The production of metaphase chromosomes from malignant cells plays a fundamental role in genetic analysis, allowing both G-banded chromosome analysis and subsequent FISH analysis. The chromosome offers a more versatile target than interphase cells since many types of FISH probes can be applied. In leukemia and lymphoma, gene fusions are relatively frequent and well characterized at the molecular level. These novel disease-associated fusion events arise through chromosomal translocations, inversions or insertions and are usually visible by routine karyotype analysis, although subtle abnormalities do exist and some of the recurrent rearrangements can be cryptic. FISH probes mapping to the unique sequences involved in these fusions are readily available and detect their respective abnormalities by one of two methods. In the first strategy, probes mapping to the two genes involved are labeled in two distinct colors; as an example, the BCR-ABL (break cluster region-Abelson) fusion associated with the t(9;22)(q34;q11.2) is illustrated in Plate 2.1. BCR is represented by the green fluorescence and ABL by the red signal. The t(9;22) translocation results in both BCR-ABL and ABL-BCR fusions, and since the probe extends beyond the breakpoint for both genes, two fusion signals (red and green juxtaposed) are generated (dual fusion probes), one on the der(9), the other on the der(22). A normal 9 and a normal 22 (single red and green signal) will also exist. To further complicate the analysis, however, deviations from this pattern may exist since some patients carry deletions around the breakpoint and some harbor cryptic insertions of part of one gene, thereby generating only one of the fusion sequences (Plate 2.2). The next generation of FISH probes look likely to use four fluorescent probes to enhance the sensitivity and specificity to simultaneously detect translocations and deletions around the breakpoint, which may confer independent prognostic value. Plate 2.3 shows a cryptic insertion of part of the RARA gene (chromosome 17) into the PML locus (chromosome 15) in a patient with acute promyelocytic leukemia. The t(15;17)(q21;q11) translocation is the hallmark of acute promyelocytic leukemia and is cytoge-netically visible in 90% of patients.
The second common type of FISH strategy is the 'break apart' probe, specifically designed to detect abnormalities affecting one specific gene which rearranges with multiple partner loci, such as MLL (11q23). Over 60 different MLL gene translocations have been cytogenetically reported, and the FISH probe used most often for diagnosis consists of a probe mapping above the breakpoint labeled with one color and a second probe mapping below the breakpoint in another color. Translocations involving MLL therefore result in the separation of one set of probes (Plate 2.4) and the displaced MLL signal will map to the partner chromosome. Single-color probes extending across the breakpoints can also be used, resulting in a split signal.
Unique sequence probes can also be used to screen for copy number changes, particularly in cases with evidence of additional genetic material, by karyotyping such as double minute chromosomes (dm), homogeneously staining regions (hsr) or additional pieces of chromosomes. Dm and hsr are manifestations of gene amplification and in certain malignant diseases, particularly solid tumors, are well recognized mechanisms for oncogene activation. FISH probes mapping to the genes commonly associated with amplification can very quickly confirm the presence of multiple copies of genes; an example is N-MYC in neuroblastoma. Plate 2.5 shows a bone marrow aspirate infiltrated by neuroblastoma and multiple copies of N-MYC. Alpha satellite probes are often used to determine chromosome number. Hyperdiploidy is a frequent phenomenon in ALL and is associated with a common pattern of gain, namely chromosomes 4, 6, 10, 14, 17, 18, 21 and X. Using a selected cocktail of alpha satellite probes mapping to these chromosomes, hyperdiploidy can be detected in both metaphase and interphase cells (Plate 2.6). Metaphase cells derived from leukemic blasts of patients with ALL can often have poor morphology and be difficult to fully characterize. In such situations FISH can be of particular value since it may help elucidate chromosomal gains and losses.
Whole-chromosome painting probes (WCP), consisting of pools of DNA sequences mapping along the full length of a particular chromosome and labeled with a fluorochrome, can be used individually or in combination with other WCPs to characterize abnormalities whose origin is uncertain by G-banding. In simple karyotypes, requiring confirmation of a suspected rearrangement, two-color chromosome painting might be the most useful option (Plate 2.7). In more complex karyotypes, such as those associated with therapy-related leukemia, a mixture of paints mapping to all 24 human chromosomes (24-color karyotyping) is probably the most informative. M-FISH/SKY is not used routinely for diagnostic purposes but has revealed cryptic rearrangements in several studies. M-FISH/spectral karyotyping (SKY) uses a combinatorial labeling approach such that each individual chromosome paint is labeled with a unique combination of not more than five fluorochromes. The 24 differentially labeled paints are then applied in a single hybridization assay and visualization is achieved using one of two strategies. M-FISH uses a series of optical filters to collect the images from the different fluorochromes, which are then merged into a composite image; a pseudocolor is then assigned to each chromosome on the basis of its fluorochrome combination (Plate 2.8). SKY uses an interferometer with Fourier transformation to determine the spectral characteristics of each pixel in the image, and assigns a pseudocolor.
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