Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma.

W Xia, Y K Lau, H Z Zhang, et al. Clin Cancer Res 1997;3:3-9.

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Vol. 3, 3-9,

January

1997

Clinical

Cancer

Research

Advances

in Brief

Strong Survival
Weiya Ai-Ru Gary Xia, Liu,

Correlation of Patients
Yiu-Keung Lei Li, Lau, Nobutaka Ruth

between with
Hua-Zhong Kiyokawa, and

c-erbB-2 Oral
Zhang,

Overexpression Cell

and

Overall

Squamous
Introduction

Carcinoma

The WHO
common cancers

has described
in the 91%
with

oral cancer
(1). Oral

as one of the 10 most

SCC3 is the most

L. Clayman, Hung

L. Katz,

world

Mien-Chie
Department

common
approximately
sis of

histopathological
patients oral SCC

type

of oral cancer,
poor; the

accounting
5-year survival

for

of all oral

malignancies
remains

(1, 2). The

progno-

of Tumor Biology and Breast Cancer Basic Research 1W. X., Y-K. L., N. K., M-C. H.}, Sections of Cytology and Image Analysis, Department of Pathology [H-Z. Z., R. L. K.], and Department of Head and Neck Surgery [G. L. C.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Department of Oral Pathology, Shanghai Stomatology College, Shanghai Second Medical University, 200016 Peoples Republic of

Program

rate averages

about

50%

and has not changed

is interest occur in behavior. prognostic

much

over

the

past 10 years (3). Therefore, there whether specific gene alterations

in determining oral cancer and Such markers studies and

whether
may

they

are more

correlated accurate

to disease and useful

provide

China

[A-R.

L., L. L.]

Abstract
Overexpression has been of c-erbB-2 (also known as HER-2lneu)

eventually more effective treatment. The c-erbB-2 gene (also known as HER-2/neu) encodes a transmembrane protein of Mr 185,000 (l8S), with extensive
sequence homology to EGFR (4-6). Like EGFR, c-erbB-2 has with collaintrinsic tyrosine kinase activity many different cellular proteins, (7-10) and can interact such as src-homologous

neck
med

found squamous expression

samples

tumor
stages

in many human cancers, including head and cell carcinoma (SCC). We therefore examof the oncoprotein in oral SCC primary and compared its relationship with clinical

gen, phospholipase
mediate of c-erbB-2 promoting metastatic may play pression several stomach has been between
reported

C-rny, and GTPase-activating

transduction shown (13). This role multiple pathway to increase and suggests adhesion metastatic invasion that the

protein,
potential steps c-erbB-2 survival lung, c-erbB-2

which
by the gene overexof and

a signal the cascade

(1 1, 12). Overexpression of

has been

and survival rate. Out of 80 cases of oral SCC, high expression level (+ + or + + +) of c-erbB-2 was found in 41 cases. Of the 80 cases with follow-up information, 39 were further investigated for the correlation of expression level of c-erbB-2 and survival rate. Overexpression of the oncoprotein was significantly correlated with shorter overall survival, and the patients with low and no expression of cerbB-2 had much higher survival rates. Overexpression of c-erbB-2 was also significantly correlated with nodal stage and metastasis. We found that high expression level of cerbB-2 was frequently detected in oral cancer cell lines but not in the other head and neck SCC cell lines. Thus, we conclude that overexpression of c-erbB-2 is a frequent event in oral SCC and is correlated with poor survival and may be
used as a poor prognostic factor.

an important

in carcinogenesis.

has been found to correlate with poor human cancers, including breast, ovarian, cancers detected c-erbB-2
in oral

(14-20). in head SCC.

Although and neck and

c-erbB-2 SCC (21-23), survival

overexpression a correlation has not been

overexpression

The purposes
of the c-erbB-2

of this study
in oral tumor and

were to examine
specimens as well SCC.

the expression
as in head For the and latter,

neck

SCC

cell lines
expression

and to determine
survival

the relationship
in oral

between

the protein

we investigated retrospectively a group of patients who had been treated by surgery with or without postoperative chemotherapy, radiotherapy, or both. Our results indicate that overexpression of c-erbB-2 is significantly correlated with shorter

overall
Materials

survival
and

in oral SCC.
Methods Eighty Shanghai specimens Second of of Oral Medical hospital primary University, oral from the Department Pathology,

Patient
Received 6/24/96; revised 10/4196;
of this

Specimens.
obtained Hospital, surgical primary

accepted
article were

10/9/96.

