[CANCER RESEARCH 48, 5825-5830, October 15, 1988]
Detection of Chromosome Aneuploidy in Interphase Nuclei from Human Primary
Breast Tumors Using Chromosome-specific Repetitive DNA Probes1
Peter Devilee, Rémi
F. Thierry, Tim Kievits, Rukmini Kolluri, Anton H. N. Hopman, Huntington F. Willard,
Peter L. Pearson, and Cees J. Cornelisse
Departments of Human Genetics [P. D., T. K., R. K., P. L. P.] and Pathology [R. F. T., C. J. C.], University of Leiden, The Netherlands; Department of Pathology,
University ofNijmegen, Nijmegen, The Netherlands [A. H. N. HJ; and Department of Medical Genetics, Toronto, Ontario, Canada [H. F. W.]
ABSTRACT
We have used in situ hybridization with chromosome specific repetitive
DNA sequences as a probe to reveal particular chromosomes as distinct
spots or clusters of signal within interphase nuclei. Using karyotypically
defined cells and cell lines, we show that the number of signals obtained
per nucleus correlates with the number of particular chromosomes present
in that nucleus. Further, admixtures of karyotypically different cell lines
could be detected. In situ hybridization of nuclei and metaphase spreads
derived from the breast cancer cell line MCF-7 shows that a deviant
number of spots/nucleus indicates a numerical and/or structural chro
mosomal aberration. In seven primary breast tumors studied, we detected
numerical aberrations of the target sites of chromosomes 1 and/or 18.
Although all had a single peak in DNA flow measurements, six of the
cases appeared to be heterogeneous with respect to their spots/nucleus
content.
INTRODUCTION
Karyotyping procedures of solid tumors normally present
difficulties such as a low mitotic index, reluctance of the cells
to grow in vitro, and poor quality metaphase figures, in which
complex chromosome rearrangements are difficult to identify
(1). These drawbacks have hampered the cytogenetic character
ization of extended series of solid tumor cases. As a conse
quence, it has been difficult to establish representative chro
mosomal aberrations or to gain insight into the existence of
cytogenetic inter- or intratumor heterogeneity.
It has been demonstrated (2-4) that ISH2 of chromosome
specific repetitive DNA probes, the target sequences of which
lie mainly in the (peri)centromeric region, can reveal those
chromosome regions as distinct clusters of signal or spots
within the interphase nucleus. Thus, this method, which is fast
and relatively easy, allows counting of the number of target
sites in a large number of tumor cells harvested directly from
the primary cancer, without any cultivation step. We show here
that the number of spots/nucleus (S/N) indicates chromosomal
ploidy and/or aberrations. With regard to cellular and cytoge
netic heterogeneity of solid tumors, the technique importantly
allows identification of subpopulations of karyotypically aber
rant cells. Analysis of seven primary breast tumors indicate that
"interphase cytogenetics" will prove to be a powerful tool in
the study of cytogenetic heterogeneity in solid tumors.
MATERIALS AND METHODS
Cells and Cell Lines. Normal diploid cells used in this study were
either male or female blood lymphocytes or fibroblasts from skin
Received 1/15/88; revised 6/10/88; accepted 7/20/88
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.
' This work has been supported by the Royal Dutch Cancer Fund (Koningin
Wilhelmina Fonds, IKW 86-15).
2The abbreviations used are: ISH, in situ hybridization; S/N, spots/nuclei;
PBS, phosphate-buffered saline; 2x SSCP, 0.3 M NaCl-30 HIMsodium citrate-20
mM phosphate buffer, pH 6.0; FCM, flow cytometry measurements; DI, DNA
index.
biopsies. Cells with trisomy 18 as their only aberration were obtained
from an amniotic fluid cell culture after amniocentesis at the 17th week
of pregnancy. MCF-7 is a described breast cancer cell line (5). Cells
from primary solid tumors were collected by scraping from the freshly
cut tumor surface (6).
Metaphase Preparations. Metaphase spreads from MCF-7 were ob
tained after 1-3 h Colcemid treatment and 15 min hypotonie shock in
50 mM KC1-5 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid,
pH 8.0-10 mM MgCU-3 mM dithiothreitol, followed by routine meth
ods.
DNA Probes. LI.84 and pBamXS belong to a class of repetitive
sequences referred to as a satellite (7, 8). They are located in the
centromeric regions of chromosomes 18 and X, respectively. PUC1.77
represents a Satellite III repetitive DNA family located in the heterochromatic region of chromosome 1 (9, 10).
