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Int. J. Cancer (Pred. Oncol.): 69, 165-169 (1996)
0 1996 Wiley-Liss, Inc.
Publicationof the InternationalUnion Against Cancer
Publicationde I'llnion lnternationaleContre le Cancer
ASSESSMENT OF EGFR AND TGF-ALPHA EXPRESSION
IN RELATIONSHIP TO HPV STATUS AND KI-67 DISTRIBUTION
IN CERVICAL INTRAEPITHELIAL NEOPLASMS
Athanassios DELLAS1s3,Elisabeth SCHULTHEISS~,
Alfonso C. ALMENDRAL',
Joachim TORHORST?
and Fred G U D A T ~
Department of Obstetrics and Gynecology and 21nstituteof Pathology, University of Basel, Basel, Switzerland.
'
Expression of epidermal-growth-factor receptor (EGFR),
transforming growth factor alpha (TGF-a) and Ki-67 proliferation antigen in cervical intra-epithelial neoplasms were analyzed. To examine the interrelationshipof TGF-a, EGFR, Ki-67
and HPV status in dysplasia and carcinoma in situ, formalin-fixed
tissue sections of 92 women were immunostainedwith monoclonal antibodies to EGFR, TGF-a and Ki-67. The presence of HPV
was assessed by in situ DNA hybridization. The highest positive
TGF-a expression was seen in the group of mild dysplasia. The
difference was significant between the relatively high expression in mild dysplasia and the low occurrence in severe dysplasia
and carcinoma in situ as well. The same relation could be found
between TGF-a expression in papilloma-virus-negative dysplasia and those with the presence of HPV 16/ 18. In contrast to
these findings, the Ki-67 proliferation marker was intensely
detectable in severe dysplasia and carcinoma in situ. Ki-67stained neoplastic cell n d e i were found in a significantly higher
percentage of HPV-positive than in HPV-negative lesions. TGF-a
over-expression is obviously combined with low proliferating
activity and vice versa. Irrespective of the grade of dysplasia or
HPV status, EGFR was expressed abnormally as compared with
normal squamous epithelium. Over-expressionof TGF-a in mild
dysplasia could be associated with the autocrine pathway of
cell-growth regulation. In the presence of HPV 16/ 18 the
EGFR/TGF-a pathway for growth stimulation is probably not
involved.
o 1996 Wiley-Liss,Inc.
The concept that simultaneous production of a growth
factor and expression of its specific receptor by the same cell
could result in self-stimulation is based on the landmark
description by De Larco and Todaro (1978) of the autostimulation pathway of EGF-receptor activation in cultured
tumor cells (Sporn and Todaro, 1985).
Although TGF-a and EGF act primarily by stimulating cell
proliferation, they also can have inhibitory effects. In high
concentrations, they inhibit the growth of cultured tumor cell
lines that express extraordinarily high levels of EGF receptors.
The mechanisms that determine these 2 opposed pathways are
not yet determined (Mendelsohn and Lippman, 1993). Many
types of epithelial malignancies, such as cancers of the lung,
breast and bladder, display increased EGF receptors on their
cell-surface membranes (Harris et al., 1989; Neal et aL, 1985;
Ozanne et al., 1986; Sainsbury et al., 1985; Veale et al., 1989).
Increased receptor expression is often associated with increased production of TGF-a by the same tumor cells (Luetteke and Lee, 1990). Additional data are derived from using
anti-EGF-receptor monoclonal antibodies (h4Abs) in nonmalignant and malignant cell lines.