SCC Ninth

were

defrayed in part by the This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I The research was supported in part by United States Department of Health and Human Services Cancer Center Core Support Grant CA16672 from the National Cancer Institute, NIH Grants CA58880 and CA60856 (to M-C. H.), and Grant no. DAMD 17-94-J4315 (to M-C. H.) from the Department of Defense. 2 To whom requests for reprints should be addressed, at Box 079, Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-3668; Fax: (713) 794-4784. The costs of publication payment of page charges.

Peoples underwent The

Peoples
female) 54 years).

Republic

of China.

These
treatment

patients
in the graded

(60 male

and
between

20

1989 and 1990. Their

ages ranged
tumors

from

10 to 76 years

(median,
clas-

were

by the WHO

3 The abbreviations used are: 5CC, squamous cell carcinoma; epidermal growth factor receptor; MOD, mean optical density; tumor-node-metastasis.

EGFR,
ThM,

c-erbB-2

Expression

in Oral SCC

Ta ble 1

Relationship

betw een

histological

gra de and Level

c-erbB-2

expression

in oral

SCCs

of expressionb
+ + +1%

Histological I
II III
a b

grade

No. of cases 63 (78.75) 14 (17.5)

3 (3.75)

+ + +

positive 85.70
92.90

8(10)

7 (8.75)
0 (0)

Total p < 0.01 by the

No. of cases (%).

80(100)

15 (18.75)

19(23.75) 4 (5.00) 3 (3.75) 26 (32.5)

36(45)

3 (3.75)
0 (0) 39 (48.75)

100.00
86.40

x2 test.

sification were
tissues cases),

of histological as grade
of the involved floor in tumor mouth

differentiation I, 14 as grade
included (8 cases), tongue palate

(24). II, and

(40

Sixty-three 3 as grade
cases),

cases Ill.
gum

cytoplasmic
with strong

and
staining

membrane
(>50%

staining
cells

were
stained)

observed.
were scored

The

cases

defined

The
(11

as high,

(5 cases), (1 case), specimens sectioned

cheek and were

(10 rou-

those with moderate mediate, those with low, and those with was with Products
The

staining (20-50% cells weak staining (up to-20% no staining as negative.

stained) as intercells stained) as

cases), tinely

mucosa fixed

of lip (3 cases), molar areas embedded

mandible The

mucosa serially

of mandible

(2 cases).

Quantitative
immunostaining image analysis tem (Imaging

Computerized
objectively a SAMBA International,
image analyzer

Image
quantified 4000 Cell Inc.,
was

Analysis.
by Image Chantilly,
an integrated

c-erbB-2
computerized Analysis VA),
system

in formalin,

in paraffin, H&E. was Accurate available. the after They

every 5 p.m, and stained with information on 39 of 80 patients tients overall ment. were therefore and used c-erbB-2 survival Since

and detailed These 39 pabetween either treatof the primary received surgical died of the have

Sysand a
of

to analyze expression. or both died

correlation their

20 objective.

Windows sitometric, intensity of Nikon JVC

(Microsoft, morphometric, colorimetric microscope KY-17U

Redmond, and analysis with video

X

WA)-based red-green-blue of cells and The X40

software to hue

for densaturation and a image with a Syquest microcolor video capaseveral for the stroma, values was with of negative

chemotherapy

or radiotherapy the surgical one has

treatment,

10 patients of metastasis The from

deparaffinized

and tissues SAMBA

and consisted objectives 4000

primary tumor and tumor to the lung. Immunohistochemical dase staining

Briefly,

10, X20, camera.

