applications of sky in cancer cytogenetics

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APPLICATIONS OF SKY IN CANCER CYTOGENETICS
Jane M. Bayani and Jeremy A. Squire*
Ontario Cancer Institute, Princess Margaret Hospital, University Health Network,
and Department of Laboratory Medicine and Pathobiology, and Medical
Biophysics, Faculty of Medicine, University of Toronto, Ontario, Canada.
*Corresponding Author:
JA Squire, Ph.D.
Ontario Cancer Institute
Division of Cellular and Molecular
Biology
610 University Ave. Room 9-721
Toronto, Ontario, Canada
M5G 2M9
E-mail: [email protected]
Key words: FISH, CGH, marker chromosomes, M-FISH, molecular cytogenetics
Acknowledgements:
The authors express their gratitude for the contributions of Ajay Pandita, Paula
Marrano, Ben Beheshti, Jana Karaskova, Paul Park and Zong Mei Zhang at the
Ontario Cancer Institute as well as Margaret Skokan, George McNamara and
Randy Knudtson at Applied Spectral Imaging for technical information. Our
gratitude also to Mark Israel for permission to cite recent findings concerning the
murine oligodendroglioma model.
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Abstract:
Clinical and cancer cytogenetics is a rapidly evolving discipline. The past
decade has seen a dramatic change in molecular biology and fluorescence
microscopy.
The use of fluorescence in situ hybridization (FISH)
technologies has enabled the rapid analysis of cytogenetic specimens as
an adjunct to classical cytogenetic analysis. Spectral Karyotyping (SKY) is
a 24-color, multi-chromosomal painting assay that allows the visualization
of all human chromosomes in one experiment. The ability for SKY analysis
to detect equivocal or complex chromosomal rearrangements, as well as to
identify the chromosomal origins of marker chromosomes and other extrachromosomal structures, makes this a highly sensitive and valuable tool
for identifying recurrent chromosomal aberrations. SKY has been applied
to various tumor groups including hematological malignancies, sarcomas,
carcinomas and brain tumors, with the intent of identifying specific
chromosomal abnormalities that may provide insight to the genes involved
in the disease process as well as identifying recurrent cytogenetic markers
for clinical diagnosis and prognostic assessment. SKY has also been
applied for the mouse genome, enabling investigators to extrapolate
information from mouse models of cancer to their human counterparts.
This review will address the advances that SKY has facilitated in the field of
cancer cytogenetics, as well as its variety of application in the cancer
research laboratories.
Introduction
Cytogenetics is a discipline, like so many in molecular biology today, that
is undergoing dynamic change in both technology and application. Hungerford et
al.(1), made the first karyotype from peripheral blood cultures in 1959 using, by
present standards, relatively crude techniques, but demonstrated the value of
chromosome analysis in both the clinical and research laboratories. Over the
years the use of cytogenetic analysis has enabled the identification of recurrent
chromosomal abnormalities that are diagnostic markers for cancers including
hematological malignancies and solid tumors (2). Early karyotype analysis of
many types of malignancies identified complex structural rearrangements and
extra-chromosomal structures that were left unidentifiable and simply termed
"marker chromosomes" (Figure 1). By the late 1980s, cytogenetics was a mature
discipline and underwent more extensive technological change as molecular
genetic technologies were applied to cytogenetic preparations.
Fluorescence in situ hybridization (FISH) quickly replaced radioactive in
situ assays by the late 1980s (3,4). The hybridization of fluorescently labeled or
fluorescently detectable probes from known genes and specific chromosomal loci
enabled the genome to be studied in a completely new way. The advancement
of recombinant DNA technology permitted newly identified genes to be mapped
to chromosomal regions, and mapped relative to other genes (5).
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Figure 1. G-banding analysis of an astrocytoma cell line (U373). Arrows
indicate abnormal chromosomes detected by banding analysis as well as marker
chromosomes. The banded karyotype is as follows:
73,XY,+Y,+Y,+Y,der(1)t(1;?)(p11;?)t(1;?)(q43;?)1,
der(1)t(1;?)(p11;?)t(1;?)(q43;?)2,add(5)(p10),-6,+7,+der(7)t(7;?)(q32;?),
der(9)t(9;?)(q10;?),-10,dup(10)(?p12p15),+der(12)t(12;?)(p10;?),+13,-14,
del(15)(q26.1),der(16)t(16;?)(p10;?),der(18)t(6;18)(p11.2;q11.2),-22,mar1,
mar2,mar3.
