PART ONE
General Cytology
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Fig. 2.6 Multicolor FISH (SKY)
allowing the
identification of every human chromosomal
pair with an individual color using 24 different
painting probes (22 pairs of autosomes plus the
two sexual chromosomes). Normal chromosomes
are uniform in color, whereas rearranged
chromosomes will display two or more colors.
This method makes it possible to detect cryptic
rearrangements and marker chromosomes in
complex karyotypes as demonstrated here.
displaying a specific immunophenotype but is of little value for
minimal residual disease.
Multicolor Metaphase FISH
Multicolor-FISH includes mainly two different methods called
spectral karyotyping (SKY) and multiplex-FISH (M-FISH). In
SKY, the chromosomes are first stained with a mixture of 24
chromosome-specific painting probes; each one being labeled
with a different combination of five fluorochromes. The spec-
tral pattern of chromosomes is then classified using computer
software to identify individual chromosomes. M-FISH uses a
combinatorial labeling scheme with only five fluorochromes
having different emission spectra. Those fluorochromes are
similar to that used for SKY but the method for detecting and
discriminating the different combinations of fluorescence
signals is different.
Both methods are useful in characterizing complex chromo-
somal rearrangements and in documenting ambiguous marker
or ring chromosomes10 (Fig. 2.6).
Comparative Genomic Hybridization (CGH)
This method has the advantage of circumventing the need
for tumor cell metaphases. Total genomic tumoral DNA is
labeled in green and the normal reference DNA in red. Both
differentially labeled tumor and normal DNA will be hybrid-
ized together to normal human metaphases and will compete
with one another. The ratio of the fluorescent green and red
intensities is measured along every chromosome, making it
possible to give an overview of DNA sequence copy number
changes—gains and losses—in the neoplastic cells mapped on
normal chromosomes. CGH is thus able to detect amplified
and deleted genomic regions harboring oncogenes or tumor
suppressor genes, respectively. The limitation of this method is
that it can identify DNA imbalances but not balanced chromo-
somal translocations (hence, without loss or gain of chromo-
somal material subsequent to translocation).
Acquired Chromosomal Aberrations
in Cancer
Introduction
It has long been agreed that tumor cells carry chromosomal aber-
rations, but their causes have only recently been more deeply
explored.11,12 Until the seventies, cytogeneticists were dealing
with malignant effusions or long-term cell cultures yielding
roughly recognizable multiple chromosome changes, with very
large amounts of rearranged DNA in complex aneuploidies.
Consequently, this situation led to disillusion in the litera-
ture seeded by a plethora of reports with confusing malignant
karyotypes, suggesting to most scientists that the chromosomal
rearrangements observed were just epiphenomena accompa-
nying the process of malignancy. At that time, no method was
able to show molecular changes at the gene level. Karyotype
analysis based on banding techniques renewed interest in the
characterization of cytogenetic abnormalities in malignant
tumors. It appeared evident that nonrandom primary changes
involved specific chromosome regions, and were subsequently
overwhelmed by secondary more massive variations affecting
randomly all chromosomes. This state of overall genomic insta-
bility developed during the malignant clonal progression.
Cytogenetic investigations focused initially on leukemias.
They identified a constantly increasing number of character-
istic chromosomal patterns after the Ph/CML association was
detected. The FAB classification of leukemias was consequently
enriched by the addition of prototypic karyotypic profiles.
Beside leukemias, other cytogenetic and molecular informa-
tion emerged with studies of lymphomas and sarcomas. In
those tumors, relatively simple balanced rearrangements often
appeared as fingerprints for a unique tumor type. These specific
chromosomal abnormalities were rapidly considered as reli-
able diagnostic, prognostic, and predictive parameters on daily
routine. The importance of chromosomal identification in the
diagnosis of human leukemias, lymphomas, and mesenchymal
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