[Cancer Research 61, 1214-1219, February 1, 2001]
© 2001 American Association for Cancer Research
Chromosomal Rearrangements and Oncogene Amplification Precede Aneuploidization in the Genetic Evolution of Breast Cancer1
Karin Rennstam2,
Bo Baldetorp,
Soili Kytölä,
Minna Tanner and
Jorma Isola
Department of Oncology, Jubileum Institute, University Hospital, S-221 85 Lund, Sweden [K. R., B. B., M. T., J. I.]; Institute of Medical Technology, Tampere University and University Hospital, Tampere, FIN-33101 Finland [S. K., M. T., J. I.]; and Department of Molecular Medicine, Karolinska Hospital, CMML 8:01, Stockholm, S-17176 Sweden [S. K.]
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ABSTRACT
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Breast carcinoma is thought to arise because of multiple successive
changes in the genome of the normal epithelial cells. However, little
is known of the order of appearance of different types of genetic
aberrations. We studied the ERBB2 (Her-2/neu) and
CCND1 (cyclin D1) oncogene amplification in flow
cytometrically sorted diploid and nondiploid tumor cell populations by
fluorescence in situ hybridization (FISH). The purity of
the cell sorting was confirmed by static DNA image cytometry. Spectral
karyotyping was used to define differences in a genome-wide manner
between two distinctly different aneuploid cell clones found in each of
two breast cancer cell lines. FISH indicated the presence of gene
amplification both in diploid and nondiploid cell clones in 17 of the
21 amplification-containing tumors analyzed. The oncogene copy numbers
remained unchanged throughout aneuploidization in 11 of 17 tumors. The
remaining six tumors showed an increase in oncogene copy number as well
as the number of chromosome 11 or 17 centromeres (the original location
of CCND1 and ERBB2, respectively). Breast
carcinoma cell lines MDA-157 and MDA-436 showed a significant number of
chromosomal rearrangements in the near-diploid clones, which were
present in duplicate in the corresponding aneuploid (polyploid) clones.
These results indicate that ploidy shift, i.e.,
aneuploidization, in breast cancer is a late genetic event, which is
preceded by both oncogene amplifications as well as many chromosomal
rearrangements.
 |
INTRODUCTION
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Breast carcinoma, as well as other carcinomas, arises because of
multiple changes in the genome of the normal epithelial cells. These
changes include single nucleotide point mutations, amplifications, or
deletions of single genes, insertions and translocations, gains and
losses of entire, or parts of, chromosomes and chromosome arms, and
eventually gross changes in chromosome number (aneuploidization; Refs.
1, 2, 3
). A widely accepted model depicted from colorectal
carcinoma suggests that genetic aberrations occur in stepwise manner,
correlating well with morphological change from adenoma to
carcinoma (4)
. However, studies in early breast
lesions are not fully concordant with this model. Atypical ductal
hyperplasia and
DCIS3
have repeatedly been shown to contain numerous genetic aberrations,
similar to invasive carcinomas (5
, 6)
. Thus, the
relationship between genetic pathogenesis and the morphological
progression in breast cancer has remained obscure.
Relatively little is known also of the order of appearance of different
types of genetic aberrations. Studies based on cytogenetic data suggest
indirectly that chromosomal imbalances may occur earlier than gross
ploidy shifts (aneuploidization), indicating the latter to be a late
genetic event (7
, 8)
. These observations, in turn, are
contradicted by observations of aneuploidy being commonly present in
early breast lesions, such as DCIS (9, 10, 11, 12)
. Even less is
known on the appearance of oncogene amplifications, which are crucial
in determining the clinical outcome and treatment response. The most
commonly amplified oncogenes in breast cancer involve oncogenes
ERBB2, MYC, and CCND1, all of which
have been found amplified in DCIS (13, 14, 15)
.