3-CCD

Staining. used described was modified by Hsu

were

immunoperoxithe avidin-biotin (25) and Hsu

and dehy-

method
tissue

analyzer consisted of an IBM 80-386/33-MHz clone VGA graphics board, 80-MB hard disc, 8 MB of RAM, et 88-MB scope, video removable cartridge system, Nikon Labophot-2 Hewlett Packard PaintJet color printer, Mitsubishi copy processor (printer), and Polaroid freeze-frame recorder with 35-mm film processing and instant prints bility. The fields best software enabled slides, Similarly, averaging in which the between the operator, to set a density cell results nuclei from antibody a threshold the primary after and for evaluating value cytoplasm, positive 10 fields on control discrimination by tissues threshold

complex al. (26).

technique

and Raine

sections

drated in a graded series of alcohol. Then, they were digested in 0.05% trypsin for 15 mm, blocked in 0.3% H202 in methanol for 15 mm, and treated with 1% (vlv) normal horse serum for 30 mm. with Then, 0.003 the slides were pg/ml c-neu incubated for 3 h at room temperature (Ab-3) monoclonal antibody diluted PBS, with in PBS. the slides biotinylated The slides were goat were with The
ami-

1:3200. incubated antimouse

After extensive washing with for 30 mm at room temperature IgG antibody diluted 1:200

or background. obtained control

subsequently incubated avidin-biotin-peroxidase

peroxidase-catalyzed

for 60 mm at room temperature complex diluted 1 :100 in PBS.

product was visualized with 0.125%

was replaced

an isotype-matched irrelevant antibody. The threshold value was the minimum absorbance at which the image analyzer would discriminate that value performed negatively from positively stained cytoplasm. After was read, background on every tissue after tissue, we measured (labeled nuclear score (labeling subtraction was automatically measuring an empty field on the the cytoplasmic area/total nuclear index X MOD). area, area To MOD, measpermit the techas an

no-ethyl Co.). (pH 7.6)

carbazole Between three and steps,

chromogen the slides After (Sigma times. mounted.

stock were light

solution rinsed

(Sigma for 2 minutes with the in which

Chemical in PBS Mayers were was PBS

counterstaining Chemical controls, Co.), were run strongly

modified dehydrated used instead of staining.

hematoxylin

slides

Negative antibody, identified

slide. For each labeling index ured), and

of the primary A previously

with each batch staining tumor and posThe Those were immunoreviewed of Both

quick

tissue section was used as a positive control. Inter-assay intra-assay consistency was maintained by including these itive and negative controls with each batch of slides stained. prepared tumor considered staining c-erbB-2 and were was independently staining cells slides that were were examined immunostained and two those negative. pathologists. into four groups of positively cells The stained by light with microscopy. red granules any were

simultaneous measurement of c-erbB-2 immunostaining, samples were immunostained by avidin-biotin complex nique (25, 26), with amino-ethyl carbazole chromogen indicator and Mayer s hematoxylin solution as a counterstain. Statistical Analyses. contingency tables, with
significance. Survival

The x2 test was used for analysis of P < 0.05 as the criterion of statistical
were calculated by the method of

positive, considered by ranked intensity

without

curves

All the cases according

Kaplan lyzed lines

and

Meier,

and

differences test.

between

curves and neck

were SCC and

anacell Neck

immunoreactivity to percentage cells. tumor

by the Wilcoxon

Cell Lines
were

and Culture.
from the

Seven
Department

head

obtained

of Head

Clinical

Cancer

Research

t I
.

,.

p
..

.
4

I
-

1.

,*p
#

c ..

,v

-
.i.

*?
:

I,

Is
I

#{149}

.#{248}:.
A,

I
C,

Fig.

Immunohistochemical

staining

of oral SCC for c-erbB-2.

high;

B,

intermediate;

low;

D,

no expression.

X400.

Surgery Center. the tongue.

of

the The

University was other derived cell

of Texas from were lines

M. D. lymph described

Anderson node previously

Cancer of (27).

with

10%

fetal

bovine incubator 90% with buffer 1%

serum

and

antibiotics.

Cells 5% CO2

were in air.

grown

6861LN-l

metastasis

in a humidified

at 37#{176}C under

Cell Lysis
grown washed of RIPA-B 1% Triton to about twice lysis

and Western
confluence cold PBS (20 mt aprotinin,

Blot
and

Analysis.
harvesting. lysed pH 7.4,

The cells
They 150 mi on ice with

were
were 0.5 ml NaCI,

The human overexpression immunoblotting Fl2 (Life

breast cancer of c-erbB-2, experiment. Technologies,

cell line MDA-MB-453, was used as a positive All Inc., cells were Island, grown NY) Grand

which has control in the in DMEM/ supplemented

before then Na2PO4, 1 mM

X-lOO,

phenylmethysulfonyl

c-erbB-2

Expression

in Oral SCC

fluoride, scraped debris samples torphoresis specific buffer was then (5% was

100 mri NaF, from were and membrane nonfat incubated antibody, by incubating antibody the tissue removed

and

2 nmi Na3VO4). culture dishes, For

The and

cells the

were

then cell the elecNon-

Table

Characteristics

of 39 oral SCCs No. of patients 22 17 29 10

19

residual gel

Patient

characteristics

by centrifugation. by 6% was primary

immunobloning, membrane. with The (c-neu

separated transferred

SDS-polyacrylamide minimized 20-PBS). antibodies

to a nitrocellulose in Tween

binding dry milk with

a blocking membrane (Ab-3) NY) anti-

monoclonal followed mouse IN).