The SKY karyotype (data not shown) is as follows: 73,XXY,+Y,+Y,+Y,
inv(1)(p21;q44),+der(1)del(1)(p22)t(1;8)(q41;?),der(4)t(4;5)(p11;p12)t(4;15)
(q31;?q24),der(5)t(5;12)(p12;p12),6,+7,+der(7)del(p13;p15)t(7;8)(q32;?),
t(9;16)(p21;p11),-10,dup(10)(?p12?p15),+der(12;17)(p12;q12),+13,-14,del(15)
(q26.1),+del(17)(q12),der(18)t(6;18)(p11.2;q11.2),-22.
Chromosome descriptions in bold describe the revised SKY/cytogenetic
description. Underlined descriptions indicate aberrations determined by SKY
analysis, not previously detected by banding.
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As interphase FISH techniques were applied, the genomic information
sequestered in the nuclei could now be accessed, and provide important
information on the status of locus/gene specific probes when metaphase spreads
were few or not available (6,7), thus overcoming one of the many limitations of
classical cytogenetics: the requirement for good quality metaphase preparations
(Figure 2).
Figure 2. Fluorescence in situ hybridization (FISH) on a primary neuroblastoma
specimen. A. An interphase nucleus showing amplification signals using the
MYCN gene. The amplification signal pattern seen in this nucleus is indicative of
double minute chromosomes as seen in B. B. A metaphase preparation from
the same case showing the paired extra-chromosomal structures hybridized with
the MYCN probe.
Interphase FISH also enabled retrospective study of large archives of
histopathologic paraffin sections for analysis of aneusomies and for subsequent
correlative comparisons with established tumor markers (8-10). One of the
technically most demanding of the various new FISH technologies is
Comparative Genomic Hybridization (CGH) (11-14). Since only DNA is required,
CGH is ideal for large retrospective surveys of genomic imbalance and has the
potential to locate regions that harbor potential oncogenes in regions of gain, and
novel tumor suppressor genes in regions of loss (15,16). Furthermore, it has
also been shown to be a useful adjunct to classical cytogenetic methods.
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Spectral Karyotyping (SKY)
The advent of the various multicolor FISH techniques, generically termed
M-FISH, (17-19) has greatly improved the certainty with which cytogeneticists are
able to identify abnormal chromosomes. Presently, there are two methods of
performing M-FISH, one based on the use of specific filter sets (17) and the other
based on the spectral signature of the fluorochromes or dyes used and termed
Spectral Karyotyping (SKY) (18,19). Currently the most popular method of
performing this type of analysis is Spectral Karyotyping (SKY) (19). SKY
involves the use of 24-color, whole chromosome-painting permitting visualization
of each chromosome in one experiment. This technology is based on the
principles of spectral imaging (20) and Fourier spectroscopy (21). Flow sorted
chromosomes are PCR-labeled (22), either directly or indirectly, with
fluorochromes or haptens. Five pure dyes that are spectrally distinct are used in
combination to create the unique chromosome cocktail of probes. This probe
cocktail is hybridized to metaphase preparations and detected using
sophisticated image analytical methodologies.
Image acquisition is
accomplished by conventional fluorescence microscopy and the use of a
specially designed triple filter (SKY CUBE, Applied Spectral Imaging). In this
design, light passes through a Sagnac interferometer focussed on a chargedcoupled device (CCD) (Figure 3). Spectral images are acquired and analyzed
with the commercially available SD 200 Spectral Bio-Imaging System (ASI Ltd.,
MigdalHaemek, Israel). The generation of a spectral image is achieved by
acquiring ~100 frames of the same image that differ from each other only by the
optical path difference (OPD). The collected images are Fourier Transformed
and the data sorted in the software (SKYVIEW™). Each chromosome has a
unique spectral "signature", generated by the specific combination of one or
more of the five pure dyes. Once a spectral image is acquired, the SKYVIEW™
software compares the acquired spectral image against the combinatorial library
containing the fluorochrome combinations for each chromosome and generates a
"classified" image. The classified image pseudo-colors the chromosomes to aid
in the delineation of specific structural aberrations where the RGB (Red-GreenBlue) display image, which displays the fluorescent colors of the chromosomes
may appear quite similar. For every chromosomal region, identity is determined
by measuring the spectral emission pixel by pixel. Regions where sites for
rearrangement or translocation between different chromosomes occur are
visualized by a change in the display color at the point of transition.
