ERBB-2 (HER2/neu) Gene Copy Number, p185HER-2

Overexpression, and Intratumor Heterogeneity in Human Breast

Cancer
Jnos Szllsi, Margit Balzs, Burt G. Feuerstein, et al.
Cancer Res 1995;55:5400-5407.

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[CANCER RESEARCH55, 5400-5407. November 15, 19951

ERBB-2 (HER2Ineu) Gene Copy Number,

Intratumor
Jnos

@185HER2Overexpression,

and

Heterogeneity in Human Breast Cancer'

Szllosi,2

Margit

Ba1IZS,2

Burt

G. Feuerstein,

Christopher

C. Benz,

and

Frederic

M. Waldman3

Division of Molecular Cytometry, Department of Laboratory Medicine If. S., B. M., B. G. F., F. M. W.]. Brain Tumor Research institute (B.G.F.], and Cancer Research Institute
(C. C. B.], University of California, San Francisco, California 94143-0808

ABSTRACT

amplified ERBB-2 in tumor DNA, rare breast tumors overexpress

Amplification of the ERBB-2 (HER-2/neu) gene Is accompanied by

overexpression

of Its ceH surface receptor

neity

observed

has been

for both

product,

the gene copy

pl85@2. Heteroge

number

and the level of

overexpresslon ofits proteIn product. To better understand their relation

ship, correlation between the level of cellular expression of pf85@@an2and

ERBB-2 gene amplification was studied in four human breast cancer cell
lines (BT-474, SK-BR-3, MDA-453, and MCF-7) and in a primary
breast

tumor

sample.

The relative

expression

of pl85@'t2

human

was measured

by hnmunofiuorescence by using flow and/or image cytometry while

correlated

DNA analysis was performed

on the same cells by fluorescence

in situ hybridization to determine ERBB-2 gene and chromosome 17 copy

numbers. Marked heterogeneity was observed in both protein expression
and ERBB-2 copy number. Despite this heterogeneity, and in accordance
with previous

studies,

the average

levels of p1ss@R2

expression

corre.

lated well with average ERBB-2 gene copy numbers in the four lines
examined (r = 0.99). When the relationship between copy number and
proteIn expression

was studied on a cell-by-cell

basis, p185@2 expres

sion correlated with both the absolute number ofERBB-2 gene copies/cell
(r 0.590.63)
and chromosome 17 copy number (r 0.450.61).
It Is of
Interest that there was weak or no correlation between p185@2protein
expression and the ERBB-2 copy number:chroinosome
17 copy number
ratio (r = 0.0-0.25). In more than one-half of cells expressing a high level

of p185@@E@@2,
the chromosome 17 copy number was
(two or three
times the average copy number), whereas <2% of an unselected popula
tion had a high chromosome 17 copy number. Bromodeoxyuridine incor
poration indicated that the S-phase-labeling index was homogeneous
across various

p185'@2-expressing

subpopulations

In the SK-BR-3 cell

line. Analysis of the primary breast tumor sample showed results similar
to the cell lines, supporting the strong possibility of a mechanistic link
among p185@2
overexpresslon,
mosome 17 copy number.

ERBB-2 amplification,

and high chro

INTRODUCTION
A characteristic feature of cancer cells is the unregulated expression
of genes involved in cellular growth control. One of these genes is the
ERBB-2 (HER-2/neu) proto-oncogene, which encodes a Mr 185,000
transmembrane glycoprotein
@l85@.@@2)
that belongs to a subfamily
of growth factor receptors having intrinsic tyrosine kinase activity,
including the epidermal growth factor receptor and the receptors
HER-3 and HER-4 (1-3).
Amplification and overexpression of ERBB-2 is found in 2530%
of primary human breast cancers and is associated with a poor clinical
outcome (46).This suggests that overexpression of pl85}@1t2 plays
a role in the pathogenesis of some human breast cancers (5, 6).
Although overexpression of p185HER2 is usually accompanied by