Oncogene Science, with horseradish Mannheim

Inc., Uniondale, peroxidase-goat Corp., Indianapolis,

(Boehringer

Results
Immunohistochemical Staining of immunostaining are shown of c-erbB-2 expression Fifteen and of Oral 5CC 1 . There showed 26 low/no levels for oral was high (32.5%) of in Fig. 1. I disease and all 3

c-erbB-2.
SCCs wide oral levels showed variation SCC of

The

results in the level

of 80 primary in Table of them expression staining, showed

for c-erbB-2 specimens. cytoplasmic medium

in the primary

( 1 8.8%)
membrane

levels,

and 39 (48.8%)

immunoreactivity. For c-erbB-2 (85.7%), 13 of cases of grade ing. Statistical grade c-erbB-2 was

A representative expression, 54 of 14 cases of grade

example 63 cases II disease

is shown of grade (92.9%)

III disease (100%) demonstrated analysis by the x2 test showed associated with 1).

positive stainthat histological level of

significantly expression

expression

Correlation

(P < 0.01; Table between c-erbB-2

Expression

and

Poor

Survival. The significance tein in human oral SCC was

of the expression of the oncoprostudied retrospectively in 39 cases Table The

that had been followed up since their surgical treatment. 2 provides clinical data for the 39 oral SCC patients. SAMBA 4000 cell image analysis system was used tively quantitate cytoplasm and membrane specimens to determine the exact amount expression discussed under a light in oral previously microscope SCC. (28). The The reliability tumor with

Age, yrs 50 <50 Sex Male Female Primary tumor site Tongue Gum Floor of mouth Other Histological grades I II ThM Tumor size T1 I2 T3 NOdal stagec N0 N1 N2 N3 Metastasis M0 M1 c-erbB-2 expression Low or none Intermediate High Survival, months after diagnosis <12 12-36 >60 a All patients received surgical treatment. b T1, tumor 2 cm in greatest dimension; cm; T3, tumor >4 cm. C N0, no regional lymph node metastasis;

7 5 8 33 6

20 15 4 29 2 2 6 28 11 20 8 11 4 6 29 T2, tumor >2 cm but 4 to a single nodes (>2

to objec-

N1, metastasis

staining of the tumor of c-erbB-2 protein method were has been observed image

of the specimens from level

lymph node (2 cm); N2, metastasis to one or more lymph cm but 5 cm); N3, metastasis to a lymph node (>5 cm). d distant metastasis; M1, distant metastasis.

connected

a computerized

analysis system. Ten imens were analyzed. immunostaining divided with (20.51%) (51.28%) lation protein into a MOD with with three of

microscopic fields The oncoprotein Next, high expression,

each of the specwas measured as levels (28.2 8 were 1%) cases 1 1 cases

survival test).

rates,

as shown

in Fig.

2 (P

<

0.001 and that cell that

by the Wilcoxon Neck a high lines

absorbency. groups: 26-70;

the expression expression,

Western Lines. c-erbB-2 (Tu 138, Western was

Blot
blot found

Analysis
analysis and Tu in all three

of Head
revealed oral and cancer 167)

SCC
level

Cell
of lines

intermediate

examined four cancer cell c-erbB-2

a MOD a MOD

of 12-26; of 0-10.

and low expression, 20 cases There was a significant corre-

686/LN-l,

the others

between the MOD expression levels relationship is shown with nodal

reading and visual quantification of in the tumor specimens (P < 0.001). TNM staging and 3. The expression as well as metastasis, c-erbB-2 exof c-erbB-2 with statistical

had only low or no expression line, MDA-MB-453, which (Fig. 3). This overexpression expression may result confirms in oral SCC

relative is known the high and also

to the breast to overexpress frequency suggests head and

The pression correlated

between in Table stage,

of c-erbB-2 that c-erbB-2 neck SCCs.

