Assessment of trisomy 12 in chronic lymphocytic leukemia by three methods: conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and Laser Scanning Cytometry (LSC)

Abstract Details: Chronic lymphocytic leukemia (CLL) is
the most common adult leukemia in Western countries
and has an extremely variable clinical course, presumably
related to its genetic heterogeneity. It is highly
recommended to conduct genomic analysis before
initial treatment, including del(13q), trisomy 12,
del(11q) and del(17p), which are identified by FISH.
Conventional FISH protocols are labour intensive and
analyse low total numbers of cells. We compared the
following 3 technical approaches for specificity, sensitivity
and cost/time efficiency using FISH as the accepted
standard:
(i) Conventional FISH assesses the probes by fluorescence
microscopy and visually scores up to 200
nuclei. This technique is considered the diagnostic
standard assay for CLL despite its limitation of the
low number of cells targeted and its labour
intensive demand.
(ii) FISH in suspension (FISH-IS) incorporates the standard
FISH technique with the high-throughput
detection afforded by flow cytometry, enabling
the quantification of genetic abnormalities in up to 1000 cells/second with high accuracy. Particularly,
the spot count program in the IDEAS software
enables accurate scoring of the spots within a cell.
(iii) Laser scanning cytometry (iCys) is a microscopebased
cyto-fluorometer that allows quantitative
measurements on single cells at a high resolution
with computer controlled analysis of thousands of
cells. Automated spot counting can solve the
problem of fatigue caused by many hours of
microscope viewing, which can lead to mis-interpretation
and scoring inconsistencies.
We compared the above methods in the analysis of
trisomy 12 using the Vysis commercial probe (Abbott
Molecular) in CLL samples. Centromere 12 probes are
repetitive sequences that provide very high fluorescence
intensity at the target site due to a large number of
bound probes. We were able to confirm that the FISH-IS
method was able to accurately detect disomy and
trisomy in CLL cells with centromere 12 probes
(Figure 1). In addition, iCys analysis is also able to
confirm the conventional FISH results with nearly 2000
cells-counted. This research is being continued; the next
phase is to apply these three methods to locus specific
probes (13q, 11q, 17p) in order to further evaluate the
FISH technique before making any conclusions on
prognosis; and more importantly, its potential use in
monitoring clonal evolution in CLL.