pl85F@1t2protein or c-ERBB-2 mRNA levels in the absenceof

detectable gene amplification (7).
Although amplification of ER.8B-2 is generally considered to be a
significant prognostic indicator in patients with breast cancer, its
applicability continues to be controversial, in part because of analyt
ical discrepancies associated with the methods traditionally used to
evaluate its amplification and/or overexpression. These techniques
include Southern blotting, slot blot analysis, and FISH4 for detection
of amplification, while ELISA, Western blotting, immunohistochem
istry, and immunofluorescence are used to evaluate overexpression
(815).Because FISH allows the observer to distinguish small sub
populations of amplified cells, it is more sensitive than blotting
techniques. In addition, FISH allows one to identify particular loca
tions where aberrations exist in single tumor specimens (10, 14).
Similarly, because immunohistochemically stained slides are difficult
to quantify and because ELISA and Western blotting data do not
provide information concerning heterogeneity, immunofluorescence
has advantages over these other methods (13, 14).
Marked heterogeneity has been described in primary breast cancers
in both the copy number of ERBB-2/cell and in the level of p185@@E1t2
protein (512).Although cell-to-cell differences may be due in part to
analytical variation, genetic and epigenetic dispersion may also play
significant roles. This heterogeneity provides a potential source for the
selection of subclones with increased malignant and metastatic poten
tial, especially in the context of therapeutic targeting based on
ERBB-2 expression.
Although amplification of ERBB-2 correlates well with overexpres
sion of pl85@@E1t2
protein in cell populations (5, 6, 9, 11, 1416),the
correlation has not been made on a cell-by-cell basis. The present
communication describes our analysis of the extent to which ERBB-2
gene amplification relates to the expression of p185@1t2 on a single
cell basis. We have found that although pl85@@1t2 expression corre
lates with the ERBB-2 copy number/cell, p185@@1t2expression cor
relates poorly with the ERBB-2 copies:ch.romosome 17 copies ratio.
Surprisingly, there was correlation between p185@1t2 expression and
chromosome 17 copy number, suggesting that hyperploidy may be
related to the p185HER2 expression.
MATERIALS

AND METhODS

Cell LInes. Humanbreastcancercell lines, BT-474, SK-BR-3, MDA-453,

and MCF-7 were obtained from the American Type Culture Collection (Rock
ville, MD) and grown according to their specifications. The four cell lines were
characterized

previously for ERBB-2 gene amplification

(10). For flow cyto

metric immunofluorescence measurements, cells were harvested either by

Received 6/8/95; accepted 9/14/95.
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.
I This

research

States-Hungarian
2 Present

was

supported

by

NIH

Grants

CA-49056

and

CA-44768

and

by

United

Joint Fund for Science and Technology (JF292/92B).

address:

Department

of Biophysics,

Medical

University

School

of Debrecen,

Nagyerdei krt. 98, H-4012 Debrecen, Hungary.

3 To

whom

requests

for

reprints

should

be addressed,

trypsin

or 25 mM EDTA

in PBS (pH 7.2; Ref. 17). For slide-based

fluorescence measurements,

immuno

cells were cultured in slide chambers (Nunc, Inc.,

Naperville, IL). For BrdUrd (Sigma Chemical Co., St. Louis, MO), labeling
cells were pulsed with 100 @M
BrdUrd for 60 mm. Cells were washed three
times with PBS containing 1 mMCaC12before immunofluorescence labeling.
Tumor. A biopsy froma node positive, T2 tumor,was frozenimmediately
after resection. Imprint preparations were made after thawing by gently touch

at Department

of

Laboratory

Medicine, MCB-230, Box 0808, University of California at San Francisco, San Francisco,
CA 94143-0808.

Phone:

(415)

4763821; Fax: (415)

4768218;

E-mail:

waldman@

4 The

abbreviations

used

are:

FISH,

fluorescence

in

situ

hybridization;

modeoxyuridine; chr, chromosome; F!, fluorescence index.

dmc.ucsf.edu.

5400

Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.

BrdUrd,

bro

ERBB.2 EXPRESSION

AND AMPLIFICATION

ing the slide surface with tumor material. Slides were then fixed in 1%
formaldehyde for 60 mm at room temperature and subsequently fixed and

images acquired; they were then hybridized for gene copy number and scored
after relocating cells analyzed previously. After immunofluorescence analysis,

stored in 70% ethanol. The autofluorescence of air-dried touch imprint prep

arations was too high for reliable immunofluorescence analysis. Fresh fixation
of slides in 1% formaldehyde and subsequently in 70% ethanol reduced

slides

autofluorescence

3.0 mm, dehydrated in graded ethanols, treated with 0.25 @g/ml

proteinase K
(Sigma) in 20 mMTRIS buffer (pH 7.5) containing 2 mt@i
CaCl2 for 7.5 min at
37C,and again dehydrated. The hybridization mixture was denatured at 73C
for 5 min, reannealed for 30 mm at 37C,and applied to warmed slides. Ten
p@l
of hybridization mixture contained 6 ng of fluoresceinated chromosome 17
centromeric probe, 34 ng of rhodaminated ERBB-2 probe, and 10 ng of
unlabeled, sonicated human placental DNA (Sigma) in 50% formamide, 2X
SSC, and 10% dextransulfate. Hybridizationwas overnightat 37C.Slides
were washed three times for 10 mm each in 55% formamide-2X SSC, once in
2X SSC at 45C,and once in 2X SSC at room temperature. Nuclei were

significantly.

Immunolabeling. For flow cytometry,unfixed trypsinizedcells were in

cubated with 5 ,.tg/ml mAbi (Triton, Alameda, CA) raised against the extra
cellular domain of p18SHER2,in the presence of 1% BSA on ice for 45 mm,
washed three times with PBS, and incubated with fluoresceinated rabbit
antimouse

IgG (1:100

dilution;

Sigma)

at 0Cfor 45 mm. After washing

with

PBS, cells were fixed in 1% formaldehyde solution and stored for not more
than 3 weeks

at 4C before

analysis.

