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Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WLHuman breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science (Wash DC) 235: 177-182

Authors:
  • Gary Clark Statistical Consulting, LLC

Abstract and Figures

The HER-2/neu oncogene is a member of the erbB-like oncogene family, and is related to, but distinct from, the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines. In the current study, alterations of the gene in 189 primary human breast cancers were investigated. HER-2/neu was found to be amplified from 2- to greater than 20-fold in 30% of the tumors. Correlation of gene amplification with several disease parameters was evaluated. Amplification of the HER-2/neu gene was a significant predictor of both overall survival and time to relapse in patients with breast cancer. It retained its significance even when adjustments were made for other known prognostic factors. Moreover, HER-2/neu amplification had greater prognostic value than most currently used prognostic factors, including hormonal-receptor status, in lymph node-positive disease. These data indicate that this gene may play a role in the biologic behavior and/or pathogenesis of human breast cancer.
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DOI: 10.1126/science.3798106
, 177 (1987); 235Science et al.DJ Slamon,
oncogene
survival with amplification of the HER-2/neu
Human breast cancer: correlation of relapse and
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BIOM 255 (Leffert) – Discussion Feb. 1, 2007
Human
Breast
Cancer:
Correlation
of
Relapse
and
Survival
with
Amplification
of
the
HER-2lneu
Oncogene
DENNIS
J.
SLAMON,*
GARY
M.
CLARK,
STEVEN
G.
WONG,
WENDY
J.
LEVIN,
AxEL
ULLRICH,
WILLiAM
L.
McGuIRE
The
HER-2/neu
oncoFene
is
a
member
of
the
erbB-like
oncogene
family,
and
aS
related
to,
but
distinct
firom,
the
epidermal
growth
factor
receptr.
This
gene
has
been
shown
to
be
amplified
i
human
brt
cancer
cell
lines.
In
the
current
study,
alterations
of
the
gene
in
189
primary
human
breast
cancers
were
instigated
HER-2/
neu
was
found
to
be
amplified
frm
2-
to
eater
than
20-
fold
in
30%
of
the
tumors.
Correlation
ofgene
amplifica-
tion
with
several
disease
parameters
was
evaluated
Am-
plification
of
the
HER-2/neu
gene
was
a
significant
pre-
dictor
of
both
overall
survival
and
time
to
relapse
in
patients
with
breast
cancer.
It
retained
its
significance
even
when
adjustments
were
made
for
other
known
prognostic
factors.
Moreover,
HER-2/neu
amplification
had
greater
prognostic
value
than
most
currently
used
prognostic
factors,
incuding
hormonal-receptor
status,
in
lymph
node-positive
disease.
These
data
indicate
that
thbis
gene
may
lay
a
role
in
the
biologic
behavior
and/or
pathogenesis
human
breast
cancer.
HE
EVIDENCE
LINKING
PROTO-ONCOGENES
TO
THE
INDUC-
tion
or
maintenance
of
human
malignancies
is
largely
cir-
cumstantial,
but
has
become
increasingly
compelling.
This
circumstantial
evidence
is
derived
from
studies
of
animal
models,
tumor
cell
lines,
and
actual
human
tumors.
Data
from
animal
models
and
cell
lines
include:
(i)
sequence
homology
between
human
proto-
oncogenes
and
the
viral
oncogenes
of
transforming
retroviruses
that
are
known
to
be
tumorigenic
in
some
species
(1,
2);
(ii)
transfction
studies
showing
the
transforming
potential
of
proto-oncogenes
m
NIH
3T3
cells
and
primary
embryo
fibroblasts
(3-5);
and
(iii)
the
central
role
of
certain
proto-oncogenes
in
tumorigenesis
by
chronic
transforming
retroviruses
such
as
avian
leukosis
virus
(6).
Data
from
human
tumors
include:
(i)
increased
expression
of
specific
proto-
oncogenes
in
some
human
malignancies
(7,
8);
(ii)
localization
of
proto-oncogenes
at
or
near
the
site
of
specific,
tumor-associated
chromosomal
translocations
(9);
and
(iii)
amplification
of
proto-
oncogenes
in
some
human
tumors
(10,
11).
Additional
data
linking
proto-oncogenes
to
cell
growth
is
their
expression
in
response
to
certain
proliferation
signals
(12,
13)
and
their
expression
during
embryonic
development
(14,
15).
More
direct
evidence
comes
from
the
fact
that,
of
the
20
known
proto-
oncogenes,
three
are
related
to
a
growth
factor
or
a
growth
factor
receptor.
