Elsevier

European Journal of Cancer

Volume 38, Issue 12, August 2002, Pages 1564-1579
European Journal of Cancer

Review
Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours

https://doi.org/10.1016/S0959-8049(02)00094-1Get rights and content

Introduction

During the 5 years since the publication of the first international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid tumours [1], new concepts and mechanisms of tumour vascularisation have been described with a major impact on the prognostic/predictive value of angiogenesis quantification and on the strategies of development of anti-angiogenic and vascular-targeting agents in oncology. The second consensus report aims at integrating these important novelties in updated guidelines for the estimation of the amount of blood vessels in a solid tumour and for the estimation of the ongoing angiogenesis. Qualitative aspects of tumour blood vessels will also be addressed, since these aspects are associated with a tumour vasculature established by mechanisms other than angiogenesis.

Redundancy appears to be characteristic to the development of vertebrates and of tumour growth, as demonstrated by several knock-out mice models. This is also illustrated by the alternative mechanisms solid tumours apply to obtain blood vessels. The statement of Judah Folkman that tumours need angiogenesis to grow, invade and metastasise [2] is no longer applicable to all tumours. Tumours, as all tissues and organs do, need blood vessels, but these blood vessels are not necessarily derived by sprouting of new blood vessels from pre-existing ones as in angiogenesis.

In primary non-small cell lung carcinomas, an alveolar growth pattern was observed [3]. In this growth pattern, tumour cells filled the alveolar spaces, entrapping, but not destroying, the alveolar septa with the co-opted blood vessels. In the tumour cell nests, no associated desmoplastic stroma or new blood vessels were present. This putatively non-angiogenic growth pattern was also observed in lung metastases [4]. The lung tumours expressing this growth pattern were more often poorly differentiated than tumours expressing other growth patterns.

Holash and colleagues [5] have studied early vascularisation of gliomas in a rat model. Although the prevailing view was that malignancies and metastases initially reside as small avascular masses, 1 week after implantation of tumour cells, well-vascularised tumours were observed in their model. The tumour cells had co-opted the blood vessels of the surrounding brain tissue. There was no angiogenic response in the tumours. As a consequence of tumour growth in the absence of angiogenesis, the centre of the tumours became hypovascular. Later on, frank vessel regression led to tumour cell death. This tumour cell loss and hypoxia resulted in intense angiogenesis at the tumour margins. Autocrine expression of Angiopoietin-2, the endogenous Tie-2 receptor antagonist, marked the co-opted vessels of 2-week gliomas. At this stage, vascular endothelial growth factor (VEGF) expression in tumour cells was absent or low. After vessel regression and subsequent hypoxia, VEGF upregulation induced growth of the Angiopoietin-2 expressing and therefore destabilised blood vessels.

In this model of tumour vascularisation, co-option is restricted to the early phase of tumour growth. Compelling morphological evidence in human tumours suggests that co-option of pre-existing blood vessels might persist during the growth of primary tumours and of metastases, especially in well-vascularised organs such as the lungs and the liver. In liver metastases of colorectal adenocarcinomas, three growth patterns were observed [6]. In one of the patterns, tumour cells replaced the hepatocytes of the liver cell plates, while conserving the supportive tissue including the sinusoidal blood vessels. The endothelial cells of these co-opted sinusoids did not express the full spectrum of endothelial cell markers, which is comparable to the lack of expression of endothelial cell antigens on sinusoids in the normal adjacent liver parenchyma. The fraction of proliferating endothelial cells was small. Other investigators identified a subgroup of squamous cell lung carcinomas with low microvessel density and high tumour cell proliferation characterised by a high take rate in nude mice and by a short survival of patients [7].

A second alternative mechanism of tumour vascularisation, and a matter of vigorous debate [8], was described by Maniotis and colleagues [9] in aggressive human uveal and cutaneous melanomas. The tumour cells were found to line vascular channels forming a perfused network in the absence of endothelial cells, and expressing genes associated with vascular development, such as the endothelial receptor kinase Tie-1. In the mosaic blood vessels described by Chang and colleagues [10], the luminal surface is formed by both endothelial cells and tumour cells. Approximately 14% of the investigated blood vessels in human colorectal cancer biopsies showed mosaicism. The formation of mosaic vessels is distinct from the ‘vasculogenic mimicry’ described by Maniotis and colleagues [9] as the tumour cells of mosaic blood vessels do not show an endothelial phenotype.