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Figure 3. Schematic representation of image acquisition using an interferometer
housed in the optical head (Applied Spectral Imaging, Carlsbad, CA). Images
from the hybridized specimen are collected, Fourier transformed and processed.
SKY has enabled the elucidation of complex or equivocal structural
aberrations that may otherwise have been left undetected by classical
cytogenetics or FISH alone (Table 1).
Each assay has particular strengths, but many also possess limitations in
certain situations. One of the most obvious limitations of SKY is its inability to
readily detect deletions or other intrachromosomal structural changes such as
inversions. However there are some compelling reasons for the systematic use
of SKY in the analysis of abnormal chromosome preparations of the type
encountered in cancer cells. The ease of interpretation of the clearly assigned
color patterns means that it is not essential to have a highly experienced
metaphase analyst to perform the microscopy. In addition the acquired images
can be analyzed objectively so that subtle translocations as small as ~2
megabases of DNA can be detected (23). Such a small chromosomal region
would be difficult to detect with certainty by conventional banding analysis. SKY
therefore provides a method for rapid high-resolution screening of the cancer
karyotype and has applications both in the research and clinical cytogenetics
laboratory. In the four years since the first SKY report (19), there have been
almost 100 publications illustrating how SKY has helped delineate aberrations
and improve our understanding of human disease processes.
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Description
Identifies chromosomal aberrations
•translocations (balanced)
•translocations (unbalanced)
•copy number changes
•additions (>10MB)
•deletions (>10MB)
•deletions of specific genes/loci
•inversions
•insertions
•identifies presence of markers
•distingushes between double
minutes and homogeneously
staining regions
•identifies chromosomal origins
•identifies specific p/q arms/bands
Requires specifically labelled probes
•more than 2 probes at one time
Requires viable material
Requires metaphase spreads
Applicable to interphase nuclei
Applicable to DNA extracted from
paraffin embedded tissue
Affected by normal tissue
contamination
Identifies tumour heterogeneity
Applicable for studies in other species
G-Banding
Analysis
FISH*
SKY
CGH
yes
yes
yes
yes
yes
no
yes
sometimes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
sometimes
yes
yes
yes
no
yes
yes
yes
yes
sometimes
no
no
sometimes
no
sometimes
yes
no
yes
yes
no
no
yes
yes
yes
sometimes
sometimes
yes
yes
yes
yes
no
yes
yes
yes
yes
no
no
sometimes
sometimes
yes
yes
no
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
yes
*FISH requires the use of loci specific probes to detect the aberration of interest.
Table 1. Comparison between molecular cyotgenetic techniques.
The advantages and limitations of conventional banding, Spectral Karyotyping,
Comparative Genomic Hybridization and Fluorescence in situ Hybridization.
Hematological Malignancies
The vast majority of cytogenetic data derived from human diseases has
come from the study of hematological malignancies (2,24,25). With the relative
ease of access to blood and bone marrow samples, for both initial diagnosis and
monitoring, a wide variety of different analyses can be performed. The
cytogenetic study of hematological malignancies is particularly important since
the presence of recurrent and specific chromosomal aberrations are etiologically,
diagnostically and prognostically significant (2,25). Among the first papers,
describing SKY analysis, to be published was a study of hematological
malignancies, including CML, AML, MDS, and ALL (26). In this report, SKY
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analysis confirmed initial rearrangements, identified the chromosomal origins of
marker chromosomes as well as uncovered translocations not detected by
banding analysis.
The improved sensitivity in detecting these subtle
chromosomal translocations demonstrated the immediate value of SKY analysis.