not be common

in other

significance (P < c-erbB-2 expression

0.05 by the x2 test). may be associated experiments increased level overall

The results suggest that with oral cancer metasthat indicated that metastatic potential was rate, signifand the higher survival

Discussion
We association previously between demonstrated that there was and we used between a significant oral cancer a different c-erbB-2 TNM of the

tasis, consistent with animal enhanced c-erbB-2 expression (13). icantly patients Also, high expression with low and associated with

c-erbB-2

overexpression

of the oncoprotein had significantly

progression (23). cohort to further expression stage. The

In the study reported here, investigate the relationship histological was expressed

a shortened no expression

and patient survival, c-erbB-2 gene product

grade, and in 86.4%

Clinical

Cancer

Research

Table

Relationship

be tween

ThM

staging

a nd c-erbB-2

expression Level

in 39 oral SCCs

of expression Low/none 14 (35.90) 6 (15.38) 0(0) P<0.l25

TNM Tumor T1 T2 T3 size

No. of cases 21 (53.85) 13 (33.33) 5(12.82)

(%)

High 4 (10.26) 4 (10.26) 3(7.69)

Intermediate 3 (7.69) 3 (7.69) 2(5.13)

Nodal stage N0
N3

28 (71.79)
2(5.13)
2(5.1)

5 (12.82)
2(5.13)

8 (20.51)
0(0)

15 (38.46)
0(0) 2(5.13)

N2 N3 Metastasis M0 M1 Total
a b p

7(17.95)

0(0) 4(10.26)

0(0) 0(0)

3(7.69) P<0.028

28 (71.79) 11 (28.21) 39(100) were calculated by the

5 (12.82) 6(15.38) 11(28.21)

8 (20.51) 0(0) 8(20.51)

15 (38.46) 5(12.82) P<0.0l5 20 (51.28)

No. of cases (%).

values

x2 test.

Low/none

(n=20)

modimers other

or heterodimers

might

be correlated 5CC. both

to survival cytoplasmic tumor

or

L.

clinicopathological In the study reported membrane We (data staining obtained 1, Vector protein has c-erbB-2 with some argue the

parameters of oral here, we observed of c-erbB-2 protein by using Inc.) validity results

Intermediate
80

(n=8)

and imens.

in our another

spec-

similar The been

antibody staining tumors On the could reports a secreted receptor of the recepfunctions antibodies of have

(NCL-CB1 protein
60
I-

Laboratories,

specific of cytoplasmic Some

to the c-erbB-2 researchers in breast (32). staining several to have kinase members

not shown).

c-erbB-2 demonstrated that

questioned. c-erbB-2

ci) 40

cytoplasmic that c-erbB-2 (33-35).

staining amplification cytoplasmic However, can be detected was reported tyrosine that these

correlates hand,

other
High (n=ll)

be an artifact indicated (36-38). form, (39). These

or nonspecific c-erbB-3 strongly

that EGFR Furthermore, as well as being findings

and c-erbB-2 a transmembrane

in the nucleus

20

suggest

20

40

60

EGFR family not only tors but are also capable inside gave toplasmic It was or even the same outside results;

function as membrane-associated of performing other unknown the cell. therefore, demonstrated In addition, we believe is not multiple that

Months

Fig. 2 Correlation between c-erbB-2 expression and survival rate. Survival curves were calculated by the method of Kaplan and Meier. P values: high versus low, P < 0.001 ; high versus intermediate, P < 0.002. p values were analyzed by the Wilcoxon test.

c-erbB-2

cyof cthe 22). other as well

staining

of tumor

specimens

nonspecific.

previously

that overexpression

erbB-2 could be detected expression of c-erbB-2 These 80 may, c-erbB-2 survival reported significant stage ing was c-erbB-2, and oral SCC gene (P
<

in head and neck 5CC. However, did not correlate to survival (21, included our Western tumor specimens and pharynx, results, from such as the larynx

earlier and neck

reports regions, cavity.

have From

specimens expression 0.001).

examined. was

c-erbB-2 of oral SCC.

