The non-dividing cell population can be examined using FISH probes to yield both diagnostic and prognostic molecular cytogenetic information. Interphase cells from the sample sent for cytogenetic analysis (peripheral blood, bone marrow, lymph node, etc.), tumor touch imprints, PETS and bone marrow smears can be used as the target for FISH. In these instances screening for a specific chromosomal abnormality is performed, thereby allowing detection or exclusion of a single event per FISH assay. Techniques that involve the use of whole or chromosome-specific probes cannot readily be applied to interphase cells. PETS are sometimes the only tumor sample available for analysis since the paraffin treatment preserves the morphology of the tumor, thereby enabling histological diagnosis. However, if histology is equivocal, FISH for a tumor-associated chromosomal abnormality can be extremely valuable for diagnostic purposes. FISH on PETS does have inherent technical problems not found with FISH on other sample types; for example, probe accessibility is reduced, the thickness of the section means that the resulting FISH signals may not all be visible in the same focal plane, and the cells may be very tightly packed, making analysis more difficult. Interphase cells can be used to assess the copy number of unique sequences/alpha satellites or to look for chimeric fusion genes. Plate 2.9 illustrates two PETS screened for the presence of the EWS/FLI1 rearrangement associated with Ewing's sarcoma/ primitive neuroectodermal tumor.
CGH provides a global assessment of copy number changes, revealing regions of the chromosome that are either gained or lost in the tumor sample. A key feature of this technique is that dividing tumor cells are not required. DNA is extracted from the tumor, labeled (usually) with a green fluorochrome and compared with DNA from a normal reference labeled with a red fluorochrome. Labeled test and reference DNA are combined and hybridized to normal chromosomes and the resulting ratio of the two signals along the length of the chromosomes reflects the differences in copy number between the tumor and reference DNA samples (Plate 2.10). Regions of gain in the tumor DNA are represented by an increased green/red ratio whereas deletions are indicated by a reduced ratio. CGH requires 50% abnormal cells to be present within the tumor sample for reliable detection of genomic imbalance and will not easily detect regions involving less than 10 Mb of DNA unless it involves high-level amplification. Nevertheless, CGH is particularly applicable to the analysis of solid tumors since DNA can be readily extracted from them and the karyotype frequently involves loss or gain of whole or partial chromosomes.
The use of genomic DNA arrays as the hybridization target essentially allows much higher resolution for the detection of copy number changes. Array CGH is based on the metaphase CGH method but uses mapped sequences as the hybridization target rather than metaphase chromosomes, and hence the resolution is limited only by the density of the sequences spotted on the slides. Array CGH was first introduced in 1997 (Solinas-Toldo et al., 1997), and since then a number of groups have set up their own facilities for spotting sequences onto slides. In addition, some companies have produced commercial chips ranging from specialist arrays containing 287 targets (Vysis, Inc., Downers Grove, IL, USA) to 1 Mb arrays (e.g. Spectral Genomics, Inc., Houston, TX, USA) containing around 3000 target clones. Array CGH has the potential to provide a highly sensitive global assessment of gene copy number, simultaneously screening hundreds of individual gene sequences, although its reproducibility and robustness need to be thoroughly validated before it is used in the diagnostic setting. Figure 2.1 clearly demonstrates the sensitivity of array CGH. This figure shows regions of simultaneous gain and loss along chromosome 13 and demonstrates how the technique gives more precise definition than metaphase CGH.
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