In Situ Hybridization. Plasmid DNA of PUC1.77, LI.84, and
pBamXS was purified (11) and nick-translated either with [3H]dTTP
(4) or with biotin-11-dUTP (12) or modified with 2-acetylaminofluorene (13). Harvested cells were washed twice in PBS and given hypotonic treatment in 75 mM KC1 at 37°Cfor 30 min. After three cycles
of centrifugation (5 min, 1500 rpni) and resuspending in inclini
nohacetic acid (3:1, v/v), nuclei were spotted on ethanol-cleaned slides.
Twenty /il of hybridization mix containing 60% formamide, 2x SSCP,
0.4 mg/ml of single-stranded salmon sperm DNA and 2-10 ng of probe
DNA, were applied to each slide and covered with 32- x 24-mm
coverslips. Probe and target DNA were denatured by incubating the
slides at 80°Cfor 5 min. Hybridization was allowed overnight at 37"C
in a moist chamber. Slides were washed twice in 2x SSCP, twice in
60% formamide/2x SSCP, once in 2x SSCP (15 min each), and once
in PBS containing 0.05% Tween 20 (5 min), all at room temperature.
Slides were then incubated for 30 min with 40 /il of PBS containing
bovine serum albumin and 0.05% Tween 20 under coverslips and
dehydrated.
Detection of Hybrids. Detection of target sites of 3H-labeled probes
or 2-acetylaminofluorene-modified probes was performed as described
earlier (2, 7, 13). The hybridization sites of biotinylated probes were
detected with an avidin-fluorescein isothiocyanate complex (4). Slides
were scored independently by two observers who had no advance
knowledge of results of flow cytometry or histological examination. In
the case of primary tumors, ~400 nuclei were scored by each observer
in order to avoid possible bias because of clonal heterogeneity.
Flow Cytometry. FCM were carried out on an ICP-22 flow cytometer
as described (6, 14). The DI of a tumor cell population is operationally
defined as the ratio between the modal channel number of the Go.i peak
of the tumor cell population and the GO.Ipeak representing nonneoplastic cell types that are usually present in tumor samples. Tumors
with a DI of 1.0 are classified as diploid and those with a distinct GO.I
population with a DI 1.0 as aneuploid.
Histological Examination of Tissue Sections. The percentage of non
tumorous cells was visually estimated by scoring representative histo
logical sections at low magnification. In aneuploid tumors, this fraction
was also calculated from DNA flow histograms by integrating peak
areas.
Statistical Methods. Kendall's Tau-B test was applied in all compar
isons. If the distributions pertained two variables with an intrinsic
ordering between the levels (number of S/N), this test is equivalent to
the Mann-Whitney test. If the variable of interest was a dichotomy, it
is equivalent to the x2 test for an R x C contingency table. In all
instances the text refers to Kendall's Tau-B test.
5825
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/A SITI' HYBRIDIZATION
OF INTERPHASE
RESULTS
Number of S/N Corresponds to the Number of Particular
Chromosomes Present. Fig. 1 shows typical male, female, and
49,XXXXY nuclei obtained after ISH with 3H-labeled
pBamXS. The number of grain clusters reflects the number of
X-chromosomes present in each nucleus. When probes L 1.84
(chromosome 18) or PUC1.77 (chromosome 1) were used on
nuclei from normal diploid fibroblasts, two S/N were typically
observed (not shown). Scoring a large number of nuclei of
diploid and aneuploid cells (Table 1, top) yields frequency
distributions of S/N that are typical in two ways: (a) the peak
always indicates the modal number of particular chromosomes
present in that cell population; (b) the percentage of nuclei with
one spot less than the peak value is always higher than the
percentage of nuclei with one spot more than the peak value,
although the former was slightly more variable between differ
ent observers (not shown). When distributions obtained with
PUC1.77 on phytohemagglutinin-stimulated
versus peripheral
lymphocytes were compared, no significant differences were
observed (not shown); i.e., distributions were not influenced by
the presence of higher fractions of cycling cells.
Since samples from tumor specimens would probably contain
nontumorous cells, we addressed the question of whether subpopulations within a heterogeneous population of cells can be
reliably detected. A 25% admixture of trisomy 18 cells and
euploid cells gave rise to a significant (P< 0.01) increase in the
percentage of nuclei with three S/N relative to the same fraction
in a pure diploid population (Table 1, bottom). When mixed in
a 50:50 ratio, the number of nuclei with two spots almost
equaled the number of nuclei with three spots.
