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GENES, CHROMOSOMES & CANCER 27:418–423 (2000)
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BRIEF COMMUNICATION 333333333333333333333333333333333333333333333333333333333333
Spectral Karyotyping Combined With Locus-Specific
FISH Simultaneously Defines Genes and
Chromosomes Involved in Chromosomal
Translocations
Giovanni Tonon, Anna Roschke, Kristen Stover, Yaping Shou, W. Michael Kuehl, and Ilan R. Kirsch*
Genetics Department, Medicine Branch, National Cancer Institute, Bethesda, Maryland
Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal
breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and
expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence
in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement
of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase
cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the
FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as
Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be
queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this
technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach
that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from
cancer gene expression studies. Genes Chromosomes Cancer 27:418–423, 2000.
Published 2000 Wiley-Liss, Inc.†
In the past three decades, cytogenetics has greatly
advanced our understanding of tumor biology. Chromosomal banding techniques, the central tool of
standard cytogenetics, have identified gains or
losses of specific chromosomal fragments and have
defined chromosomal rearrangements in metaphase cells from tumor cell lines and patient
samples. In particular, the cloning of DNA proximate to the breakpoints of chromosomal translocations has led to the discovery of many genes
involved in critical steps in the initiation and
progression of cancer (Rowley, 1998).
Cytogenetics has been particularly helpful in the
identification of rearranged genes associated with
hematologic tumors. This has been most easily
accomplished when only one or a very few rearrangements are present. However, most solid tumors and
some hematologic malignancies have complex
karyotypes with many translocations (Bardi et al.,
1993; Mertens et al., 1997). A great challenge is the
specific definition of these breakpoints and of the
genes involved in these karyotypically complex
tumors, the vast majority of cancers in man. Classical cytogenetics, through G-banding, is not up to
the task of accurately and reliably defining these
complex karyotypes (Schrock et al., 1997).
Comparative genomic hybridization (CGH) (du
Manoir et al., 1995), spectral karyotyping (Garini et
Published 2000 Wiley-Liss, Inc.
†This article is a US Government work
and, as such, is in the public domain in the United States of America.
al., 1996; Schrock et al., 1996; Veldman et al., 1997;
Macville et al., 1999), and fluorescence in situ
hybridization (FISH) (Raap, 1998) have greatly
increased the resolution of cytogenetics, introducing a new era for the study of the genetics of cancer.
Thanks to these techniques, the analysis of the
genome has become much more informative, comprehensive, and accurate.
Spectral karyotyping and multiplex-fluorescence
in situ hybridization (M-FISH) (Speicher et al.,
1996) involves the simultaneous hybridization of 24
chromosome-specific painting probes labeled with
different fluorochromes or fluorochrome combinations. A spectral classification then assigns a discrete pseudo-color to all pixels with the identical
spectrum. Therefore, a specific color defines each
chromosome. Locus-specific FISH is able to locate
at which site on a specific chromosome a probe
hybridizes. The introduction of fluorescent dyes for
labeling DNA probes has greatly improved the
spatial resolution and the sensitivity of detection of
in situ hybridization (Wiegant et al., 1991). Moreover, when more than one fluorochrome is used,
*Correspondence to: Ilan R. Kirsch, M.D., Genetics Department,
Medicine Branch, National Cancer Institute, NNMC, 8901 Wisconsin Avenue, Bldg. 8, Room 5101, Bethesda, Maryland 20889-5105.
E-mail: [email protected]
Received 16 July 1999; Accepted 16 September 1999
COMBINATION OF SPECTRAL KARYOTYPING AND FISH
multiple loci can be detected simultaneously in the
same metaphase cell.
Even with these new approaches, delineating the
chromosomes and, at the same time, the genes
affected by chromosomal rearrangements, is very
time- and labor-intensive. Multiple rounds of FISH
and chromosomal painting are often necessary, the
task being additionally complex if a gene is present
in multiple copies and involved in multiple translocations.
To try to solve this problem, we developed a
technique that combines spectral karyotyping and
FISH. This approach retains the refinements of
each technique, but it adds the critical feature of
defining, in a single step, both the involvement of a
specific gene in a translocation and the chromosomes involved. Moreover, the use of different dyes
for different probes, in a single hybridization, allows
the analysis of different genes in a single metaphase
cell.
For the preparation of the metaphase cells, we
used standard cytogenetic procedures (Kirsch et al.,
1982). Dr. Abraham Karpas kindly provided Karpas
620 cells. Slides of metaphase cells from peripheral
normal blood lymphocytes were obtained from
Vysis (Downers Grove, IL).
