FISH is a technique that uses
fluorescent labelled probes to bind to part of a chromosome which is then
analysed with a fluorescence microscope to see where the probe has bound(1).
There are different types of probes to
detect translocations. Dual fusion probes are differently labelled probes (e.g.
red and green) that hybridise to the two chromosomal loci involved in
translocations(1,12). A normal result would be two red and two
green signals, but a translocation would show the derivative chromosome
producing a yellow fusion signal, and the other no longer labelled. Break apart
probes involve differently labelled probes on each side of a translocation
break point. A normal result would be two fusion signals and a translocation
would show separate red and green signals. This has a higher sensitivity for
Advantages of using FISH are the
maximum use of difficult to obtain tissue, such as clinical biopsies(13). This is because multiple different
hybridisations can be performed on the same tissue sample, which also means
tissue libraries can be formed and tested later. Disadvantages include the
difficulty in identifying targets that have low DNA and RNA copies(13).
Applications in Burkitt Lymphoma (BL)
Burkitt lymphoma (BL) is a mature
B-cell neoplasm defined by activation of the MYC gene through a translocation,
normally t(8;14)(q24;q32)(14). Alternative translocations are t(8;22) and t(2;8)(15). These all juxtapose the MYC gene next to an
immunoglobulin gene, leading to deregulated expression of the oncogene MYC.
This in turn leads to uncontrolled cell proliferation, genomic instability and
escape of immune surveillance(12). BL is an extremely aggressive disease with
rapidly growing cells, so prompt diagnosis, staging and treatment with high
dose chemotherapy is vital.
BL can be extremely hard to differentiate from
diffuse large B-cell lymphoma (DLBCL). The border between two conditions is
hard to determine as they are morphologically and molecularly extremes of a
continuum. This is called the intermediate or grey zone(15).
Intermediate lymphomas are DLBCL that acquire MYC
mutations and secondary events. Double or triple hits involve MYC, BCL2 and
BCL6(15). As MYC mutations are
associated with aggressive clinical behaviour in lymphomas other than Burkitt
lymphoma is it important to find these translocations as treatment strategies
and outcomes will be different(16). The WHO classifications of lymphoid neoplasms 2016
split these intermediate cases into the subgroups high grade b cell lymphoma
(HGBL) with MYC and/or BC2 and/or BCL6 and HGBL, NOS(17).
FISH can be used to detect translocations involving
MYC, BCL2 and BCL6 to distinguish BL from DLBCL. Break apart probes can be used
for this as they can test the integrity of MYC, but they give no information on
the translocation partner involved(12). To do this, dual fusion probes are needed, as
they can detect known translocation partners. These probes are available
commercially so are a useful diagnostic tool. However, FISH cannot detect gene
expression on transcriptional and translational levels like IHC(16). This means an approach using both IHC and FISH
with a scoring system is needed(12).
FISH can also detect minimal critical regions
(MCRs) localised on chromosome 1q harbouring genes such as BCA2, PIAS3, MDM4
and AKT3. These genes are critically involved in BL prognosis(12).
Expression microarrays allow the
genotyping on thousands of different loci at a time and can assess thousands of
molecular markers on a single patient sample.
Microarrays work by analysing mRNA to
assess gene expression, as described in Govindarajan et al. (2012)(18). A DNA ‘chip’ is produced by
depositing many DNA sequences on a glass slide, identifiable by their location.
The mRNA is converted to cDNA then cut with restriction endonucleases and
fluorescent markers are attached. These are then mixed with the probes on the
DNA chip and hybridise. The fluorescent signals resulting from the
hybridisation identifies which RNAs are present.
An advantage is that it allows the
analysis of many more genes than PCR and also the genes examined are not
influenced by the preselection of genes(18).
Comparative genomic hybridisation (CGH)
arrays involve using test and reference sample on the same chip, but with
different fluorescent markers(e.g. red and green)(19). The fluorescent signals will then mix
for the DNA sequences where both samples hybridise (producing a yellow signal),
for the sequences where the test sample expresses more it will appear red and
where the test sample expresses less it will appear green.
Single nucleotide polymorphism (SNP)
arrays only use a test sample and can detect copy number variations by
measuring probe signal intensities(19).