Cytogenetic analysis from DNA by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.     

Analysis of genetic aberrations in uterine adenomyosis using comparative genomic hybridization

OBJECTIVE: To use comparative genomic hybridization (CGH) to analyzefrozen tissuesfrom adenomyosis cases to discover and map genomic regions for chromosomal gains and losses. STUDY DESIGN: In a retrospective study, upper and lower threshold values of 1.20 and 0.80, respectively, were used to define positive findings. RESULTS: No positive recurrent gene copy number alterations were detected in the 25 cases of pathologically proven adenomyosis. CONCLUSION: Although CGH is extremely useful in investigating candidate genes in the development of adenomyosis, CGH was not useful in this study. Genetic changes might be indeed extremely rare in adenomyosis, or CGH was not sensitive enough to detect candidate genes.     

Molekularzytogenetische Methoden und Array-Diagnostik in der Pr?natalmedizin

In addition to the widely used cytogenetic standard approaches, molecular methods are being increasingly used in prenatal diagnostics. While molecular cytogenetics, e.g., fluorescence in situ hybridization (FISH), has been used for many years in invasive prenatal diagnostics, array-based diagnostics are only now being implemented in this field. FISH is prenatally applied for determination of size of a mosaic cell clone, for exclusion of a microdeletion, or for further clarification of structural chromosomal aberrations. Array CGH (comparative genomic hybridization) is used more conservatively in prenatal diagnostics, mostly for further clarification in sonographically abnormal fetuses and to diagnose breakpoints in cases with proven chromosomal changes. In the future, array CGH will gain further importance, but already provides a valuable supplement to the diagnostic approaches of the cytogenetic and the molecular-based methods.     

Comparative analysis of algorithms for identifying amplifications and deletions in array CGH data

MOTIVATION: Array Comparative Genomic Hybridization (CGH) can reveal chromosomal aberrations in the genomic DNA. These amplifications and deletions at the DNA level are important in the pathogenesis of cancer and other diseases. While a large number of approaches have been proposed for analyzing the large array CGH datasets, the relative merits of these methods in practice are not clear. RESULTS: We compare 11 different algorithms for analyzing array CGH data. These include both segment detection methods and smoothing methods, based on diverse techniques such as mixture models, Hidden Markov Models, maximum likelihood, regression, wavelets and genetic algorithms. We compute the Receiver Operating Characteristic (ROC) curves using simulated data to quantify sensitivity and specificity for various levels of signal-to-noise ratio and different sizes of abnormalities. We also characterize their performance on chromosomal regions of interest in a real dataset obtained from patients with Glioblastoma Multiforme. While comparisons of this type are difficult due to possibly sub-optimal choice of parameters in the methods, they nevertheless reveal general characteristics that are helpful to the biological investigator.     

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions

 The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels. Accepted: 27 June 1997     

Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays

Chromosomal imbalances such as deletions and amplifications are common rearrangements in most tumors. Specific rearrangements are consistently associated with specific tumor types or stages, implicating the role of the genes in a region of chromosomal imbalance in tumor initiation and progression. The development of comparative genomic hybridization (CGH) has obviated the need to obtain metaphase spreads from tumors, so that the chromosomal imbalances in many solid tumors may be revealed using an extracted genomic DNA sample. However, the resolution of the cytogenetic method remains and the extreme technical difficulty of CGH has restricted its use. Conceptually, DNA microarray-based CGH is an obvious solution to all of the limitations of conventional CGH. Although arrays have been used for CGH studies, their success has been limited by poor specific signal-to-noise ratios. Here we demonstrate a microarray-based CGH method that allows reliable detection of chromosomal deletions and amplifications with high resolution. Our microarray system is fundamentally different from most current microarray technologies in that activated DNA is printed on natural glass surfaces while other systems almost exclusively focus on activating the surfaces, a strategy that invariably introduces hybridization backgrounds. The concept of using pre-modification may be generally applied for making arrays of other biological materials, as modifying the substrates will be more controllable in solution than on surfaces.     