Recent publications describing the cytogenetics of chronic myeloid
leukemia (CML) have shown the presence of other cytogenetic abnormalities in
addition to the hallmark Philadelphia translocation (9;22) (26-28). Markovic et. al.
(27), examined a series of variant Philadelphia translocation cases by
cytogenetics, FISH and SKY. Among the patients studied, none indicated the
presence of the classic (9;22) translocation together with a variant translocation,
suggesting that these variant rearrangements developed de novo and concurrent
with the onset of disease. Figure 4 shows an example of a variant CML case.
Studies of acute myelogenous leukemia (AML) by SKY have revealed the
frequent involvement of chromosomes 5, 7 in rearrangements and translocations
(26,29,30). Interestingly, a similar result was found among myelodysplastic
syndrome (MDS) cases (26,31-33). MDS is a myeloid disorder usually resulting
as a secondary malignancy following chemo- or radiotherapy (34,35). Giemsa
banding (G-banding) analysis of MDS has detected the frequent losses of
chromosomes 5 or 5q as well as losses of 7 or 7q. The application of SKY
analysis to these tumors, have revealed that these losses, while frequent, are
also involved in unbalanced and cryptic translocations. Kakazu et al.. (31),
published 20 MDS cases comparing banding analysis and SKY. G-banding
analysis revealed the expected frequent losses of 5/5q and 7/7q. However, SKY
analysis revealed that in some samples, the deletions were not simple interstitial
losses, but were shown to be cryptic, unbalanced translocations or additions,
leading to deletion of 5q or 7q.
Figure 4. SKY analysis of a variant CML case. A. Shown is the Red-Green-Blue
(RGB) display of the hybridized metaphase spread. Arrows indicate aberrations
detected by SKY. B. The SKY karyotype using the classified (pseudo-colors)
colors and the detected aberrant chromosomes.
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The use of SKY has been applied to other hematological malignancies
including the B- and T-cell neoplasms (36-39) and multiple myelomas (40,41).
Sawyer et al. (41) identified 6 multiple myeloma patients with a novel recurrent
t(14;16)(q32;q22 approximately 23). In a similar study, Rao et al. (40) also
identified the recurrent breakpoint site at (14)(q32) in their patient group. The
use of SKY analysis, therefore, demonstrates the ability to refine karyotypic
analysis and identify potentially new recurrent translocations.
Sarcomas
Sarcomas are defined as tumors arising from bones, muscles, fibrous
tissues, and some organs. As with hematological malignancies, specific
chromosomal aberrations are often associated with a particular histological
subtype leading to fusion oncoproteins. The ability to detect additional acquired
and/or recurrent aberrations associated with recurrence, survival, response to
therapy or stage is invaluable. To date, SKY analysis has only been applied to a
small number of sarcomas.
Rhabdomyosarcoma (RMS) is a tumor derived from skeletal muscle and is
comprised of two major subtypes (42). Pandita et. al.(12), compared established
embryonal and alveolar cell lines by SKY and determined that the alveolar cell
line SJRH30 exhibited a higher degree of chromosomal rearrangement than the
embryonal cell line RD. This suggested that the alveolar cell line possessed an
increased level of genomic instability, possibly explaining the less favorable
outcome in this subtype.
More rare tumors such as alveolar soft part sarcomas (ASPS) have also
been studied using SKY. The literature cites chromosome 17q25 as a site of
frequent cytogenetic classification of an ASPS with the sole abnormality of an
add(17)(q25) to der(17)t(X;17)(p11.2;q25) following SKY analysis. A previous
report by Heimann et.al(44) also identified a t(X;17)(p11;q25) in an ASPS, using
a series of two and three-color FISH experiments to confirm their results. This
example highlights the advantages of SKY in enabling the identification of all
translocations in one experiment, rather than in a series of experiments that are
both time consuming and exhaustive of precious cytogenetic samples.