An Aberrant Number of S/N Indicates Numerical and/or
Structural Chromosome Abnormalities. The breast cancer cell
line MCF-7 has been cytogenetically characterized by WhangPeng et al. (5). Our DNA flow measurements (Fig. 2A) indicated
a DI of 1.78. ISH with biotinylated PUC1.77 of interphase
nuclei revealed a major population with three spots (Fig. 2, B
and £),although nuclei with four or more spots were occasion
ally noted. In metaphase spreads, PUC1.77 typically detects
three chromosomes. Two are apparently normal chromosomes
1; one has a largely deleted q arm (Fig. 2C, arrow). Similarly,
L 1.84 detected three chromosomes, two of which were struc
turally distinct from a normal chromosome 18 (data not shown).
NUCLEI FROM BREAST TUMORS
Distributions of S/N in Solid Tumors. We have examined the
nuclei obtained from six primary infiltrating ductal breast car
cinomas and one benign tumor (giant fibroadenoma, case 4).
In FCM, cases 1-4 are diploid (DI = 1.00), cases 5 and 6 are
hypotetraploid (DI =1.51 and 1.59, respectively), and case 7 is
tetraploid (DI = 2.00).
Diploid Cases. After ISH with PUC1.77, the majority of
nuclei from case 1 contained either two or three spots (Fig. 3,
A and Q. The DNA flow histogram does not show any evidence
of aneuploid subpopulations (Fig. IB). Case 1 was estimated to
contain approximately 40% nontumorous cells (Table 2).
Hence, virtually all tumor cells probably contain three S/N.
Note that the obtained S/N distribution is largely independent
of the labeling method (Fig. 3C). The peak fractions detected
after ISH with either PUC1.77 or L1.84 of cases 2 and 3
contained two S/N (Table 2). However, in these cases the
fraction with three S/N consistently exceeds the fraction with
one S/N. In this respect, all distributions differ significantly (P
< 0.001) from those obtained with both PUC1.77 and L 1.84
on normal cells and therefore indicate the presence of minor
cytogenetically aberrant subpopulations. In case 4, one observer
noted a similar significant increase in the fraction with three
S/N; the other observer, however, did not (Table 2).
Aneuploid Cases. All three cases gave one aneuploid peak in
FCM (Fig. 4B for case 7; not shown for cases 5 and 6). In case
7, however, two major populations of nuclei with either three
or four S/N were observed with both PUC1.77 (Fig. 4, A and
C) and L 1.84 (Table 2). The percentage of nuclei with two S/
N correlates well with the expected number from FCM (~20%
versus 16%; Fig. 4B). Typically, two observers arrive at virtually
similar distributions (Fig. 4C). Only once (Table 2, case 4) did
the obtained distributions differ significantly between the two
observers. Nuclei with either four or five S/N were prevalent in
case 6 after ISH with PUC1.77. Probe L 1.84 did not hybridize
to case 6 nuclei in three successive experiments, despite clear
signals in parallel hybridizations of lymphocyte nuclei or nuclei
from other tumors. Heterogeneity with respect to S/N content
was also seen in case 5. Although this sample contains ~75%
tumor cells with a DI of 1.51, only 36% were found to contain
three S/N after ISH with PUC1.77 (Table 2), indicating that a
substantial fraction of the tumor cells contains two S/N.
DISCUSSION
Earlier studies (2-4) have shown that repetitive DNA se
quences located predominantly in the (peri)centromeric region
' .
.
46, XY
46, XX
49,XXXXY
Fig. 1. In situ hybridization with 3H-labeled pBamXS of nuclei isolated from (At peripheral blood lymphocytes from a healthy male, (B) a healthy female, and (Q
a 49.XXXXY cell line. Giemsa counterstaining.
5826
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Iff SITI' HYBRIDIZATION
OF INTERPHASE
of one particular chromosome will, under proper hybridization
conditions, reveal only their cognate chromosomes as distinct
spots in an interphase nucleus. We show here that the distri
bution of the number of S/N peaks at the modal number of
chromosomes present in that population of cells in a typical
way. Of relevance for the detection of cytogenetic heterogeneity,
a 25% admixture of trisomy cells to euploid cells could be
reliably detected. Further, our results with MCF-7 demonstrate
Table 1 Distribution of spots/nucleus (percentage of total count N)
Probes and
nucleiPBNL"
source of
46.XYpBamXS,
[3H]dTTPPBNL
46.XXpBamXS.
[3H]dTTPCell
XXXXYpBamXS.
line 49,
[3H]dTTPFibroblasts
spots/nucleus19526214424701080163
of
413
N10514410
126
525
157ISO1
46.XYLI.84.
173
AAFCell
line.No.
