Comparative genomic hybridisation

Abstract

Comparative genomic hybridisation (CGH) is a technique that permits the detection of chromosomal copy number changes without the need for cell culturing. It provides a global overview of chromosomal gains and losses throughout the whole genome of a tumour. Tumour DNA is labelled with a green fluorochrome, which is subsequently mixed (1:1) with red labelled normal DNA and hybridised to normal human metaphase preparations. The green and red labelled DNA fragments compete for hybridisation to their locus of origin on the chromosomes. The green to red fluorescence ratio measured along the chromosomal axis represents loss or gain of genetic material in the tumour at that specific locus. In addition to a fluorescence microscope, the technique requires a computer with dedicated image analysis software to perform the analysis. This review aims to provide a detailed discussion of the CGH technique, and to provide a protocol with an emphasis on crucial steps. (J Clin Pathol: Mol Pathol 1999;52:243-251) Keywords: comparative genomic hybridisation; methodology; protocol; paraYn wax embedded material Comparative genomic hybridisation (CGH) is a technique that permits the detection of chromosomal copy number changes without the need for cell culturing. It gives a global overview of chromosomal gains and losses throughout the whole genome of a tumour. Thus, CGH is a relatively fast screening technique that can point at specific chromosomal regions that might play a role in the pathogenesis or progression of tumours. Guided by CGH results, more specific molecular biological techniques (such as fluorescence in situ hybridisation, loss of heterozygosity analysis, and sequencing) can be used to identify oncogenes and/or tumour suppressor genes in these regions.Kallioniemi et al at the University of California, San Francisco were the first to report CGH as a new chromosome analysis technique in 1992, 1 shortly followed by du Manoir et al. 2In short, tumour DNA is labelled with a green fluorochrome, mixed (1:1) with red labelled normal DNA, and hybridised to normal human metaphase preparations. The normal reference DNA and the metaphases are obtained from a healthy volunteer and do not need to be from the patient. The green and red labelled DNA fragments compete for hybridisation to their locus of origin on the chromosomes. The green to red fluorescence ratio measured along the chromosomal axis represents loss (ratio < 1) or gain (ratio > 1) of genetic material in the tumour at that specific locus (figs 1 and 2). In addition to fluorescence Figure 1 Principle of comparative genomic hybridisation (CGH

Comparative Genomic Hybridization of Microdissected Familial Ovarian Carcinoma: Two Deleted Regions on Chromosome 15q Not Previously Identified in Sporadic Ovarian Carcinoma

“…Chromosomes 16 and 19 were excluded from the analysis because of known interpretation difficulties (Kallioniemi et al, 1994;Weiss et al, 1999). We found 12 of 36 (33%) losses in this region; previously, Arnold et al (1996) also detected frequent loss of this region (11 of 47; 23%).…”

“…Using archival material and microdissection, we were able to accurately isolate pure (Ͼ80%) tumor DNA, whereas Tapper et al (1998) selected tissue containing more than 50% tumor cells without microdissection. Weiss et al (1999) demonstrated that copy losses easily become undetectable at 50% contamination of normal tissue. As a result, our technique using microdissected material is more sensitive to the detection of single copy chromosomal losses.…”

“…In our analysis we have excluded gains and losses of chromosome 16 and 19 because CGH data of these chromosomes is notoriously difficult to interpret due to the repetitive nature of the DNA surrounding the centromeres (Kallioniemi et al, 1994;Weiss et al, 1999).…”

Simultaneous chromosome 1q gain and 16q loss is associated with steroid receptor presence and low proliferation in breast carcinoma

“…In order to reduce the quantity of falsenegative results, samples showing a percentage of neoplastic cells fewer than 50% were not used for DNA extraction and CGH analysis. 10,11 The percentage of neoplastic vs stromal and inflammatory cells was defined by one pathologist (DS), scoring the control hematoxylin-eosin-stained sections of each sample.…”

“…In total, 9-12 metaphases were analyzed for each sample and the average green-tored intensity ratio profiles were calculated and plotted with 95% confidence interval (CI). 11 Any relatively small, distinct locus showing a green-tored fluorescence ratio greater than 1.5 was regarded as high-level amplification. Chromosomes 19, 1p32-ter and 16p-were excluded from the analysis since it has been demonstrated that the green-to-red fluorescence ratio in these regions cannot be reliably measured.…”