Summary The concentration of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml-' for bFGF and 500 pg ml-1 for VEGF, were identified as 'elevated'. One measurement during follow-up was included into the analysis per patient. For 19 treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic factors. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with 15% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic factors compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas.

SummaryWe have compared the platelet number and the serum concentration of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6) in 80 blood samples of 50 patients with advanced cancer. We have also measured the mitogenic effect of patient sera on endothelial cells in vitro in order to estimate the biological activity of serum VEGF. Serum VEGF concentration correlated with platelet number (r = 0.61; P < 10 -4 ). Serum IL-6 levels correlated with platelet count (r = 0.36; P < 10 -3 ), with serum VEGF levels (r = 0.55; P < 10 -4 ) and with the calculated load of VEGF per platelet (r = 0.4; P = 3 × 10 -4 ). Patients with thrombocytosis had a median VEGF serum concentration which was 3.2 times higher (P < 10 -4 ) and a median IL-6 serum level which was 5.8 times higher (P = 0.03) than in other patients. Serum bFGF did not show an association with any of the other parameters. Patient sera with high VEGF and bFGF content stimulated endothelial cell proliferation significantly more than other sera (P = 4 × 10 -3 ). These results support the role of platelets in the storage of biologically active VEGF. Platelets seem to prevent circulating VEGF from inducing the development of new blood vessels except at sites where coagulation takes place. IL-6, besides its thrombopoietic effect, also seems to affect the amount of VEGF stored in the platelets. This is in accordance with the indirect angiogenic action of IL-6 reported previously. The interaction of IL-6 with the angiogenic pathways in cancer might explain the stimulation of tumour growth occasionally observed during IL-6 administration. It also conforms to the worse outcome associated with high IL-6 levels and with thrombocytosis in several tumour types and benign angiogenic diseases.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Improved understanding of the mechanisms responsible for the differences between IBC and non-IBC might provide novel therapeutic targets. We studied 35 consecutive patients with IBC, biopsied prior to the initiation of chemotherapy. Angiogenesis was evaluated by Chalkley counting and by assessment of endothelial cell proliferation (ECP) and vessel maturity. The presence of fibrin, expression of the hypoxia marker carbonic anhydrase IX (CA IX) and epithelialcadherin (E-cadherin) expression were immunohistochemically detected. The same parameters were obtained in a group of 104 non-IBC patients. Vascular density, assessed by Chalkley counting (Po0.0001), and ECP (P ¼ 0.01) were significantly higher in IBC than in non-IBC. Abundant stromal fibrin deposition was observed in 26% of IBC and in only 8% of non-IBC (P ¼ 0.02). Expression of CA IX was significantly less frequent in IBC than in non-IBC with early metastasis (P ¼ 0.047). There was a significant positive correlation between the expression of CA IX and ECP in IBC (r ¼ 0.4, P ¼ 0.03), implying that the angiogenesis is partly hypoxia driven. However, the higher ECP in IBC and the less frequent expression of CA IX in IBC vs non-IBC points at a role for other factors than hypoxia in stimulating angiogenesis. Strong, homogeneous E-cadherin expression was found at cell -cell contacts in all but two IBC cases, both in lymphovascular tumour emboli and in infiltrating tumour cells, challenging our current understanding of the metastatic process. Both the intense angiogenesis and the strong E-cadherin expression may contribute to the highly metastatic phenotype of IBC.

This study assessed the ability of real-time reverse transcription -polymerase chain reaction (RT -PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n ¼ 116/78%) and metastatic (stage M þ , n ¼ 32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT -PCR detection method. The mean follow-up time was 786 days ( þ /À 487). Kaplan -Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P ¼ 0.0045 (CK-19) and P ¼ 0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P ¼ 0.551 (CK-19) and P ¼ 0.329 (MAM)). Separate analyses of the M0 and M þ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M þ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.