65
160
trisomy 18
L 1.84, AAF
Admixture 50:50
10
45
38
100
(trisomy 18:46.XY)
LI.84. AAF
Admixture 25:75
15
61
17
100
(trisomy 18:46.XY)
LI.84, AAF
" PBNL, peripheral blood nonstimulated lymphocytes; AAF. 2-acetylaminoflu-
NUCLEI FROM BREAST
TUMORS
that a deviant number of S/N is indicative of a chromosomal
aberration, which may be either numerical or structural or both.
Similar observations have recently been reported with PUC1.77
and L 1.84 on neuroectodermal tumor cell lines (10), and with
pBamXS on an X-autosome translocation in an incontinentia
pigmenti patient (15). The structural abnormalities of chro
mosomes 1 and 18 in MCF-7 found by us were also noted by
Whang-Peng et al. (5) in an MCF-7 variant termed "1975."
Five breast tumors (cases 2, 3, and 5-7) showed different
degrees of heterogeneity with respect to their S/N content
obtained with either PUC1.77 or L1.84. For reasons discussed
below, the two observers were in conflict about the presence of
cytogenetic heterogeneity in the giant fibroadenoma (Table 2,
case 4). Since hybridizations were carried out separately, the
question whether the nuclei with three spots detected by each
probe in case 3 derive from one subpopulation or not remains
unanswered. Simultaneous ISH with the two probes (10, 16)
would clarify this. The frequent involvement of chromosome 1
is not surprising since a number of reports describe an overrepresentation of the long arm of chromosome 1 (Iq) in human
breast cancer via simple chromosome addition (trisomy) or
structural alterations (17, 18). Our results provide further evi
dence that such aberrations may be present in subpopulations
of tumor cells. Notably, this heterogeneity was not seen in flow
cytometry, in which all cases showed a single peak, with either
a diploid or aneuploid DNA value. Cytogenetic evidence sug
gests that chromosome abnormalities can occur in breast cancer
without associated shifts in DNA content or total number of
Fluorescence
MCF-7
I Observer R. N= 702
» 2
3
Õ
5
S
Number of soots/nucleus
Fig. 2. (.-I). DNA profile of MCF-7 obtained by DNA cytometry, using propidium iodide-stained trout RBC (IKll< ) as internal ploidy standard. The G0.i peak
had a Dl of 1.78. (B) interphase nuclei and (f') a metaphase spread of MCF-7 after ISH with biotinylated PUC1.77. Fluorescein isothiocyanate, fluorescence,
propidium iodide-counterstaining. Arrows in R. blurred spot morphology, presumably representing one target site; arrow in C, structurally aberrant chromosome 1.
(D) Distribution of S/N in 702 MCF-7 nuclei after ISH with biotinylated PUC1.77.
5827
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IN SITU HYBRIDIZATION OF INTERPHASE NUCLEI FROM BREAST TUMORS
Fluorescence
AAF, N=642
(percentag
cy
Biotm,N=337
Lt
1231,56789
41-n
10
Number of spots/nucleus
Fig. 3. (A) A field of nuclei isolated from case 1 after ISH with biotinylated PUC1.77. Presumed minor binding site and "double" structure can be noted (left and
right arrows, respectively). (B) DNA profile of case 1. (C) Distributions of S/N in a population of case 1 nuclei after ISH with either 2-acetylaminofluorene (AAF)modified or biotinylated PUC1.77. Data from two observers were accumulated to form one histogram. TRBC, trout RBC.
chromosomes (18, 19). Further, even if trisomy 1 was the sole
cytogenetic abnormality, it would probably go undetected in
FCM. At best, a minimal DNA difference of about 4% is
required for obtaining a split peak in heterogeneous cell popu
lations (20), about the DNA content of a complete chromosome
are shown separately. In case 4 the distributions obtained with PUC1.77 differ
1 (21). A similar discrepancy between FCM and ISH results
significantly (P< 0.001) between the two observers and are therefore also shown
has been noted by Hopman et al. (16) in a series of solid bladder
separately. Case 5 did not yield enough material for hybridizations with L 1.84 to
be performed on it.
tumors. In this context, ISH data may be more helpful in
spots/nucleusCase1234567DI1.001.001.001.001.511.592.00%NT"
No. of
further stratifying DNA ploidy data, especially in breast cancer,
Probe/label*40
where the relationship between ploidy and survival is more
>634
complex (22) than it is for other types of carcinomas (23, 24).