The present study was initiated by our findings from CGH, which detects
clonal chromosomal copy number imbalances (gains and losses) when
present in at least 60% of the cells from which the DNA is extracted
(16)
. We have, however, repeatedly found breast tumors
showing numerous copy number changes, despite DNA flow cytometry
(performed from adjacent tissue sections), indicating only a small
fraction (<30%) of nondiploid cells. This suggests that chromosomal
changes may be present also in the diploid cell population. In the
present study, we examined this aspect directly by analyzing diploid
and nondiploid cell populations separately after flow cytometrically
sorting them by their DNA content. Originally, we tried to perform CGH
on these sorted cells. This was, however, unfruitful, because the
normal cell contamination within the diploid peak was too high to allow
any of the aberrations in the nonnormal diploid cells to be detected.
(All aberrations seen in the aneuploid peak were also seen in the whole
tumor CGH.) For this reason, we chose FISH, which allows the analysis
to be made on a cell-by-cell basis. The cell populations were analyzed
separately with probes identifying gene amplifications of
CCND1 and ERBB2 oncogenes. DNA probes for the
chromosomes on which the oncogenes are located (chromosomes 11 and 17,
respectively) were used as reference, separating the true oncogene
amplifications from multiplication of the entire chromosomes. SKY was
used to further clarify the order of appearance of chromosomal
rearrangements in clonally heterogeneous breast cancer cell lines,
where high-quality metaphase preparations could be prepared easily.
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MATERIALS AND METHODS
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Tumor Samples.
Tumor specimens from 21 primary invasive breast cancers were obtained
from the frozen tumor tissue bank. The specimens have been collected
for the routine analysis of steroid hormone receptors, and DNA flow
cytometry was performed at the Department of Oncology, Lund University,
Sweden. The tumors selected had all been determined previously as
having amplification of the 11q13 region (tumors 110) or
ERBB2 oncogene (tumors 1121) by Southern blot
hybridization (17
, 18)
. For the SKY analysis, two breast
cancer cell lines (MDA436 and MDA157) were obtained from American Type
Culture Collection (Rochester, Michigan) and cultured in recommended
conditions.
DNA Flow Cytometry.
Freshly frozen tumor samples (100200 mg) and cell lines were prepared
for flow cytometric analysis as described previously (19)
.
Calculation of the DNA index was done after zero point adjustment of
the DNA histogram using the modal values of chicken and trout RBCs. The
mean channel numbers of all
G0-G1 peaks were then used
for the calculation of the DNA index, with chicken and trout RBCs as
reference standard (19)
. The number of cells in the
aneuploid G0-G1 peak was
divided by the total number of cells in the DNA histogram to get the
aneuploid population fraction. The CVs of the diploid
G0-G1 peaks of 20 of the 21 tumors
ranged between 2.2 and 5.2. The CV of tumor number one was 7.4. All CVs
were well under the recommended maximum value published previously
(20)
.
Flow Cytometric Sorting.
Simultaneous with the DNA analysis of the tumors, nuclei from the two
different cell populations were separated by flow cytometric sorting.
As sorting criteria, narrow electronic gates were set in the DNA
histogram around the G0-G1
peaks, defined by the CVs. PBS (pH 7.0) was used as sheath fluid for
sorting. The analysis rate was
150 nuclei/s. Electronic controls for
the sorting were set as follows: droplet frequency, 30 KHz; three
droplets/sorted event; and coincidence check of five droplets, this
yielding an efficiency close to 100% and purity >95%. Up to 300,000
sorted nuclei was collected onto microscope slides for each peak.
Slides were immediately fixed in 50% Carnoys solution (3:1
methanol:acetic acid in water) and air dried.
DNA Image Cytometry.
Before analysis, the slides with flow cytometrically sorted cells were
fixed in 4% phosphate-buffered formaldehyde for at least 30 min and
then Feulgen stained as described earlier (21
, 22) .
Integral optical densitometric measurement of nuclear DNA content was
performed with a LabEye 3PC image analysis system (Innovate Vision AB,
Linköping, Sweden). For each specimen, well-preserved nuclei were
selected randomly, and integrated absorbance was measured at a
wavelength of 540 nm. Nuclei from human cerebellum (fresh autopsy
material) were used as diploid external reference cells for ploidy
assessment and as a control for Feulgen staining (23)
.
FISH.