These
genes
include
c-sis,
which
is
homologous
to
the
9
jANUARY
I987
transforming
gene
of
the
simian
sarcoma
virus
and
is
the
1B
chain
of
platelet-derived
growth
factor
(PDGF)
(16,
17);
c-fis,
which
is
homologous
to
the
transforming
gene
of
the
feline
sarcoma
virus
and
is
closely
related
to
the
macrophage
colony-stimulating
factor
receptor
(CSF-1R)
(18);
and
c-erbB,
which
encodes
the
EGF
receptor
(EGFR)
and
is
highly
homologous
to
the
transforming
gene
of
the
avian
erythroblastosis
virus
(19).
The
two
receptor-
related
proto-oncogenes,
c-fins
and
c-erbB,
are
members
of
the
tyrosine-specific
protein
kinase
family
to
which
many
proto-onco-
genes
belong.
Recently,
a
novel
transforming
gene
was
identified
as
a
result
of
transfction
studies
with
DNA
from
chemically
induced
rat
neu-
roglioblastomas
(20).
This
gene,
called
neu,
was
shown
to
be
related
to,
but
distinct
from,
the
c-erbB
proto-oncogene
(21).
By
means
of
v-erbB
and
human
EGFR
as
probes
to
screen
human
genomic
and
complementary
DNA
(cDNA)
libraries,
two
other
groups
indepen-
dently
isolated
human
erbB-related
genes
that
they
called
HER-2
(22)
and
c-erbB-2
(23).
Subsequent
sequence
analysis
and
chromo-
somal
mapping
studies
revealed
all
three
genes
(neu,
c-erbB-2,
and
HER-2)
to
be
the
same
(22,
24,
25).
A
fourth
group,
also
using
v-
erbB
as
a
probe,
identified
the
same
gene
in
a
mammary
carcinoma
cell
line,
MAC
117,
where
it
was
found
to
be
amplified
five-
to
ten-
fold
(26).
This
gene,
which
we
will
call
HER-2/neu,
encodes
a
new
member
of
the
tyrosine
kinase
family;
and
is
dosely
related
to,
but
distinct
from,
the
EGFR
gene
(22).
HER-2/neu
differs
from
EGFR
in
that
it
is
found
on
band
q21
of
chromosome
17
(22,
24,
25),
as
compared
to
band
pll.-p13
of
chromosome
7,
where
the
EGFR
gene
is
located
(27).
Also,
the
HER-2/neu
gene
generates
a
messenger
RNA
(mRNA)
of
4.8
kb
(22),
which
differs
from
the
5.8-
and
10-
kb
transcipts
for
the
EGFR
gene
(28).
Finally,
the
protein
encoded
by
the
HER-2/neu
gene
is
185,000
daltons
(21),
as
compared
to
the
170,000-dalton
protein
encoded
by
the
EGFR
gene.
Conversely,
on
the
basis
of
sequence
data,
HER-2/neu
is
more
closely
related
to
the
EGFR
gene
than
to
other
members
of
the
tyrosine
kinase
family
(22).
Like
the
EGFR
protem,
HER-2/neu
has
an
extracellular
domain,
a
transmembrane
domain
that
includes
two
cysteine-rich
repeat
dusters,
and
an
intracellular
kinase
domain
(21),
indicating
D.
J.
Slamon,
S.
G.
Wong,
and
W.
J.
Levin
are
in
the
Division
of
Hematology-
Oncokogy,
Department
of
Medicine
and
Jonsson
Comprehensive
Cancer
Center,
UCLA
School
of
Medicine,
Los
Angcels,
CA
90024.
G.
AM.
Clark
and
W.
L.
McGuire
are
in
the
Division
of
Oncology,
Department
of
Medicine,
University
of
Texas
Health
Science
Ccnter
at
San
Antonio,
San
Antonio,
TX
78284.
A.
Utlrich
is
in
the
Deparmenet
of
Molecular
Biology,
Gcnentech,
Inc.,
South
San
Francisco,
CA
94080.
*To
whom
correspondence
should
be
addressed.
ARTICLES
177
on January 15, 2007 www.sciencemag.orgDownloaded from
BIOM 255 (Leffert) – Discussion Feb. 1, 2007
that
it
too
is
likely
to
be
a
cellular
receptor
for
an
as
yet
unidentified
ligand.
As
a
result
of
the
published
data
showing
amplification
of
HER-
2/neu
in
a
human
mammary
carcinoma
cell
line,
and
as
part
of
an
ongoing
survey
in
our
laboratory
of
proto-oncogene
abnormalities
in
human
tumors,
we
evaluated
alterations
of
the
HER-2/neu
gene
in
a
large
series
of
human
primary
breast
cancers.
Our
results
show
that
amplification
of
this
gene
occurs
relatively
frequently
in
breast
cancer,
and
that
it
is
associated
with
disease
relapse
and
overall
patient
survival.
Factors
that
are
known
to
be
important
in
the
prognosis
of
breast
malignancies
in
individual
patients
include:
size
of
the
primary
tumor,
stage
of
disease
at
diagnosis,
hormonal
receptor
status,
and
number
of
axillary
lymph
nodes
involved
with
disease
(positive
nodes)
(29).
