Comparative genomic hybridization (CGH) is a powerful new method for molecular cytogenetic analysis of cancer. In a single hybridization, CGH provides an overview of DNA sequence copy number changes (losses, deletions, gains, amplifications) in a tumor specimen and maps these changes on normal chromosomes. CGH is based on the in situ hybridization of differentially labeled total genomic tumor DNA and normal reference DNA to normal human metaphase chromosomes. After hybridization and fluorescent staining of the bound DNAs, copy number variations among the different sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target metaphase chromosomes. CGH is in particular useful for analysis of DNA sequence copy number changes in common solid tumors where high-quality metaphase preparations are often difficult to make, and where complex karyotypes with numerous markers, double minutes, and homogeneously stained chromosomal regions are common. CGH only detects changes that are present in a substantial proportion of tumor cells (i.e., clonal aberrations). It does not reveal translocations, inversions, and other aberrations that do not change copy number. At present, CGH is a research tool that complements previous methods for genetic analysis. CGH will advance our understanding of the genetic progression of cancer and highlight important genomic regions for further study. Direct clinical applications of CGH are possible, but will require further development and validation of the technique. We describe here our recent optimized procedures for CGH, including DNA labeling, hybridization, fluorescence microscopy, digital image analysis, data interpretation, and quality control, emphasizing those steps that are most critical. We will also assess sensitivity and resolution limits of CGH as well as discuss possible future technical improvements.

Comparative genomic hybridization was applied to 5 breast cancer cell lHes and 33 primary tumors to discover and map regions of the genome with increased DNAsequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copynumber affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression.Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.Increased expression of specific genes plays an important role in the pathogenesis of solid tumors (1-3). Gene amplification, characterized by distinct cytogenetic structures, such as homogeneously stained regions, double-minute chromosomes (1-7), is commonly found in tumor cells and is considered an important mechanism by which tumor cells gain increased levels of expression of critical genes. Increased copy-numbers also occur as a result of extensive chromosomal rearrangements, such as duplications, isochromosomes, extra marker chromosomes, and acentric chromosomal fragments that may affect the gene dosage of numerous genes simultaneously. In breast cancer, cytogenetic evidence of increased DNA-sequence copy-number is common (4-7). For example, homogeneously stained regions have been found in 60% of primary breast carcinomas (7). Although genetic analysis has found amplification of oncogenes, such as ERBB2 (17q12), MYC (8q24), PRADII CYCLIN D (11q13), FLG (8p12), BEK (10q24), and IGFR-1/FES (15q24-q25) (8-12), in most cases these do not explain the presence of large homogeneously stained regions (13). Thus, amplification of currently unknown genes may often occur in breast cancer.We have recently developed a method, comparative genomic hybridization (CGH), for surveying entire genomes for DNA-sequence copy-number variation (14, 15). In CGH, the relative intensities of tumor DNA (detected using green fluorescence) and normal reference DNA (detected with red fluorescence) after hybridization to normal metaphase chromosomes is used to reveal and map regions of increased DNA-sequence copy number (14-16). These loci are visualized as chromosomal region(s) with predominantly green fluorescence ( Fig. 1) and quantified by digital image analysis as an increased green-to-red fluorescence intensity ratio (Fig. 2). As no specific probes or previous knowledge of aberrations is required, CGH is especially suitable for identification and mapping of previously unknown DNA copy-number changes that may highlight locations of important genes. In the present study, we have used CGH to identify and map increases in DNA-sequence copy number in 15 breast cancer cell lines and 33 uncultured primary breast tumors. MATERIALS AND ME...

We describe and evaluate the image-processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818,1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes.The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented. ti 1995 WiIey-I.iss, Inc.Key terms: Amplification, background correction, deletion, fluorescence in situ hybridization, genomic DNA, image analysis, molecular cytogenelics, intensity profile, ratio profile, simulation, tumor cytogenetics Since its recent introduction ( 4 ) , comparativc genomic hybridization (CGH ) has become an important technique for genetic analysis. CGH provides a rapid method for comparing DNA sequence copy number throughout two (or more) genomes. It has found major application in the analysis of genetic aberrations in cancer (3-6,9). Detection and chromosome localization of copy number changes such as deletions and amplifications may highlight the locations of inactivated tumor suppressor genes and activated oncogenes, respectively. Because CGH does not require cell culture, it can be applied to many situations where standard cytogenetics yields little or n o information, such as in solid tumors and archival (fixed) tumor specimens. Comparison of tumor and normal genomic DNAs is a major application o f CGH, and, in this paper, we focus on the analysis of this type o f experiment. However, CGH is also expected to have significant utility in perinatal analysis of whole and segmental chromosome ancuploidies such as Down syndrome and possibly also i n microdeletion syndromes.In a typical CXH analysis, an abnornial tcst genome and a normal reference genome are simultaneously hybridized to normal nietaphase "target" chromosomes ( Fig. I ) and detected with different fluorochromes. The ratio of the staining intensities at a particular location on a niet:iphase chromosome reflects the ...

Procedures for fully automatic location of chromosome axis and centromere in metaphase chromosomes are described for a practical interactive chromosome analysis system that omits the usual stages of interactive axis and centromere correction. Accuracy of centromere finding and consequential determination of a chromosome's polarity, i.e., which end is which, is measured experimentally. The saving in interaction by not correcting centromeres is compared to the increase in errors at the classification stage and the consequent increase in interaction needed to correct these errors. Some previously unreported features for banded chromosome classification are described, and in particular a set of global shape features is introduced. The discrimination capability of the feature measurements is evaluated by use of simple statistics and by reference to the performance of classifiers trained with various feature subsets. Class discrimination capability of the global shape feature set is shown to be comparable to that of centromere position, a widely used local shape feature. The variability of feature measurements that might occur in data from different laboratories on account of differing tissue, preparation methods, and digitiser hardware is assessed using three data bases of G‐banded human metaphase cells. It is shown that the differences can be considerable and that appropriate feature selection and classifier training substantially improve classification performance.

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