A total 33 formalin-fixed, paraffin-embedded specimens of ICC were collected. Using a 4 µm hematoxylin and eosin stained adjacent section as a guide, tumor-containing areas were microdissected from sectioned 10 µm specimens with a sterile 25-gauge needle under a microscope. Normal DNA from human peripheral blood and tumor DNA from microdissected cells were prepared by standard protocols.

DOP-PCR was performed in two separate steps according to the published protocol with some modifications (15, 16). Briefly, in the preamplification step, the four initial cycles were performed at low stringency conditions (denaturation at 95℃ for 3 min followed by 4 cycles of 94℃ for 1 min; 25℃ for 1 min; 56℃ for 3 min; 74℃ for 2 min), followed by 35 cycles in high stringency conditions (at 94℃ for 1 min; 56℃ for 1 min; 72℃ for 2 min). Labellings of tumor DNA with biotin-16dUTP and control DNA with digoxigenin-11dUTP were performed by nick translation (Nick Translation System, Gibco/BRL, Rockville, MD, U.S.A.). Equal amounts of labeled normal control and tumor DNA including 40 µg of Cot-1 DNA (Gibco/BRL) and 1 µg salmon sperm DNA were precipitated with ethanol. Labeled DNA probes were mixed with hybridization buffer, denatured at 75℃ for 5 min and then applied onto the denatured metaphase slides. After incubation for 3 days, the slides were washed and the hybridization signal was detected using an avidin-fluorescein isothiocyanate (FITC, 1:400) and mouse anti-digoxigenin (1:100). Metaphase slides were counterstained with DAPI at a concentration of 0.1 µg/mL in an antifade solution (Oncor, Gaithersburg, MD, U.S.A.) for chromosomal identification. Mean ratio profiles were determined from analyses of 10-15 metaphase cells. Thresholds for gains and losses were defined as the theoretical value of 1.25 and 0.75, respectively. The following regions were excluded for the analyses; centromere, acrocentric short arms, telomere, and heterochromatin-rich region (17).