The data from these studies are best presented by considering 3 types of response to anti-EGF-receptor MAbs (Bates et
al., 1990; Ennis et al., 1989). Cells that can proliferate without
exogenous TGF-a and are strongly inhibited by MAbs presumably have an active and obligatory autocrine pathway. Second,
other cell lines proliferate in response to exogenous TGF-a
and are inhibited by MAbs. The third type of response
represents cells that express EGF receptor, do not respond to
TGF-a and ignore the presence of MAb. It is suggested that
they do not require receptor activation for growth. Similar
pathways could be demonstrated using MAbs against TGF-a
in cultures with EGF/TGF-a-dependent malignant epithelial
cells (Ciardiello et al., 1990). To explore the role of the
TGF-a/EGF-receptor pathway in the growth of cervical cancer, Brown et al. (1994) analyzed 4 cervical cancer cell lines for
EGFR content, TGF-a-gene expression and growth response
to treatment with an anti-EGFR MAb that blocks TGF-a
binding to EGFR. Their findings provide evidence for an
autocrine-growth-stimulatory pathway involving TGF-a/ EGFR.
In clinical material, the immuno-histochemical evidence of the
EGFR/TGF-a pathway is particularly lacking.
A MAb, Ki-67, has been utilized to demonstrate proliferating cells in the GI, S, G2 and M phases of the cell cycle (Gerdes
et al., 1984). Cells in quiescent phase Go consistently lack this
antigen. Ki-67 stained an antigen present in the nucleoli of
proliferative interphase cells as well as the condensed chromatin in mitotic cells. This MAb has since been applied to study
the growth fraction and cytokinetic activities in various cancers, e.g., lung cancer (Gatter et al., 1986), breast cancer
(Gerdes et al., 1986) and cervical cancer (Brown et al., 1988).
Its use has provided useful prognostic information. It was
found that a high Ki-67 score correlates with histological tumor
grade and with early recurrence in cancer. The present study
describes results for clinical material. We attempted to identify
EGF receptor and TGF-a expression in dysplasia and in
carcinoma in situ of the uterine cervix. We also compared the
presence of EGF receptor and TGF-a with histological grade
and HPV status and assessed neoplastic-cell proliferation by
analyzing the Ki-67 staining.
SUBJECTS AND METHODS
Samples of 20 specimens with normal cervical squamous
epithelium and 92 specimens of dysplasia and carcinoma in situ
(CIS) of the uterine cervix were obtained prior to surgical
treatment or from hysterectomy specimens in the Department
of Gynecology. The intraepithelial neoplasms were classified
and graded according to the WHO criteria.
Immunostaining of EGFR and TGF-a
The tissue samples were fixed in 4% buffered formalin,
embedded in paraffin and routinely stained to determine
histology. Serial sections 4-pm thick were cut from each
selected block, de-waxed and processed for enzyme digestion
with 0.1% pronase (protease type XIV; Sigma, St. Louis, MO)
for 5 min at 37°C in a water bath. To abolish endogenous
peroxidase activity, sections were immersed in 0.3% H202
methanol. They were buffered in 0.3 M PBS at pH 7.3 and then
incubated sequentially with normal horse serum (Vector,
Burlingame, CA) for 30 min at room temperature, and
afterwards with the primary MAb (mouse) at 4°C overnight.
The primary mouse MAbs used were obtained from Triton
Diagnostics (Alameda, CA; anti-EGFR, clone 31G7; diluted
to 1:150) and from Oncogene Science [Uniondale, NY; anti3To whom correspondence and reprint requests should be sent, at
Department of Obstetrics and Gynecology, University of Basel,
Schanzenstrasse 46, CH-4031 Basel, Switzerland. Fax: (+41) 61-3259191.
Received: October 20,1995 and in revised form January 24,1996.
166
DELLAS ETAL
TGF-a (Ab-2), clone 213-4.4; diluted to 1:1000]. After overnight incubation, the slides were incubated with biotinylated
anti-mouse IgG (1:200) as secondary antibody, for 30 min at
room temperature. The streptavidin-biotin-peroxidase preformed complex (ABC Elite Standard, Vector) was subsequently applied for 30 rnin at room temperature, diluted at
150. In order to obtain a negative control, appropriate slides
were incubated with PBS instead of primary mouse MAbs. The
immunologic reactions for the EGFR and TGF-(Y.antibodies
were developed with 3,3’-diamino-benzidine (DAB) tetrahydrochloride (Sigma). To obtain this solution, 3 tablets of DAB
tetrahydrochloride (1 tablet = 10 mg) were dissolved in 60 ml
0.05 M Tris-buffered saline (pH 7.6). To DAB, 40 pI 30%
hydrogen peroxide were added. The slides were then incubated with this solution for 6 rnin at room temperature. For
additional chromogen enhancement, osmium tetroxide (Simec,
Birsfelden, Switzerland) was applied for 2 min at room
temperature, diluted at 1 : l O in PBS. Sections were counterstained with hematoxylin (Shandon, Zeist, The Netherlands),
dehydrated and mounted with Eukitt (0.Kindler, Freiburg,
Germany).
