**3. COMBO-FISH probe sets for genes and breakpoint regions in oncology**

Cancer cells are mutations of former "normal" cells. One reason for cancer cell malfunctions are over-expressions of genes, which are mainly caused by two reasons, either one gene is over-expressed or one gene is amplified and additionally to the original gene the copies also express. The latter frequently occurring in solid tumors leads to measureable copy number increases which can be used as diagnostic parameter in tumor biology and medicine. In contrast to solid tumors, blood cell tumors show structural aberration-like translocations in the early stages which can then be followed by numerical aberrations of larger chromosome parts or even whole chromosomes. Translocation chromosomes are resulting by fusion of two parts of different chromosomes that were broken very exactly at breakpoints within a certain breakpoint region; for example, for CML (chronic myeloid leukemia) a famous hallmark is the Philadelphia chromosome with the translocation ABL-BCR t(9,22)(q34,q11), in which a fusion of a part of the abl-region on chromosome 9 with part of the bcr-region on chromosome 22 takes place (for review [38]). Since the breaks occur very exactly at certain breakpoints the fusion region can be transcribed into a functioning protein that does normally not exist in a cell and that is involved in CML-induction.

Although appropriate target sites for homo-purine or homo-pyrimidine probes only make about a few percent of the genome, some prominent tumor genes and breakpoint regions can be specifically labeled by *uniquely co-localizing* sets of COMBO-FISH probes. Others can only be labeled by a set overlapping on neighboring regions. Probe sets for Her2/neu, abl, and bcr have been published elsewhere [29–31, 34]. In the following we will show further examples.

It has been observed that in various types of cancer such as breast, ovarian, and squamous cell cancer an amplification of 20q13 occurs. When analyzing such cancers it has been found that often the region which encodes ZNF217 is amplified and an increased expression of the specific region of ZNF217 has been observed. In some cases also neighboring gene encoding sequences are also amplified. It has to be mentioned that the detection of the copy number of ZNF217 can be done by standard FISH, but the shortest available sequence to detect ZNF217 has a length of about 245 kb [39], which means—compared to the length of ZNF217 of about 16 kb—that further amplifications within the probe-binding sequence might not have been visualized using this method. To further reduce the length of the detected target, a COMBO-FISH probe set was designed (**Table 3**). As a result, we obtained 25 oligonucleotide probes within a range of 133 kb. When reducing the number of sequences to 21 by removing the first and the last three probes the length of the considered DNA region can be downscaled to 91 kb.

The gene TP53 (sometimes called p53) encodes the "tumor protein 53" (P53). Its purpose is to maintain genomic stability and to control cell growth. Moreover, it is important for the induction of apoptosis and the coordination of repair processes. Labeling of this gene can be obtained by five COMBO-FISH probes only (**Table 4**).


#### **Table 3.**

*List of COMBO-FISH probe targets for ZNF217 and its surroundings (target sequences are written from left to right in the 5*<sup>0</sup> *-XXXX-3*<sup>0</sup> *direction).*


#### **Table 4.**

*List of COMBO-FISH probe targets for TP53 (target sequences are written from left to right in the 5*<sup>0</sup> *-XXXX-3*<sup>0</sup> *direction).*
