CGH analysis
From each case, 20–30 paraffin sections (thickness 3–4 μm) were prepared for DNA extraction using the QIAamp Tissue Kit (QIAGEN GmbH, Germany). The yield of DNA was maximized with prolonged proteinase-K digestion according to previously published protocols [12]. CGH analysis was performed as previously described [11]. In brief, tumor DNA samples were labeled with FITC-dUTP (DuPont, Boston, MA, USA) by nick translation, whereas normal reference DNA was labeled with Texas Red (Vysis Inc., Downers Grove, IL, USA). In all cases, the tumor and reference DNA samples were sex-matched. Tumor and reference DNA were mixed with unlabeled Cot-1 DNA (Gibco, BRLTM), denatured, and applied onto slides with denatured metaphases of normal lymphocytes (Vysis Inc.). After hybridization at 37°C for 48 h, the slides were washed in 0.4 × SSC/0.3% NP-40 at 74°C for 2 min and in 2 × SSC/0.1% NP-40 at room temperature for 1 min. After air-drying, the slides were then counterstained with DAPI (Vysis Inc.). Two control hybridizations were also performed. In the first one, DNAs from a normal female and a normal male were labeled and hybridized to normal male metaphases. For the second experiment, DNA from a previously characterized breast cancer cell line (MPE 600, Vysis Inc.) and DNA from a normal female were labeled and hybridized to normal male metaphases. Six to 10 three-color digital images (DAPI, FITC, and Texas Red fluorescence) were collected from each hybridization using a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss Jena GmbH, Jena, Germany) and a Sensys charge-coupled-device camera (Photometrics, Tucson, AZ, USA) interfaced to an IPLab Spectrum 10 workstation (Signal Analytics Corporation, Vienna, VA, USA). Interpretation of CGH results was carried out as previously described. Green-to-red ratios < 0.80 were considered to signify losses of genetic material, ratios > 1.2 gains, and ratios >1.5 high-level amplifications. Heterochromatic regions, the short arms of the acrocentric chromosomes, and the sex chromosomes were not included in the evaluation.