Ki-67 immunostairiing
The tissue samples were de-waxed and rehydrated. They
were placed for 60 min at 90°C in 10 mM citrate buffer (pH
6.0). This step was performed using a H2500 microwave
processor (Energy Beam Sciences, Agawam, MA). Following
the microwave procedure, all slides were treated with 0.3%
H 2 0 2methanol and subsequently with normal horse serum for
30 min at room temperature. To assess the nuclear Ki-67
proliferation antigen, the mouse MAb MIB 1 (Dianova,
Hamburg, Germany) was used, diluted to 15300. With the
applied MIB 1, the slides remained overnight at 4°C in a humid
chamber. Thereafter, the slides were incubated with the
bridging anti-mouse antibody, the avidin-biotin-peroxidase
complex, and then with 3-amino-9-ethylcarbazole (AEC) chromogen (BioGenex, San Ramon, CA) to obtain strong red Ki-67
staining. The sections were counterstained with hemalum.
This procedure leads to blue-stained Ki-67-negative nuclei.
We chose the AEC chromogen to obtain a better contrast
between red- and blue-stained nuclei. Finally, all sections were
mounted with Crystal/Mount (Biomeda, Foster City, CA).
Assessment of stainingfeatures
Immunohistochemical staining of TGF-a and EGFR were
scored as follows: 0, negative; 1, sporadic reaction on single
cells; 2, heterogenous pattern with a less than 50% of positive
cells; 3, staining present in the majority of neoplastic cells; 4,
homogeneous staining in all neoplastic cells. The score was
converted in values expressed by percentage for better presentation purposes. For EGFR, only membrane but not cytoplasmatic staining was scored; for TGF-a, cytoplasmatic and
perinuclear staining was scored. Over-expression of EGFR
was defined as staining in excess of normal cervical epithelium,
which shows staining only in basal cells and the lower third of
squamous epithelium. Staining of TGF-a was considered as
positive when immunoreactivity occured only in neoplastic
cells.
A quantitative method was used to count the proportion of
Ki-67-positively stained nuclei. In cervical intraepithelial lesions, 500 neoplastic cells were counted at randomly chosen
fields at ~ 4 0 0magnification. The growth fraction of a neoplasm can be expressed as the percentage of positively stained
neoplastic nuclei in the total number of nuclei counted.
Analysis of the HPVstatus
For the detection and typing of human papillomavirus
(HPV) DNA in the specimens we used an in situ DNAhybridization procedure (PathoGene DNA Probe Assay, Enzo,
Farmingdale, NY) to identify HPV types 6/11, 16/18 or
31/33/51. The formalin-hed, paraffin-embedded tissue sections were de-paraffinized, rehydrated and treated with
0.0125% proteinase K (to make the specimen DNA accessible
to the biotinylated DNA probes) and with 3% hydrogen
peroxide in buffered NaCI/EDTA (to eliminate endogenous
peroxidase activity). In small biopsy specimens, 0.0125%
proteinase K was applied for 5 rnin on a 37°C heating block,
whereas on relatively large tissue samples the incubation time
was extended to 15 min. The sections were then dehydrated
and dried at room temperature. In accordance with instructions for the assay, one drop (40 KI) of each used vial of HPV
DNA probe reagent was applied to the slides. They were
placed on a 95°C heating block for 3 min and then moved to the
37°C slide warmer for 15 min. They remained overnight in a
humid chamber at 37°C to obtain a stronger signal. During the
post-hybridization procedure, 0.5 ml buffered NaCl containing
formamide were applied to each slide for 10 min. Before
application of the detection reagent, the slides were gently
washed in buffered NaCI/EDTA. Specific hybridization between the HPV DNA probes and DNA in the specimen was
determined by the detection of biotin. Detection of biotin was
accomplished in 2 steps. First, a streptavidin-horseradishperoxidase complex was bound to the biotin of the hybridized
HPV DNA probes. In the second step, the entire complex was
visualized after conversion of the substrate and chromogen
into a localized brick-red precipitate within the nuclei of
epithelial cells. As chromogen we used AEC mixed with
hydrogen peroxide in acetate buffer. The slides were incubated
with chromogen for 10 min at room temperature. Following
counterstaining with hemalum and mounting with Crystal/
Mount (Biomeda), the stained cells were observed by light
microscopy. For a specimen to be identified as positive for
HPV, at least one positive brick-red nucleus must be present.