Synovial sarcomas, characterized by a t(X;18)(p11.2;q11.2) and Ewings
sarcomas, characterized by a t(11;22)(q24;q12) have also been analysed by
SKY(47,48) to detect recurrent aberrations in addition to those described. An
interesting report by Cohen et al. (47), described a bone tumor that was originally
diagnosed as a primitive neuroectodermal variant of a Ewings sarcoma by
immunohistochemistry and histopathology. The G-banding analysis revealed an
unbalanced translocation between chromosomes 1 and 22 as well as additional
material on the X chromosome. SKY confirmed the der(1)t(1;22)(p13;q12?) and
identified the aberrant X as a balanced translocation between X and 18:
t(X;18)(p11.2;q11.2). SKY analysis, therefore, helped to identify the bone tumor
as a synovial sarcoma, rather than the original description of a primitive
neuroectodermal variant of a Ewings sarcoma.
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The analysis of osteosarcomas and other tumors of the bone will benefit
greatly from SKY analysis. It is well known that the karyotypes of osteosarcomas
are frequently complex and highly aneuploid(49-51). In many cases, only partial
karyotypes can be accomplished due to the presence of numerous structural
aberrations and extra-chromosomal structures including double minute
chromosomes (dms), homogeneously staining regions (hsrs) and ring
chromosomes. The lack of identifiable banding patterns makes traditional
cytogenetic analysis challenging. The lack of a consistent chromosomal
aberration makes FISH analysis also difficult. SKY studies of osteosarcomas in
our laboratory and others (Zielenska et al. submitted, Boehm et al. submitted)
have identified numerous complex rearranged chromosomes often involving
more than 5 different chromosomes within the structure. It has enabled the
identification of ring chromosomes and small marker chromosomes. Although
every chromosome appears to be involved in chromosomal translocations, there
is an apparent preferential occurrence of rearrangements involving 1q21-22,
1q41-44, 11p15, 12p13, 17p, 19q13 and 22q11-13.
Among the more benign bone tumors, chondromyxoid fibromas are rare
tumors that are histopathologically difficult to distinguish from other cartilaginous
neoplasms. A cytogenetic and SKY study of two chondromyxoid fibromas by
Safar et al.(52), identified clonal abnormalities of chromosome 6 [t(6;9)(q25;q22)]
but at a breakpoint on the long arm distal to a pericentric inversion of
chromosome 6 [inv(6)(p25q13)], that has been proposed as a specific genetic
marker for chondromyxoid fibroma (53).
Carcinomas
Carcinomas, like the sarcomas, reveal highly aneuploid and grossly rearranged
karyotypes. The SKY literature of both primary tissues and established
carcinoma cell lines is rapidly increasing. A number of publications have used
SKY for the study of oral carcinoma (54), hepatocellular carcinoma (55,56),
colorectal carcinoma (57), bladder cancer (58), pancreatic carcinomas (59),
breast (60,61) and cervical carcinoma (62). The use of cell lines for SKY studies
is facilitated by the ease of culture conditions and optimizing sample quality.
However many cell lines are intrinsically unstable and clonal isolates may exhibit
quite divergent or heterogeneous karyotypes at higher passage numbers.
Prostate carcinoma has been intensively studied over the past few years,
particularly in the field of early detection. Chromosomal aberrations that are
detected in early stage prostate cancer could serve as markers for diagnostic
testing. Many studies make use of established cell lines from high grade and
metastatic tumors. Two publications describe the cytogenetic changes in the
prostate cancer cell lines PC-3, DU145 and LNCaP (63,64) by SKY. Relatively
consistent results were obtained between the two studies and additional changes
not apparent by G-banding were present but neither study was able to identify a
recurrent or consistent aberration common to all three cell lines. Macoska et al.
(65) examined the karyotypes of 4 virally transformed low-grade prostate tumors
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using SKY and screened for loss of heterozygosity of 8p by allelotyping. The
results indicated the preferential loss of 8p sequences as well as gains or
aberrations of 8q among the tumors. These losses of 8p sequences could be
directly correlated to the disruption of chromosome 8p as determined by SKY
analysis. Other common chromosomal aberrations involving 11q13,q22,q23 were
detected in four HPV-immortalized cell lines (normal epithelium and prostate
tumors), believed to be a result of the viral transformation.
