Defining a 0.5-mb region of genomic gain on chromosome 6p22 in bladder cancer by quantitative-multiplex polymerase chain reaction

Abstract

Metaphase-based comparative genomic hybridization (CGH) has identified recurrent regions of gain on different chromosomes in bladder cancer, including 6p22. These regions may contain activated oncogenes important in disease progression. Using quantitative multiplex polymerase chain reaction (QM-PCR) to study DNA from 59 bladder tumors, we precisely mapped the focal region of genomic gain on 6p22. The marker STS-X64229 had copy number increases in 38 of 59 (64%) tumors and the flanking markers, RH122450 and A009N14, had copy number gains in 33 of 59 (56%) and 26 of 59 (45%) respectively. Contiguous gain was present for all three markers in 14 of 59 (24%) and for two (RH122450 and STS-X64229) in 25 of 59 (42%). The genomic distance between the markers flanking STS-X64229 is 0.5 megabases, defining the minimal region of gain on 6p22. Locus-specific interphase fluorescence in situ hybridization confirmed the increased copy numbers detected by QM-PCR. Current human genomic mapping data indicates that an oncogene, DEK, is centrally placed within this minimal region. Our findings demonstrate the power of QM-PCR to narrow the regions identified by CGH to facilitate identifying specific candidate oncogenes. This also represents the first study identifying DNA copy number increases for DEK in bladder cancer.

Figure 1

Scores for 1-copy (white), 2-copy (gray) or genomic gain (black) of STS markers for 70 retinoblastoma tumors (A) and 59 bladder tumors (B). The most frequent genomic gain in bladder cancer was 64% for marker 229 (green box) with 56% for marker 450, in contrast to retinoblastoma where the most frequent genomic gain was 59% for marker 450 (red box) and 46% for marker 229. One retinoblastoma with i(6p) and one with no genomic gain were tested simultaneously with the bladder samples as external controls. C: Distribution across the 16.0-Mb region on chromosome 6p22 covered by the nine STS markers used in our study and four markers studied by Bruch et al. Four candidate oncogenes in the region are indicated by orange boxes (RBKIN, DEK, S0X4, and PRL). (STS marker abbreviations: 608 – SHGC-130608; 718 – RH113718; 208 – WI-19208; 450– RH122450; 229 – STS-X624229; N14 – A009N14; 629 – WI-22629; 319 – STS-U60319; 660 – WI-660.)

Figure 2

Interphase FISH analysis on paraffin-embedded tissue sections, using a BAC probe (RP11–686D16) specific for the region 6p22.3–24.3, either indirectly detected by FITC-Avidin (A) or directly labeled with spectrum orange (B and C). A: Prostate cancer with no amplification of 6p. B: Bladder tumor showing copy number gain of 6p. An average of 4.5 signals per nucleus was seen (range, 0 to 11, with 50% of the nuclei showing 5 to 11 signals). C: Lymphocytes in the core of a papillary frond in a bladder tumor serve as an internal 2-copy control. The tumor nuclei covering the papillary frond show copy number gain of 6p, with an average of 3 signals per tumor cell nucleus.