For image analysis, cells were first fixed in 0.5% formaldehyde solution for
20 mm at room temperature

and in 70% ethanol

at 4C overnight.

Cells on

were

refixed

in methanol:acetic

acid (3:1;

Carnoys

solution)

and air

dried. FISH was performed as described previously (19) with modifications.

Briefly, cells on slides were denatured in 70% formamide-2X

SSC at 73Cfor

slides could be stored in ethanol at 4Cfor not more than 2 months. Slides were

counterstained by using 4',6-diamidino-2-phenylindole

then preblocked

ular Probes, Eugene, OR) at 0.01 g@g/m1

in antifade solution (20).
Simultaneous detection of BrdUrd incorporation and dual FISH staining
was performed with three fluorescent dyes (fluorescein- and rhodamine-la
beled probes and a Cascade Blue-conjugated antibody; Ref. 21). Cells and

in 5% Carnation

dry milk, 0.1% Triton

X-100 in 4X SSC (1X

SSC is 0.15 M NaCl and 0.015 M sodium citrate) for 10 mm at room

temperature. Staining was at room temperature for 45 mm. Samples were first

incubated with CB1I antibody (BioGenex, San Ramon, CA) specific to the
intracellular domain of the p185HER.2protein, diluted (1:200) in the blocking
buffer,

washed

twice with the blocking

buffer,

and incubated

with fluorescein

ated rabbit antimouse IgG (1:100; Sigma). After washing, samples were
refixed in 1% formaldehyde solution in PBS and kept at 4Cfor not more than
3 weeks

before

microscopic

analysis.

During

this time, no significant

deteri

oration of the fluorescence signal was observed.

To control for nonspecific staining, cells were preincubated with irrelevant
monoclonal antibody of the same isotype before staining with fluorescein
conjugated rabbit antimouse IgG. We also compared immunofluorescence
in PBS.

Trypsinization

caused

as described above. After washing,

slides were preblocked in 5% Carnation dry milk and 0.1% Triton X-100 in 4X
SSC for 10 min at room temperature.All staining reactionswere at room
temperature for 30 min. Slides were incubated with IU4 mouse anti-BrdUrd
(1:400; Caltag, La Jolla, CA), diluted in blocking buffer, washed twice with
blocking buffer, and incubated with Cascade Blue-antimouse IgG (1:300;

Molecular Probes), and coverslipped with antifade solution alone.

Scoring of Interphase Nuclei. Cells analyzed previously for cell surface
expression of p185HER2 protein were relocated on the basis of their coordi

labeling of MDA-453 and SK-BR-3 cells harvested with either trypsin or 25

mM EDTA

probes were denatured and hybridized

hydrochloride (Molec

a 1015% loss in fluorescence

nates and scored for chromosome 17 and ERBB-2 signals by using a X100
NA:1.3 Plan Neofluar oil immersion objective and a computer-controlled
stage. ERBB-2 doublets

were counted

as separate

signals.

Broken,

torn,

intensity as compared to cells harvested with 25 mr@iEDTA (data not shown).

Because this loss was not significant, and the two other cell lines could not be
harvested with 25 mM EDTA, we used trypsin to harvest cells for flow

was accompanied by a control hybridization using normal lymphocytes. The

cytometric analysis. Results from monoclonal antibody (mAbi) raised against

scoring results were expressed both as an absolute ERBB-2 copy number/cell

the extracellular

and as the ERBB-2copy number relative to the 17 centromere copy number.

Three color images were acquired by using the digital imaging analysis
system described previously. A triple-band-pass beam splitter and emission

monoclonal
protein.

domain

antibody

With

of

(CB11)

@l85H@.2 protein
raised

mAbI , prefixation

against

was

were

similar

the intracellular

unnecessary,

resulting

to those
domain

from
of the

in lower

non

specific binding.
Flow Cytometry. Cell suspensions were filtered through a 35-g.@m
nylon
mesh to remove aggregates before flow cytometric analysis. Analysis was
performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
equipped with a 15 mW argon laser (488 nm) and pulse-width doublet
discrimination. A total of 10,000 events were recorded in list mode after
logarithmic amplification of the fluorescence signal.

Digital Image Analysis. The fluorescence of cells stained on slides was

analyzed by using a digital image analysis system based on a Zeiss Axioplan
microscope equipped with the Microlmager 1400 Digital camera (Xillix Tech
nologies Corp., Vancouver, British Columbia, Canada). Images were captured
through a fluorescein excitation filter, beam splitter, and emission filter by
using

a X 20, NA: 0.5 Plan Neofluar

quantitatively

analyzed

objective.

with a Sun IPX workstation

Images

were

processed

using Scil-Image

and

software

(National Research Institute, Delft, The Netherlands). Local background flu

orescence was determined for each image, and the average autofluorescence of

the isotypic control cells was subtracted from the total fluorescence intensity of
labeled cells. The Fl was defined as a ratio of the corrected total fluorescence
intensity of labeled cells to the mean autofluorescence

of the isotypic control

cells.

