Breast, bladder, colon, and ovarian carcinomas show frequent low level 20q gain and less frequently high level 20q13.2 amplification, but the significance of these 20q amplifications in transformation has not been defined. Using karyotypic and comparative genomic hybridization (CGH) analyses, chromosome losses and gains were analysed in six newly immortalized human uroepithelial cell (HUC) lines transformed by Human Papillomavirus 16 (HPV16) E7. Results showed clonal chromosomes with 20q11->qter gain in all six lines. CGH revealed a peak of 20q13.2 amplification in two cell lines. FISH with whole chromosome 20 paint showed expanded chromosome regions (ECRs) and double minute chromosomes (DMs) that contained chromosome 20 material in cell lines with 20q13.2 amplification. FISH with probes from the center of the 20q13.2 human breast cancer amplicon showed as many as 24 signals in cells with 20q13.2 amplification. The acquisition of genome instability in these E7-HUCs did not correlate with TP53 mutation, as all E7-HUCs contained only wildtype TP53. These results suggest that low level 20q gain is associated with overcoming cellular senescence in E7 transformed cells (P-value=2 x 10(-7)), but does not confer genome instability, while high level 20q13.2 amplification is associated with chromosome instability. Loss of 10p (P-value = 3 x 10(-5)) was also important in immortalization of E7-transformed HUCs. Thus, these results have profound implications for interpreting the significance of high versus low level 20q gains in human cancers.