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839
E D I T O R I A L
Interphase Cytogenetics for
Studying Solid Tumors
Hon Fong L. Mark,
Ph.D.
Lifespan Academic Medical Center Cytogenetics Laboratory and Brown University School of
Medicine, Providence, Rhode Island.
C
See referenced original article on pages 977– 88,
this issue.
The author thanks Dr. Roger Mark for reading the
article. The support of the staff of the Lifespan
Academic Medical Center Cytogenetics Laboratory
is also acknowledged.
Address for reprints: Hon Fong L. Mark, Ph.D.,
Cytogenetics Laboratory, Lifespan Academic Medical Center, Rhode Island Hospital, APC Building
11th floor, 593 Eddy Street, Providence, RI 02903.
Received March 31, 1998; accepted March 31,
1998.
© 1998 American Cancer Society
onventional cytogenetic analysis is a powerful, established technique that can provide a picture of the human genome at a
glance. Most laboratories use G-banding using trypsin and Giemsa
stain (GTG-banding) for conventional cytogenetic analysis. In a routine cytogenetic study, a short term culture is either established in the
presence of a mitogen—such as phytohemagglutinin (PHA), where it
is called a stimulated culture— or grown without such an agent, in
which case it is called an unstimulated culture. The former is used for
peripheral blood cultures to rule out constitutional abnormalities,
whereas the latter is used for the study of neoplastic tissues. Longterm tissue cultures are usually established for solid tumor studies.
Harvesting chromosomes for conventional cytogenetics1 is a rather
lengthy and tedious process. Colcemid, a derivative of colchicine, is
usually used to block spindle fiber formation and arrest the chromosomes in metaphase. This is followed by a hypotonic treatment to
cause the cells to take up water and swell so that the chromosomes
will spread well when dropped onto glass slides at a later step. After
the hypotonic treatment step, the cell pellet is fixed with a fixative
consisting of three parts methanol to one part glacial acetic acid. After
repeated rinsings, the cells are then dropped onto glass slides and
airdried. Slides are aged for a variable amount of time, then banded
and stained according to one of the banding protocols. GTG-banding
seems to be the most popular method in the U.S.,2,3 probably because
of its simplicity. Prior to the advent of modern-day imaging systems,
photographs were taken of the best metaphase spreads, and the
photographs were enlarged and hand-cut to separate the images of
the chromosomes for identification. Most cytogenetics laboratories
now own computer-assisted karyotyping systems. Karyotyping involves arranging the 46 chromosomes in the human genome according to shape, size, and banding patterns. Thus, conventional cytogenetics is a labor-intensive process requiring highly trained personnel.
In addition, conventional cytogenetics depends entirely on the availability of high quality metaphases, thus excluding from analysis the
vast majority of cells that are in interphase.4
Conventional Cytogenetics of Solid Tumors Has Been Hampered
The conventional cytogenetics of solid tumors has been hampered by
a number of factors,5–9 making an already lengthy and tedious process
even more challenging. One is the difficulty of establishing a cell
culture. Another is the susceptibility to culture contamination despite
840
CANCER September 1, 1998 / Volume 83 / Number 5
adherence to sterile techniques. The tumor tissue may
grow at a very slow rate. The culture sometimes has a
low mitotic index, resulting in insufficient cells for
karyotyping. The banding of the metaphases is at
times suboptimal, with fuzzy chromosomes. Even if all
these factors could be overcome, the resultant metaphases could all be normal, leading one to wonder
whether the tumor cells have been overgrown by normal stromal cells. The resultant karyotype, on the
other hand, could be extremely complex, with multiple numeric and structural abnormalities leading to
difficulties in interpretation. The possibility that the
abnormal karyotype could be an artifact of culture
also cannot be completely ruled out. Due to these
various factors, conventional cytogenetics of solid tumors has not kept pace with that of hematopoietic
disorders, and few tumors are well characterized from
a conventional cytogenetic standpoint.
In Situ Hybridization
In situ hybridization with radionuclide-labeled probes
has been reported since the late 1960s.10 Autoradiography, however, requires long periods of exposure that
are not practical for most applications. Biotinylated
DNA probes and probes modified with other reported
molecules were introduced in the 1980s.5,11–13,14 –16
These early colorimetric in situ hybridization assays
were performed with immunocytochemical stains,
such as horseradish peroxidase, and hybridization was
detected with a light microscope. Some laboratories
today still adhere to these systems because of preference, whereas others have little choice because of
financial constraints. With the advent of fluorescencebased methods, termed fluorescent in situ hybridization (FISH),17 many laboratories began to use this
protocol for both clinical and research applications.
