The Principles of Clinical Cytogenetics - Extra Materials - Springer

426 Jonathan Fletcher
and more sensitive than CISH, but CISH is visualized on an ordinary brightfield microscope, whereas
FISH requires a high-quality fluorescence microscope and appropriate imaging software (see Chapter 7).
The CISH detection reactions, generally performed using peroxidase or alkaline phosphatase strategies
(9–11), are also extremely stable and can, therefore, be archived for many years.
The FISH/CISH analyses are most conveniently performed using cytogenetic preparations, but they
are increasingly being applied to paraffin sections and other archival pathology preparations as well.
One substantial advantage in the use of paraffin sections is that the well-preserved cell morphology can
be used as a guide to enable evaluation of the chromosomal events in only the relevant cell populations.
An example is the evaluation of ERBB2 (HER-2/neu) gene amplification in breast cancer, where hybridization
against paraffin sections permits scoring of the ERBB2 gene signals precisely in the invasive
carcinoma components of the tumor biopsy (12,13; see also Chapter 17, Fig. 14). A drawback in the use
of paraffin tissue sections is that the nuclei are generally incomplete, having been sliced during preparation
of the sections, which are typically no more than 4 µm in thickness (14). ISH can also be carried
out against nuclei disaggregated from thick (50–60 µm) paraffin sections (15), but the author has found
that this often results in substantial damage to the nuclei. Alternate methods, including disaggregation
of cells from thin core biopsies of the paraffin block, could circumvent these limitations (16).
Most FISH studies performed in clinical cytogenetics laboratories address focused questions, such as
whether there are deletions, rearrangements, or amplifications of particular gene loci or of particular chromosomes.
However, various molecular cytogenetic methods have expanded the capabilities of solid-tumor
molecular cytogenetics by enabling fluorescence evaluation of the entire karyotype or of the entire genome.
Examples include comparative genomic hybridization (CGH) (17,18) and multiplex FISH (M-FISH) (19),
both of which permit genomewide evaluation of chromosomal aberrations (see Chapter 17).
Comparative genomic hybridization is performed by extracting total genomic DNAs from a tumor
of interest and from a non-neoplastic control cell population. These DNAs are differentially labeled
(e.g., tumor DNA with fluorescein and control DNA with rhodamine), and are then cohybridized
against normal metaphase cells (metaphase CGH) or against arrays of genomic or cDNA clones
(array CGH). Chromosomal regions that are overrepresented (amplified) or underrepresented (deleted)
in the tumor DNA will be manifested as color shifts when the metaphase cells, or arrays, are visualized
under fluorescence. An advantage of CGH, compared to conventional karyotyping, is that the
tumor DNA can be isolated from frozen or paraffin specimens, without need for cell culture. In
addition, the array CGH methods can detect very small deletions, which would be overlooked by
traditional cytogenetic banding assays. However, CGH does not detect balanced chromosomal rearrangements
(e.g., balanced translocations that are diagnostic markers in many soft tissue tumors and
in some carcinomas) (see Chapter 17, Figs. 16 and 17).
Genomewide molecular cytogenetics can also be performed using M-FISH, in which panels of
DNA probes are cohybridized against tumor metaphase cells (19). Whereas conventional FISH techniques
involve hybridization of one or two fluorescence-tagged probes, M-FISH can utilize probes
for each chromosome or chromosome arm (24 or more probes). Each probe is detected combinatorally,
using different ratios of fluorescence markers. By varying the ratio of each fluor, each chromosome
can be given a unique color. Thus, M-FISH enables a comprehensive ISH screen of the
entire tumor cell karyotype. M-FISH is a powerful research tool in solid-tumor cytogenetics and has
been particularly useful in elucidating complex karyotypes and in identifying recurring deletion or
amplification regions within those karyotypes (20,21). However, it has not been adopted widely for
routine clinical applications, given that it requires specialized equipment and is relatively timeconsuming
and costly (see Chapter 17, Fig. 18).
CYTOGENETIC MECHANISMS IN SOLID TUMORS
The cytogenetic aberrations in solid tumors vary from extremely simple, involving loss or rearrangement
of a single chromosome, to highly complex. Complex abnormal karyotypes, which typi-