After air drying, slides were fixed with 50, 70, and 100% Carnoys
solution, 10 min each. Samples were then further fixed with 1%
paraformaldehyde in PBS (10 min at 4°C), dehydrated in graded ethanol
series (70, 85, and 100%), air dried, and baked at 80°C for 30 min
in a hybridization oven. Two-color FISH was carried out as described
previously (8)
with minor modifications. Slides were
denatured in a 70% formamide-2x SSC at 72°C for 3 min. The directly
labeled dual-color probes for CCND1 (and chromosome 11
centromere) and ERBB2 (and chromosome 17 centromere) were
obtained from Vysis, Inc. (Downers Grove, IL). The hybridization
mixture for each slide contained 3.4 µl of master mix (70% formamide
and 10% dextran sulfate in 2x SSC), 0.4 µl of placental DNA, and
0.25 µl of the probe solution. The probe mixture was denatured at
75°C for 5 min and applied onto slides. The hybridization was carried
out overnight at 42°C. Posthybridization stringency washes were done
at 72°C (0.4x SSC for 2 min) and room temperature (2x SSC for 1
min). After a short rinse in distilled water, the slides were air dried
and counterstained with 0.2 µM DAPI in an
antifade solution (Vectashield; Vector Laboratories, Burlingame, CA).
Hybridization signals were analyzed using a Zeiss Axioplan 2
epifluorescence microscope equipped with dual band-pass fluorescence
filter (Chromatechnology, Brattleboro, NV), which enables
simultaneous detection of both green (500600 nm) and red (600700
nm) fluorescence. Hybridization signals from at least 50 nuclei were
scored to assess the chromosome centromere and oncogene copy numbers.
The nuclei was determined to carry an amplification if the number of
gene probe signals divided by the number of centromere signals was
1.5. Digital images were taken with a cooled CCD camera (Sensys;
Photometrics, Tucson, NV) operated via Quips FISH image analysis
software (Vysis, Inc.).
CGH.
CGH was done according to a protocol published previously (16
, 24) . Briefly, tumor DNA was extracted from freshly frozen tumor
tissue (Qiagen, Hilden, Germany) and labeled with FITC-dUTP and
FITC-dCTP (DuPont, Boston, MA) using standard nick translation. Labeled
DNAs (400800 ng each, labeled reference DNA; Vysis) and 10 µg of
unlabeled Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD) were
hybridized onto commercially available normal metaphase chromosomes
(Vysis, Inc.). The hybridizations were evaluated using the QUIPS
digital image analysis system (Vysis, Inc.). At least five metaphases
from each tumor were analyzed.
SKY.
The probe mixture containing 24 differentially labeled,
chromosome-specific painting probes and Cot-1 blocking DNA (SKY kit;
ASI Applied Spectral Imaging, Migdal HaEmek, Israel) was
denatured and hybridized to denatured tumor metaphase chromosomes
according to the protocol recommended by ASI. After hybridization and
washing, the chromosomes were counterstained with DAPI. Image
acquisitions were performed using a SD200 Spectracube system (ASI)
mounted on a Zeiss Axioskop microscope with a custom designed optical
filter (SKY-1; Chroma Technology, Brattleboro, VT). The emission
spectra were then converted to the pseudocolors matching the
fluorochrome combinations of each chromosome. For each cell line and
clone, at least seven metaphases were analyzed (25)
.
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RESULTS
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This study was initiated by our CGH findings of breast
tumors containing only a small proportion of aneuploid cells when
studied by flow cytometry. An example of such a tumor is given in Fig. 1
. The flow cytometric DNA histogram (Fig. 1A)
, shows only a
small proportion (23%) of nondiploid cells (DNA index, 2.8); yet, the
CGH made from the same tumor specimen reveals a large number of
chromosomal gains and losses (Fig. 1B)
. CGH typically
detects clonal chromosomal copy number imbalances (gains and losses)
when present in at least 60% of the cells from which the DNA is
extracted. This suggests the presence of genetically deviant diploid
cells, because the small fraction of nondiploid cells could not
possibly be detected by CGH.