Statistical analysis
Results are given as mean and standard deviation (SD).
One-way ANOVA was used to compare the groups of Ki-67,
EGFR and TGF-a scores in relationship to histological grade
and HPV status.
RESULTS
The expression of EGFR, TGF-a and Ki-67 proliferation
antigen in the different histological grades of cervical intraepithelial neoplasia is shown in Table I. The EGF receptor is
over-expressed in dysplasia, irrespective of the grade, and in
carcinoma in situ of the uterine cervix. In contrast to dysplasia,
EGF-receptor expression is lost in the upper two thirds of
normal epithelium. Intense cell-surface expression of the
receptor was demonstrated by the anti-EGFR MAb used,
which only rarely presented a diffuse cytoplasmic pattern. No
significant differences in EGFR expression could be found
between different grades of intraepithelial neoplasms. Receptor immunoreactivity was not related to HPV status (Table 11).
The highest positive TGF-a expression was seen in the
group of mild dysplasia. Compared with expression in the
carcinoma in situ, the difference was significant (Fig. la). The
higher the grade of intraepithelial neoplastic cell transformation, the lower was the TGF-a immunoreactivity ( p = 0.001).
The expression pattern was staining in a few selected dysplastic cell groups, and the pattern of TGF-a immunoreactivity
showed cytoplasmic fine granularity, in most cases combined
with intense perinuclear staining. In normal epithelium, TGF-a
was not detectable; it was found particularly in HPV-negative
cervical intraepithelial neoplasms (Fig. l b ) . The higher the
incidence of HPV in intraepithelial neoplasms, the lower was
the TGF-a expression (Table 11). We found a correlation
between TGF-a expression in HPV-negative intraepithelial
167
TGF-ALPHA, EGFR, HPV AND KI-67 IN CERVICAL CANCER
neoplasms and pre-cancerous lesions infected with HPV type
16/18 (Fig. 3a).
No correlation was found between EGFR and TGF-a
positivity (results not shown).
In contrast to the results of the TGF-a analysis, we found a
high density of Ki-67-positively-stainedneoplastic nuclei in
severe dysplasia and carcinoma in situ (Fig. Za). The area of
nuclear staining varied from whole nuclei to the nucleoli and to
small clusters along the nuclear membrane. Cytoplasmic staining by Ki-67 was not observed. In normal cervical epithelium,
Ki-67 was present in the nuclei of the basal-cell layer, as the
most active cell layer. A relatively low count of Ki-67-positive
cells occured in mild and moderate dysplasia (Table I).
HPV-positive cervical intraepithelial neoplasms were associated with a significantly higher percentage of Ki-67-stained
nuclei, compared with HPV-negative lesions (Table 11, Fig. 2b).
Of 92 probes with cervical intraepithelial neoplasms, 63
were HPV-16/18-positive, 12 were HPV-31/33/51-positive
and 2 were HPV-6/1l-positive. The analysis of Ki-67 staining
showed a clear difference between HPV-negative and HPV16/18 probes (Fig. 3b).
