NEXT-GEN FISH technology improving deletion detection

The CymoGen Dx Four color FISH for deletion detection also uses individually labeled probes. Each distinctly colored probe will bind to the region of a chromosome containing a tumor suppressor gene of interest. The probes are designed to span an appropriate genomic interval that is frequently deleted in human cancers. The tumor suppressor gene is placed in the middle region of the three probes and is detected with a specific color. This gene probe is flanked by differently colored probes on either side (Figure 3A).

FIGURE 3 Schematic depiction of DDP probe set configurations (panel A) and typical interpretations (panel B) using three color interphase FISH analysis of nuclei. In this example a BAC probes that encompasses a tumor suppressor gene (Tum Sup) is labeled with red fluorescence and flanking BAC probe "A" is labeled with green fluorescence while flanking BAC "B" with blue fluorescence. The upper "A" panel shows the configuration on a schematic chromosome. In panel B a normal nucleus (left) will have three pairs of each color signal. For simple hemizygous loss (center nucleus) of the Tum Sup gene the red BAC is deleted so only one signal is present, but probes "A" and "B" flanking the tumor suppressor gene are present in duplicate. For homozygous loss (right nucleus) both red signals are deleted while flanking probes are retained. Note that in comparison to Figure 2B and 2C in which randomness of signal distribution within the nucleus is apparent, the spot distribution in Panel 3B suggest a spatial relationship, or "traffic signal configuration" (assay implications discussed below).

In interphase nuclei without a deletion or rearrangement of the tumor suppressor gene, two separate clusters comprising three different colored signals will be visible (Figure 3B –left). In the simplest case involving a deletion there will be loss of the gene-specific signal, while the control flanking probes are still present. Sometimes both copies of the gene are deleted and this results in no signals. This is called a homozygous deletion (Figure 3B-right). If only one copy of the gene is lost, this deletion is called hemizygous (Figure 3 B –center).

In addition to the simple deletion configurations shown in Figure 3, more complex patterns involving gain or loss of signals may be seen because of additional rearrangements close to the regions containing the flanking control probes. Some of the PTEN gene losses observed in advanced prostate cancer appear to also involve genes closely linked to PTEN that are associated with deletion events (discussed below). In addition, complex signal configurations bearing additional spots may also arise from complex chromosomal gains due to unbalanced translocations, polysomy, or polyploidy. Any pattern differing from the simple patterns observed in normal nuclei is usually considered abnormal if it appears in a significant proportion of cells. The number and location of signals in aberrant patterns should be carefully evaluated and interpreted as it can provide valuable information of underlying chromosomal change.