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Fig. 1. A flow cytometric DNA histogram (A) and
the CGH copy number profiles of chromosomes showing gains and losses
(B) from a primary breast tumor that contained only a
small proportion (23%) of nondiploid cells of the total cell count
(nondiploid DNA index, 2.8). In panel A, C and
T refer to chicken and trout erythrocytes, respectively,
used as internal controls. D and Non-D,
diploid and nondiploid cell populations, respectively. In panel
B, the curves shown are mean values of gains/losses from at
least six chromosomes (bold) ± 95%
confidence interval. The threshold values for losses and gains were set
to 0.85 and 1.15, respectively. This tumor was not further analyzed by
FISH.
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Analysis of ERBB2 and Cyclin D1 Copy
Numbers in Flow Cytometrically Sorted Diploid and Nondiploid Tumor Cell
Populations.
To study diploid and nondiploid tumor cell clones separately, the
propidium iodide-stained tumor cell suspensions were sorted by their
DNA content with a flow cytometer and collected onto microscope slides.
Static DNA image cytometry was performed to confirm the purity of
sorted cell clones. The cell clones with the DNA indexes matching flow
cytometry were found by image cytometry, and no evidence for
significant impurity or contamination was found (Fig. 2
and Table 1
).
The FISH hybridizations, performed on the sorted cells, indicated that
both diploid and nondiploid cell clones often showed amplification of
ERBB2 and CCND1 (Fig. 3)
. The gene copy numbers from 21 sorted tumors are presented in Tables 1
and 2
. Eight of 10 tumors (80%) with CCND1 amplification
were amplified in both diploid and nondiploid cell (copy number
relative to 11 centromeres,
1.5). In two tumors (18%), only the
nondiploid clone was found amplified (Table 1)
. In the 11 tumors
previously determined as having ERBB2 oncogene
amplification, 9 (82%) showed amplification in both clones and two in
the nondiploid clone only (18%; Table 2
). Tumors with amplification in the diploid clone only were never seen.

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Fig. 3. A (diploid) and B
(nondiploid) show FISH of ERBB2 oncogene
(red) and chromosome 17 centromere
(green; tumor 14). C (diploid) and
D (nondiploid) show FISH of CCND1 oncogene
(red) and chromosome 11 centromere
(green; tumor 4). The amplifications of both oncogenes
are clearly distinguishable in both cell clones when dividing the
number of gene probe signals (red) by the number of
centromere probes (green; amplification if 1.5).
Lower right corners, average probe:reference ratio for
each sample. DAPI (blue) was used as a nuclear
counterstain.
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The mean copy number of CCDN1 and ERBB2 (per
cell) remained approximately the same in diploid and nondiploid cells
in 11 of 17 tumors with amplification in both clones (tumors 1, 2, 8,
10, 15, 16, 17, 18, 19, 20, and 21; see Tables 1
and 2
). In the
remaining cases (tumors 3, 4, 6, 7, 13, and 14; see Tables 1
and 2
),
the gene copy number, as well as the chromosome copy number of the
respective reference centromere, increased during aneuploidization.
When counting the ratio between gene copy number and chromosome copy
number, we found that it remained stable throughout aneuploidization.
We analyzed the copy numbers of the reference probes (pericentromeric
probes for chromosomes 11 and 17, analyzed separately). Surprisingly,
multiple signals were found in the diploid clones of 7 tumors (tumors
1, 2, 10, 16, 17, 18, and 21; see Tables 1
and 2
). All of these tumors
showed ERBB2 or CCND1 amplification both in
diploid and nondiploid clones. The corresponding flow cytometric DNA
histograms showed a small CV (<5.2% in all but one tumor) for the
diploid DNA peaks, which is generally considered as a sign of little or
no genetic instability.
Duplication of Chromosomal Changes as Evidenced by SKY.
The order of appearance of chromosomal rearrangements was further
studied with breast cancer cell lines MDA-157 and MDA-436, which both
contain two distinct nondiploid cell clones despite tens of passages in
culture. The flow cytometric DNA indexes were 1.31 and 2.50 (79 and
21% of cells in G0-G1
peaks) for MDA-157 and 0.85 and 1.70 for MDA-436 5 (77 and 23% of
cells in G0-G1 peaks; Fig. 4
). The modal chromosome numbers in these clones when analyzed by SKY
where 54 and 95 for MDA-157 and 39 and 80 for MDA-436, respectively.