DNA Probes and Probe Labeling. Two contiguous ERBB-2 cosmid

clones (cRCNeul and cRCNeu4), together spanning 55 kb of genomic DNA
(10), were used in combination

pericentromeric

sequence

with a probe specific

(p17H8;

for the chromosome

Ref. 18) for two-color

17

FISH analysis.

Probes were directly labeled with fluorescein-11-dUTP or tetramethylrhodam

inc-I 1-dUTP

(Boehringer

Mannheim,

Indianapolis,

squashed,

IN) by nick translation

by

using commercially available kits (Bethesda Research Laboratories, Gaithers

burg, MD).
fluorescence in Situ Hybridization and Staining for BrdUrd. Dual
analysis of gene copy number and protein expression was done as a two-stage

procedure. Slides were first stained for protein expression and fluorescence

smeared,

or overlapping

nuclei

were

ignored.

filters were used (22). Excitation of each fluorochrome

Each

hybridization

was accomplished

by

using single-band-pass excitation filters in a computer-controlled filter wheel.

This made it possible

to collect

sequential,

properly

registered

images

of the

three fluorochromes (4',6-diamidino-2-phenylindole hydrochloride or Cascade

Blue, fluorescein, and rhodamine). The three-color images were processed
with a Sun IPX workstation using Scil-Image software for pseudocolor display.
Statistical
ber between

Analysis.
the

Significance levels for differences in gene copy num

@185HER.2bright

and total cell populations

were determined

by contingency table analysis.

RESULTS
ERBB-2 Amplification and Expression in Breast Cancer Cell
Lines Four breast cancer cell lines, MCF-7, MDA-453, SK-BR-3,
and BT-474, known to have various levels of amplification of the
ERBB-2 gene (10) were studied for distribution of ERBB-2 gene copy
number and chromosome 17 centromere copy number (Fig. 1). Am
plification of the ERBB-2 gene can be expressed as copy number/cell
or as copy number relative to chromosome 17 copy number. Using a
relative measure is especially important for those cell lines that are
aneusomic for chromosome 17. Amplification of ERBB-2 gene was
observed in MDA-453, SK-BR-3, and BT-474 cell lines, using either
the definition of amplification as total ERBB-2 copies/cell or the ratio
of ERBB-2 copy number to chromosome 17 copy number. There was
marked heterogeneity for ERBB-2 copy number, chromosome 17 copy
number, and their ratios in the three cell lines with ERBB-2 amplifi
cation. In MCF-7, the ERBB-2 gene was deleted (ERBB-2 gene copy
number was less than the chromosome 17 copy number/cell) and there
was less heterogeneity in ERBB-2 gene copy number/cell and in the

5401

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Research.

@. Lu1

.@

ERBB.2 EXPRESSION AND AMPLIFICATION

ERBB-2:chromosome 17 ratio. The mean values and the SDs of the

copy number distributions are summarized in Table 1.
We next characterized the expression levels of the ERBB-2 gene
product p185HER2 by flow cytometry. Fig. 2 shows the fluorescence
intensity histograms of the four cell lines labeled with mAbi against
p185@@E@2. Heterogeneity

of expression

of p185'@@2

was similar

I-..

in

the four cell lines. The MCF-7 cell line was the least positive, only
twice background, whereas the BT-474 cells were the most positive.

L)

U BT-474

U MDA-453
0

SK-BR-3

MCF-7

10
@

,n

@
@

an ,,@

In

e@ ri

$fl 0

V@

in

@i
@
@
Si
in

Fluorescence Intensity

m in
N
t'@

Fig. 2. Frequency distribution of fluorescence intensity after immunofluorescence

ERBB-2 Signals/Cell

c)

100

staining for p1@5HER.2.Trypsinized

cells were labeled with mAbi raised against the

extracellular domain of @185UER.2

and then with fluorescein-conjugated rabbit antimouse

IgG.Anirrelevant
primary
antibody
ofthesameisotype,
followed
byfluorescein

conjugated rabbit antimouse IgG, was used for the blank control (SK-BR-3 cells). The
mean values of these distribution curves are summarized in Table 1. Note that the level
of heterogeneity (width of the intensity profiles on this log intensity scale) is similar in the
four cell lines, although the absolute amount of p185'@'@2 varies greatly from line to line.

40

20
@
.

