Biomedical Engineering Reference
In-Depth Information
16.4.1
Reverse Transcriptase-PCR Based Detection
One of the more commonly used screening strategies has been RT-
PCR on archival tissue. Given that
map in opposite
orientations within the chromosome 2p, primers designed to amplify
the fusion points of
EML4
and
ALK
would not be expected to yield products
from normal cells or cancer cells without the inv(2)(p21p23). In
theory, this is a sensitive and rapid method for potentially detecting
mutant translocations. Initially, only a few primer sets were tested,
but with the recent discovery of at least 9
EML4-ALK
variants, a
number of multiplex assays incorporating different primer sets were
designed to detect all in-frame fusions between
EML4-ALK
EML4
and
ALK
[9,
10, 29]. Despite the fact that different breakpoints in the
gene
may be detected, novel fusions proteins, like TFG and KIF5B, may not
be. Depending on the frequency of these non-EML4 fusion partners,
the sensitivity of RT-PCR for detecting ALK rearranged NSCLC would
be reduced. Another disadvantage of this technique is that RT-PCR
requires good-quality mRNA. Most biopsy specimens are stored as
formalin-fixed paraffin embedded (FFPE) tissues. The RNA obtained
from these samples is highly degraded and makes PCR difficult. Even
though PCR from FFPE tissue has been challenging, newer assays are
being developed for
EML4
fusion gene transcripts suitable for
use with commonly available FFPE tissues of limited size [47]. Lastly,
there are published data indicating that RT-PCR may yield positive
results for the EML4-ALK fusion in 15% of non-tumor lung tissue
[33] as well as positive results without a corresponding chromosome
2p rearrangement as detected by FISH [33, 34]. Although the data
are subject to interpretation, it suggests the possibility of obtaining
false positives and hence a reduced specificity of this test. Despite
the disadvantages, many still advocate RT-PCR in detecting
EML4-ALK
EML4-
ALK
given the advent of newer high-throughput multiplex assays
[10, 47].
16.4.2
Immunohistochemistry Based Detection
Immunohistochemistry (IHC) is a reliable and relatively easy
diagnostic tool to detect pathologic proteins in paraffin-embedded
tissues without the loss of the cytologic and architectural features.
Several antibodies specific for ALK have been developed to diagnose
ALK rearranged (NPM-ALK) anaplastic large cell lymphomas
