The Principles of Clinical Cytogenetics - Extra Materials - Springer

Cytogenetics of Solid Tumors 425
Solid-Tumor Cytogenetics: Art or Science?
Various factors determine the success of solid-tumor cytogenetics, not least of which being the
experience of a given laboratory in processing and analyzing such specimens. Four of the main considerations
are as follows:
1. Unpredictable growth of the neoplastic cells in tissue culture. Benign solid tumors generally contain few
mitotic cells, and one must often wait for a week or more before such specimens begin to proliferate
actively in tissue culture. In the meantime, the neoplastic cells might be overgrown by non-neoplastic
cells (see point 2). Surprisingly, there are many highly malignant solid tumors that also grow poorly in
tissue culture, despite the fact that they were growing rapidly in the patient. Such tumors can sometimes
be stimulated in culture by use of relevant culture media and growth factors, but it is impractical in the
clinical cytogenetics laboratory to stock the various different growth factors and media that are optimal
for many varieties of solid tumors. Therefore, in practice, it is challenging to culture certain types of solid
tumors in the clinical laboratory. A notable example of this is prostate cancer, which, although a very
common type of cancer, requires extremely specialized methods for successful tissue culture. It is also
important to recognize that only a minority of the overall neoplastic population in a given sample might be
capable of growing under a particular set of tissue culture conditions. Therefore, one cannot assume that a
clonally abnormal karyotype is representative of the overall neoplastic process. For example, in a given
tumor, the final karyotype might be representative of the components of the tumor that were either more or
less clinically aggressive, depending on which component was best suited to growing under the particular
tissue culture conditions used for that case.
2. Overgrowth of neoplastic cells by “reactive” non-neoplastic cells. All solid-tumor biopsies contain mixtures
of neoplastic and non-neoplastic cells. The non-neoplastic elements can include fibroblasts, normal
epithelial cells, endothelial cells, or glial cells (in the case of brain tumors), depending on the type and
location of the tumor. Any of these reactive cell types, in a given specimen, can grow more successfully
that the neoplastic cells in culture. Therefore, culture overgrowth by reactive cells is the most common
explanation for a normal diploid karyotype in solid-tumor cytogenetics. For this reason, it is crucial to
learn the morphology of the common sorts of reactive cells, which can be distinguished from the neoplastic
cells by daily evaluation of the cultures via phase-contrast inverted microscopy. Metaphase cells should
be harvested at the first signs of reactive cell overgrowth, even in cases where the neoplastic population
has not yet begun to grow actively. Otherwise, the finding of a spurious normal karyotype, deriving from
the overgrowth of reactive cells, will be the predictable end result of the cytogenetic analysis.
3. Destruction of tumor cultures by bacterial or fungal infection. Loss of solid-tumor cytogenetic cultures to
infection should be an uncommon event, providing that the specimen transport and culture media contain
broad-spectrum antibiotics (e.g., penicillin/streptomycin) and antifungals (e.g., amphotericin). Infectious
contamination generally occurs when specimens have been crudely handled in the pathology department,
either by cutting a sample with a previously used blade or by placing the sample directly on a dirty cutting
surface. Infectious contamination can be unavoidable in the case of specimens from body regions that are
extensively colonized by bacteria (e.g., in the case of a colorectal carcinoma whose surface is within the
intestinal lumen).
4. Failure of tumor cultures to grow because of nonviable tumor. Many solid tumors, particularly those that
are highly malignant, are largely composed of nonviable regions, or regions with few neoplastic cells.
Such regions can be extensively necrotic, because the tumor cells have died, having outstripped their
blood supply. Other regions of a tumor mass can be composed largely of blood (hemorrhage) or scarred
tissue (fibrosis). Therefore, it is crucial that the pathologist select a maximally viable tumor region for the
solid-tumor cytogenetic analysis.
Molecular Cytogenetics
Whereas conventional cytogenetic analyses are performed using various staining techniques that
highlight chromosome bands, the various molecular cytogenetics methods involve evaluation of relevant
chromosome regions using DNA probes (8,9). Most molecular cytogenetic methods are based
on ISH (in situ hybridization); that is, the DNA probes are hybridized and evaluated in the cellular, in
situ, context. ISH assays can be performed with fluorescence or enzymatic detection, which are referred
to as FISH (fluorescence in situ hybridization) (see Chapter 17) and CISH (chromogenic in situ
hybridization), respectively. There are pros and cons to both of these methods. FISH assays are simpler