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Fig. 4. DNA flow cytometry histograms of the two cell lines
MDA-157 (A) and MDA-436 (B). In both
panels, C and T refer to chicken and
trout erythrocytes, respectively, used as internal controls.
Near-D and Non-D, near-diploid and
nondiploid cell populations, respectively. In A
(MDA-157), the near-diploid population has a DNA index of 1.31, and it
constitutes 79% of the counted G0-G1 cells.
The nondiploid population has a DNA index of 2.50 and constitutes of
21% of the counted G0-G1 cells. In
B (MDA-436), the near-diploid population has a DNA index
of 0.85 (hypodiploid), and it constitutes 77% of the counted
G0-G1 cells. (This is the same DNA index as for
the trout erythrocytes. In the histogram, the two populations are
merged into one peak.) The nondiploid population has a DNA index of
1.70 and constitutes 23% of the counted G0-G1
cells.
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The different clones were analyzed separately by SKY, as shown in Fig. 5
. In both cell lines, the aneuploid (polyploid) clone contains a
majority of the aberrant chromosomes of the near-diploid clone in
duplicate. For example, derivative chromosomes containing material from
chromosomes 3, 5, 12, and 20 in MDA-157 and chromosomes 1, 7, 8, and 21
in MDA-436 (Fig. 5)
were duplicated in the polyploid clone. Several
"new" aberrations (not present in the near-diploid clone) were also
found in the polyploid clones. These include chromosomes 1, 4, and 9 in
MDA-157 and chromosomes 2, 3, and 6 in MDA-436.

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Fig. 5. SKY of the different cell clones of breast cancer cell
lines MDA-157 (A) and MDA-436 (B).
Chromosomes are seen in SKY classification colors. A:
the upper left panel shows the near-diploid cell clone
(mean number of chromosomes from 10 metaphases, 54), and the
lower panel shows the nondiploid (polyploid) clone (mean
number of chromosomes from 10 metaphases, 95). Note that many
chromosomes containing rearrangements in the near-diploid clone were
duplicated in the polyploid clone (e.g., chromosomes 3,
5, 12, and 20, extrapolated). B: the upper left
panel shows the near-diploid cell clone (mean number of
chromosomes from 10 metaphases, 39), and the lower panel
shows the nondiploid (polyploid) clone (mean number of chromosomes from
10 metaphases, 80). Note also here that many chromosomes containing
rearrangements in the near-diploid clone were duplicated in the
polyploid clone (e.g., chromosomes 1, 7, 8, and 21,
extrapolated).
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DISCUSSION
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This study was initiated by our findings made by CGH in which we
were able to show copy number imbalances in breast tumor samples
containing only a small fraction of aneuploid cells, as evidenced by
DNA flow cytometry (26)
. CGH generally requires the sample
to contain at least 6070% of genetically deviating cells to detect
aberration (16)
. We have found several tumors with an
aneuploid cell fraction of <30%, and yet they show multiple
chromosomal aberrations by CGH. This suggests indirectly that a
significant fraction of the diploid cells must be genetically deviant
and contain at least partly the same genetic aberrations as the
aneuploid cells.
To explore the order of appearance of genetic aberrations directly, we
sorted tumor cells for their DNA content by flow cytometry and analyzed
the different clones by FISH. In 17 of 21 tumors, the amplification of
ERBB2 and CCND1 was present both in DNA-diploid
and nondiploid cell clones. When the diploid clone was found amplified,
typically only 2550% of the cells showed oncogene amplification. The
presence of cells without amplification can be best explained by the
presence of nonmalignant cells, i.e., stromal, inflammatory,
and benign epithelial cells that are present in every breast tumor. The
presence of nonepithelial cells in breast carcinomas has been shown
previously (27)
. The contamination of the diploid cell
sort by nondiploid cells was excluded by analyzing the DNA content of
the cells after sorting, using DNA image cytometry. These experiments
showed clearly that there was no contamination of nondiploid cells on
the diploid sorted slides (or vice versa). On the basis of
the image cytometry data, we feel that the relatively high proportion
of cells with oncogene amplification among the diploid cells (2550%)
is very unlikely due to nondiploid cell contamination. Thus, we
conclude that aneuploid primary breast tumors often contain DNA diploid
cell clone(s) that have undergone oncogene amplification.