The
current
study,
which
was
conducted
in
two
parts,
involved
the
evaluation
of
tissue
from
189
separate
breast
malignan-
cies
that
were
part
of
a
breast
cancer
study
ongoing
at
the
University
of
Texas,
San
Antonio.
This
cohort
of
tumors
was
of
interest
because
considerable
information
was
available
on
the
majority
of
the
specimens
including
size
of
the
primary
tumor,
estrogen
recep-
tor
status,
progesterone
receptor
status,
age
of
patient,
disease
stage,
and
status
of
the
axillary
lymph
nodes.
In
the
initial
survey,
tissue
from
103
primary
breast
cancers
was
evaluated
for
alterations
in
the
HER-2/neu
gene.
DNA
from
individual
tumors
was
prepared
as
described
(30),
digested
with
Eco
RI,
and
subjected
to
Southern
blot
analysis
with
a
32P-labeled
HER-2/neu-1
probe,
which
is
known
to
detect
a
13-kb
hybridizing
band
in
human
DNA
(22).
Examples
of
tumors
from
the
initial
survey
are
shown
in
Fig.
1.
Of
the
103
samples
examined,
19
(18%)
showed
evidence
of
HER-2/neu
gene
amplification.
The
degree
of
amplification
in
individual
cases
was
determined
by
dilution
analysis
(Fig.
2A),
as
well
as
soft
laser
densitometry
scanning.
To
determine
that
the
amount
of
DNA
loaded
in
each
lane
was
equivalent,
all
filters
were
washed
and
rehybridized
with
a
32P-labeled
arginase
gene
probe
(31).
This
probe
identifies
a
15-kb
hybridizing
band
on
Eco
RI-digested
human
DNA,
and
was
selected
as
a
control
because
it
more
appropriately
assesses
the
relative
amount
and
Fig.
1.
Analysis
of
alterations
of
the
HER-2/neu
gene
in
human
breast
cancer.
Shown
are
79
of
the
189
breast
tumors
used
in
this
analysis.
Tumors
with
a
single
copy
of
HER-2/neu:
3,
4,
10
to
15,
20,
23
to
25,
27
to
29,
31,
38,
42
to
46,
48,
49,
52,
55,
61,
65,
66,
71,
72,
and
74.
Tumors
with
two
to
five
copies
of
HER-2/neu:
1,2,
5,
7,
9,
16,
17,
19,
21,
22,
32,
35,
36,
47,
50,
54,
56
to
58,
60,
62,
70,
and
75
to
77.
Tumors
with
5
to
20
copies
of
HER-2/neu:
6,
8,
26,
34,
37,
39
to
41,
51,
53,
63,
64,
67,
69,
73,
and
79.
Tumors
with
more
than
20
copies
of
HER-2/neu:
18,
30,
33,
59,68,
and
78.
Examples
of
tumors
77
to
79
have
rearrangements
in
the
HER-2/neu
gene.
DNA
was
extracted
from
tissues
and
digested
with
Eco
RI
as
described
(30).
A
total
of
12
,ug
of
Eco
RI-
digested
DNA
was
loaded
onto
0.8%
agarose
gels,
separated
by
electrophoresis,
and
transferred
onto
nylon
filter
papers
(Biodyne)
(30).
All
filters
were
baked
in
a
vacuum
oven
for
3
hours
at
80°C,
prehybridized
in
5
x
SSC
(standard
saline
citrate)
containing
50%
formamide,
10%
dextran
sulfate,
0.1%
SDS,
denatured
salmon
sperm
DNA
(1
mg/
mnl),
and
4x
Denhardts
solution
for
12
hours,
then
hybridized
in
the
same
solution
containing
32P-labeled
nick-translated
HER-2
probe
(21)
specific
activity
of
1
x
108
cpm
per
microgram
of
DNA;
2
x
106
cpm/ml.
Hybridization
occurred
at
42°C
for
48
hours,
followed
by
washing
of
filters
under
the
following
conditions
in
succes-
178
Table
1.
Association
between
HER-2lneu
amplification
and
disease
parame-
ters
in
103
breast
tumors.
Number
of
tumors
Factor*
Single
2
to
5
5 to
20
>20
Total
Pt
copy
copies
copies
copies
Hormonal
receptor
status
ER+
53
2
9
1
65
0.99
ER-
31
1
2
4
38
PgR+
42
2
6
2
52
0.85
PgR-
42
1
5
3
51
Tumor
size
(centimate,r)
c<2
13
1
1
0
15
0.82
2-5
34
1
5
1
41
>5
17
1
2
2
22
Unknown
20
0
3
2
25
Age
at
diagnosis
(yeats)
c50
21
1
2
1
25
0.83
>50
52
2
7
4
65
Unknown
11
0
2
0
13
Number
of
positive
Iymh
nodes
0
30
0
3
1
34
0.11
1-3
20
0
1
1
22
>3
17
2
4
2
25
Unknown
17
1
3
1
22
*Receptor
status
was
analyzed
as
described
(39).
