Detection of angiogenic cytokines in serum and plasma of patients with breast cancer

Tumour growth depends on angiogenesis, although some tumours can grow by co-opting preexisting microvessels. We evaluate the clinical significance of some angiogenic cytokines in serum and plasma of patients with breast cancer. We compared prospectively serum and plasma concentrations of vascular endothelial growth factor (VEGF), serum concentrations of basic fibroblast growth factor (bFGF), Interleukin-6 (IL-6) and Interleukin-8 (IL-8)levels in healthy controls, patients with localized breast cancer and in patients presenting with untreated and thus progressive metastatic disease. Furthermore, static clinico-pathological parameters, e.g. hormone receptor status, and more dynamic parameters such as tumor doubling time are recorded and associations with these angiogenic cytokines are studied. The prognostic importance of these levels of angiogenic cytokines are also documented in these patients.

Methods:

Sample handling
Blood is collected in a serum separator tube (Vacutainer, Becton-Dickinson) and allowed to stand for 30 min at room temperature to ensure full clotting. Samples are subsequently centrifuged at 3000g for 5 minutes. The supernatant is aliquoted and stored at -80°c until further analysis. Plasma samples are collected in sterile glass tubes containing sodium citrate (0,129M) as anticoagulant (Vacutainer, Becton-Dickinson). Samples are centrifuged at 3000g for 10 minutes and stored in the same manner as the serum samples.

Detection method:

Serum and plasma concentrations of VEGF, serum concentrations of bFGF, IL-6 and IL-8 are determined with enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN, USA). The half of the detection limit value of the patient samples is used for statistical analysis in case the measured values did not reach the detection limit of the assay. The detection limit of the assays are 9 pg/ml for VEGF, 0,22 pg/ml for bFGF, 0,70 pg/ml for IL-6 and 10.0 pg/ml for IL8.

Cut-off values: The 95th percentile of serum or plasma concentration measured in blood from healthy controls is used as cut-off value. pVEGF: cut-off = 30 pg/ml sVEGF: cut-off = 250 pg/ml BFGF: cut-off = 5 pg/ml IL-6: cut-off = 1,6 pg/ml IL-8: cut-off = 10 pg/ml

Relevant publications:
• Benoy IH, Salgado R, Van Dam P, Geboers K, Van Marck E, Scharpé S, Vermeulen PB, Dirix LY. Increased serum Interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clin Cancer Res. 2004 Nov 1;10(21):7157-62
• Salgado R, Benoy I, Vermeulen P, Van Dam P, Van Marck E, Dirix L. Circulating Basic Fibroblast Growth Factor is Partly Derived from the Tumour in Patients with Colon, Cervical and Ovarian Cancer. Angiogenesis. 2004;7(1):29-32.
• Salgado R, Vermeulen P, Dirix LY. Elevated perioperative serum vascular endothelial growth factor levels in patients with colon carcinoma. Cancer. 2004 Jul 15;101(2):431-2; author reply 432-3.
• Salgado R, Junius S, Benoy I, Van Dam P, Vermeulen P, Van Marck E, Huget P, Dirix LY. Circulating interleukin-6 predicts survival in patients with metastatic breast cancer. Int J Cancer. 2003 Feb 20;103(5):642-6.
• Salgado R, Benoy I, Weytjens R, Van Bockstaele D, Van Marck E, Huget P, Hoylaerts M, Vermeulen P, Dirix LY. Arterio-venous gradients of IL-6, plasma and serum VEGF and D-dimers in human cancer. Br J Cancer. 2002 Dec 2;87(12):1437-44.
• Benoy I, Salgado R, Colpaert C, Weytjens R, Vermeulen PB, Dirix LY. Serum interleukin 6, plasma VEGF, serum VEGF, and VEGF platelet load in breast cancer patients. Clin Breast Cancer. 2002 Jan;2(4):311-5.
• Salgado R, Vermeulen PB, Van Marck E, Benoy I, Dirix L. Correspondence re: M. L. George et al., Correlation of plasma and serum vascular endothelial growth factor levels with platelet count in colorectal cancer: clinical evidence of platelet scavenging? Clin. Cancer Res., 6: 3147-3152, 2000. Clin Cancer Res. 2001 May;7(5):1481-3.