From microscopes to microarrays: dissecting recurrent chromosomal rearrangements

Submicroscopic chromosomal rearrangements that lead to copy-number changes have been shown to underlie distinctive and recognizable clinical phenotypes. The sensitivity to detect copy-number variation has escalated with the advent of array comparative genomic hybridization (CGH), including BAC and oligonucleotide-based platforms. Coupled with improved assemblies and annotation of genome sequence data, these technologies are facilitating the identification of new syndromes that are associated with submicroscopic genomic changes. Their characterization reveals the role of genome architecture in the aetiology of many clinical disorders. We review a group of genomic disorders that are mediated by segmental duplications, emphasizing the impact that high-throughput detection methods and the availability of the human genome sequence have had on their dissection and diagnosis.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

Comparative genomic hybridization (CGH): ten years of substantial progress in human solid tumor molecular cytogenetics

Data from ten years of research using comparative genomic hybridization (CGH) for the detection of chromosomal alterations in human solid tumors are concisely reviewed. By use of a basic methodology with some variations more or less specific patterns of genomic imbalances were found in a large number of tumors of various entities. Specific gains and losses of genomic material have not only opened the way to the detection of a series of cancer-related genes but also to clinical implications. Not only several areas of basic oncogenetic research, but also differential diagnosis, prognosis of disease progression, and therapeutic decisions have profited by CGH.     

Karyotyping of comparative genomic hybridization human metaphases by using support vector machines.

BACKGROUND: Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic method for detecting chromosomal imbalance. Karyotyping of human metaphases is an important step to assign each chromosome to one of 23 or 24 classes (22 autosomes and two sex chromosomes). Automatic karyotyping in CGH analysis is needed. However, conventional karyotyping approaches based on DAPI images require complex image enhancement procedures. METHODS: This paper proposes a simple feature extraction method, one that generates density profiles from original true color CGH images and uses normalized profiles as feature vectors without quantization. A classifier is developed by using support vector machine (SVM). It has good generalization ability and needs only limited training samples. RESULTS: Experiment results show that the feature extraction method of using color information in CGH images can improve greatly the classification success rate. The SVM classifier is able to acquire knowledge about human chromosomes from relatively few samples and has good generalization ability. A success rate of moe than 90% has been achieved and the time for training and testing is very short. CONCLUSIONS: The feature extraction method proposed here and the SVM-based classifier offer a promising computerized intelligent system for automatic karyotyping of CGH human chromosomes.     

Cytogenetically balanced translocations are associated with focal copy number alterations

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Estimation of Copy Number Alterations from Exome Sequencing Data

Exome sequencing constitutes an important technology for the study of human hereditary diseases and cancer. However, the ability of this approach to identify copy number alterations in primary tumor samples has not been fully addressed. Here we show that somatic copy number alterations can be reliably estimated using exome sequencing data through a strategy that we have termed exome2cnv. Using data from 86 paired normal and primary tumor samples, we identified losses and gains of complete chromosomes or large genomic regions, as well as smaller regions affecting a minimum of one gene. Comparison with high-resolution comparative genomic hybridization (CGH) arrays revealed a high sensitivity and a low number of false positives in the copy number estimation between both approaches. We explore the main factors affecting sensitivity and false positives with real data, and provide a side by side comparison with CGH arrays. Together, these results underscore the utility of exome sequencing to study cancer samples by allowing not only the identification of substitutions and indels, but also the accurate estimation of copy number alterations.     

Human and mouse oligonucleotide-based array CGH

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using ‘off-the-shelf’ oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.     

A statistical approach for array CGH data analysis

Background  

Microarray-CGH experiments are used to detect and map chromosomal imbalances, by hybridizing targets of genomic DNA from a test and a reference sample to sequences immobilized on a slide. These probes are genomic DNA sequences (BACs) that are mapped on the genome. The signal has a spatial coherence that can be handled by specific statistical tools. Segmentation methods seem to be a natural framework for this purpose. A CGH profile can be viewed as a succession of segments that represent homogeneous regions in the genome whose BACs share the same relative copy number on average. We model a CGH profile by a random Gaussian process whose distribution parameters are affected by abrupt changes at unknown coordinates. Two major problems arise : to determine which parameters are affected by the abrupt changes (the mean and the variance, or the mean only), and the selection of the number of segments in the profile.     

