ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (aCGH)

Genome-wide DNA profiling, sometimes simply referred to genomic profiling in the context of DNA characterization studies, is a powerful approach for cancer research. It allows to simultaneously identify multiple alterations at genomic level from various specimens in an unbiased manner and short time.

Array CGH has proven to be a specific, sensitive, and fast technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes and many other applications (Figure 1.3). Array CGH enables analysis of the whole genome in a single experiment and allows the global profiling of copy number imbalances in tumors and an accurate determination of the breakpoints of regions that are gained and/or lost ^{20, 21}.

Figure 1.3 Application of aCGH in Cancer (taken from Kallioniemi, A) ²²

Conventional CGH is a molecular cytogenetic technique that detects and maps alterations in DNA copy number of DNA sequences²³. In this approach, the genomic DNAs of test (patient) and reference (normal) samples are differentially labeled. The DNAs are then hybridized to normal human metaphase chromosomes where DNA sequences from both sources compete for their targets. Subsequently the ratio of the hybridization signal intensity of the test and reference sample at any interrogated locus is detected. The assumption is that the relative amount of test and reference DNA bound to a given chromosomal locus is determined by relative abundance of that sequence in the two DNA samples ²¹ (figure 1.4). After measurement of the ratio of the intensities of the two different dyes along the target chromosome, region of impaired content can be detected.

The resolution of conventional CGH is limited by the length of the metaphase chromosomes and is approximately 10 megabases and could contain hundreds of genes. Microarray based CGH has been developed, which combines microarray technology with the CGH approach ^{22, 24}.

In the present work we focused our project on the use of oligoarray CGH platforms. Different oligoarrays combine different labeling and hybridization techniques, and all have yielded high-resolution copy number measurements.

Affymetrix sells a commercial oligoarray CGH platform that contains short 25 mer oligonucleotides photo lithographically synthesized on the arrays. These are single-channel arrays, which means that only test DNA needs to be labeled and hybridized. The labeling of the test sample involves a restriction enzyme-based complexity reduction, which precludes the use of suboptimal DNA quality samples (Figure 1.5).

Figure 1.4 CGH methodologies ²⁵. (See text for detailed description)

The hybridization signal intensity (Phycoerythrin florescence) is detected by a scanner after having washed away the hybridization buffer from the array. The staining process is designed to amplify the signal of an annealed probe and comprises a first staining with streptavidin, followed by incubation with biotinylated anti-streptavidin antibody and finally with streptavidin-phycoerythrin conjugates.

250 ng Genomic DNA

RE digestion

Nsp1 Nsp1 Nsp1

Adapter ligation Single Primer

Amplification

Fragmentation and Labeling

Hyb & Scan on Standard Hardware

Affymetrix Human Mapping Array

Figure 1.5 Gene Chip mapping assay overview (Affymetrix).

The results are obtained through software applications that compare the acquired hybridization signal intensities corresponding to the analyzed genome with built in average values from normal reference samples.

The technical noise per element on the array is relatively high, which is compensated by the large amount of elements on the array, currently over 2,000,000. The Gene Chip Mapping 500k array set consists of two independent arrays (Gene Chip Human Mapping 250k Nsp and Sty arrays, also used in the present work), that enable the genotyping for more than 500,000 loci with a single primer. Every 250k Array is made of 6.5 million of 5 µm x 5 µm features, each feature consisting of more than one million copies of a 25-mer oligonucleotide probe of defined sequence. For each SNP, redundancy of probes is assured by 24 or 40 different oligonucleotides tiled around the SNP.

Furthermore aCGH is not dependent on the tumor source since tumor DNA can be obtained either from cell lines or from fresh frozen tissues. An important contribution of aCGH to cancer research has thus been in determining putative

locations of cancer genes, especially at chromosomal sites undergoing DNA amplification ²⁵. A large number of sub regional chromosomal gains and DNA amplifications have been discovered by aCGH in some cancers. For example, CGH studies have implicated amplification of oncogenes whose activation in cancer was previously known to occur by chromosomal translocation only, such as amplification of the REL proto-oncogene in non-Hodgkin lymphomas ²⁵. The discovery of novel small regions of loss by aCGH could also help the search for new tumor suppressor genes ^{4, 26-30}. In this way, aCGH is extremely useful in the analysis of the biological basis of tumor progression.

In addition to tumor progression, the high number of CN aberrations has also been linked with poor prognosis, and the identification of specific genetic aberrations associated with patient outcome has been the goal of a number of aCGH studies ^31-33. Several different aberrations have been implicated in different tumor types and both increased and decreased copy number are found to have prognostic significance. For instance, 17q23.2-qter gains and 17p13.1–p13.3 losses were indicative of poor patient outcome in medulloblastomas ³⁴, whereas gain of 1q or loss of chromosome 13, were associated with poor prognosis in multiple myeloma ^{22, 32}.

Moreover, specific genetic aberrations discovered by aCGH have also been linked to different response to various cancer therapies ^35-37. Increased number of genetic changes was shown to be associated with therapy resistance in ovarian cancer in which twice as many aberrations were observed in the treatment of the resistant tumor group as compared with the sensitive tumor group ³⁵. Furthermore, data from this study also showed that losses of 1p36.33 and gains of 17q11.2 were found more often in tumors that were resistant to treatment, whereas losses of 13q12–q13 were seen in sensitive cases ²².