2
Basic Cytogenetics and the Role of Genetics in Cancer Development
Fig. 2.18
(A)
Interphase FISH of follicular lymphoma
cells with the
use of a dual-fusion BCL2-IgH probe. The two fusion/colocalization signals
indicate the existence of a
BCL2-IgH
oncogene and its reciprocal while the
green and red signals correspond to the remaining normal
IgH
and
BCL2
genes respectively. (B)
Interphase FISH
with the use of a dual-fusion break-
apart probe specific to the
EWSR1
locus. the lower nucleus shows a normal
pattern, whereas the upper one displays split of one
EWSR1
gene copy as it
can be observed in Ewing's tumors.
signals (corresponding to two copies of nonrearranged genes),
whereas in the case of translocation involving one of the two
genes, one of the fusion signal splits, resulting in a characteristic
1 red-1 green-1 yellow fusion ("1R1G1F") signal pattern. The
break-apart strategy offers the advantage of detecting in a single
experiment all recurrent rearrangement of a gene involved in
translocations with different gene partners. A typical example is
the
EWSR1
gene which can fuse with no less than nine different
gene partners (Fig. 2.18B).
Interphase FISH is also able to identify submicroscopic chro-
mosomal deletions as well as numerical chromosomal abnor-
malities such as trisomy or monosomy. The probes used to detect
entire chromosomal gains or losses are juxtacentromeric alphoid
DNA sequences while submicroscopic deletions will be identi-
fied with locus-specific probes. To ensure the quality of hybridi-
zation (mainly the hybridization properties of the tumor cells
being analyzed), a control probe, labeled with a different fluoro-
phore and identifying any other chromosome, will be cohybrid-
ized with the probe of interest. For detection of microdeletion,
the control probe will also serve to identify the chromosome har-
boring the deleted region. Examples are trisomy 3 (Fig. 2.19) and
deletion of chromosome 7q in marginal zone lymphoma.
Application
Lymphomas
Studies demonstrating the feasibility and diagnostic utility of
FISH in FNA specimens have focused on the most frequent lym-
phoma such as FL and, to a certain extent, diffuse large B-cell
Fig. 2.19 Interphase FISH
using centromeric probes for chromosomes 7
(green) and 18 (red). Both cells show three signals for each probe indicative
of trisomies 7 and 18 as observed in nodal
marginal zone lymphoma.
lymphoma. Although less common, MCL has also been a subject
of interest because of the clinical relevance and difficulties to dif-
ferentiate it cytologically from other small cell NHLs. Among the
latter, small lymphocytic lymphoma may be difficult to diagnose
when it presents as isolated lymphadenopathy.
As mentioned earlier, FISH and PCR remain complementary
methods for detecting predictable chromosomal abnormalities
in lymphoma, but comparative studies on specimens such as tis-
sue imprints, cytospins, or smears have demonstrated a higher
qualitative sensitivity of I-FISH. In follicular lymphoma, the
detection rate of the t(14;18) translocation with PCR was 70%
at best, whereas a positive result could be achieved in around
90% of cases with FISH.65,66,70-73 The low detection rate encoun-
tered with the PCR technology is due to mutation involving
primer binding sequences and to the fact that the current PCR
method applicable in routine use is not able to detect break-
points outside the known major breakpoint region (MBR) and
minor cluster region (mcr). A similar situation is encountered in
MCL where the sensitivity of FISH analysis for the direct detec-
tion of the t(11;14) translocation largely exceeds DNA-PCR
methods; the detection rate reaching nearly 100% according to
FISH studies,64,69,72,74,75 while it falls in the range of 40% with
the second method.74,60 The lower qualitative sensitivity offered
by DNA-PCR is mainly due to the wider variation of
BCL1
gene
breakpoints that are difficult to span with primers. FISH analysis
circumvents these limitations by using
IgH/BCL2
and
IgH/BCL1
dual-fusion probes covering the entire
BCL2
and
BCL1
gene,
respectively. Moreover, these results highlight the greater appli-
cability of FISH since all known
BCL2
and
BCL1
breakpoints can
be covered and detected with a single-probe set. Among other
small-cell lymphoproliferative disorders, SLL/CLLs are charac-
terized by recurrent chromosomal abnormalities such as tri-
somy 12 or interstitial deletion involving 13q14 chromosomal
region. Interphase FISH can easily detect these aberrations63,76
and, together with negative results for
BCL2
and
BCL1
rearrange-
ments, can help in assessing an accurate diagnosis of SLL/CLL.
In FNA specimens displaying monomorphous lymphoid popu-
lation composed of medium-sized or large cells, a proper diag-
nosis of Burkitt, diffuse large B-cell, or anaplastic lymphoma
can be easily reached with the use of specific break-apart probes
targeting
C-MYC, BCL6,
or
ALK
gene, respectively.72,77 Beside the
detection of lymphoma-associated specific translocations, atypi-
cal patterns revealed by interphase FISH can help in better clas-
sifying lymphoma. For example, a current
IgH/BCL1
dual-fusion
probe can identify hypotetraploid profiles with extra copies of
BCL1
signals as observed in blastoid variants of MCL.74,78
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