Reflecting the broad variety of chromosome aberrations observed in acute leukemias in particular, FISH “screening” of interphase nuclei only covers a fraction of the mutations that may potentially be present and is therefore unable to replace classic chromosome analysis. In contrast, the FISH technique is a rapid and reliable method that may produce findings within 4 hours in response to specific questions, for instance validation of translocation t(15;17)(q22;q12) for cases of suspected acute promyelocytic leukemia.
The FISH technique can be used in progress control during therapy as a method for the determination of residual disease whenever the initial diagnosis using chromosome analysis has discovered mutation that can be identified using suitable FISH probes. This method is more sensitive than chromosome analysis, but less so than polymerase chain reaction or immunophenotyping.
Between 5 and 10 ml of bone marrow with heparin anticoagulant, i.e. peripheral blood, are needed for the FISH analysis. EDTA or citrate can be used alternatively as anticoagulants. Peripheral blood is sufficient as a test material, provided that malignant cells have passed into the blood. Therefore, bone marrow should be tested if passage has not occurred.
Specific hybridization with fluorescence-marked DNA probes
The FISH technique is based on the hybridization of DNA probes that identify specific chromosomal structures. It is possible to use probes that mark specific centromere regions of individual chromosomes, genes or entire chromosomes. The DNA of the selected probes and the patient DNA requiring analysis are denaturated, meaning that the two DNA strands of the double helix are separated. During subsequent renaturation, the DNA probes accumulate on the complementary sections of the patient DNA (hybridization). The DNA probes directly carry the fluorescent marker. Therefore, the matching chromosome structures show up as fluorescence signals.
Table 1: Advantages and limitations of FISH
Can be conducted on metaphases and interphase nuclei (cell division is not necessary).
Identifies even minor mutations below the resolution range of chromosome analysis.
The probes can only provid information about the target chromosomes/genes.
FISH can be also conducted on interphase nuclei as a purposeful addition to chromosome analysis, for screening, e.g. for CLL and multiple myeloma, and during disease progression. The FISH technique is particularly suitable for the identification of so-called sub-microscopic mutations below the resolution range of chromosome analysis, for instance extremely minor deletions like those encountered during CLL, or tiny insertions that are observed among a small proportion of patients with fusion genes.
But the FISH technique can only be used for progress control during therapy as a method for the determination of residual disease if the initial diagnosis using chromosome analysis has discovered mutation that can be identified using suitable FISH probes. Interphase FISH is more sensitive than chromosome analysis, but far less so than polymerase chain reaction or immunophenotyping.
Besides the probes used on interphase nuclei, so-called chromosome painting probes that specifically mark the entire DNA of a chromosome are suitable for metaphases. This technique is used above all to confirm chromosome analysis in difficult cases. It can also be conducted on the same metaphases and after banding analysis.
The 24-color FISH method permits the mapping of all 22 chromosome pairs, as well as the X and Y chromosome, in a single hybridization. It can only be performed on metaphase chromosomes, and is used to identify complex structural aberrations. Equally, it can be conducted on the same metaphases and after banding analysis.
ISCN for FISH on interphase nuclei
According to the ISCN nomenclature (International System for Human Cytogenetic Nomenclature), Interphase FISH findings begin with “nuc ish” for “nuclear in situ hybridization.” In the formula, the probe is specified by the chromosome band in which it is located and by its precise designation, either as the name of a gene or the designation of an anonymous locus. Furthermore, the formula indicates how many signals for a specific probe were observed and possibly its position relative to the other analyzed probes.
So a probe hybridized for the centromere region of chromosome 8, in case of a normal finding (2 chromosomes 8 in each cell nucleus), will be described by the ISCN formula “nuc ish 8cen(D8Z2x2),” while a trisomy 8 (3 chromosomes 8 in each nucleus) would be “8cen(D8Z2x3).”
If hybridization takes place with probes that bridge the breakpoints in genes RUNX1 and RUNX1T1 that are affected by the t(8;21)(q22;q22)/RUNX1-RUNX1T1 rearrangement, the ISCN finding would be, in the case of an RUNX1-RUNX1T1 rearrangement,
nuc ish 8q22(RUNX1T1x3),21q22(RUNX1x3),(RUNX1T1 con RUNX1x2).
The breaks in the RUNX1 gene and the RUNX1T1 gene cause a splitting of the probes and hence 3 signals for RUNX1 and RUNX1T1, respectively: 8q22(RUNX1T1x3),21q22(RUNX1x3); one RUNX1- and one RUNX1T1 signal in each case are located directly adjacent on the two derivative chromosomes (colocalization): (RUNX1T1 con RUNX1x2).
In case of a verified deletion, e.g. in the long arm of chromosome 5, band 5q31, the ISCN interphase FISH finding is “nuc ish 5q31(EGR1x1)” – there is only one signal for EGR1, instead of the usual two.