The spectral karyotyping hybridization protocol
has been described in detail (Schrock et al., 1996).
Slides were hybridized simultaneously with a spectral karyotyping probe mixture, containing 24 distinctly labeled chromosome-specific probes, and
FISH probes. Specific chromosomes, kindly provided by Dr. Thomas Ried, were obtained by
high-resolution flow sorting, and then amplified in
our laboratory by two consecutive rounds of degenerate oligo-primed (DOP)-PCR amplification. Subsequently, the probes were distinctly labeled with
combinations of three fluorochromes and two haptens, to create a unique spectral definition for each
chromosome. As fluorochromes, Spectrum Orange
(Vysis), Rhodamine 110 (Perkin Elmer, Foster City,
CA), and Texas Red (Molecular Probes, Eugene,
OR) were used for the direct labeling, whereas
biotin-16-dUTP and digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) were used for
indirect labeling. One hundred nanograms of each
chromosome-specific probe was mixed and ethanolprecipitated with variable amounts of the FISH
probes, 50 µg of human Cot-1 DNA (Life Technologies, Rockville, MD), and 1 µl of salmon sperm
DNA (Sigma, St. Louis, MO). The precipitate was
then resuspended in 10 µl of hybridization solution
(50% formamide (Fluka, Milwaukee, WI), 2⫻ standard saline citrate, 10% dextran sulfate). Metaphase
419
chromosome slides were denatured separately at
75°C for 2–3 min in 70% formamide/2⫻ SSC
(volume/volume) and dehydrated in an ethanol
series. The probe cocktail was applied to the slides,
and hybridization was carried out for 48–72 h at
37°C. After hybridization, biotin was detected with
Avidin-Cy5 (Amersham, Piscataway, NJ) and digoxigenin-11-dUTP with mouse anti-digoxin antibodies (Sigma) followed by sheep anti-mouse antibodies custom-conjugated to Cy5.5 (Amersham). The
slides were counterstained with 4,6-diamidino-2phenylindole (DAPI, Sigma) and covered with
antifade solution (Vector, Burlingame, CA). Spectral images were acquired with an SD200 SpectraCube system (Applied Spectral Imaging, Carlsbad,
CA) mounted on a Leica DMRBE microscope
(Leica, Wetzlar, Germany) through a customdesigned triple bandpass optical filter (SKY v.3;
Chroma Technology, Brattleboro, VT). Spectrumbased classification of the raw spectral images was
performed using SKYView 2 software (Applied
Spectral Imaging). The DNA probes that we used
included: the GS53N21 BAC (kindly provided by
Dr. Raluca Yonescu), that maps to chromosome
7q21.11 (http://www.ncbi.nlm.nih.gov/CCAP/), a
MYC plasmid that maps to 8q24.1, and a 38 IGH
BAC containing sequences that hybridize to the
two alpha constant/enhancer regions of the IGH
locus, at 14q32.3 (Gabrea et al., 1999). For the
FISH analysis, probes were labeled by nick translation according to standard procedures (Sambrook et
al., 1989). We used the following dyes or haptendUTPs for the nick translation, at 1 mM concentration: Texas Red, Spectrum Orange, Rhodamine
110, Cy5 (Amersham), biotin-16-dUTP, digoxigenin11-dUTP, Cy3 (Amersham), and Alexa 594 (Molecular Probes). Nick translation yielded fragments
primarily between 300 and 600 bp.
The technique presented here is rapid, simple,
and flexible in its applications. In both spectral
karyotyping and FISH applications, probes are
usually ethanol-precipitated, resuspended in a small
volume, and applied to metaphase cells on a slide.
We precipitated the probes for spectral karyotyping
and FISH together and then hybridized the mixture of the two probes on the same slide. A strong
gene-specific signal (FISH signal), localized to the
expected chromosome, was evident above the background label of the specific chromosome painting
probe set (spectral karyotyping signal) (Fig. 1). The
amount of total DNA needed for the FISH probes
could be relatively low. We tested amounts of DNA
as low as 0.1 µg in the hybridization, and the signal
did not decrease in intensity. Therefore, a concen-
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TONON ET AL.
Figure 1. Combination of spectral karyotyping
and FISH in a normal chromosome metaphase cell,
using as a FISH probe a MYC fragment inserted in a
plasmid, labeled with Cy3 (arrows).
Figure 3. Combination of spectral karyotyping
and FISH, using two FISH probes, one for MYC,
labeled with Cy3 and mapping to chromosome 8
(arrowheads) and another, BAC GS53N21, labeled
with Alexa 594 and mapping to chromosome 7
(arrows).