ER,
estrogen
receptor:
+
and
-
refer
to
the
prcsence
or
absence
of
:3
fmol
of
receptor
per
milligram
of
protein.
PgR,
progesterone
receptor:
+
and
-
refers
to
the
presence
or
absence
of25
fmol
of
receptor
per
milL:am
of
protein.
tStatistical
anaf,ses
for
correlation
of
HER-2/neu
amplifi-
cation
with
discase
parameters
were
performed
by
the
x2
test.
P
values
were
computed
after
combining
the
cases
with
5
to
20
and
>20
copies.
transfer
of
high
molecular
weight
species
than
a
probe
hybridizing
with
low
molecular
weight
species,
which
transfer
more
readily
on
Southern
blotting.
All
lanes
were
shown
to
contain
equivalent
amounts
of
high
molecular
weight
DNA
(Fig.
2B).
Individual
tumors
were
assigned
to
groups
containing
a
single
copy,
2
to
5
copies,
5
to
20
copies,
and
greater
than
20
copies
of
the
HER-2/neu
gene
(Fig.
1).
Assignment
of
tumors
to
the
various
groups
was
done
u_0-S;0 ,-M; 60S_tJ
~~~~~~~~~~kb
tN
~~~~~~~~~~~~~~~~-12
sion:
2x
SSC
for
20
minutes
at
room
tempera-
0.5x
SSC,
0.1%
SDS
at
650C.
Filters
were
then
ture;
two
washes
of
30
minutes
each
in
2
x
SSC,
exposed
to
XAR-5
x-ray
film
(Kodak)
for
autora-
0.1%
SDS
at
65°C;
one
wash
of
30
minutes
in
diography.
SCIENCE,
VOL.
235
on January 15, 2007 www.sciencemag.orgDownloaded from
BIOM 255 (Leffert) – Discussion Feb. 1, 2007
Fig.
2.
(A)
Example
of
dilutional
analysis
to
assess
degree
of
HER-2/neu
gene
amplification.
Lanes
a,
g,
k,
and
p
were
loaded
with
12
,ug
of
Eco
L1-
digested
breast
tumor
DNA.
Lane
a
is
DNA
from
tumor
31
(Fig.
1),
which
represents
a
tumor
with
a
single
copy
of
the
HER/2-neu
gene.
Lane
g
is
DNA
from
tumor
33,
which
represents
a
tumor
with
>20
copies
of
the
HER-2/neu
gene.
Lanes
b
to
f
are
serial
dilutions
(1:100,
1:
20,
1:10,
1:
5,
and
1:
2,
respectively)
of
the
DNA
sample
in
lane
g.
Lane
k
is
DNA
from
tumor
35
(Fig.
1),
which
represents
a
tumor
containing
two
to
five
copies
of
the
HER-2/neu
gene.
Lanes
h
to
j
are
serial
dilutions
(1:10,
1:
5,
and
1:
2,
respectively)
of
the
DNA
sample
in
lane
k.
Lane
p
is
DNA
from
tumor
34
(Fig.
1),
which
represents a
tumor
with
5
to
20
copies
of
the
HER-2/neu
gene.
Lanes
I
to
o
are
serial
dilutions
(1:
20,
1:10,
1:
5,
and
1:2,
respective-
ly)
of
the
DNA
sample
in
lane
p.
The
filter
was
prepared
and
hybridized
with
a
P-labeled
HER-2
probe
as
in
Fig.
1.
(B)
Example
of
arginase
probe
hybridization
to
demonstrate
that
equivalent
amounts
of
tumor
DNA
were
loaded
into
each
lane.
Rehybridization
of
filter
containing
lanes
30
to
40
(Fig.
1).
The
filter
was
first
stripped
of
label
by
washing
in
a
buffer
made
up
of
50%
formamide,
3x
SSC,
and
0.1%
SDS
at
65°C
for
20
minutes,
following
by
three
successive
washes
of
5
minutes
each
in
0.1
x
SSC
at
room
temperature.
Filters
were
exposed
overnight
on
XAR-5
film
(Kodak)
to
ensure
removal
of
all
radioactive
probe,
then
rehybridized
as
in
Fig.
1
with
a
32P-labeled
human
arginase
gene
probe
(31).
in
a
blinded
fashion,
in
that
they
were
made
without
knowledge
of
disease
parameters.
Analysis
of
the
data
for
association
between
gene
amplification
and
a
number
of
disease
parameters
was
then
per-
formed.
Of
103
tumors
evaluated
in