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Tumor Biology |
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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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA Flow Cytometry.
Freshly frozen tumor samples (100–200 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% Carnoy’s 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)
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FISH.
After air drying, slides were fixed with 50, 70, and 100% Carnoy’s
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 (500–600 nm) and red (600–700
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 (400–800 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 Ha’Emek, 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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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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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and Table 1
).
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. 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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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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. 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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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 25–50% 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 (25–50%) 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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FOOTNOTES |
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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-B98–01VAA and 4047-B99–02VAA 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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REFERENCES |
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