Comparative Genomic Hybridisation and Oncological Studies

Contents

• About Me

• Comparative Genomic Hybridisation and Oncological Studies

• Relationship between Virology, BSE (Bovine Spongiform Encephalopathy) and CJD (Creutzfeldt-Jakob Disease)

• Bovine Spongiform Encephalopathy

• Creutzfeldt-Jakob Disease

• Differences between BSE and CJD from other infectious diseases

• Journal of Molecular Biology

• Useful Books and Links to Biology related subjects

Comparative genomic hybridisation (CGH) was developed in 1992 by Kallioniemi, who utilized it for the identification of 16 different previously unknown regions of amplification in tumour cell lines and primary bladder tumours. A simple one-colour in situ hybridisation strategy was used initially for the genome-wide screening and detection of highly amplified DNA sequences in tumours. However, low-level gins and losses of DNA sequences are not reliably detected by this single-colour technique due to variations of hybridisation efficiency from one chromosomal region to another. Thus, an improved method using two-colour strategy was developed to overcome the above difficulties.

Historically, the first cytogenetic method available was the karyotypic analysis of tumours, which examines the number and size of chromosomes. In 1960, a small morphologically distinct chromosome was found in chronic myeloid leukaemia. It was termed the Philadelphia chromosome, and was the first tumour specific chromosomal abnormality to be identified. This was heralded as a huge scientific breakthrough and it was anticipated that this would be the first of many such specific chromosomal aberrations. However, technical difficulties meant that no other consistent chromosomal changes were found for over a decade.

The introduction of chromosomal banding in 1970 revolutionised the field of cancer cytogenetics because it meant that chromosomes could be identified precisely and reliably. Rapid progress was made in revealing consistent chromosomal aberrations in leukaemias, lymphomas, and sarcomas. The Philadelphia chromosome was shown to be derived not just from chromosome 22 material but to result from a translocation, although it would take another decade before it was found to be a reciprocal translocation t(9;22)(q34;q11), with the genes at the breakpoint identified and the molecular consequences of the translocation established. Morphologically, these tumours are often almost identical, but can now be distinguished on the basis of their specific genetic aberrations.

Comparative Genomic Hybridisation (CGH) is a recent development in molecular cytogenetics techniques that enables comprehensive, genome-wide screening of deoxyribonucleic acid (DNA) sequence copy number changes. CGH is an in situ hybridisation technique that is used in the characterisation of chromosomal abnormalities where there is a gain (duplication, insertion or amplification) or net loss (deletion of material).

Principle

For a CGH experiment, two genomic DNA samples are simultaneously hybridized in situ to normal human metaphase spreads, and detected with different fluorochromes. The two genomic DNA are extracted from a tumour (test sample) and normal tissue (reference sample), both DNA are labelled with different fluorochromes such as rhodamine and tetramethyl rhodamine isothiocyanate (TRITC) which fluoresces red and fluorescein isothiocyanate (FITC) which fluoresces green. Refer to figure 1.

Figure 1. CGH using whole-genomic DNA probes.

Labelling is done by nick translation, a procedure for making a DNA probe in which a DNA fragment is treated with DNase to produce single stranded nick. In indirect labelling the test DNA is usually labelled with biotin, the reference sample with digoxigenin. Detection is accomplished using FITC and TRITC conjugated antibodies which give a green fluorescent signal for the test DNA, and a red fluorescent signal for the reference DNA. Direct labelling of the DNA with fluorochrome conjugated dNTPs eliminates the need for the antibody detection steps. Nick translation is the preferred labelling method because the length of the DNA probes can be controlled using DNase I digestion to give an optimum probe size within the range of 0.5-2kb.

Figure 2. Illustration of indirect and direct labelling of DNA in CGH.

Equal amounts of the two labelled DNAs are mixed with an excess of human cot-1 DNA to prevent binding of repeat sequences present is both test and reference DNA. The labelled DNAs are denatured and co-hybridised for a period of 2-3 days to metaphase chromosome spreads from a normal control. The two-labelled DNAs compete with each other for hybridisation sites along the chromosomes. Tumour DNA that contains extra copies of genetic material will bind to the corresponding chromosome on the metaphase spread. Tumour DNA that lacks a portion of normal DNA will cause more binding of normal DNA to their correspondence DNA on the metaphase spread. Metaphase is a stage in the cell division cell cycle prior to division, all the chromosomes line up on the metaphase plate in no particular order.