PUC1.77/A
140 9
2
PUC1.77/B
7
4
3
214744
337467725 /// situ hybridization is a technique which relies on complete
L1.84/A+B50
Weak58Weak60
,evaluated20
not
denaturation of target sequences in conjunction with complete
penetration of the nucleus by probe sequences. If these two
PUC1.77A
7
1
L1.84/B40
,evaluated23
not
conditions are not fully met, it is conceivable that merely one
spot is lit up in a diploid nucleus. Alternatively, an observer
PUCI.77/B
may see two spots as one if the orientation of the nucleus
PUC1.77/B
10 69 16
1
535
L1.84/BPUC1.77/B35979122246
6485 178 152 115
565756 fortuitously positions them on top of each other. In case 6 no
signal at all was observed with probe LI.84 in three successive
PUC1.77/B
68 19
4
924
experiments. Although nullisomy has been reported to occur
L1.84/B22
6441345
10485179142245
183684
1082
for several chromsomes in breast cancer (19), more definitive
proof should come from simultaneous hybridization with two
PUC1.77/A14
115
probes for different chromosomes (10, 16). Another factor
PUC1.77/B
14 29 35
6
possibly disturbing distributions is variation in spot morphol
L1.84/A+B16
No
obtained2118322440393
signal
ogy. Known single hybridization sites sometimes appear
"blurred" (Fig. 2B) or as a double structure (Fig. 3/1). The basis
PUC1.77/B
2
Table 2 Distribution of spots/nucleus (percentage of total count N) in nuclei
obtained from human primary breast tumors
Whenever possible, a minimum of 400 nuclei were scored by each of the two
observers. Numbers represent accumulated and averaged data (e.g., for case 7 see
Fig. 4C~).For cases 1 and 3 the ISHs with PUC1.77 were repeated and the results
L1.84/B18
7 10N642623
" %NT, percentage of nontumorous cells in sample. See "Materials and Meth
ods" for determinations.
* Probe and labeling procedure: A, 2-acetylaminofluorene; B, biotin-11-dUTP.
for this phenomenon, which has also been noted by others (16,
25), is not known, but it may cause the observer to score two
spots instead of one. Finally, a successful hybridization with
one of the currently available chromosome specific repeats
5828
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IN SITU HYBRIDIZATION
OF INTERPHASE NUCLEI FROM BREAST TUMORS
Fluorescence
PROBE 1.77IBIOTIN1
50
40-
• Observer R.N=1003
CH
Observer H.N=1242
20-
10-
LCU
Õ
5
Ã-
7
B
9
W
11
12
Number of spots/nucleus
Fig. 4. (.-()A field of nuclei isolated from case 7 after ISH with biotinylated PUCI.77. Presumed minor binding sites are indicated (arrows). (B) DNA profile of
case 7. (Q Distributions of S/N obtained independently by two observers in a population of case 7 nuclei after ISH with biotinylated PUC1.77.
relies strongly on the stringency conditions (2). The presence
of nonhomologous hybridization may substantially interfere
with the scoring of the number of S/N, although with nonisotopic protocols "minor" hybridization sites (Figs. 3A and 4A)
Howard Cooke for making PUC1.77 available to us, and Heleen Semé
and Klaas van de Ham for helping with the preparation of this manu
script.
can often be excluded on the basis of their weaker signal
intensity. The use of a composite probe, a mixture of many
single copy sequences from a relatively small chromosome
region to constitute an artificial repeat, has been suggested to
overcome this problem (2). A different interpretation of ISH
signals by two observers may have caused the significant differ
ence in obtained distributions with PUC1.77 in case 4. Con
versely, it may have been due to sample heterogeneity. Never
theless, interobserver variability in S/N distributions in this
study was generally limited (Table 2). However, for general
applicability of this approach, it will be necessary to standardize
criteria for interpretation of spot morphology (16).
Although the idea of "intcrphase cytogenetics" is not new
REFERENCES
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human chromosome complement. This report and that of Hopman et al. (16) are the first to apply "interphase cytogenetics"
to the study of chromosomal aberrations in primary solid
tumors. Especially for this category of tumors, we expect that
this technology will provide important additional information
to classical cytogenetic and DNA flow analysis.
ACKNOWLEDGMENTS
The authors are indebted to Nel Dijkshoorn and Hans de Koning
for technical assistance, Dr. E. van de Velde for statistical analysis. Dr.
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Detection of Chromosome Aneuploidy in Interphase Nuclei from
Human Primary Breast Tumors Using Chromosome-specific
Repetitive DNA Probes
Peter Devilee, Remi F. Thierry, Tim Kievits, et al.
Cancer Res 1988;48:5825-5830.
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