues analyzed, but these markers were also found in other types of cells depending on the region evaluated (see result section and Fig.…”

Lymphangiogenesis: from the pig embryos to cancer

“…Regularly, and by analogy to angiogenesis, lymphatic immunostained vessels in tumour sections has been evaluated according to Weidner et al (24) , where LVD is determined in vascular "hot spots" or areas giving the impression, at low magnification, of containing numerous stained microvessels at 20x magnification. The reproducibility of this method is questionable, since it depends on the investigator's training and experience (23) . Therefore, Chalkley counting method was considered more useful to assess LVD in tumour setting (23) .…”

“…The reproducibility of this method is questionable, since it depends on the investigator's training and experience (23) . Therefore, Chalkley counting method was considered more useful to assess LVD in tumour setting (23) . This method involves the use of an eyepiece reticule containing 25 randomly positioned dots, which is rotated so that the maximum number of points is on or within the vessels of the vascular "hot spot".…”

“…Additionally, there is also high intra-and interobserver variability in lymphangiogenesis quantification. Recently, a preliminary effort to standardize immunohistochemical lymphangiogenesis assessment was reported (23) . This purpose is now available and its impact as a clinically useful parameter will be known prospectively.…”