12
.--

r r

;-@-

10

11

12

Chr 17 Signals/Cell
80

60

0 0.5 1

9 10 11 12 13

ERBB-2 Signals/Chr 17 Signals

Fig. 1. Number of ERBB-2and chromosome 17 centromere copies in four breast cancer
cell lines. A, frequency distribution of ERBB-2 signals/cell; B, chromosome 17 signals]
cell; C, ERBB-2:chromosome (Chr) 17 ratio. The values along the abscissa represent the
lower limits of the range of values for each category. At least 100 cells were scored to

create the distribution histograms. The mean values and their SDs are summarized in
Table 1. Note the wide heterogeneity present in all but the MCF-7 distributions.

linesBreast
Table 1ERBB-2

amplification and expression in breast cancer celi

cancer
17FPMCF-72.2
cell linesERBB-2'@Chr

17bERBB-2/Chr

0.2209MDA-45311.03.94.11.62.81.018675SK-BR-331.0
05d3.8
1.00.6

The mean values and the SDs of the fluorescence intensity histograms
are summarized in Table 1. The mean fluorescence intensity was
strongly correlated with the mean ERBB-2 copy number/cell
(r
0.99; Table 1). A strong correlation was also observed between
the mean protein expression and mean ERBB-2:chromosome 17 ratio
(r
0.99), whereas there was a weaker correlation with average
chromosome 17 copy number (r = 0.75).
ERBB-2 Gene Expression and Amplification on a Single Cell
Basis. Protein expression and copy number were measured in the
same individual cells to study their correlation on a single cell basis.
This was especially relevant given the wide range in both copy
number and immunofluorescence observed (Figs. 1 and 2). Immuno
fluorescence intensity of individual SK-BR-3 and MDA-453 cells was
studied by image microscopy, and the same cells were identified and
scored for ERBB-2 gene and chromosome 17 copy number after dual
FISH labeling. The fluorescence intensity was too low in MCF-7 to
perform quantitative image cytometry, and BT-474 cells could not be
separated from each other during image analysis because of their
piled-up growth pattern.
Correlated measurement of @185Han@2
expression and ERBB-2
copy number was performed in the same cells by consecutive analysis
(Fig. 3). The fluorescence images of cells displayed in Fig. 3B are
shown after double-target hybridization in Fig. 3C. The green signals
correspond to chromosome 17 centromere, and the red signals to
ERBB-2 signals. The heterogeneity of pl85I@@t2 expression in 5KBR-3 cells by image microscopy (Fig. 3A) was similar to that found
by flow cytometry (Fig. 2).
The linked analysis of p185H@2 expression and ERBB-2 gene
amplification

114BT-47452.0

a ERBB-2

copy

fluorescence

d Data

expressed

1.04.5

1.2326

11.36.0

1.19.0

2.3549

165

number/cell.

b Chr, chromosome
C Mean

9.06.9

17 copy number/cell.
intensity

as mean,

determined

SD.

from

flow

cytometric

histograms.

in SK-BR-3

cells is shown in Figs. 4 and 5. Note the use

of a Fl for these measurements, rather than absolute intensity (as was

used for the flow measurements), in order to control for the increased
levels of autofluorescence in these fixed samples. ERBB-2 copy
number showed a significant correlation with protein expression on a
cell-by-cell basis. The correlation was stronger using absolute
ERBB-2 copy number/cell (Fig. 4A) than when using a relative
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ERBB.2 EXPRESSION AND AMPLIFICATION

B
4/

I
a?

4,
C

,@

..,

0
4

C
7

..

Fig. 3. Linked detection of pl85@@ER2

expression and gene amplification in individual cells. A, SK-BR-3 cells display immunofluorescence staining for pi85@R2 expression (X20
objective) after staining with mAbl. B, computer magnification (X5) of the rectangle in A. C, FISH detection of ERBB-2 (red) and chromosome 17 centromeres (green) in identical

cells shown in B (X 100 objective). Cells were refixed after immunofluorescence labeling and denatured and hybridized with directly labeled ERBB-2 and chromosome 17
centromere-specific probes. Not all signals are visible in this image because the plane of focus is thinner than the specimen. Anti-BrdUrd labeling (blue) is positive in the top cells.
These are pseudocolor, contrast-enhanced digital images.

measure of ERBB-2 amplification (ERBB-2:chromosome 17 copy

to phenotypic dispersion, the genetic composition of bright
cells (with
number ratio; Fig. 4B). There was also a correlation seen between
more than four times more fluorescence intensity than the nonspecific
p185H@@@2
expression and copy number of chromosome 17 (Fig. 4C),
staining of isotypic control cells) was analyzed as a separate group. We
perhaps due to an second association between aneuploidy and ERBB-2
compared the distribution of ERBB-2 copy number (Fig. 5A), ERBB-2:
amplification.
chromosome 17 copy number ratio (Fig. SB) and the chromosome 17
A subpopulation of cells was seen, which stained especially brightly copy number (Fig. SC) of bright cells to an unselected population and
for p185H@2To test whether this was due to genetic heterogeneity or
found that these differences were all highly significant.
5403

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Research.

ERBB-2 EXPRESSION

significant difference (P = 0.29) in the average fluorescence intensity.