overexpression In this with gene study, poor

head

therefore,

be a characteristic Amplification

as the oral

blotting

we found 5CC may and not

significantly of the

correlated
EGFR

has been

that high expression of c-erbB-2 was found (Fig. 3). These results suggest that c-erbB-2 be a characteristic neck show SCCs. This there the correlation of oral may 5CC but why metastatic having resistance in non-small not the explain of c-erbB-2 are five group of c-erbB-2 to induce

only in the oral overexpression of the earlier level (5-year) cell lung other reports and in the head did

in both cell culture and fresh correlation between EGFR also reported c-erbB-3, heterodimers, be interesting and it will expression (31). and Because c-erbB-4, which to analyze

tissue samples (29-31). A levels and tumor size and the EGFR is known trigger EGFR, may family, different c-erbB-3, a subset includhosignal or of hoto form

expression patients a long

survival. low/none survival. carcinoma agents

In our study, c-erbB-2-expressing Overexpression has been shown

modimers pathways, c-erbB-4

to examine whether

to chemotherapeutic

c-erbB-2

Expression

in Oral

SCC

cy,
L()

r c
<

N.

00
I -

-J

N.

o
00
D

ef,
-

00
-

00

I.-

=
I-

00
,-

(Y)
1

I-

Fig. 3 Western blotting analysis of head and neck SCC cell lines. High level of c-erbB-2 expression
was lines 167), lines sion. cancer was found in three oral SCC cell (Tu 138, 6861LN-l, and Tu and that of the other cell had only low or no expresThe c-erbB-2 overexpressing cell line MDA-MB-453 used as positive control.

Blotted

with

anti-erbB-2

I..

(40).
survival

Low
rate

c-erbB-2
in those

expression
cases because

levels
they

may
may

contribute
be more

to longer
sensitive

10.

Yarden,

Y., and

Ullrich,

A. Growth

factor

receptor

tyrosine

kinases.

Annu.
11. Xie,

Rev. Biochem.,
Y., Pendergast,

57: 443-478,
A. M., and rat

1988.
Hung, HER-2/neu. M-C. Dominant-negative

to postsurgery sion protein is common

chemotherapy. in oral SCC, correlated

In conclusion, and with high poor survival.

c-erbB-2 level

expresof the

expression

mutants

of Grb2 induced

reversal

of the transformed

phenotypes
J. Biol. Chem.,

is strongly

by the point 30717-30724,

mutation-activated 1995.

caused 270:

Acknowledgments
We thank
pital, Shanghai

the Department
Second Medical

of Oral Pathology,
University, Shanghai,

Ninth

Peoples

HosRepub-

Peoples

12. Xie, Y., Li, K., and Hung, M-C. Tyrosine phosphorylation of Shc proteins and formation of Shc/Grb2 complex correlate to the transformation of NIH 313 cells mediated by the point-mutation activated neu. Oncogene, 10: 2409-2413, 1995. 13. Yu, D., Wang, lung S-S., Dulski, cells K. M., Tsai, C-M., Nicolson, G. L., and

lic of China,
for proofreading

for providing

the manuscript;

pher Amos for making

patients clinical information; Dr. Hong Lu and Drs. Naoto T. Ueno and Christosuggestions and comments about the manuscript.

Hung, erties.

M-C. c-erbB-2/neu
cancer

overexpression
by induction D. A.,

enhances 1994.
Nordberg,

metastatic

potential
propD. B.,

of human 14. Kern,

of metastasis-associated J. E.,
p185neu

Cancer

Res., 54: 3260-3266,

Schwartz,

References
I. Silverberg,
1995. Edwards, Institute, 3. Silverman, 1796-1799, CA Cancer B. K. 1991.

J. A.,

Weiner, Cancer

E., Boring,
J. Clin., Cancer

C. C., and Squires, 40: 9-26, 1995. B. T., Miller,

Statistics diagnosis review of oral

T. S. Cancer

statistics A. M., and

Cancer

Greene, M. I., Torney, L., and Robinson, R. A. human lung adenocarcinomas predicts shortened
50: 5184-5191, 1990.

Expression

in
Res.,

survival.