Figure 3. Illustration of metaphase spread

After hybridisation, washing was done to remove any excess DNA. The three images of hybridisation, representing both fluorescent labels as well as DNA counterstain, 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) are acquired using fluorescence microscope and digital image analysis.

 Figure 4. Ratio image FITC/TRITC

Computer software can be used to profile the relative ration of red to green fluorescence along each chromosome and used to map a gain-loss profile for each chromosome. Regions of the genome that are either gained or lost in the tumour are indicated by the differences between bindings of the two-labelled DNAs as evidenced by the fluorescence intensity ratio profiles.

Advantages

CGH has distinct advantages over the other techniques, such as DNA cytometry, tumor cytogenetics (TC), in situ hybridization (ISH), and the microsatellite assay.

1. The main advantage of the CGH method is that the whole genome can be screened in a single experiment using DNA samples.

2. A two-colour CGH was developed to overcome low-level gain and losses of DNA sequences which were not reliably detectable by the single colour technique. This is due to variations of hybridisation efficiency from one chromosomal region to another.

3. Very small amounts (ng) of DNA are required, paraffin sections, frozen tissue sections and even small numbers of cells(100-500) can be used as a source of DNA. A degenerated PCR method (DOP-PCR) can be used to amplify DNA from such minute amounts of starting material.

4. Metaphase chromosomes from the test sample are not required.

5.  CGH is a molecular cytogenetic technique that is particularly useful in the study of solid tumours, although the technique has also been applied to the characterisation of unbalanced constitutional chromosome abnormalities.

6. Only tumour DNA is required and DNA from archival material can be used.

Limitations

1. CGH cannot be used to characterise balanced rearrangements.

2. Used in isolation CGH cannot provide information about the structural arrangement of any gain of material in an unbalanced rearrangement

3. Some chromosomal regions cannot be easily analysed by CGH. E.g., the pericentromeric and heterochromatic regions are polymorphic and are blocked in varying degrees by the excess of cot-1 DNA during the hybridisation. Similarly, the p arms of the acrocentric chromosomes are excluded from CGH analysis. Some chromosomal regions (1p32-pter, 16p, 19 and 22) show consistently abnormal signal ratios in normal control, towards the telomeres the hybridisation signal fades. Results from these regions must be interpreted with caution

4. Theoretically the resolution of CGH has been estimated to be 2-4Mb. In practise the resolution for the detection of deletions is limited to ~5-10Mb. Amplifications of ~100kb can be detected when present as a 20-fold excess, or 1Mb when present as a 5-10 fold excess. In the future the resolution of CGH may be improved by the replacement of metaphase chromosome spreads with an ordered array of large insert genomic clones

5. CGH provides no information about ploidy, i.e. cannot distinguish between diploid, triploid or tetraploid tumour cells.

6. CGH cannot detect intratumor karyotypic variability or tanslocations and inversions that have a central role in tumour development and progression.

7. Specialised imaging equipment is required, which is expensive.

Oncological Studies

CGH has been particularly useful in the study of solid tumours (such as breast and cervical cancer) and has identified a tumour suppressor gene in Peutz-Jeghers disease.

The assessment of overexpressing tumours is clinically relevant because targeted treatment using a recombinant humanised monoclonal antibody directed against HER2/neu, Herceptin, is now available

Characterisation of hot spots for unbalanced rearrangements which are often found in solid tumours. CGH analysis of identical tumour types have revealed consistent, non-random genetic rearrangements and some of these changes are common in different tumour types. For example, gain of 1q, 3q and 8q; loss of 8p, 13q, 16q and 17p are common in breast, ovarian, prostate, renal and bladder cancer. A few other examples of tumour specific changes identified by CGH are shown below.

Characterisation of novel genes involved in copy number alterations. Over expression of genes that have undergone amplification is thought to be an important mechanism for cancer progression. Some examples of amplified genes found in certain cancers, identified through CGH studies are shown below.

Future Development

CGH technology is still undergoing development. Optimisation of the hybridisation is required before CGH is to become applicable in routine aberration screening.

Further improvement in the image analysis software is required to enhance the automation of karyotyping. This facilitates the analysis speed, objective assessment of hybridisation quality, improved chromosome length normalization, and automated interpretation of the results.

This new generation of CGH array technologies are still in their infancy. The technique has become established in only a few areas, and the tools remain expensive. Even though this array system only measures a relatively small quantity of gene targets, it provides useful information on those genes that are either up- or down- represented in prostate tumorigenesis.

In the future these techniques will probably be used increasingly in the clinical setting