SK-BR-3

100

c@0

10
0

AND AMPLIFICATION

S-phase
cellshada higheraverage
ERBB-2
genecopynumberand

oo

ERBB-2:chromosome

o@8o@

o@@d?

r=0.60

20

40

60

SO

100

120

1'@0

not

100

0
0

0
0

10
@
@
I

i@'6'@o@t'@ 0

F = 0.25

ERBB-2 Signals/Chr

I'2

10

ratio

than

did

non-S-phase

1'4

to

the

unselected

population.

In

general,

the

observed in this touch imprint preparation

observed in tumor cell lines.

17 Signals

100

differ.

(caseno.B372)areshowninFigs.8 and9.Positive
correlations
were

0@6'@0@

00

number

found between p185HER2 expression and ERBB-2 gene copy number

(Fig. 8A), the ERBB-2:chromosome 17 ratio (Fig. 8B), and the chro
mosome 17 copy number (Fig. 8C). There were significant differ
ences in the distribution of the ERBB-2 copy number (Fig. 9A), the
ratio of ERBB-2:chromosome copy number (Fig. 9B), and the chro
mosome 17 copy number (Fig. 9C) when bright cells were compared

000

copy

ERBB-2 Gene Expression and Amplification

on a Primary
Tumor Sample. The results of consecutive analysis of pl85H@@2
expression and ERBB-2 gene amplification in primary tumor cells

ERBB-2 Signals/Cell

17

cells (43.7 versus 38.7 and 6.3 versus 5.0, respectively), perhaps
because doublets forming during DNA synthesis were scored as two
separate gene copy numbers as described in Materialsand Methods.
The labeling index of the whole cell population (39.7% of 224 cells)
.
.
and for cells with >10 chromosome 17 copies (38.3% of 60 cells) did

correlation

patterns

were similar to those

C
00

00

8@

10

00

900

SK-BR-3

U Average

D Bright(Fl>4)

r = 0.45
0

10

40

20

20

p < 0.0001

30

Chr 17 Signals/Cell
@
@

Fig. 4. ERBB@2
gene expression and amplification in single SK-BR-3 cells. Expression
level of p185@@E@2
protein
plotted against: A, ERBB-2 copy number; B, ERBB-2:
chromosome

17 ratio; and C, chromosome

in Q in Q in in
,,
@, ,,@

17 copy number. Cells were labeled with

in in
.@

ERBB-2

in

in in

in

in in
V

Signals/Cell

antibody (CB11) against the intracellular domain of p18SHER2protein. Data from 184
@

cells are shown. There is a good correlation between p185@2 expression and copy
number of either ERBB.2 or chromosome 17 centromere (A and C). The correlation
between
@185HER.2
expression and ERBB.2:chromosome
17 copy number ratio was
weaker(B).

@
@
@
@

c)

I
I

from the unselected

population

b@

20@

The results in MDA-453 cells were similar to SK-BR-3 cells for

ERBB-2 gene amplification and protein expression (Figs. 6 and 7).
However, there was no correlation between protein expression and
ERBB-2:chromosome 17 copy number ratio (Fig. 6B), and the distribution of ERBB-2:chromosome 17 ratio of bright cells did not differ
significantly

,.@

p<0.002

@o
-I
]
I

I LI

01

if@@
lf@@
in@ in @
I(@in in@
in@
V@Q@in@
,- r-@ e@ e@ r')
in
@c r-.
ERBB.2

(Fig. 7B). In both cell

lines, the centromere 17 copy number was high (two or three times the
average copy number) in >50% of the bright cells (expressing a high
level of p185HER2), whereas <2% of the unselected population had a
high chromosome 17 copy number.
Relationship between DNA Synthesis and ERBB-2 Gene Ex
pression and AmplificatiOn. We next addressed the issue of
whether the bright cells having high pl85'@@2 expression and high
chromosome 17 copy number were proliferatively active. SKBR-3
cells were pulse labeled with BrdUrd, p185H@@@@2
expression was
determined before fixation, and then BrdUrd incorporation and dual
color FISH were detected simultaneously (for demonstration see Fig.
3C) Correlation between ERBB-2 gene amplification and protein
expression in these cells (data not shown) was similar to that found in

LL@m@

Signals/Chr

17 Signals

C
60
40

p < 0.0001
20

e@r.@

in @cr..

o _ ei

an @ar. ao

@
17 SignalS/Cell
Fig.5. ERBB.2copy number(A), ERBB.2:chromosome
(Chr) 17 ratio(B), and
chromosomei7 copy number(C) in unselectedand in highlyp185@52-expressing
(Fl > 4) SK-BR-3 cells. Distributions of bright cells were determined from cells plotted

prefixed cells (Figs. 4 and 5). When BrdUrd-positive cells (cells in S

phase) were compared with BrdUrd-negative cells, there was no

in Fig.4. Cellsthatexpressedp185HER2
at a highlevelhadsignificantlymorecopiesof
ERBB-2andchromosome17,anda higherratioof thetwo,thananunselectedpopulation.