If the gene amplification takes place already in diploid state (before
aneuploidization), one would expect that the number of oncogene copies
becomes multiplied along with chromosome multiplication. This was seen
only in 6 of the 17 tumors where both diploid and nondiploid cells were
found amplified. Thus, in these 6 tumors, the amplicon-containing
chromosomes have multiplied during aneuploidization. In the remaining
11 tumors, the mean number of copies of ERBB2 or
CCND1 was almost the same in the DNA diploid and nondiploid
cells. The same was true also for chromosome 11 and 17 centromere
counts, which neither showed any clear increase. In these tumors, the
diploid clones already showed unexpectedly more than two centromere
signals/cell. It is, therefore, possible that the multiplication of
amplification-carrying chromosomes has occurred already before gross
polyploidization. The preserved diploid DNA content by flow cytometry
can be explained by losses of other chromosomes or chromosome arms. An
alternative explanation for unaltered oncogene copy numbers is that the
amplified gene copies are present in double minute chromosomes.
However, according to cytogenetic literature, double minute chromosomes
are considered to be rare in primary breast cancer (28)
.
The endoreduplication after multiple chromosomal rearrangements was
best visualized by our data from SKY. The flow cytometry analysis
confirmed that the two different clones analyzed were not artifacts
caused by induction metaphase cells in culture (by Colcemid
treatments). Distinct G0-G1
as well as G2 phases could be seen in both cell
lines. The SKY data from both breast cancer cell lines demonstrated
many translocation events in one copy in the near-diploid clone but in
two or more copies in the corresponding aneuploid clone. This result
indicates that at least in these cell lines, polyploidization does not
give significant growth advantage over the already aneuploid cells,
which can persist in culture despite tens of passages. In fact, in
MDA-157, it seems that the proportion of cells in the near-diploid
population is increased during cell culturing (data not shown). Thus,
the growth characteristics of these cell lines are likely to be
determined by the genetic aberrations present already in the
near-diploid phase and not by aneuploidization.
Taken together, our present results demonstrate that cancer cells that
are flow cytometrically diploid in their total DNA content often
persist in primary breast cancers that have undergone aneuploidization.
These results are parallel to cytogenetic observations
(7)
, which also suggest aneuploidization to be a late
genetic event. However, cytogenetic results are indirect, and
artificial clonal selection during in vitro culture cannot
be ruled out. Our present data from uncultured interphase cells showed
more specifically that oncogene amplification, which is biologically
and prognostically a very important genetic defect in breast cancer,
takes place before aneuploidization. The presence of diploid and
nondiploid malignant cells is also a clear indication of heterogeneity
in the genetic composition of malignant tumor cells.
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ACKNOWLEDGMENTS
|
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We thank Ghita Fallenius for skillful technical assistance with
the DNA image cytometry measurements.
 |
FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by The Swedish
Cancer Foundation Grant 4237-099-01XAB; the Mrs. Berta Kamprads
Foundation; the Gunnar, Arvid, and Elisabeth Nilsson Foundation; the
John and Augusta Persson Foundation; the Inga Britt and Arne Lundberg
Foundation; the Hospital of Lund Foundation; the Franke and Margaretha
Bergqvist Foundation; the King Gustav V Jubilee Foundation Grant
99:525; the Finnish Cancer Foundation; and the Scientific Foundation of
Tampere University Hospital. J. I. received Fellowships
4047-B9801VAA and 4047-B9902VAA from the Swedish Cancer Society. 
2 To whom requests for reprints should be
addressed, at Department of Oncology, Jubileum Institute, Lund
University, S-221 85 Lund, Sweden. Phone: 46-46-177567; Fax:
46-46-147327; E-mail: Karin.Rennstam{at}onk.lu.se 
3 The abbreviations used are: DCIS, ductal
carcinoma in situ; CGH, comparative genomic
hybridization; FISH, fluorescence in situ hybridization;
SKY, spectral karyotyping; CV, coefficient of variation; DAPI,
4,6-diamino-2-phenylindole. 
Received 5/30/00.
Accepted 12/11/00.
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