Vasculogenesis, or the growth of blood vessels out of angioblasts, also seems to occur in tumour tissue. In a murine bone-marrow transplantation model, bone-marrow-derived Flk-1- or Tie-2-expressing endothelial cell progenitors or angioblasts were abundant in the highly vascularised peripheral regions of implanted colon cancer [11]. An increase in circulating endothelial cell progenitors was observed following VEGF administration in this model [12]. In patients with chronic myelogenous leukaemia carrying the BCR/ABL fusion gene, this gene was detected by in-situ hybridisation in endothelial cells generated in vitro from bone-marrow-derived progenitor cells and in vascular endothelium in myocardial tissue [13], corroborating the concept of vascular endothelium maintenance by bone-marrow-derived endothelial cells.

Intussusceptive vascular growth refers to vascular network formation by insertion of interstitial tissue columns into the vascular lumen and subsequent growth of these columns resulting in partitioning of the vessel lumen [14]. Endothelial cell division is not required for this form of vascular remodelling.

The College of American Pathologists has investigated the prognostic and predictive factors in breast cancer and has stratified these into categories reflecting the strength of the published evidence [15]. Quantification of tumour angiogenesis by counting microvessels in immunostained tissue sections was ranked in category III, encompassing “all factors which are not sufficiently studied to demonstrate their prognostic value”. The issues of methodological variation mentioned include: antibody selection, type of fixative used, methods of counting vessels, calculation of microvessel density, observer variability (especially of the selection of the field in which to count) and cut-off value for ‘increased’ vascularity. The current consensus report will address these issues and aims to lower the methodological variabilities of angiogenesis quantification in tumour tissue sections. New parameters measuring endothelial cell phenotypic characteristics of activation and differentiation and taking into account the angiogenesis-independent mechanisms of tumour vascularisation, will be presented.

Section snippets

Role of vascular differentiation and activation markers in tumour angiogenesis quantification (Table 1)

The stimulation of vascular endothelial proliferation results in upregulation of many biochemical pathways. Examples include upregulation of Kdr by VEGF [16], the urokinase receptor, CD105, which is a Transforming Growth Factor β (TGFβ)-receptor and Flt-4, which is the VEGF-receptor 3 17, 18. The VEGF-receptor 3 is normally only expressed in lymphatics but in tumour vessels it becomes abnormally upregulated. The basement membrane is not properly formed, so there may be loss of differentiation

Methodology of angiogenesis quantification in solid tumour sections

Counting microvessels in a microscopic field of a tumour tissue section gives an estimate of the net result of phases of angiogenesis and of the angioregression a tumour went through. The rationale of counting microvessels in vascular ‘hot spots’, or in areas giving the impression at low magnification of containing numerous microvessels, is that these areas originated from tumour cell clones with the highest angiogenic potential and, consequently, with the easiest access to the blood stream and

Surrogate histopathological markers of the angiogenic activity of tumours

Histological surrogate markers should give a reliable, fast and technically easy estimate of the amount of angiogenesis, ongoing and past, of a tumour. Ideally, a standard histochemical technique, such as a haematoxylin-eosin stain, should suffice. Interpretation of the slides should not rely on extensive training and should not be time-consuming. Yet, a clear pathophysiological mechanism should corroborate the association of these markers with parameters directly reflecting angiogenesis, such

Other surrogate markers of tumour-related angiogenesis

The main disadvantage of the histological surrogate markers of angiogenesis is the inherent inter-observer variability. A more objective approach is the quantification by multi-parameter flow cytometry of circulating endothelial cells [59]. In a mouse model of human lymphoma, the fraction of CD45- and Flk+ cells in the peripheral blood was found to represent circulating endothelial cells which also expressed CD31, VE-cadherin and MECA-32. In control mice, approximately 1 circulating endothelial

Quantification of lymphangiogenesis

In several types of malignant tumours, like cutaneous melanoma and head-and-neck squamous cell carcinoma, the microvascular density appears not to correlate with metastatic spread 113, 114, 115. Until now, the reason for this lack of correlation is unclear, but it may be speculated that, next to several properties of tumour cells and tumour-associated stromal cells, the presence of a lymphatic vascular bed is an important factor. It is obvious that the propensity of a tumour to spread initially

Summary and future directions

With the development of angiogenesis inhibitors—over 150 compounds are in different stages of clinical development from phase I up to phase III studies—detailed knowledge of the vascular component of a tumour in a particular patient is becoming essential. Most drugs target one component of the angiogenesis program, yet the studies reviewed above show the great inter- and intra-individual variability in the process. It is perhaps not surprising that, as of yet, marked tumour regressions in many

Acknowledgements

G. Gasparini is supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan) and the Consiglio Nazionale Ricerche (CNR, Rome). P.B. Vermeulen, C. Colpaert, E. Van Marck and L.Y. Dirix are supported by the F.W.O. Vlaanderen ‘Kom op tegen Kanker’, grant no. G.0408.00 and G.0330.02.

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