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Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
Intratumor
Jnos
@185HER2Overexpression,
and
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
neity
observed
has been
for both
product,
pl85@2. Heteroge
number
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
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
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
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,
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
AND METhODS
research
States-Hungarian
2 Present
was
supported
by
NIH
Grants
CA-49056
and
CA-44768
and
by
United
address:
Department
of Biophysics,
Medical
University
School
of Debrecen,
whom
requests
for
reprints
should
be addressed,
trypsin
or 25 mM EDTA
fluorescence measurements,
immuno
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)
4768218;
E-mail:
waldman@
4 The
abbreviations
used
are:
FISH,
fluorescence
in
situ
hybridization;
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,
slides
autofluorescence
significantly.
IgG (1:100
dilution;
Sigma)
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
at 4C overnight.
Cells on
were
refixed
in methanol:acetic
acid (3:1;
Carnoys
solution)
and air
SSC at 73Cfor
slides could be stored in ethanol at 4Cfor not more than 2 months. Slides were
then preblocked
in 5% Carnation
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
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
deteri
Trypsinization
caused
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;
hydrochloride (Molec
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,
the extracellular
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.
quantitatively
analyzed
objective.
Images
were
processed
using Scil-Image
and
software
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
cells.
pericentromeric
sequence
(p17H8;
17
FISH analysis.
(Boehringer
Mannheim,
Indianapolis,
squashed,
by
procedure. Slides were first stained for protein expression and fluorescence
smeared,
or overlapping
nuclei
were
ignored.
Each
hybridization
was accomplished
by
to collect
sequential,
properly
registered
images
of the
Analysis.
the
@185HER.2bright
were determined
5401
Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
@. Lu1
.@
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'@
ERBB-2 Signals/Cell
c)
100
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
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
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
Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
B
4/
I
a?
4,
C
,@
..,
0
4
C
7
..
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.
Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
ERBB-2 EXPRESSION
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
17 Signals
100
differ.
(caseno.B372)areshowninFigs.8 and9.Positive
correlations
were
0@6'@0@
00
number
000
copy
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
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
in Q in Q in in
,,
@, ,,@
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
population
b@
20@
,.@
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
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
in Fig.4. Cellsthatexpressedp185HER2
at a highlevelhadsignificantlymorecopiesof
ERBB-2andchromosome17,anda higherratioof thetwo,thananunselectedpopulation.
5404
Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
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).
ERBB-2
r = 0.63
@
.1
10
20
30
40
transcription
and
translation
rates,
or in the
half-lives
of RNA
50
between
genotype
and
phenotype.
ERBB-2
transcript
levels
in ampli
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
. 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
ERBB-2 Signals/Chr
10
is
entire
8-fold
(26).
@c@cP6
oo0
gc@
only
blotting)
was
p < 0.004
F]
c@ @C
ei @
@C
e@ e@ r-4e@ e-1r'@
ERBB-2 Signals/Cell
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
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
of ERBB-2:chromosome
17 ratio.
5405
Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
ERBB.2 EXPRESSION
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
of a mechanistic
link between
ERBB-2
number
100
copy
ERBB-2 Signals/Cells
17
0
0
chromosome
tumor cell DNA index. In fact, most primary tumors with p185HER2
10
and
its
between
20
havior
importance
p185HER@2
is
inferred
by
overexpression
and
the
established
aggressive
association
breast
cancer
be
8:88000
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.
5406
Downloaded from cancerres.aacrjournals.org on October 15, 2014. 1995 American Association for Cancer
Research.
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U., Levinson,
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25.
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