Routine and new cytogenetic technologies
Metaphase cytogenetics has become a routine test in the management of haematological malignancies in which the presence of specific chromosomal translocations is diagnostic, while, for other diseases, specific chromosomal aberrations are highly predictive of prognosis or responsiveness to targeted therapeutics. Invariant aberrations serve as clonal markers to detect and follow minimal residual disease and relapse. Moreover, metaphase cytogenetics facilitates the mapping of recurrent lesions to delineate minimally affected regions. However, metaphase cytogenetics is time consuming and technically demanding; its yield is related to the proportion of clonal cells in the tested sample (sensitivity) and size of the lesion (resolution). The sensitivity is relatively low; traditionally 2 abnormal metaphases of 20 tested are considered pathological. The resolution depends on the location of the lesion with regard to the banding pattern. The need for cellular proliferation to obtain chromosomal spreads is a limitation; metaphase cytogenetics determines the proportion of abnormal cells within the dividing progenitor pool and may not always correlate with the total percentage of malignant cells, a fact that may account for the quantitative differences between various cytogenetic methods. Due to its inherent limitations, it is likely that when metaphase cytogenetics is applied in the study of haematological malignancies many chromosomal defects remain undetected.
Complementary cytogenetic techniques
Fluorescence in situ hybridization (FISH) is widely applied in cytogenetics, in particular to precisely diagnose reciprocal translocations. Currently, polymerase chain reaction (PCR)-based methods are often utilized for detection of known fusion genes, reducing the need for cytogenetic analysis and allowing for better quantitative follow-up analyses. Due to the high background and inherent lower precision of FISH, its role in the detection of unbalanced defects is less well defined and identification of very small numbers of abnormal cells is of unclear clinical significance.
Novel array-based technologies
Various DNA array-based technologies have been introduced to facilitate the examination of the normal and malignant genome. Based on the availability of bacterial artificial chromosome (BAC) libraries, arrays with various densities of BAC probes have been generated, enabling array-based comparative genomic hybridization. The subsequent introduction of high-density oligonucleotide arrays (comparative genomic hybridization arrays; CGH-A) has enabled even more precise scanning of the genome for copy number changes. Using a similar microchip technology, single nucleotide polymorphism arrays (SNP-A), developed for whole genome association studies, have also been adopted for karyotyping. Unlike routine cytogenetics, arrays can be performed on interphase cells and consequently even archival samples can be examined (Table I). While these techniques only enable the detection of unbalanced defects and do not allow for the distinction between multiple large clones (clonal mosaicism versus compound lesions), they have superior resolution when compared to metaphase analysis. SNP-A also has the advantage of simultaneous genotyping, enabling detection of copy number-neutral loss of heterozygosity (CN-LOH), also referred as to as somatic uniparental disomy (UPD; see below). In addition to its diagnostic value in cytogenetic diagnostics, array-based karyotyping technologies constitute an excellent chromosome mapping tool, thereby allowing for delineation of boundaries of commonly deleted/duplicated regions.
Comparative genomic hybridization arrays rely on the difference in the copy number between differentially labelled test and reference DNA samples (Fig 1). Through competition between test (e.g. tumour) and control diploid DNA, imbalances due to copy number differences result in a shift in the fluorescence spectra. The ability to compare the hybridization signals of test and control DNA affords a high level of precision and exclusion of artefacts, a clear advantage of CGH-A over SNP-A. High-density and very precise oligo-CGH-A platforms are now available from Agilent (Barrett et al, 2004) or NimbleGen (Nuwaysir et al, 2002; Albert et al, 2003; Selzer et al, 2005). In contrast to SNP-A, CGH-A enables even or targeted distribution of probes, including areas of known copy number variants (CNVs) (Tan et al, 2007), but does not allow for detection of UPD (Table I). Of note is that using ‘standard’ control DNA, CGH-A allows for detection of germ line CNVs for which the test sample varies from the control DNA. Application of paired germ line DNA and tumour DNA from the same individual would allow for exclusion of any germ line artefacts and differences would only reflect somatic lesions.
Single nucleotide polymorphism arrays rely on oligonucleotide probes corresponding to the allelic variants of selected SNPs. Hybridization of genomic DNA to both probe variants indicates heterozygosity, while a signal for only one allele is consistent with homo/hemizygosity at any given locus. In addition, the strength of fluorescence emitted from individual probes allows for the analysis of gene copy number. As SNPs are not evenly distributed across the genome, coverage of some chromosomal regions is not possible. Several generations of chips varying in density of probes have been developed, successively resulting in increased analytic precision. The most common SNP-A platforms include Illumina (Gunderson et al, 2005) and Affymetrix (Syvanen, 2005) arrays, which utilize bead or chip technology, respectively. In the Affymetrix technology, genomic DNA is digested by restriction endonucleases, amplified and labelled. In bead-based platforms, the whole genome amplification and fragmentation steps are followed by hybridization to an oligonucleotide bead array. One of two bead types correspond to each allele in the SNP locus and allelic specificity is conferred by enzymatic (allele-specific primer or single base) extension and fluorescent staining. For both array types, the read-out includes genotyping calls and hybridization signal strength, corresponding to gene copy number. Final analysis is performed using various biostatistical and genetic software packages. The CNAG (Copy Number Analyser for GeneChips®) programme (Nannya et al, 2005; Yamamoto et al, 2007) combines copy number analysis and LOH and analytic programmes are available allowing determination of the overlap between lesions with known CNVs (Fig 1, lower panel).
A major advantage of SNP-A over metaphase cytogenetics and CGH-A is the ability to detect diploid stretches of homozygosity present throughout the genome (Fig 2). They can be a result of acquired somatic UPD, autozygosity or early embryonic UPD. While significant autozygosity inherited from both parents is non-clonal and unlikely to be clinically relevant for haematological malignancies, UPD can result from errors during mitosis leading to both copies of a chromosome or chromosomal region being derived from one parent. Acquired somatic UPD can be due to segmental deletions and subsequent replacement of the lost fragment by a copy of the remaining allele or mitotic recombination.