TANLE I RtLATIOh I3LTWFF.N HISTOI.0GIC' CiKAUL: OF C ' t K V I C " .
l\\TKAEPllHI:I.IAL StOPl..ASSIS, TGF-a, EGbK ,\NVKI-07 STAIKIYG
( V A I LJES I\' M I 3 S
= 'ill ,\RE
Histologic
EX1'KI.SPED AS I'tIR('ENT,\GE)
Number
made
Mild dysplasia
Moderate dysplasia
Severe dysplasia
Carcinoma in situ
12
20
19
41
Mild dysplasia
Moderate dysplasia
Severe dysplasia
Carcinoma in situ
12
20
19
41
Mild dysplasia
Moderate dysplasia
Severe dysplasia
Carcinoma in situ
12
20
19
41
Mean
2
p value
SD
TGF-a
45.6 ? 34.7
23.5 2 35.6
4.7 k 13.9
8.2 k 18.6
EGFR
92.9 18.89
79.4 ? 20.68
90.8 i. 18.05
76.9 k 21.76
Ki-67
22.2 2 8.6
31.5 2 9.8
42.9 2 11.4
37.1 12.4
.001
*
NS
.0004
*
TABLE I1 - RELATION BETWEEN HPV STATUS, TGF-a, EGFR AND Ki-67
STAINING (VALUES IN MEAN
HPV status
Number
Negative
Positive
15
77
Negative
Positive
15
77
Negative
Positive
15
77
2
SD EXPRESSED AS PERCENTAGE)
Mean
2
SD
p value
TGF-a
37.8 2 37.83
11.15 2 23.38
EGFR
80.83 k 19.64
82.05 k 22.11
Ki-67
25.9 ? 8.7
37.5 12.7
.0032
NS
.003
*
DISCUSSION
The growth factor TGF-a is present in cervical intraepithelial neoplasms and can be detected by immunohistochemistry.
Although EGFR and TGF-a have been found both in normal
and in neoplastic endometrium (Santini et al., 1994), and in
different cervical-carcinoma cell lines (Brown et al., 1994).
relatively little is known about the distribution and significance
of both factors among normal squamous epithelium, precancerous cervical lesions and different histotypes of cervical
carcinoma. In the present study, a significant correlation
between grade of intraepithelial neoplasms, HPV status, Ki-67
staining and TGF-a expression, detectable particularly in
HPV-negative mild dysplasia, was found. These relationships
suggest TGF-a/EGFR-dependent neoplastic autocrine growth
stimulation in some cervical pre-cancerous lesions. Any contribution of the TGF-a/EGFR pathway to autocrine growth is
probably strictly confined to mildly dysplastic lesions only.
Such growth is never substantial, since these early lesions have
a relatively low proliferative capacity. In this study, TGF-a
expression was associated with low Ki-67-proliferation-marker
activity and with HPV-negative cervical lesions.
Staining for E G F R and TGF-a was intense in the neoplastic
cells, in contrast to weaker plasma-membrane or basal-cell
distribution of E G F R and no staining for TGF-a in normal
cervical tissues. In a TGF-a-positive tissue, presumably not all
neoplastic cells are capable of producing TGF-a for autocrine
growth. W e demonstrated this heterogeneity of neoplastic
cells in TGF-a expression in cervical neoplastic epithelium.
Lack of TGF-a staining in a few moderate dysplasia, in
severe dysplasia and in carcinoma in situ suggests that the
proliferative state of neoplastic cells is maintained through
other mechanisms, possibly other autocrine pathways or oncogene amplifications.
/
90
8 80
a
80
70
60
3 50
.E
$
b
8 70
5
60
a
5
50
40
40
2
T
3
30
20
I
1
-20
-20
CIN I
CIN II
CIN I l l
CIS
'
H Pkpositive
HPV-negative
FIGURE1- ( a ) Distribution of TGF-a in relation to the histological grade of cervical intraepithelial neoplasms (CIN). CIN I, mild
dysplasia; CIN 11, moderate dysplasia; CIN 111, severe dysplasia; CIS, carcinoma in situ. (b) Distribution of TGF-a in relation to HPV
status.