5404

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Research.

ERBB.2 EXPRESSION AND AMPLIFICATION

linkage between gene copy number and protein expression was still

MDA-453

100

present within each cell line (r = 0.590.72) but was weaker than that

0
0

10

observed

@0

(r

when

cell

lines

were

compared

at

the

population

level

0.99).

Several reasons may account for the observed dissociation between

ERBB-2 copy number and protein expression. A normal dispersion in
I

ERBB-2

r = 0.63
@

.1

10

20

30

40

transcription

and

translation

rates,

or in the

half-lives

of RNA

transcripts and protein products, might lead to a weakened linkage

50

between

genotype

and

phenotype.

ERBB-2

transcript

levels

in ampli

fled SK-BR-3 and BT-474 cells are 2040times that of immortalized

ERBB-2 Signals/Cell

but nontumorigenic

HBL-100

breast

epithelial

cells,

although

the

average level of ERBB-2 gene copy number in the amplified cell lines
100
@

@
@

%oo

10
0

2
8I'@

basis

,@o

protein

r = 0.04

population

is measured.

occurring

number
is of

either

gene

by Southern
on a cell-by-cell

copy

number

or

immu

during

Asymmetric

mitosis,

which

distribution

occurs

HER-2

of p185

in exponentially

grow

and

expression.

interest

that

p185'@@2

expression

and

the

ERBB-2

copy

17 copy number ratio were not closely linked

. Average

20

o
.

10

20

e@ @000

Chr 17 Signal/Cell

for

(determined

association

Bright(Fb4)
0

10

variation,

cells
lower

30

e@
-

of the

40@

00

analytical

HBL-100

MDA-453

cause

number:chromosome

of the

8
0i0@0@

It

17 Signals

100

that
Another

ing cellpopulations (27),might also lead to less linkage between gene

copy

ERBB-2 Signals/Chr

10

is

entire

8-fold
(26).

nofluorescence intensity, which is much less of a factor when the

@c@cP6
oo0

gc@

only

blotting)

was

p < 0.004
F]

c@ @C

ei @
@C

e@ e@ r-4e@ e-1r'@

Fig. 6. ERBB.2 gene expression and amplification in individual MDA-453 cells. Fl

ERBB-2 Signals/Cell

plotted against: A, ERBB-2 copy number; B, ERBB-2:chromosome 17 ratio; and C,

chromosome i7 copy number. Cells were labeled with antibody (CB1 1) against the

intracellular domain of pi8S'@'@2protein. Data from 239 cells are shown. There is a good

correlation between p185HER2 expression and copy number of either ERBB-2 or chro
mosome 17 centromere (A and C) but no correlation between p185@@2 expression and
ERBB.2 to chromosome (Chr) 17 ratio (B).

DISCUSSION

p = 0.72

Overexpression of p185@2 can occur as a result of either DNA

amplification or by increased levels of RNA transcription. Concordant
ERBB-2 gene amplification and p185HER2 overexpression has been
found in both human mammary cancers and cell lines, with good
correlation between the level of ERBB-2 gene amplification and the
average p185HER2 protein overexpression (5, 6, 9, 11, 1416,2325).
However, there has been no prior analysis of this genotype-phenotype
association on a single-cell basis.
To investigate the cell-by-cell basis for the correlation between
ERBB-2 amplification and overexpression, several well-established
breast cancer cell lines having a wide range of gene amplification and
protein expression were studied, as was a primary breast tumor
sample. The levels of gene amplification in the cell lines, as detected
by FISH, were equivalent to those reported previously by Southern
and slot blot analysis (10). We found that the population mean values
for pl85H@2 protein expression were concordant with their average
ERBB-2 gene copy number. However, on a cell-by-cell basis, signif
icant heterogeneity was present both in gene copy number (detected
by FISH) and in protein expression level (by immunofluorescence
using two different p185HER2@specific monoclonal antibodies). The

in

in

1'1 e@

in

in

in

@e e'i @@in in

ERBB-2 Signals/Chr

17 Signals

80

60

40
p < 0.00001

20

ei

@in

I-.

0@

ei

@in

I'.

Chr 17 Signals/Cell
Fig. 7. ERBB-2copy number(A), ERBB-2:chromosome17 ratio (B), and chromosome
(Chr) 17 copy number (C) in unselected and in bright (Fl > 4) MDA-453 cells.
Distribution of bright cells was determined from cells plotted in Fig. 6. Cells that express
p185M5@@.2

at a high

level

have

more

copies

of both

ERBB-2

and chromosome

17 than

does

an unselected population. There was no significant difference between unselected and

bright cells in the distribution

of ERBB-2:chromosome

17 ratio.

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Research.

ERBB.2 EXPRESSION

The positive correlation observed between pl85@@2overexpres

Touch Imprint # B372

100

0
0

0
@o 0
0

AND AMPLIFICATION

6o@@o

sion

o8

000

@r@0:

r = 0.72
.