Regardless of whether the in situ hybridization is fluorescence-based, it has the advantage of making analysis of nondividing cells possible. This capability of
performing cytogenetic analysis on interphase cells,
called interphase cytogenetics, has indeed revolutionized the field of cytogenetics.
Interphase Cytogenetics Complements Conventional
Cytogenetics
Interphase cytogenetic analysis can be performed using a variety of DNA probes, the most popular of
which are alpha-satellite DNA probes.18 Alpha-satellite DNA sequences are organized as tandem repeats
of unique 171 base-pair sequences that are present in
as many as 5000 copies. For interphase studies, a DNA
probe to the pericentromeric alpha-satellite DNA of a
specific chromosome is biochemically modified by
nonisotopic methods, such as nick translation with
biotinylated dUTP, and then hybridized to cells using
routine hybridization techniques. Because of the high
copy number of the target DNA, highly sensitive, albeit
cumbersome, radioactive detection methods are not
required, and rapid, simple-to-use, nonisotopic detection methods can be employed. Commonly used for
the detection of a biotinylated probe are fluoresceinated avidin and avidin conjugated with a detector
enzyme, such as alkaline phosphatase. Both produce
punctate signals that can be recognized microscopically. The advent of digital imaging microscopy has
brought further improvements in signal detection and
signal-to-noise ratios. The multiple applications of interphase cytogenetics have been discussed elsewhere.7,15
As already mentioned, the advantages of interphase cytogenetics include the fact that it is not restricted to cells arrested in metaphase, thus allowing
analysis of large numbers of cells. The ability to analyze large numbers of cells is significant in that it
permits the detection of low frequency abnormalities
that are otherwise difficult to detect.4 Thus, specimens
that would not otherwise be suitable for analysis by
conventional techniques could still yield useful cytogenetic information. Interphase analysis could thus be
used to study cells from tumors with low proliferative
activity or from tumors that are difficult to maintain in
short term cultures. In addition, interphase cytogenetics is rapid because it does not require special training
for interpretation. Interphase cytogenetics allows
some correlation of cytogenetic findings with morphology, which is not possible with conventional cytogenetic study because all nuclear details are lost in a
metaphase cell. When used as an adjunct, interphase
cytogenetics offers benefits in both the study of malignant cells and the management of patients with
malignant disorders. It is ideal for archival tumor tissues that are amenable to immunocytohistochemical
studies but not amenable to conventional cytogenetic
analysis.
For the above reasons and possibly others, interphase cytogenetics has been used to examine solid
tumors, such as bladder tumors,19 –21 gestational trophoblastic disease,22 breast carcinoma,9,23–26 neuroectodermal tumors,27 carcinoma of the testis,28 prostate
carcinoma,29 –31 and rhabdomyosarcoma.32
Interphase Cytogenetics Is Ideal for Studying Aneuploidy,
a Potential Cancer Biomarker
Aneuploidy has been explored as a biomarker for
stratifying many cancers. Traditionally, flow cytometry has been the method of choice for ploidy analysis.
Many studies suggest that DNA ploidy analysis using
flow cytometry and static image cytometry can pro-
Interphase Cytogenetics and Solid Tumors/Mark
vide independent prognostic information in addition
to stage and histologic grade.33,34 However, flow cytometry for detecting aneuploidy has its own limitations regarding sensitivity.35–37 For example, Visakorpi
et al.36 found that FISH using three selected chromosomes specific probes was two to three times more
sensitive than cytometric DNA content analysis in aneuploidy detection. DNA flow cytometry, especially
when performed on paraffin embedded tumors, cannot distinguish aneuploid cell clones that have only a
few numeric chromosomal changes from diploid
clones. Early stage tumors that are often nearly diploid
or show balanced chromosomal abnormalities are not
detected as DNA abnormal. Mesker et al.,38 for example, noted that the diagnostic value of DNA ploidy
measurements by flow or image cytometry with respect to the detection of DNA aneuploidy is limited,
because only relatively significant changes in total
DNA content can be detected (i.e., at best 2%, depending on the accuracy of the measurements). Generally,
the equivalent of a gain or loss of one large or several
small chromosomes (a 4% change in total DNA content) is required for adequate detection by flow cytometry.25,39,40 Thus, although the ability to evaluate large
numbers of nuclei readily is a strength of flow cytometry, a small population of abnormal cells may be
difficult to detect within a much larger population of
normal cells.