2. Ries,

L. A. G., Hankey,

B. A., Hartman,
1973-88. cancer. National Cancer

S. J. Early 1988.

(Phila.),

62:

15. Sozzi, G., Miozzo, M., Tagliabue, E., Calderone, C., Lombardi, L., Pilotti, S., Pastorino, U., Pierotti, M. A., and Della Porta, G. Cytogenetic abnormalities and overexpression of receptors for growth factors in normal bronchial epithelium and tumor samples of lung cancer patients. Cancer Res., 51: 400-404, 1991. 16. Weiner, D. B., Nordberg, J., Robinson, R., Nowell, P. C., Gazdar, A., Greene, M. I., Williams, W. V., Cohen, J. A., and Kern, J. A. Expression of the neu gene-encoded protein (p1 85neu) in human nonsmall cell carcinomas of the lung. Cancer Res., 50: 421-425, 1990.

4. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N., Miyajima, N., Saito, T., and Toyoshima, K. Similarity of protein encoded by the human c-erbB-2 gene to epidermal growth. Nature (Lond.), 319: 226-230, 1986. 5. Bargmann, gene encodes C. I., Hung, M-C., and epidermal growth factor 226-230, 1986. Weinberg, R. A. The neu oncoreceptor-related protein. Nature

(Land.),

x. Y.,
gene

17. Schneider, P. M., Hung, M-C., Chiocca, Fang, K., and Roth, J. A. Differential
in human M-C. small cell 1989. The neu and non-small cell

S. M., Manning, J., Zhao, expression of the c-erbB-2

lung and cancer. breast Cancer cancer. Res., Cancer

319:

6. Schechter, A. L., Hung, Yang-Feng, T. L., Francke,

gene: an erbB-homologous encoding the EGF receptor. 1985.

M-C., Vaidyanathan, L., Weinberg, R. A., U., Ulirich, A., and Coussens, L. The neu
gene distinct from and Science (Washington unlinked to the gene DC), 229: 976-978,

49: 4968-4971,
I 8. Hung,

proto-oncogene

Bull.,

40:

300-303,

1988.

7. Akiyama, Yamamoto,

T., Sudo, C., Ogawara, H., Toyoshima, K., and T. The product of the human c-erbB-2 gene: a 1 85-kilodalton glycoprotein with tyrosine kinase activity. Science (Washington DC), 232: 1644-1646, 1986.
8. Coussens, L., Yang-Feng, T. L., Liao, Y-C., Chen, E., Gray, A.,

19. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and McGuire, W. L. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science (Washington DC), 244: 707-712,
1989. 20. Zhang, X., Silva, tion and rearrangement E., Gershenson, D., and of c-erbB proto-oncogenes Hung, M-C. in cancer Amplificaof human J. R.,

McGrath, Francke,

J., Seeburg, P. H., Libermann, T. A., Schlessinger, U., Levinson, A., and Ullrich, A. Tyrosine kinase receptor

J., with

female
21.

genital
J.

tract. Oncogene,
K., Spandidos, head and neck.

4: 985-989,
D. A.,

1989.
M., Gosney,

extensive homology to EGF receptor neu oncogene. Science (Washington

shares chromosomal location with DC), 230: 1132-1139, 1985.

Field,

Yiagisis,

Papdimitriou,
carcinoma

K., and Stell,

of the

P. M. c-erb-2
Anticancer

expression
Res.,

in squamous 12: 613-620,

cell
1992.

9. Stem, D. F., Heffernan, P. A., and Weinberg, R. A. p185, a product of the neu proto-oncogene, is a receptor-like protein associated with tyrosine kinase activity. Mol. Cell. Biol., 6: 1729-1740, 1986.

22. Craven, M., Kelly,

J. M., Pavelic, Z. P., Stambrook, P. J., Pavelic, L., Gapany, D. J., Bapany, S., and Gluckman, J. L. Expression of

Clinical

Cancer

Research

c-erbB-2
2273-2276, 23. Hou,

gene

in human 1992.

head

and neck

carcinoma.

Anticancer

Res.,

12:

32. ization

Gusterson, of

B. A., Guillick, c-erbB-2 in human

W. J., Vener, breast

D. J., Powles, Br.

T. J., Elliott, J. Cancer,

C., Ashley,
D., Tu, S-M.,

S., Tidy, A., and Harrison,

1988.

S. Immunohistochemical
carcinomas.

L., Shi, Lett.,

Oral
Cancer 24.

cancer

progression 65: 215-220,

Zhang, H-Z., Hung, M-C., and c-erbB-2/neu proto-oncogene

1992.

and Ling, D. expression.

local58:

453-457, 33. Dc Pauwels, lular cells. Int.

