Eppenberger et al. BMC Cancer 2010, 10:93 http://www.biomedcentral.com/1471-2407/10/93 invasion of tumour cells. Although significantly less is known about VEGFR1 (FLT1), it appears to function as a negative regulator of angiogenesis [5]. VEGF-A is expressed on vascular cells and binds to VEGFR1 with an affinity that is much higher than that for VEGFR2. However, VEGFA seems to induce much weaker tyro- sine kinase activity in VEGFR1 possibly because of an inhibitory sequence in the juxtamembrane domain that represses VEGFR1 activity [6]. In keeping with this observation, a model for VEGFR1 has been developed whereby it could act as a decoy receptor to modulate angiogenesis through its ability to sequester VEGFA thereby reducing signaling through VEGFR2. VEGF-B has also been found to bind to VEGFR1, although the role of this interaction remains to be completely eluci- dated. VEGFR3 is the specific receptor for VEGF-C and -D and is predominantly found on lymphatic, but also to a lesser extent, on vascular endothelial cells and also on tumour cells [7]. Interestingly, VEGF-C along with VEGF-A and a variety of pro-angiogenic cytokines have been shown to be released from tumour associated macrophages, whose infiltration is thought to be, at least in part, responsible for the angiogenic switch in tumours whereby the balance of pro- and anti-angiogeneic factors favour a pro-angiogenic phenotype [8-10]. In 1971, the pioneering work by Folkman and collea- gues led to the hypothesis that anti-angiogenic com- pounds could be successfully applied as anti-cancer therapies [11,12]. In fact, blocking of VEGF has been shown to lead to normalization of the vasculature, thus increasing the efficacy of both radiotherapy (by increas- ing the partial oxygen pressure of cells) and also the delivery of chemotherapeutic agents to target cells (by decreasing vascular permeability) [13]. Currently, the humanized monoclonal antibody Bevacizumab approved for the treatment of patients with metastatic colorectal cancer has been successful in improving overall survival times in several randomized controlled studies while other approaches such as the use of tyrosine kinase inhi- bitors continue to be investigated [14,15]. VEGFR1 immunoreactivity in tumour cells has been correlated with poor prognosis, metastasis and recurrence in a vari- ety of tumour types including breast and lung cancers [16-18]. Inhibitors of VEGFR1 activity, such as VEGFR1 antibodies or soluble VEGFR1 traps have been developed for preclinical and clinical evaluation and have been shown to suppress tumour growth by inhibiting expres- sion of VEGF on both tumour and stromal cells [5]. Although several studies have evaluated one or more of these VEGF ligands or their receptors by immunohis- tochemistry and their potential prognostic value, still lacking is a comprehensive analysis performed on a large number of tumours from patients with full clinico- pathological data taking into consideration the different Page 2 of 10 expression ratios between the VEGF ligands and their receptors. Such an evaluation may provide a more pro- found understanding of the involvement of these angio- genic proteins in colorectal tumour progression, particularly considering the known differences in bind- ing affinities of VEGF ligands to their receptors. The aim of this study was therefore to elucidate the prognos- tic role of the VEGF ligand to receptor ratios and their effects in tumour progression and metastasis on 387 patients with mismatch repair-proficient colorectal cancers. Methods Patients 489 non-consecutive, unselected colorectal cancer patients treated at the Stadtspital Triemli, Zürich, Swit- zerland were initially entered into this study. Histomor- phologic features were systematically re-reviewed from the corresponding hematoxylin and eosin (H&E) stained slides in all cases and included TNM stage (according to the 6th edition of the American Joint Committee on Cancer staging manual), tumor grade, the presence of vascular invasion, the tumor border configuration, the presence of peritumoral lymphocytic infiltration, the his- tologic tumor subtype and mismatch repair status. Patients with mismatch repair-deficient tumours (n = 102) were excluded from the study. Clinical data were retrieved from the patient records and included age, gender, tumor location, follow-up and disease-specific survival time, information on local recurrence and dis- tant liver metastasis. Of the 387 patients with mismatch repair-proficient colorectal cancers, 88 received post- operative therapy. All patients were pre-operatively untreated and all resections were complete. Patient characteristics are summarized in Table 1. Specimen characteristics Formalin-fixed paraffin-embedded tissue blocks were retrospectively retrieved from the archives of the Insti- tute of Pathology, Stadtspital Triemli, Zürich, Switzer- land and a tissue microarray including all 387 patients was constructed from each colorectal cancer resection specimen. From each patient, one representative tumor block was punched from the tumor centre using a tissue cylinder 0.6 mm in diameter. Tissue was brought into one recipient paraffin block (3 × 2.5 cm) using a home- made semi-automated tissue arrayer as described else- where [19] 71 tissues from normal colorectal mucosa were included as a control. Assay Methods Immunohistochemistry Immunohistochemistry for VEGF ligands and receptors was carried out. Briefly, the tissue microarray was