Figure 2. (a) Combination of spectral karyotyping and
FISH applied to a rearranged chromosome, labeled with
Spectrum Orange and Rhodamine 110. As FISH probe, a 38
IGH BAC (see text), labeled with Cy3 with a fluorescence
spectrum similar to that of Spectrum Orange, maps at the
breakpoint (arrows). (b) FISH image of the same derivative, from the same metaphase, using a filter specific for
tetramethylrhodamine-5-isothiocyanate (TRITC).
tration of probe similar to that used for routine
FISH can be used here, without reduction in the
signal intensity. Probes with inserts of different
sizes and in different vectors have been tested. We
used plasmids, cosmids, P1, and BACs, and all of
them were effective in yielding a strong signal.
More importantly, the size of the insert, which
hybridizes to the template, can cover a wide range.
COMBINATION OF SPECTRAL KARYOTYPING AND FISH
We used as a FISH probe an insert as small as 7.5
kb, and we were still able to visualize easily the
gene-specific probe (data not shown).
Different dyes or haptens have been used for
labeling of the gene-specific probes. Some of them
were detected more efficiently than others. Spectrum Orange, Cy3, Alexa 594, Texas Red, and
Rhodamine 110 all gave strong signals. In contrast,
indirect labeling, using biotin and digoxigenindUTP as haptens, and antibodies or avidin labeled
with Cy5 or Cy5.5, respectively, for detection,
failed to give signals of enough intensity to be
easily visualized. Cy5-dUTP also was not bright
enough to be visualized.
Only five chromosomes are labeled with a single
dye in the standard spectral karyotyping technique
that we used, chromosomes 2 (Cy5.5), 8 (Rhodamine 110), 11 (Spectrum Orange), 14 (Texas Red),
and 17 (Cy5). All of the other chromosomes are
defined by a combination of dyes, which give them
each distinct colors. Therefore, if the probe, which
is labeled with just one dye, hybridizes to a chromosome painted with more than one dye, the filter and
the software easily distinguish between the signal
coming from the probe and the signal coming from
the chromosome. Obviously, the FISH probe may
hybridize to a chromosome labeled uniquely with
the same or related dye (for example, Fig. 2). Here,
the signals coming from the FISH probe were
located at the juxtaposition of two different chromosomes, one of which was labeled with Spectrum
Orange, which has a wavelength similar to that of
the Cy3-labeled FISH probe. Therefore, in this
case, in some metaphase cells, it was difficult to
detect the FISH probe on this rearranged chromosome (Fig. 2a). However, we could distinguish the
FISH probe from the chromosome painting by
analyzing the metaphase using a filter with a
narrower spectrum (Fig. 2b). The FISH signals
were much brighter than the chromosome painting
and were easily distinguishable. Therefore, we
suggest the sequential use of the filter for the
spectral karyotyping and a filter with a more defined spectrum. It is possible that in certain cases
confirmation of an analysis using a second independent FISH alone would be required. However, in
approximately two dozen separate cases in which
we have compared FISH alone versus simultaneous
spectral karyotyping in combination with FISH, we
have not yet encountered a situation where the
independent FISH analysis gave us more or different information than the method described above
using different filters. The FISH probes usually do
not interfere with the spectral karyotyping analysis,
421
and the normal two ‘‘dots’’ of the FISH hybridization are easily distinguishable from the small chromosomal insertions identified by the spectral karyotyping. Therefore, it is not necessary to perform
separate hybridization of the spectral karyotyping
probes, without the FISH probes, to obtain reliable
spectral karyotyping data.
The possibility of using more than one FISH
probe labeled with different dyes will be of great
advantage for the analysis of complex karyotypes.
For example, we hybridized two probes, one for
MYC, labeled with Cy3, and a BAC, GS53N21,
mapping to chromosome segment 7q21.11, labeled
with Alexa 594. Signals specific for the two probes
were easily detectable in the metaphase cell (Fig.
3).
The major challenge in the future for cytogenetics will be the analysis of complex karyotypes, both
of solid and hematopoietic tumors. To evaluate the
ability of this technique to detect the involvement
of genes at chromosomal breakpoints and to define
the chromosomes involved in a complex karyotype,
we hybridized slides of metaphase cells of a myeloma cell line, Karpas 620 (Fig. 4a and b). Gbanding-like images were obtained from DAPI
staining of the slides (Fig. 5). A 38 IGH BAC was
labeled with Cy3-dUTP. Dysregulation of oncogenes by translocations to the IGH locus, at 14q32,
is a critical event in the pathogenesis of B-cell
tumors. The multiple translocations present in the
karyotype of this cell line were defined by spectral
karyotyping analysis. The karyotype that we describe here is partly different from the one previously described (Nacheva et al., 1990). Eight cells
were analyzed with DAPI staining, and spectral
karyotyping and FISH in combination. A representative example is shown in Figures 4 and 5. The
modal chromosome number was 68 (range, 60–68).
