short-term culture. Direct preparations tend to have
only a few metaphase cells for study. Tumor cell
cultures usually yield a satisfactory number of
metaphase cells but culturing of the cells can select
against the more dysfunctional cancer cells in the
tumor population and can select for normal cells
co-inoculated from the biopsy material (Ketter
et al.
1996). This decreases the sensitivity of the method.
Karyotyping by fluorescence
in situ
hybridization
suffers from the same problems. The use of fluores-
cence
in situ
hybridization for the detection of
defined chromosome abnormalities, on the other
hand, has many advantages: it can be performed on
interphase nuclei, so tumor cell culture is not neces-
sary, and paraffin-embedded biopsy material
prepared for histologic study can serve as the source
of cells. It has proven to be an informative and
accurate method for the directed cytogenetic analysis
of solid tumors (Werner
et al.
1997) and hemato-
logic malignancies (Kearney 1999).
Chromosome deletions and translocations can
also be studied in fresh, frozen, and even paraffin-
embedded material using a variety of molecular
diagnostic techniques (see Chapter 10). Consider
the t(9;22) translocation that results in the Philadel-
phia chromosome of chronic myelogenous leukemia
(Faderl et al. 1999). It consistently arises from
translocation of the Abelson oncogene,
abl
, on
chromosome 9 to the breakpoint cluster region,
bcr
,
on chromosome 22. The presence of the
bcr/abl
fusion DNA sequence can be demonstrated by
Southern blot hybridization or preferential PCR
amplification. The translocation can also be demon-
strated by detection of either the bcr/abl fusion
protein or the
bcr/abl
fusion gene transcription
product. The fusion mRNA is detected using North-
ern blot hybridization or reverse transcription-PCR
specific for the bcr/abl mRNA sequence. Deletions
and translocations that show extensive heterogeneity
in the site of the chromosome abnormality are not
readily studied using molecular diagnostic methods.
Gene amplification is another genetic abnormality
contributing to the development and progression of
cancer. Amplification usually involves a region of
DNA larger than one gene and can produce a struc-
tural alteration large enough to be detected by
chromosome analysis either as double minutes
(separate small DNA fragments) or as a homogene-
ously staining region (DNA segment inserted into a
chromosome). Smaller amplifications can be
demonstrated using quantitative modifications of
fluorescence
in situ
hybridization, blot hybridization,
and PCR amplification (Crotty
et al.
1994).
Cancer-associated point mutations of oncogenes
and tumor suppressor genes may be restricted to
only a few base positions in the gene, such as
ras
oncogene mutations, but more typically they occur at
diverse bases in the gene, as seen with the p53 tumor
suppressor gene (Loda 1994). Allele-specific oligo-
nucleotide hybridization is most commonly used to
detect point mutations when the mutations in a gene
consist of only a few recognized nucleotide substitu-
tions. That technique is not applied to genes that
have a large catalog of nucleotide substitutions
because of the large number of probes that have to
be used. A different technique, single strand confor-
mational polymorphism analysis, is used then
(Nollau and Wagener 1997). In this technique, the
gene’s DNA is amplified by PCR using labeled
primers or labeled nucleotides (so that all of the
amplification products are labeled). The products
are digested using restriction endonucleases to
produce fragments smaller than 200 bp, denatured to
yield single-stranded DNA, and electrophoretically
separated in nondenaturing polyacrylamide gels.
Single-stranded DNA fragments form secondary
structures that depend upon the base composition of
the fragment; the structure can vary as a result of
even a single base alteration. The different single
strand structures migrate with different speeds in the
gel leading to their separation. Mutated DNA is
recognized by the abnormal migration position of its
single strand structure. Mutations at any position in
the gene can be detected using this method.
Cancer
11-3
Table 11.1
Some Genetic Alterations Seen in Cancer
Alteration
Example
Cancer
Chromosome deletion
del 8p12
prostate carcinoma
Chromosome translocation
t(15;17)
acute promyelocytic leukemia
Gene amplification
erbB2
breast carcinoma
Gene point mutation
K-
ras
pancreatic carcinoma
