Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a key role in several pathological processes, including tumour vascularization. Our preliminary observations indicated higher VEGF concentrations in serum samples than in matched plasma samples. We have now demonstrated that this difference is due to the presence of VEGF within platelets and its release upon their activation during coagulation. In eight healthy volunteers, serum VEGF concentrations ranged from 76 to 854 pg ml(-1) and were significantly higher (P < 0.01) than the matched citrated plasma VEGF concentrations, which ranged from < 9 to 42 pg ml(-1). Using platelet-rich plasma, mean (s.d.) platelet VEGF contents of 0.56 (0.36) pg of VEGF 10(-6) platelets were found. Immunocytochemistry demonstrated the cytoplasmic presence of VEGF within megakaryocytes and other cell types within the bone marrow. From examination of the effects of blood sample processing on circulating VEGF concentrations, it is apparent that for accurate measurements, citrated plasma processed within 1 h of venepuncture should be used. Serum is completely unsuitable. The presence of VEGF within platelets has implications for processes involving platelet and endothelial cell interactions. e.g. wound healing, and in tumour metastasis, when platelets adhering to circulating tumour cells may release VEGF at points of adhesion to endothelium, leading to hyperpermeability and extravasation of cells. Images Figure 4 Figure 3

Real-time RT-PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22-to 2.8 ؋ 10 5 -fold above normal mean expression, whereas PIP was overexpressed from 30-to 2.2 ؋ 10 6 -fold above normal in 13 lymph nodes. Real-time RT-PCR analysis of pathology-negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.

This is the first report to show that overexpression of breast cancer-associated genes in breast cancer subjects with pathology-negative ALN correlates with traditional indicators of disease prognosis. These interim results provide strong evidence that molecular markers could serve as valid surrogates for the detection of occult micrometastases in ALN. Correlation of real-time RT-PCR analyses with disease-free survival in this patient cohort will help to define the clinical relevance of micrometastatic disease in this patient population.

Angiogenesis is essential in physiological processes including ovulation, implantation and pregnancy. One of the most potent regulators is the cytokine vascular endothelial growth factor (VEGF). We provide evidence for a novel pregnancy-associated soluble variant of the VEGF receptor Flt-1. VEGF ranged from undetectable to 157.3 pg/ml (mean 49.9 pg/ml, SD 48.4 pg/ml) in plasma samples from normal volunteers (n = 10), but was undetectable in plasma from pregnant women (n = 12) and amniotic fluid (n = 10). Recoveries of spiked VEGF were poor in pregnancy-related samples, indicating the presence of VEGF-binding activity which was confirmed using biosensor and chromatographic techniques. Partial purification and protein sequencing indicated a novel soluble form of Flt-1 with a subunit size of 150 kDa. Normally present as a multimeric structure of approximately 400-550 kDa, complexes of 600-700 kDa were formed following binding of multiple VEGF molecules. Reverse transcriptase polymerase chain reaction of Flt-1 in placenta, amnion, chorion, human umbilical vein endothelial cells and cord blood samples produced bands of the predicted sizes but failed to identify any additional RNA species, and possible reasons for this are discussed. Soluble Flt-1 may be important in regulating the actions of VEGF in angiogenesis and trophoblast invasion and may have therapeutic implications in diseases with inappropriate angiogenesis such as proliferative retinopathies and cancer.

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