In the majority of the cells, three copies of chromosomes 3, 5, 6, 17, 20, 21, and 22 and four copies of
chromosomes 15, 16, 18, and 19 were present. We
found two copies of chromosomes 2, 4, 7, 8, 9, 10, 12,
and X, one copy of chromosome 13, and no normal
chromosome 1, 11, and 14. The following rearrangements were present in the majority of the cells: a
reciprocal translocation t(1;11)(q32;q13), with two
copies of der(1) and one copy of der(11); one copy
each of der(7)t(4;7)(q31;p15), der (7)t(7;1)(q22;p31)
and der(13)t(8;13)(q23;q31); two copies in each cell
of der(8)t(8;11)(q24;q13), der(11)t(11;13)(q13;q14),
der(14)t(1;14)(q11;q32), and der(14)t(8;14)(q24;
q32).
Both conventional FISH and the combination of
spectral karyotyping and FISH detected six copies
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TONON ET AL.
Figure 4. a: Combination of spectral karyotyping and FISH in Karpas
620 cell line. The FISH probe is a 38 IGH (see text), labeled with Cy3. Six
separate areas of hybridization of the FISH probe were evident in all the
metaphase cells analyzed. Larger arrows: der(14)t(8;14); smaller arrows:
der(14)t(1;14); arrowheads: der(8)t(8;11). b: Classified image of the
same metaphase cell.
Figure 5. Karyotype table, showing the DAPI-stained, G-banded-like karyotype and the classified image
of the Karpas 620 cell line. For a composite description of the karyotype, see text below.
of the 38 IGH probe. In two chromosomes, a
translocation der(14)t(8;14)(q24;q32) was evident,
and the 38 IGH probe was located at the breakpoint.
The 38 IGH probe also localized to the breakpoint
of two other pairs of chromosome markers: two
copies of der(14)t(1;14)(q11;q32) and two copies of
der(8)t(8;11)(q24;q13). In the latter case, the presence of the 38 IGH probe at the breakpoint (Figs. 2,
4, and 5) suggests a small insertion of part of
chromosome 14 previously undetected by conventional cytogenetic, spectral karyotyping, or more
specific chromosome analysis. A conventional FISH
analysis, using the 38 IGH probe and chromosome
14-specific painting had also shown the presence of
38 IGH, but not of more extensive chromosome 14
material in this derivative (not shown).
Based on this analysis a composite karyotype is:
60-68, XX-X, -1,t(1;11)(q32;q13), der(1)t(1;11)(q32;
q13),-2,-4,der(7)t(1;7)(p31;q22),der(7)t(4;7)(q31;
p15),⫹8,der(8)t(8;11)(q24;q13)x2,-9,-10,der(11)t(11;
13)(q13;q14)x2,-12,-13,der(13)t(8;13)(q23;q31),der(14)
t(1;14)(q11;q32)x2,der(14)t(8;14)(q24;q32)x2,⫹15,
⫹16,⫹18,⫹19[8].ish der(8)t(8;11)ins(14)(q32.3)
(IGH)x2, der (14) t(1; 14) (IGH) x2, der (14) t (8;14)
(IGH)x2.
The human DNA sequence will be determined
in the next 2–5 years (Collins et al., 1998). In
addition, efforts are under way to establish a standard set of BAC clones spaced at 1–2 Mb intervals
across the genome (C-CAP; World Web Site: http://
www.ncbi.nlm.nih.gov/CCAP/). Using these resources, the combination of spectral karyotyping
and FISH will allow screening for genes and chromosome involvement in tumors in a much more
straightforward manner than can be achieved by
separate FISH and spectral karyotyping analyses.
COMBINATION OF SPECTRAL KARYOTYPING AND FISH
In summary, we describe a simple and rapid
technique that allows the definition of genes and
chromosomes involved in chromosomal translocations in a single step. This approach is particularly
useful for the analysis of complex karyotypes and
may help to test hypotheses coming from gene
expression studies, like cDNA microarrays, SAGE,
and CGAP (Strausberg et al., 1997; Wang and
Rowley, 1998).
ACKNOWLEDGMENTS
We thank Dr. Q. Ying for preparing Karpas 620
slides, Dr. A. Dutra for preparing some normal
metaphase cells on slides, and Dr. T. Ried for
helpful discussions.
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