In vivo measurement of tumor estradiol and Vascular Endothelial Growth Factor in breast cancer patients

Ten postmenopausal women, ages 51–86, participated in the study. Postmenopausal status was defined by having no spontaneous menstrual bleeding for at least a year in women older than 50. None of the women had ongoing hormonal treatment. The study was approved by the local ethical review board in Linköping, Sweden, (reference number 03–081) and performed in compliance with the Helsinki Declaration, and all women gave informed consent. Patient characteristics are provided in Table 1.

Microdialysis was performed on the day before or the same day as surgery using microdialysis catheters with a molecular weight cut-off of 100,000 Da (CMA Microdialysis AB, Stockholm, Sweden). Each catheter consists of a tubular dialysis membrane (length 10 mm; diameter 0.52 mm) attached to a double-lumen tube (length 100 mm; diameter 0.8 mm). Prior to microdialysis, mepivacaine (0.3 mL; 5 mg/mL) was administered intracutaneously as a local anesthetic. Two microdialysis catheters were thereafter inserted in the affected breast, one intratumorally and one in adjacent, macroscopically normal breast tissue. The insertion was guided by catheters for intravenous use (Venflon, 1.4 mm; BOC Ohmeda AB, Helsingborg, Sweden). The microdialysis catheters were connected to a microinfusion pump (CMA 107, CMA Microdialysis AB) and perfused with NaCl (154 mM) and dextran-70 (40 g/L) at a rate of 0.5 μL/min. After an equilibration period of 30 minutes, the outgoing dialysate was collected and stored together with plasma samples at -70°C for subsequent analysis. No complications occurred during or after the microdialysis experiments.

Microdialysis allows sampling of the extracellular fluid by passive diffusion of substances over a semi-permeable membrane. The recovery i.e. the amount of substances that diffuse into the perfusion fluid depends on the tissue and membrane properties, the flow rate, and the size of the compound of interest [22]. Diffusion of low molecular substances over the dialysis membrane is almost complete at low flow rates using a 30 mm long dialysis membrane [23]. However, for larger molecules the recovery over the membrane decreases and the measured levels in the microdialysis sample may not be absolute concentrations in the tissue. The recovery of certain substance may be measured in vitro, however, this in vitro recovery can only be an estimate of the in vivo recovery since other factors such as tissue pressure and temperature will affect the diffusion of substances. For the in vivo recovery several calibration techniques such as the no net flux technique, the stop flow techniques, and the endogenous reference technique have been developed for low molecular substances unbound in the extracellular space. However, when measuring high molecular weight proteins these techniques may not be applicable because the proteins of interest may be bound to binding proteins in vivo, recombinant proteins may not have the same properties as the natural proteins in vivo, and plasma and tissue levels of the protein are not equal. We have previously found that in the case of VEGF tissue homogenate, unlike plasma VEGF, correlate significantly with VEGF sampled by microdialysis. We have previously reported an in vitro recovery of VEGF of 8% and an in vitro recovery of estradiol from plasma of 30% and these data were confirmed in the present study [7, 20]. In the present study all microdialysis values are given as original raw data without any re-calculations.

Measurements of estradiol in plasma and microdialysis dialysate were conducted using a commercial quantitative immunoassay kit (estradiol ELISA, DRG Instruments GmbH, Marburg, Germany) according to the manufacturer's instructions. VEGF was quantified using a commercial quantitative immunoassay kit for human VEGF (Fluorokine MAP human VEGF kit, R&D systems, Abingdon, UK) and analyzed by the Luminex 100 IS System (Luminex B.V., Oosterhout, Netherlands). According to the manufacturer, the VEGF kit measures the VEGF₁₆₅ and VEGF₁₂₁ isoforms, and the mean minimum detectable dose is 0.81 pg/mL. The intra-assay precision for this kit is 3.7–7% which was confirmed during the experiment. All microdialysis values are stated as raw data.

Formalin-fixed, paraffin-embedded tumors were sectioned, deparaffinized and subjected to anti-human VEGF immunohistochemistry (monoclonal mouse anti-human VEGF, specific for the VEGF₁₆₅ and VEGF₁₂₁ isoforms, dilution 1:20, R&D systems, with Envision detection, DAKO). Sections were counterstained with Mayer's hematoxylin. Negative controls did not show staining. In a blinded manner, entire sections were first scanned to determine the range of intensity of the staining. VEGF staining was thereafter scored as weakly or strongly positive. Estrogen receptor (ER) and progesterone receptor (PR) were determined by immunohistochemical staining using the Ventana Benchmark system (Ventana Medical Systems Inc., Arizona, USA) and defined as the fraction of positive nuclei. Monoclonal rabbit anti-human estrogen receptor alpha antibody (prediluted 1:50, Lab Vision Ltd, Suffolk, UK) and monoclonal mouse anti-human progesterone receptor antibody (prediluted 1:50, Novocastra Laboratories Ltd, Newcastle, UK) were used for staining.

Tumor histology, size, stage (T of the TNM classification), and Nottingham histological grade (NHG) according to Elston Ellis scoring system were determined at the Department of Pathology and Cytology, University Hospital of Linköping.

Shapiro-Wilks' W test for normality was used. Wilcoxons' signed rank test for paired observations was used and normally distributed data was calculated using Pearson's correlation coefficient. Results are given in median [25–75 precentile].

Ten patients were included in the study, 7 with invasive ductal carcinoma and 3 with invasive lobular carcinoma. All of the tumors were ER-positive; 5 of 10 were PR-positive. Tumors were stage pT1-3 (TNM) and NHG 2–3. All tumors showed positive scoring for mitosis (not shown). Clinicopathological data is provided in Table 1.

Using microdialysis, levels of estradiol and extracellular VEGF were measured in tumors and in adjacent normal breast tissue in vivo. Levels of intratumoral VEGF were significantly higher than in normal breast tissue, 6.0 pg/mL [4.1–6.5] in tumors compared to 2.4 pg/mL [2, 3] in breast tissue, p = 0.005, Wilcoxon signed rank test, Figure 1). Plasma levels of VEGF were 3.67 pg/mL [0.8–5.7], range 0.62–7.59 pg/mL. In 7 of the 10 patients tumor estradiol levels were higher than in normal breast tissue, however, when comparing the whole group no significant differences were found between the levels of tumor estradiol, 106 pM [72–136], and normal breast tissue estradiol, 87 pM [48–150], Figure 2. Plasma levels of estradiol were 60 pM [40–84], range 24–198 pM.

A significant positive correlation was found between plasma levels and normal breast tissue levels of estradiol, Figure 3. No correlations were found between tumor estradiol and tumor VEGF or between plasma VEGF and tumor VEGF. ER, PR, and tumor size did not correlate with VEGF or estradiol measured in microdialysis dialysate or plasma.

All tumor sections showed positive intracellular staining for VEGF. Inter-individual differences in intratumoral VEGF quantified in microdialysis dialysate were not reflected in the intensity of staining for VEGF.