.1
0

20

40

60

80

120

100

factors

140

@
@

10
0

s/@0o 0

oo@

0c@%@c@o

000

000

supports

the

likelihood

of

and increased

that

predispose

a tumor

cell

to

a DNA

17 polysomy,

index

above

also predispose

diploid,

to the gen

increasing
theopportunity
forselection
of highlytumorigenic
cells
with polyploid DNA content. The fact that we detected cells with
1224chromosome 17 copies, having high p185H@2 expression, is

00
0
0
0
0

@0ck@0a@0
c@l0

consistent

r
0.39

unknown,

10

with the possibility

of a mechanistic

link between

ERBB-2

gene amplification, polyploid DNA content, and chromosome 17

polysomy. Although the nature of this biological link remains largely

number

eration of ERBB-2 gene amplification with p185HE@@2

overexpression.
ERBB-2 amplification also may be associated with mitotic nondis
junctional events, destabilizing the genetic integrity of the cell and

100

copy

together with chromosome

ERBB-2 Signals/Cells

17

overexpression have DNA indices in the triploid to tetraploid range

(13, 2830).Only a few cases have been reported where @185Han@2
overexpressing cells had DNA content close to diploid, and even these
cases had aneuploid elements (13, 28). It is possible that the same

0
0

chromosome

tumor cell DNA index. In fact, most primary tumors with p185HER2

10

and

biological link between ERBB-2 gene amplification

its

between

20

havior

importance

p185HER@2

is

inferred

by

overexpression

and

the

established

aggressive

association

breast

cancer

be

(9, 15, 2932).

ERBB-2 Signals/Chr 17 Signals

100
0

8:88000

Touch Imprint # B372

20'

10

U Average

0@
@

101

EL@@i

@j.

Bright

r = 0.49
@

@
@

10

12

p < 0.0002
14

@
@

@1
.

,-

Chr 17 Signals/Cell
Fig. 8. ERBB-2 gene expression and amplification in single interphase cells of a touch
preparation from a primary breast tumor (case no. B372). Fl plotted against: A, ERBB.2
copy number; B: ERBB-2:chromosome (Chr) 17 ratio; and C, chromosome 17 copy
number. Cells were labeled with antibody (CB11) against the intracellular domain of
pl85'@51@2protein. Data from 129 cells are shown. p185hlFl@2expression correlated well
with copy numbers of both ERBB-2 or chromosome 17 centromere (A and C); p185HER2
expression and ERBB-2:chromosome 17 ratio (B) correlated only weakly.

(Fb.4)

(r
0.000.25). Because chromosome 17 copy number generally
reflects the total DNA content (ploidy) of the ERBB-2 amplified cells
(data not shown), ERBB-2:chromosome 17 ratio is a measure of
ERBB-2 amplification
as traditionally characterized by Southern
blotting analysis. Also, we have reported previously that much of the
ERBB-2 amplification in SKBR3 and BT474 was not located on
chromosome 17 (10), thus, reducing the utility of the ERBB-2:chro
mosome 17 ratio. The weak correlation we observed between ERBB
2:chromosome 17 ratio and protein expression is consistent with
discrepancies reported previously between ERBB-2 Southern analysis
and p185HER2 immunohistochemistry (11). Thus, ERBB-2 amplifica
tion is dependent on the specific method of analysis used and may not
always reflect the absolute copy number of the gene.
Surprisingly, cellular expression correlated with increasing chro
mosome 17 copy number (r = 0.450.61). The brightest cells by
immunofluorescence, representing <2% of the entire cell population,
had two to three times the overall population's mean chromosome 17
copy number. This small subpopulation of highly p185'@@2 express
ing, highly aneuploid cells had the same fraction of S-phase cells as
the remainder of the population. A similar highly overexpressing and
chromosome 17 polysomic subpopulation was also observed in the
primary tumor sample, suggesting that this is not an in vitro artifact.

,-

r@

@;lFt@ .1@

inin
i

ni
nininin
i
n
in

in

@O @C I'-

t'-

0@ 0@

@l

ERBB-2 Signals/Cell
30

c)
0

20
p < 0.0002
10
0 .

SignalS40Q

I I

1;'.1
.@...

e't I'-ERBB-2
@in CI'r-1

O@

Signals/Chr

r@ r@ @in

@C

17

.J@ii,j@
@@
@C

Chr 17 Signals/Cell
Fig. 9. ERBB-2 copy number (A), ERBB-2:chromosome (Chr) 17 ratio (B), and
chromosome 17 copy number (C) in unselected and in bright (Fl > 4) interphase cells of
a touch preparation from a primary breast tumor (case no. B372). Distributions of bright

cells were determined from cells plotted in Fig. 8. Cells that express p185@'2at a high
level have significantly more copies of ERBB-2 and chromosome
of the two, than an unselected population.

17, and a higher ratio

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ERBB.2 EXPRESSION AND AMPLIFICATION

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