Comparative genomic hybridization was performed as described by du Manoir et al. with minor differences [10].

Tumor Samples. 26 transitional cell carcinomas of the urinary bladder from Bulgarian patients were collected as fresh tumor samples. According to their tumor stage the samples can be grouped as follows: pT1 (16 samples) and pT2-4 ( 10 samples ). Representative for the tumor areas for further analysis were selected by a pathologist.

DNA extraction and labeling. Tumor and normal reference DNA from peripheral blood of healthy individuals was isolated by standard proteinase K and phenol chlorophorm extraction procedure. Control DNA was labeled with digoxigenin-11-dUTP while tumor DNA - with biotin-16-dUTP by standard nick translation procedure.

Metaphase spreads preparation. Metaphase spreads for CGH were prepared from phytohemaglutinin-stimulated lymphocytes of healthy individuals by standard procedures of colcemid arrest, hypotonic treatment, and methanol/acetic acid fixation. Prior to hybridization the slides were pepsin pretreated (25 ml 10% pepsin, in 50 ml dH₂O with 250 ml 2N HCl) and denatured in 70% deionized formamide in 2xSSC (standard saline citrate) for 3 minutes at 73^o C and dehydrated through a cold ethanol series (70%, 85%, 96%) for 3 minutes each.

Hybridization. The hybridization mix consisted of 400 ng digoxygenin labeled tumor DNA, 400 ng biotin labeled control DNA and 30mg of Cot-1 DNA dissolved in 10 ml hybridization buffer (60% formamide, 10% dextran sulphate in 2xSSC, pH 7.0). The mix was denatured at 73 ^oC for 6 min, transferred for 2 min on ice and allowed to pre-anneal at 37^oC for 30 min. The DNA mixture was than applied to the denatured normal methaphase chromosomes under a coverslip, sealed with rubber cement and hybridized for 2-5 days at 37⁰C in a humified chamber.

Hybridization wash and detection. Non-specifically bound DNA was washed from the slides (5 min in 2XSSC, 15 min in 50% formamide/2XSSC at 42^o C water bath, 15 min in 2XSSC at 42^oC water bath, 1min 1XPBS). The hybridization was detected with a mix containing 35ml FITC-avidin and 35ml Rhodamine-antidigoxigenin (20 min at 37⁰ C). The chromosomes were counterstained with DAPI.

Controls and threshhold definition. In each experiment sex-mismatched normal DNA was used. Only cases with a ratio between the fluorescence intensities at the chromosome X of ³ 1.25 were accepted (internal control). Threshholds for DNA sequence copy number were defined as follows: a gain was assumed at chromosomal regions where the hybridization resulted in a tumor-to-normal ratio of ³ 1.25; loss (deletion) was presumed at chromosomal regions where the tumor-to-normal ratio was £0.80. As recommended by some authors in a similar study [9] aberrations at 1p, 16p, 19 and 22, G-C-rich regions, known to produce false positive results by CGH, were excluded from all analyses.

Digital Image Analysis. Different fluorescence imagens were taken separately with an epifluorescent microscope (Olympus BX60) equipped with DAPI, FITC and TRITC fluorescent filters connected to a computer system for analysis of fluorescent images (ISIS, MetaSystems, Germany) through CCD imaging PCO camera VC45. Five to ten metaphase spreads were analysed for each tumor sample.

Statistics. One way ANOVA was applied to compare the number of genomic alterations between tumors of different stages and grades. Contingency table analysis was used to analyse the relationship between the frequency of specific genomic alterations between tumors of different stages.