Another tumor group under intensive study, are the lung cancers. Dennis
and Stock (66) analysed a primary small cell lung carcinoma (SCLC) and 5
SCLC cell lines by FISH and SKY. Their results describe the consistent
breakpoint on the long arm of chromosome 3 at band 3q13.2 in a variety of
aberrations. Luk et al. (67) compared a non small cell squamous lung carcinoma
(NSCLC) and adenocarcinoma cell line by SKY and identified the presence of
more complex chromosomal rearrangements that were detected by CGH or
banding alone. The squamous cell line was characterized by more chromosomal
aberrations involving chromosomes 3, 5, 6, 7, 8, 10, 11, 13 and 16 as compared
to the adenocarcinoma cell line, which generally exhibited numerical changes.
Ovarian carcinoma is an aggressive tumor with a poor prognosis (68,69).
Cytogenetic analysis of these tumors demonstrate highly aberrant karyotypes
(70-72) with many random and non-random changes. A new molecular
cytogenetic study in our laboratory on primary ovarian carcinomas has revealed
a pattern of complex clonal chromosomal aberrations consistent with tumors
presenting as advanced disease (Figure 5). A study by Bible et. al (73), using an
ovarian cell line, investigated the use of Flavopiridol. Currently undergoing Phase
II testing, it is the first inhibitor of cyclin-dependent kinases to enter clinical trials.
The study attempted to discover the mechanisms of resistance in a high passage
(hp) ovarian cancer cell line OV202. SKY was used to confirm the relatedness
between the high passage line and the parental line. In a similar experiment,
Knutsen et. al. (61) conducted molecular cytogenetic studies on three multidrugresistant cancer sublines (two derived from MCF7 and one from LS174) that
were highly resistant to the chemotherapeutic agent mitoxantrone, and
anthracenedione. The amplification of 4q21-q22 and the MXR gene in
independently derived mitoxantrone-resistant cell lines was identified as well as
the participation of 4q21-22 in translocations.
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Figure 5. SKY analysis of a primary ovarian carcinoma specimen. A. Shown is
the RGB display of the hybridized metaphase with arrows indicating the
rearranged chromosomes. B. This panel illustrates the inverted DAPI banding
of the same metaphase to help identify chromosomes in a manner similar to Gbanding. The right panel shows the aberrant chromosomes as detected by SKY
analysis.
Shown from left to right, are the RGB, DAPI, and classified
chromosomes as well as their chromosomal origins. The classified colors help to
better identify chromosomal aberrations when the RGB display colors are similar
(as in the case of the t(4;8)).
Brain Neoplasms and Tumors of Neuronal Origin
Brain tumors are a poorly understood and diverse group of tumors
presenting with extraordinary difficulties in management compounded by a
relatively rudimentary understanding of their genetic basis. Prognosis is
frequently difficult to predict and the diagnosis itself is often equivocal due to the
variable nature of presentation. To date, no recurrent chromosomal aberration
has been identified in brain neoplasms and tumors of neuronal origin. SKY
studies of brain tumors are relatively few (74), but evidence suggests more
consistent cytogenetic abnormalities will be detected by this approach. Bayani et
al. (75) analysed 19 medulloblastomas and 5 supratentorial PNETs in a
retrospective and prospective study using classical banding, CGH and SKY.
They revealed that these histologically similar tumors were more heterogeneous
than previously thought, with SKY providing clearer evidence of intratumor
cytogenetic heterogeneity suggestive of chromosomal instability (see below).
The combined data from all three techniques identified chromosomes 7 and 17
as most frequently altered. Chromosomes 3, 6, 10, 13, 14, 18 and 22 were also
found to be frequently involved in gains, losses or chromosomal rearrangements.
No consistent chromosomal aberration/translocation was detected in this series,
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however this study demonstrated the presence of more aberrant karyotypes
among the supratentorial PNETs, desmoplastic and large cell variants of
medulloblastoma in comparison to the classical medulloblastoma subtype.