Washi, P. N., Cohen, B., Lutrhra, U. K., and Torloni, H. Histological typing of oral and oropharyngeal tumors. In: International Histological Classification of Tumors, No. 4, pp. 17-18. Geneva: WHO, 1971. 25. Hsu, vulgaris S. M., and agglutinin 77: 396-400, S. M., Raine, L. Discovery of a high (PHA) with gastrosecreting 1982. L., and Fanger, in immunoperoxidase H. Use affinity cells. of phaseolus Am J. Clin.

Potter, C. R., C., Verhofstede, of the J. Cancer,

Quatacker, J., C., Eechaute,

neu

Maertens, W., and in human

G., Rods,

Van Daele, S., H. The subcel-

localization

protein

normal

and neoplastic
J., and for haemat-

44: 969-974,

1989.

34. De Potter, C., Beghin, Roels, H. J. The neu-oncogene ogenous metastases M. F., in breast Hung, G.,

A. P., Makar, D., Vandekerckhove, protein as predictive factor cancer Godolphin, patients. W., Int. and J. Cancer, Slamon,

Pathol.,
26. Hsu,

45: 55-58,
D. J. Sensisource Cantyrosine Biochem.

Raine,

idase complex (ABC) between ABC and unlabelled

Cytochem., 29: 577-580,

of avidin-biotin-peroxtechnique: a comparison

1990.
35. Press, tivity of her-2/neu antibodies in archival of error in imrnunohistochemical studies cer Res., 54: 2771-2777, 1994. 36. Xie, Y., and Hung, kinase and its association Biophys. Res. Commun., 37. Marti, U., Burwen, nuclei. M-C. with tissue samples: of oncogene potential expression.

antibody
1981.

(PAP) procedures.

J. Histochem.
M-C., oncoArch.

27. Beckhart, R. N., Kiyokawa, N., Xi, L., Liu, T-J., Hung, El-Naggar, A. K., Zhang, Z-H., and Clayman, G. L. HER-2/neu gene characterization in head and neck squamous cell carcinoma. Otolaryngol. Head & Neck Surg., 121: 1265-1270, 1995.

28. Esteban, J. M., Ahn, C., Mehta, P., and Battifora, H. Biologic significance of quantitative estrogen receptor immunohistochemical assay by image analysis in breast cancer. Am. J. Clin. Pathol., 102: 158-162, 1993. 29. Eisbruch, A., Blick, M., Lee, J. S., Sacks, P. G., and Gutterman, J. Analysis of the epidermal growth factor receptor gene in fresh human head and neck tumors. Cancer Res., 47: 3603-3605, 1987. 30. Ishitoya, J., Toriyama, M., Oguchi, N., Kitamura, K., Ohsima, Asano, K., and Yamamoto, T. Gene amplification and overexpression EGF receptor in squamous cell carcinomas of the head and neck. Cancer, 59: 559-562, 1988. M., of Br. J.

Nuclear localization of pl8S transcriptional transactivation. 203: 1589-1598, 1994. A., Barker,

S. J., Wells, Hepatology,

A. M., and Jones,

in hepatocyte

A. L. Localization

of epidermal
13: 15-20,

M. E., Huling, S., Feren, growth factor receptor

1991.

38. Holt, S. J., Alexander, P., Imman, C. B., and Davies, D. E. Epidermal growth factor induced tyrosine phosphorylation of nuclear proteins associatied with translocation of epidermal growth factor receptor into the nucleus. Biochemical Pharmacol., 47: 1 17-126, 1994. 39. Katoh, M., Yazaki, Y., Sugimura, T., and Terada, encodes secreted as well as transmembrane receptor Biochem. Biophys. Res. Commun., 192: 1189-1197, M. c-erbB-3 tyrosine gene kinase.

1993. N.
Can-

31. Santini, J., Formento, J. L., Francoual, M., Milano, G., Schneider, M., Dassonville, 0., and Demard, F. Characterization, quantification, and potential clinical value of the epidermal growth factor receptor in head and neck squamous cell carcinomas. Head & Neck, 13: 132-139, 1991.

40. Tsai, C-M., Yu, D., Chang, K-T., Wu, L-H., Peng, R-P., Ibrahim, K., and Hung, M-C. Enhanced chemoresistance by elevation of p185e
levels in HER-2/neu-transfected cer Inst., 87: 682-684, 1995. human lung cancer cells. J. Natl.