BMC Cancer 2005, 5:77 http://www.biomedcentral.com/1471-2407/5/77 applied onto the spotted area of the array under a cover- slip and hybridized in a humid chamber containing 50% formamide (FA)/2XSSC at 37°C for 72 hrs. After hybridi- zation, the arrays were washed 3X in 50%FA/2XSSC at 40°C for 10 min/wash, followed by four 5 min. washes in 1XSSC at room temperature. Finally, the arrays were briefly rinsed in distilled water, mounted and counter- stained in the dark for 45 min. with DAPI (4,6-diamino- 2-phenylindole). Image capture and analysis Array analysis was performed immediately after counter- staining using the GenoSensor scanner and software. This system generates a "genomic analysis report", indicating which chromosome regions in the array are involved in copy number changes, as well as a spreadsheet containing the data generated by a single experiment. In order to compare all the experiments, a database was created using the normalized, bias corrected, tumor/normal ratio value of each experiment [see additional file 1]. Since each spot in the array is present in triplicates, the median of the three spots of each probe in the array was calculated and its log2 transformed value was used for further analysis. A fluorescence ratio >1.25 (log2 = 0.32) was considered as a DNA gain, while DNA losses were scored when the ratio was <0.75 (log2 = -0.41). A ratio >2 (log2 = 1) was consid- ered as a high copy number amplification. Results HPV detection and typing One of the premalignant lesions was positive for HPV16 infection; one for HPV31 and the other for HPV58. In the invasive tumors, seven were positive for HPV16 and in three cases, we were not able to detect HPV sequences with the oligonucleotides we used for PCR amplification. As expected, CasKi and SiHa were positive for HPV16, while HeLa, INBL, CaLo, ViVo and RoVa were positive for HPV18. Microarray comparative genomic hybridization All our samples, except one pre-malignant lesion, pre- sented alterations, ranking from 1/287 (alterations/total targets in the array) in a pre-malignant lesion to 175/ 287alterations in the cell line RoVa. We found almost twice the number of DNA gains than DNA losses (571 vs. 298) and the average number of copy number alterations (ANCA=total number of alterations in the sample collec- tive/total number of cases) was 43.45 per case. One of the pre-malignant lesions did not show any alter- ation, while amplifications at MSH2-KCNK12 (2p22.3- 2p22.1), TCL1A (14q32.1) and TOP1 (20q12) were found in a second pre-malignant lesion and DMBT1 (10q25.3), ERBB2 (17q12), and 4qTEL11 (4qtel) amplifi- cation was found in the third sample from this group. In the invasive tumors and the cell lines, the most com- mon amplifications (58.8% of the samples) were found at the clones RBP1-RBP2 (retinol binding protein 1 and 2, 3q21-q22), present as a high copy number amplification (HCNA) in two samples; DAB2 (disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila), 5p13; C84C11/T3 (5ptel) and D5S23 (5p15.2), followed by gains of Tp63 (3q27-q29, 2 HCNA); EGFR (Epidermal growth factor receptor, 7p12.3-p12.1, 4 HCNA) and D5S2064 (5p15.2), in 52.9% of the invasive samples and amplification of INS (Insulin, 11ptel) in 47% of the samples. The most common deletion was found at the clone corre- sponding to the FHIT (Fragile histidine triad) gene (3p14.2), present in 47% of the invasive samples, fol- lowed by deletions at D8S504 (8p23.3), CTDP1-SHGC- 145820 (18qtel), KIT (4q11-q12), D1S427-FAF1 (1p32.3), D9S325 (9qtel), EIF4E (eukaryotic translation initiation factor 4E, 4q24), RB1 (13q14), and DXS7132 (Xq12) present in 29.4% of the samples. A histogram of the DNA copy number alterations detected in the tumor samples analyzed by array CGH is presented in figure 1. The results of these experiments can be accessed through the Progenetix CGH database http://www.pro genetix.com[25]. Discussion Previous studies using chromosomal CGH have delimited a specific pattern of chromosomal imbalances in cervical carcinoma. However, there is little knowledge regarding the identity of particular genes that might be the targets for these copy number changes, making microarray CGH an attractive method in order to define these particular gene targets. There was concordance between the alterations detected by microarray CGH and the pattern of chromosomal alterations already described by chromosomal CGH. Although the array that we used did not cover the entire genome, we were able to detect alterations at particular genes and genetic markers that might be related to the transformation process in the cervical epithelium. It is important to notice that the limited number of pre- malignant lesions analyzed did not allowed us to detect any particular region that might be related with this stage of the disease. However, an interesting candidate gene amplified in one pre-malignant sample and in 5 invasive tumors was MSH2-KCNK12 (2p22.3-2p22.1). This gene is the human homolog of the E. coli mismatch repair gene mutS, and has been found mutated in hereditary nonpoly- posis colon cancer. Higher MSH2 expression has been described in cervical intraepithelial neoplasias and inva- sive cervical carcinomas than in non-neoplastic cervical Page 3 of 7 (page number not for citation purposes)