Analysis of glial tumours including astrocytomas, glioblastoma multiforme
(GBM), glial cell lines, gangliogliomas and an ependymoma has also been
carried out by (Squire et al. In press). SKY helped to refine complex karyotypes
and identified chromosomes 3, 5, 7 and 11 to be frequently involved in
chromosomal rearrangement, although no consistent chromosomal translocation
was detected. Furthermore, SKY helped to illustrate that the astrocytic tumours
showed more karyotypic complexity as compared to the gangliogliomas and
ependymomas.
There is a better understanding of the cytogenetics of neuroblastoma but
there still remain some uncertainties in assessing prognosis (76). Trakhtenbrot et
al. (77), described a patient with stage IV neuroblastoma and bone marrow, liver,
bone and subcutaneous metastasis. SKY analysis enabled the refinement of
misclassified chromosomes by G-banding, including the detection of an
unbalanced translocation +der(1)t(1;12)(p13;q11-12) that was originally
described as del(1)(p34.1).
SKY Analysis of Genomic Instability
The stability of the genome is governed by cell cycle checkpoint pathways
and defects in the genes involved can lead to chromosomal instability. Many of
the cytogenetic changes in carcinomas are thought to arise as a result of
segregation defects during mitosis leading to an increase in the number of
numerical changes in the karyotype (78,79). SKY can provide more precise
information concerning both numerical and structural changes when genomic
instability is suspected. A recent detailed study of in vitro evolution in prostate
cancer cell lines, demonstrated that SKY could distinguish considerable tumour
heterogeneity and identify de novo chromosomal aberrations that arise during
maintenance in vitro (Beheshti et al. Accepted).
The effects of ionizing radiation, on both tumor cells and surrounding
normal tissues, are also well suited for detailed SKY analysis. Zitzelsberger et
al..(80) analysed childhood thyroid carcinomas that were induced after the
Chernobyl nuclear accident in 1986 as well as those that resulted after
radiotherapy. Their study detected chromosomes 1, 2, 9, and 13 to be most
commonly affected, with more frequent breakpoints at 1q, 4q, 5q, 6p, 10q, 12q,
13q, and 14q. Similar results were obtained in our laboratory (unpublished),
where normal human lymphocytes from a number of individuals were exposed to
low- level ionizing radiation. The cells were analysed by SKY to gauge intrinsic
levels of radiosensitivity. In many cases, complex chromosomal changes were
detected (Figure 6), ranging from balanced and unbalanced translocations, to
insertions and deletions. These studies illustrate the growing application of SKY
in both sporadic and induced genomic alteration, and the field of radiosensitivity.
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Figure 6. SKY analysis illustrating radiosensitivity experiments carried out on normal
male lymphocytes. Normal male lymphocytes were cultured, exposed to therapeutic
levels of radiation and continued in culture for 42 hours. The cells were harvested and
fixed for cytogenetic preparations and hybridized to the SKY Paints™. Shown in the
inset are the inverted DAPI and classified chromosomes that were found to be aberrant.
Murine SKY Analysis and Detection of Recurrent Aberrations
SKY procedures and specific probe cocktails is available for murine
analysis (81). SKY can be used to analyze any metaphase target, provided pure
populations of sorted chromosomes can be obtained. The differential labeling of
mouse chromosomes greatly facilitates karyotyping of the similar looking,
acrocentric mouse genome (Figure 7). The mouse has been an important model
organism for understanding human diseases and cancer. The ability to create
transgenic animals to recapitulate the mouse form of specific human diseases
has given investigators excellent in vivo model systems for investigating novel
therapies and to study the disease process. The progress of the Human
Genome Project has also facilitated the sequencing of the mouse genome.
Syntenic maps between the mouse and human genomes have been developed
and are continuously being refined. Mapping of mouse genes have recognized
that gene clusters found in humans have identical gene orthologues in the same
configuration in the mouse (82). The manipulation of genes in the mouse can
15
have profound effects on development as well as on specific tissues and organs
often resulting in a greatly increased incidence of cancer. The consequences of
these alterations may be alluded to in their karyotypes, thus giving us an insight
as to the potential aberrations that could be expected in the human genome. A
number of investigators have taken advantage of this technology (81,83-91) for a
variety of studies.
