Original articlesDetection of Genetic Alterations in Bladder Tumors by Comparative Genomic Hybridization and Cytogenetic Analysis
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.
Section snippets
Cytogenetic Analysis
Sixteen transitional cell carcinomas were obtained from the operating room of the Department of Urology at Chungnam National University Hospital, Taejon, Korea. The tumors were classified according to the World Health Organization guidelines. Half of the tumor tissues were cultured in RPMI 1640 medium with 10% fetal calf serum for 5–10 days for cytogenetic study, and then harvested after colcemid treatment. Abnormal clones were counted and analyzed according to the recommendation of the ISCN
Results
The interpretation of CGH data was guided by the control experiments. Comparisons among five normal DNA specimens were used to establish normal levels of green to red fluorescence intensity ratio variations along the length of all human chromosomes, while a cell line (MCF-7) with known amplifications was used to assess sensitivity.
The clinical and pathological data for each patient are shown in Table 1. Most cases were of a mixed grade and stage of transitional cell carcinoma. Two cases were
Discussion
Comparative genomic hybridization is a recent molecular cytogenetic technique that allows the detection of chromosomal imbalances in tumor DNA prepared from fresh or archival materials. It is a powerful method for the identification of genomic gains and losses in DNA from tumor samples 13, 14, 15, 16, 17, 18; thus, it may have diagnostic and prognostic application in cancer. Comparative genomic hybridization can map all loci of genetic alterations with hybridization at one time, as compared
Acknowledgements
This work was supported by grants from the Korean Education Ministry, Genetic Engineering (GE-97-169).
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