In short, interphase cytogenetics based on the
hybridization of DNA probes on interphase cells has
the potential to revolutionize the field of surgical pathology because of its increased sensitivity over flow
cytometry and static image analysis in aneuploidy detection.35,41,42 Previous studies have found that polymerase chain reaction (PCR) techniques may not be as
sensitive as FISH due to tumor heterogeneity.43
Interphase Cytogenetics for Studying Aneuploidy as a
Prognostic Marker in Prostate Carcinoma Illustrates the
Utility of This General Approach
Prostate carcinoma is a common disease affecting
American men, causing 40,400 deaths each year.44 The
cause of prostate carcinoma is currently not known.
No specific genetic alterations associated with prostate carcinoma have been established so far.45
To date, tests measuring serum prostate specific
antigen (PSA) levels combined with digital rectal examinations and transrectal ultrasound– guided biopsies have been the most reliable form of early detection. Unfortunately, there are no available markers to
predict clinical outcome accurately.43
Current prognostic indicators for prostate carcinoma are limited to Gleason grade and clinical staging. As 244,000 new cases are diagnosed each year, the
841
need for more accurate methods to assess the extent
of an individual patient’s cancer and define prognostic
subgroups has become urgent.
The optimal treatment for patients with prostate
carcinoma remains controversial. The failure to stratify patients into prognostic subgroups has hampered
the recognition of the efficiency of aggressive local
therapeutic measures in comparison with watch-andwait strategies.
In this issue of Cancer, a clearly written article
based on a well-designed and scientifically significant
study is presented by Henke et al.46 The study was
conducted to evaluate the relative role of interphase
cytogenetics with chromosome enumeration probes
and of conventional pathologic characteristics in the
preoperative prediction of postoperative tumor classification and the recurrence of elevated serum concentrations of PSA.
In the study of Henke et al., interphase cytogenetics was used to determine the chromosome copy
number. Chromosome enumeration probes for chromosomes 7, 17, and X were selected based on the
frequency of numeric chromosomal abnormalities
found in the authors’ previous studies of prostate carcinoma patients.47,48 Six-micron sections of core biopsies from 75 patients with clinically localized adenocarcinoma of the prostate were used. A postoperative
increase in PSA was used as an endpoint and an index
of outcome. Kaplan–Meier analysis of results showed
that the PSA recurrence ($ 0.4 ng/mL) was more frequent and observed earlier in patients with detected
chromosomal aneusomies compared with those who
had eusomic and tetrasomic chromosome numbers
(P , 0.0001). Cox regression analysis indicated that
interphase cytogenetics was the most valuable independent factor in predicting PSA recurrences. The authors concluded that the detection of numeric chromosomal aberrations in preoperative core prostate
biopsies is an adverse prognostic sign that is important for predicting prognosis and for selecting the
appropriate form of therapy.
As Henke et al. noted, no molecular marker to
date meets the criteria of a clinically relevant prognostic factor as defined by the College of American Pathologists.49 A reliable and consistent biomarker, or a
panel of biomarkers, capable of defining therapy selection among subgroups is urgently needed. Genetic
grading based on assays of chromosome aneuploidy
may ultimately prove to be the marker of choice for
stratifying patients with prostate carcinoma and other
cancers, although the number of patients studied to
date has been relatively small and their follow-up time
has been short. The results obtained thus far are nev-
842
CANCER September 1, 1998 / Volume 83 / Number 5
ertheless encouraging and should be confirmed and
extended.
CONCLUSIONS
As Henke et al.46 noted, cancer development and progression are complex processes involving multiple genetic alterations. Whereas the mechanisms in colorectal carcinoma have been elucidated, details of the
genetic events that influence carcinogenesis and malignant progression in prostate carcinoma and most
other cancers remain poorly understood. Interphase
cytogenetics using FISH with chromosome enumeration probes is ideally suited for the study of cancer
using archival materials.
Recent advances in molecular technology have
now led to the development of newer techniques that
combine the sensitivity and specificity of FISH with
the global screening ability of conventional cytogenetics. Notably, spectral karyotyping, or SKY,50 is an
evolving molecular cytogenetic technique that permits
examination of the entire genome in a single hybridization. However, because techniques such as comparative genomic hybridization, or CGH,51–52 and SKY
require special instruments and trained personnel, the
exact roles that these new emerging technologies will
play in the average clinical cytogenetic laboratory in
the current climate of managed care and cost containment53 is yet to be determined. Meanwhile, interphase
cytogenetics using commercially available chromosome enumeration probes is increasingly being utilized for the identification of potential biomarkers,
such as chromosome aneuploidy and oncogene amplification, which are both considered manifestations
of genetic instability that play pivotal roles in the
genetics and progression of human cancer.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
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