Detection of Genetic Alterations in Bladder Tumors by Comparative Genomic Hybridization and Cytogenetic Analysis

Abstract

Comparative genomic hybridization (CGH) and conventional cytogenetic karyotyping were used to screen for losses and gains of DNA sequences along all chromosome arms in 16 bladder tumors. Cytogenetic results were highly complex. The most frequently affected chromosomes were 5, 8, 9, 21, and Y as determined by karyotyping. There was close correlation between the CGH data and cytogenetic results in near-diploid tumors with simple karyotypes. However, some unexpected results were observed by CGH in tumors with several composite clones. Common amplification of copy numbers of DNA sequences by CGH were seen at 1q, 3q, 4q, 5p, 6p/q, 7p, 8q, 11q, 12q, 13q, 17q, 18q, and 20p/q (more than 20% of cases). High level amplification was noted at 1p32, 3p21, 3q24, 4q26, 8q21-qter, 11q14∼22, 12q15∼21, 12q21∼24, 13q21∼31, 17q22, and 18q22. Deletions were noted at 2q21∼qter, 4q13∼23, 5q, 8p12∼22, 9p/q, and 11p13∼15 (more than 20% of cases). Although most amplifications and deletions have been previously described in the literature, our study showed some intriguing and uncommon regions, different from those found in past studies. These were the amplification of 7p, 8q, 11q14∼qter 12q24∼24, 13q21∼31, and 18q22, and deletion on 4q13∼23, even though loss of heterozygosity was not detected at this locus. In spite of the very complex pattern of genetic changes in bladder tumors, most of these uncommon aberrations have to be implicated in bladder tumors, and further molecular genetic methods are necessary to establish whether the chromosomal regions contain candidate genes which contributed to the initiation and progression of bladder tumors.

Introduction

Genetic changes of tumors are known to be related to initiation and progression of cancers. Molecular genetic techniques have been used for the detection of genetic changes. Loss of heterozygosity (LOH) and Southern hybridizations can be used to identify chromosomal regions harboring putative tumor suppressor genes and oncogenes. They are limited to the screening of all chromosomal arms with only one or two markers at one time, and are therefore impractical for the detection of unknown genetic changes in entire tumor genomes. In transitional carcinoma of the bladder, LOHs have been detected at 9p21, 11p, 13q14, and 17p 1, 2, 3, 4. Oncogenes, such as the MYC gene, studied by Southern hybridization, have been reported to be amplified in about 10% of bladder tumors [5]; however, the target genes in other chromosomal regions remain unknown. Genome-wide surveys of LOH and oncogene studies are very labor intensive. Cytogenetic hallmarks of gene amplification such as homogenously staining regions or double minutes are seen in bladder tumors [6].

Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic method that detects and localizes gains or losses of genetic material across the entire tumor genome [7]. For CGH, two-color fluorescence in situ hybridization (FISH) is applied to reference metaphase spreads using a mixture of differentially-labeled tumor DNA and normal genomic DNA. Fluorescence ratio changes reflect copy number changes of chromosomes within tumor cells; it is similar to conventional cytogenetics in that the entire genome of individual tumors is screened for changes; however, it does not require cell culture. Karyotyping of solid tumors is not easy because primary cultures are not readily obtained, and it is difficult to get optimal metaphases; however, its important advantages over standard molecular techniques are that probes or markers are not needed to map changes. Comparative genomic hybridization is also used in the detection of chromosomal abnormalities of congenital malformation in genetic diseases [8].

Bladder tumors comprise a highly heterogeneous group of malignancies. They show a wide range of chromosomal numbers associated with a large number of structural and numerical chromosomal changes, suggesting variety in the biology of these cancer cells [9]. Cytogenetic data have shown that although no specific abnormality has been identified, the pattern of changes is clearly nonrandom. In this study, we used cytogenetic techniques and CGH to analyze 16 primary bladder tumors for the detection of genetic alterations.