Coleman et al. (84) used SKY analysis to look at the divergent cytogenetic
natures of the prototypical bilineage lymphoblastic pre-B lymphoma cell line,
P388, and the progenitor macrophage-like tumor line P388D1. The apparently
complex and triploid karyotype of the P388D1 cell line was interpreted, by the
authors, to indicate that the myeloid differentiational program in the bipotential
pre-B cell lymphoma P388 is characterized by genomic instability.
Figure 7. SKY analysis of a murine oligodendroglioma cell line. This tetraploid
line exhibited clonal aberrations, among them, the loss of chromosomes 7, 8 and
a translocation between chromosomes 11 and 15. A. Cytogenetic preparations
from the mouse line were hybridized with the Murine SKY Paints™ and analysed
using the SKYView software. Arrows indicate chromosomal translocations. B.
RGB and inverted DAPI of the aberrant chromosomes. Shown is a non-clonal
aberration detected involving chromosomes 5 and 19, and the clonal t(11;15)
aberration.
Preliminary studies using an oligodendroglioma mouse model (personal
communication, Dr. Mark Israel) suggest that the murine system accurately
16
represents human oligodendroglioma both histologically and cytogenetically.
Using SKY (Figure 7), a mouse oligodendroglioma cell line was analyzed to
detect chromosomal aberrations. These abnormalities serve as an entry point for
identifying syntenic regions in the human for specific disease loci. Human
oligodendrogilomas are characterized by losses of genomic sequences at 1p and
19q(92,93). The corresponding chromosomal locations in the mouse map to
mouse chromosomes 4 (human 1p) and mouse chromosome 7 (human 19q).
The illustrated cell line is hypotetraploid with clonal losses including the loss of
mouse chromosome 7, consistent with the loss of 19q in the human tumor.
Additional structural changes were detected, whose significance are yet to be
determined.
Evolutionary Studies
SKY has not only been used for the cytogenetic analysis of aberrant cell
lines, tumors, transgenic mouse cells or for sensitivity to ionizing radiation and
chemo-toxins, but also for comparative studies among other primates (94,95).
Best et. al. (94) used the human SKY probes for hybridization to baboon
metaphase spreads. Their results showed that the SKY results were consistent
with the majority of comparative gene mapping data between the two species
and that these data were also compatible with earlier studies comparing
macaque and human chromosomes. In another comparative study Moore et al..
(95), conducted cytogenetic studies of a rheboon, the result of a mating between
a male rhesus macaque and a female baboon, to determine whether one
numbering system for the cytogenetics of baboons and macaques should be
adopted. The rheboon karyotype showed the expected 42 chromosomes with
each homologue identical to each other, despite the difference in parentage.
Furthermore, hybridization with the human SKY probes was identical to the SKY
karyotype generated for baboons by Best et. al(94). This data supported the
inferences regarding chromosomal homology between the primates. From an
evolutionary standpoint, it is noteworthy that most of the rheboon chromosomes
hybridized to probes derived from a single human chromosome with only a few
rheboon chromosomes comprised of more than one human chromosome.
Conclusions
Cytogenetics is the study of chromosomes and the detection of
chromosomal aberrations.
In neoplasia, diseases of inheritance and
constitutional aberrations, genetic changes occur, and in some cases, these
genetic changes can be detected at the resolution of cytogenetic analysis. The
use of advanced molecular cytogenetic assays such as FISH, CGH and SKY,
have allowed an increased sensitivity for the detection of such aberrations. The
use of SKY, is primarily in a research application (Figure 8) with a growing
presence in translational research programs. As the costs of equipment and
reagents decline, SKY will become an integral part of the routine cytogenetic
17
diagnostic laboratory. From the selection of diverse publications that have been
reviewed, it is obvious that molecular cytogenetic analysis has an impact on the
study of human cancer, disease progression/outcome, drug and radiosensitivity.
The technology has enabled researchers to view the entire genome at one time
and this comprehensive analysis provides important clues concerning genome
stability and the acquisition of cytogenetic aberrations at both the initialization
and progression phases of tumorigenesis.
Figure 8. The use of Spectral Karyotyping and other FISH-based technologies
in the research and clinical laboratories. Molecular cytogenetic analysis helps to
bridge the findings from varying disciplines within routine diagnostics and
research.
18
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