168
DELLAS E T A L .
a
60]
b
P
P
50
-
&- 4 0
-
8
.-C
.L
Y
8 30 -
c
C
8 20 -
H
-
3 100 '
0 '
CIN II
CIN I
CIN Ill
HP w o s i t i v e
CIS
H PV-negative
FIGURE 2 - (a) Distribution of Ki-67 in different histological grades of cervical intraepithelial neoplasms (CIN). CIN I, mild dysplasia;
CIN 11, moderate dysplasia; CIN 111, severe dysplasia; CIS, carcinoma in situ. (b) Relation between Ki-67 counts and HPV status
( p = 0.002).
60
-..
P
4)
E.
20-1
.........................
-20
b
'
0 '
HPV-negative
HPV 16/18
HPV-negative
HPV 16/18
F~GURE
3 - (a) TGF-a expression in HPV-negative and HPV-16/ 18-positive intraepithelial neoplasms. The difference between the 2
groups is significant ( p = 0.006). (b) Difference in Ki-67 counts of HPV-negative and HPV-lh/l8-positive intraepithelial neoplasms
( p = 0.0017).
The infection with HPV, particularly type 16/18, possibly
inhibits the process of TGF-a/EGFR-dependent autocrinegrowth stimulation. It seems that neoplastic cells in HPVinfected squarnous epithelium do not need this pathway. Our
results show that Ki-67 scores have a significant correlation
with histological grade of intraepithelial neoplasms and with
HPV status, with highest scores present in the poorly differentiated pre-cancerous lesions and in HPV-positive, particularly
HPV-16/18-infected, pre-cancerous lesions. A-Saleh et al.
(1995) have described significantly higher densities of Ki-67antigen-positive cells in HPV-16/18-positive cervical biopsies
than in low-risk HPV types. These results suggest that HPV,
particularly HPV 16/ 18, induces intense proliferation activity
in intra-epithelial neoplasms. Furthermore, HPV may induce
growth stimulation by a viral-defective version of the erbBproto-oncogene, which encodes the EGF receptor (Mendelsohn and Lippman, 1993). Two modifications characterized in
its structure explain its malignant potential. The oncogene
encodes a receptor lacking the extracellular domain that is the
binding site for TGF-a or EGF; it also lacks the carboxylterminal residues, which are presumed to have a regulatory
function. It is hypothesized that the viral-encoded EGF
receptor permanently mediates uncontrolled signal transduction to the cell nucleus (Watson et al., 1993). On the other
hand, the HPV-16 E5 gene product can interact with the EGF
receptor. This gene can stimulate the transforming activity of
the receptor in the presence of exogenous ligand (Leechanachai et al., 1992). Pim et al. (1992) showed that the HPV-16
E5 protein renders cells more sensitive to lower concentrations
of growth factors. The earlier observation that the HPV-16 E6
and E7 genes are preferentially retained in cervical carcinomas
implies that any role E5 may have in the development of these
tumors is in the early stages or in the pre-cancerous lesions.
Possibly E5 sensitizes the cells to lower levels of growth factors
early in the infection. Later, as the lesion progresses and E5 is
often deleted, this may be overcome by the observed overexpression of EGFR. The activity of E5 may therefore be one
of the first steps on the way to virus-mediated cell transformation, with the more apparent effects of E6 and E7 being later
functions in maintenance of the transformed phenotype (Pim
et al., 1992).
We therefore suggest that there is no evidence of a TGF-a/
EGFR-dependent autocrine-growth-stimulation pathway in
HPV-positive cervical carcinomas. Further investigations on
invasive cervical carcinoma may provide additional understanding of growth-stimulation mechanisms.
ACKNOWLEDGEMENTS
This work was supported by a research grant from the
Department of Obstetrics and Gynecology, University of
Basel.
TGF-ALPHA, EGFR, HPV AND KI-67 IN CERVICAL CANCER
169
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