Comparative M-FISH and CGH analyses in sensitive and drug-resistant human T-cell acute leukemia cell lines

Cell lines of human T-cell acute lymphoblastic leukemias (T-ALL) have gained high interest for study of mechanisms of cytostatic drug resistance. However, they should also be suited to examine the validity and reliability of molecular cytogenetic techniques in detecting genomic alterations in neoplastic cells. Therefore, comparative genomic hybridization (CGH) and 24-color-fluorescence-in-situ-hybridization (M-FISH) were applied to eight sublines of CCRF-CEM leukemia cells selected in vitro for drug resistance and to their drug-sensitive parental counterparts. All cell lines were characterized by altered chromosome numbers and by a variety of chromosomal structural aberrations as shown by M-FISH. The great majority of anomalies detected by this technique were confirmed by CGH. Interestingly, a considerable number of the rearrangements found were imbalanced. Amplifications of 5q13 in the six methotrexate-resistant cell lines, a del(9)(p21pter) in all lines examined, and a gain of chromosome 20 in 9 of the 10 lines examined were readily detected by both techniques. The same held true for losses of chromosomes 17 and 18 in the near tetraploid cell lines which could also be confirmed by CGH. Some imbalances of genomic material detected by CGH were, however, not observed by means of M-FISH, possibly due to the limited extension of the corresponding chromosomal segment involved or the small subpopulation of cells affected. On the other hand, reciprocal translocations, balanced isochromosomes, and small deletions remained mainly undetected by CGH. A comparison of chromosomal alterations in drug-resistant and parental cell lines showed not only amplifications of chromosomal segments harboring well-known drug resistance genes, e.g., the dihydrofolate reductase gene, but also chromosomal changes which may involve novel genes associated with drug resistance. Thus, the present study has clearly unveiled the strengths and weaknesses of both techniques which can excellently complement each other. Their combination allowed a distinct improvement of the definition of the complex karyotypes of drug-resistant cell lines.     

Comparative genomic hybridization arrays in clinical pathology: progress and challenges

Array-based comparative genomic hybridization (array CGH) genome scanning is a powerful method for the global detection of gains and losses of genetic material in both congenital and neoplastic disorders. When used as a clinical diagnostic test, array CGH combines the whole genome perspective of traditional G-banded cytogenetics with the targeted identification of cryptic chromosomal abnormalities characteristic of fluorescence in situ hybridization (FISH). However, the presence of structural variants in the human genome can complicate analysis of patient samples, and array CGH does not provide morphologic information about chromosome structure, balanced translocations, or the actual chromosomal location of segmental duplications. Identification of such anomalies has significant diagnostic and prognostic implications for the patient. We therefore propose that array CGH should be used as a guide to the presence of genomic structural rearrangements in germline and tumor genomes that can then be further characterized by FISH or G-banding, depending on the clinical scenario. In this article, we share some of our experiences with diagnostic array CGH and discuss recent progress and challenges involved with the integration of array CGH into clinical laboratory medicine.     

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.     

Digital karyotyping: an update of its applications in cancer

DNA copy number alterations, including entire chromosomal changes and small interstitial DNA amplifications and deletions, characterize the development of cancer. These changes usually affect the expression of target genes and subsequently the function of the target proteins. Since the completion of the human genome project, the capacity to comprehensively analyze the human cancer genome has expanded significantly. Techniques such as digital karyotyping have been developed to allow for the detection of DNA copy number alterations in cancer at the whole-genome scale. When compared with conventional methods such as spectral karyotyping, representational difference analysis, comparative genomic hybridization (CGH), or the more recent array CGH; digital karyotyping provides an evaluation of copy number of genetic material at higher resolution. Digital karyotyping has therefore promised to enhance our understanding of the cancer genome. This article provides an overview of digital karyotyping including the principle of the technology and its applications in identifying potential oncogenes and tumor suppressor genes.