Biomedical Engineering Reference
In-Depth Information
available LSI ALK Dual Color break-apart rearrangement probe
(Abbott Molecular, Abbot Park, IL) [33, 40, 50]. This assay employs
one probe at the 3
end of ALK (Spectrum Orange (red)-labeled
250 kb probe) and a second probe at the 5
′
end of ALK (Spectrum
Green (green)-labeled 300 kb probe) that when hybridized against
normal nuclei yield a merged (green-orange fluorescent) signal
detected through light microscopy. However, any interchromosomal
or intrachromosomal rearrangement involving
′
(or
any other fusion partner) would be detected by a “split” (green and
orange fluorescent) signal. Although FISH is a sensitive and specific
diagnostic test to detect these
ALK
and
ELM4
rearrangements, it is not entirely
flawless. The interpretation of a “split” signal through the introduction
of a gap between the two probes can be difficult since the
ALK
EML4
and
ALK
loci are closely mapped on 2p and the chromosome inversion
is often associated with deletion around the ALK 5
′
breakpoint
resulting in the 5
probe failing to hybridize [29, 34]. This pattern is
different than the widely split pattern of interchromosomal lesions
found in anaplastic large cell lymphoma. In addition to observer
error and aberrant probe hybridization (where intended targeted
sequences do not hybridize with the probe or the probe hybridizes
to nonspecific sequences), nuclear truncation on sectioning leading
to the loss of signals and background noise where weak signals
mimic nonspecific noise are other drawbacks that contribute to false
positive and false negative results. Also, this technique would not be
able to differentiate between the various
′
fusion variants
however the significance of this remains to be seen. Currently, FISH
remains the diagnostic method used as an eligibility criterion in
current clinical trials that are accruing patients positive for ALK-
rearrangements [51]. These studies deem a specimen to be positive
if 15% of the scored tumor cells have split
EML4-ALK
ALK
5
′
and 3
′
signals
or have isolated 3
signals. This cutoff was recently validated in a
study by where the positivity via FISH was seen at a minimum of
approximately 22% of cells in ALK-positive tumors [52]. This study
also noted that both the sensitivity and specificity of this assay
improved to 100% when 4 or more tumor areas were examined,
including approximately 60 tumor cells [52]. Further studies will
need to be performed to validate these results and to standardize
the criteria for the detection of
′
ALK
rearrangements by FISH.
