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513
Correlation of Cervical Carcinoma c-erb B-2 Oncogene
with Cell Proliferation Parameters in Patients Treated
with Radiation Therapy for Cervical Carcinoma
Takashi Nakano, M.D.
Kuniyuki Oka, M.D.
Atsuko Ishikawa, M.D.
Shinroku Morita, M.D.
BACKGROUND. Although c-erb B-2 oncoprotein expression (CerbB-OPE) is believed
to be associated with tumor cell proliferation and prognosis, the correlation between CerbB-OPE and cell proliferation parameters has not been fully analyzed.
METHODS. Immunohistochemical studies were performed on 64 cervical carcinoma
Division of Radiation Medicine, National Institute of Radiological Sciences, Chiba, Japan.
patients treated with radiation therapy. Prognosis was analyzed by CerbB-OPE,
growth fraction determined with Ki-67 immunohistochemistry (Ki-GF), and the
mitotic index of proliferating cell population (pMI).
RESULTS. CerbB-OPE was observed on the cell membrane of carcinoma cells. Positivity of CerbB-OPE, which was 42.4% in total, increased significantly with stage
progression. No significant differences were observed among histologic subtypes.
Mean total Ki-GF and pMI were 36% and 2.5%, respectively. Mean Ki-GF for
CerbB(/) patients was 26.2%, which was significantly lower than the 38.3% for
CerbB(0) patients (P õ 0.01). The mean pMI for CerbB(/) patients was 3.7%, which
was significantly higher than the 2% for CerbB(0) patients (P õ 0.05). The 5-year
survival rates of CerbB(/) patients and CerbB(0) patients were 45.1% and 75.6%,
respectively, indicating that CerbB(/) patients showed significantly poorer survival
than CerbB(0) patients (P õ 0.01). The difference in survival was due mainly to
local recurrence rather than distant metastasis. There were significant correlations
between prognosis and Ki-GF and pMI.
CONCLUSIONS. The poor prognosis of patients with cervical carcinoma with CerbBOPE was due to local recurrences after radiation therapy. The correlations of CerbBOPE with Ki-GF and pMI suggest that c-erb B-2 oncoprotein may play an important
role in the cell proliferation status of cervical carcinoma. Cancer 1997; 79:513–20.
q 1997 American Cancer Society.
KEYWORDS: c-erb B-2, Ki-67, cervical carcinoma, radiation therapy, growth fraction.
T
The authors thank Professor H. Niibe, Radiation
Oncology, Gunma University, for his helpful scientific support.
Address for reprints: Takashi Nakano, M.D. Division of Radiation Medicine, National Institute
of Radiological Sciences, 9-1, Anagawa-4chome, Inage-ku, Chiba-shi 263, Japan.
Received July 24, 1996; revision received October 4, 1996; accepted October 4, 1996.
he c-erb B-2 oncogene is a protooncogene that encodes for a transmembrane protein (p185) with tyrosine kinase activity.1,2 This oncogene product has homology with the epidermal growth factor receptor (EGFR)3 and is thought to be a functional growth factor receptor. This oncogene is amplified or overexpressed in a variety of human
adenocarcinomas,4 including adenocarcinoma of the breast,5 – 8 gastrointestinal tract, lung, and renal cell carcinoma. In addition to adenocarcinoma, squamous cell carcinoma of the cervix9 – 12 also expresses the c-erb B-2 oncoprotein. The immunohistochemical expression of c-erb B-2 oncoprotein (CerbB-OPE) was believed to be
associated with the amplification of the c-erb B-2 oncogene, and was
reported to be highly associated with poor prognosis.11,12 Although
CerbB-OPE is believed to correlate with tumor cell proliferation, the
correlation with cell proliferation parameters has not been fully ana-
q 1997 American Cancer Society
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CANCER February 1, 1997 / Volume 79 / Number 3
TABLE 1
Characteristics of Patients
Variables
Stages
I
II
III
IV
Histology
Keratinizing
Nonkeratinizing
Small cell
Large cell
Tumor volume
Small
Medium
Large
Total
tively. As for histologic subtype, the numbers of patients with keratinizing, small cell nonkeratinizing,
and large cell nonkeratinizing types were 10, 14, and
40, respectively.
No. of patients
2
13
42
7
10
14
40
12
33
19
64
lyzed. Especially with radiation therapy, tumor cell
proliferation activity affects the local control probability, which is essential for long term prognosis after
radiation therapy. Many biologic parameters including
cell cycle factors13,14 and histologic features15 have
been analyzed for the prediction of local control probability after radiation therapy. However, little is known
regarding the implications of the oncogene expression
of cancer cells in regard to radiation resistance and
local control probability with the use of radiation therapy.
Therefore, the current study was performed to
evaluate the correlation between CerbB-OPE and cell
proliferation parameters including growth fraction determined by Ki-67 immunohistochemistry (Ki-GF ) and
the mitotic index of proliferating cell population (pMI)
as well as their impact on local control and prognosis.
MATERIALS AND METHODS
Eighty-one patients with invasive squamous cell carcinoma of the uterine cervix received radiation therapy
alone at the National Institute of Radiological Sciences
Hospital (Chiba, Japan) between 1988 and 1990. Of
these, 64 patients with frozen cervical specimens available for immunohistochemical staining were analyzed.
The minimum follow-up period was 4 years, and most
of the patients were followed longer. Clinical stages
and histologic subtypes are summarized in Table 1.
The clinical staging and histologic classification were
based on the criteria of the International Federation
of Gynecology and Obstetrics (FIGO) classification16
and the World Health Organization (WHO) classification,17 respectively. The numbers of patients with
Stages I, II, III, and IV were 2, 13, 42, and 7, respec-
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Radiotherapeutic Protocol
Patients were treated with a combination of external
and high dose rate intracavitary irradiation. Details of
the protocol have been reported elsewhere.18 Briefly,
external whole pelvic irradiation was performed with
anteroposterior and posteroanterior parallel opposing
ports, with a dose of 1.8 Gray (Gy) per fraction, 5 times
per week, to a total dose of 30.6 Gy. This was followed
by a central shielding pelvic field, with a dose of 2 Gy
per fraction, 5 times per week, to a total dose of 20
Gy. Along with the central shielding irradiation, these
patients also received intracavitary irradiation by remote afterloading system using 60Co sources; they received 4 insertions (1 per week) with fraction doses of
550 Centigray (cGy) to 600 cGy at point A. The total
doses ranged from 22 Gy to 24 Gy.
Histopathologic Study
The immunohistochemical staining method was described elsewhere.11,19 All specimens were excised from
cervical tumors by punch biopsy before radiation therapy. The biopsy was performed by an expert clinician
who could differentiate between necrosis and fresh
tumors. All the fresh biopsy specimens were divided
into two parts; one was fixed with 10% formaldehyde
solution for conventional hematoxylin and eosin staining and the other was quickly frozen for immunostaining with c-erb B-2 oncoprotein and Ki-67 antibodies.
The specimens were cut with a cryostat at 6-mm thickness, air-dried, and fixed with cold 4% paraformaldehyde solution for 30 minutes. The sections were then
reacted with polyclonal anti-c-erb B-2 oncoprotein antibody (Dako, Copenhagen, Denmark) and Ki-67 antibody (Dako) for 1 hour at room temperature. The sections were followed by reaction with biotinylated second antibodies for 30 minutes and treated with the
avidin-biotin complex (ABC)20 method (Vector Laboratories, Burlingame, CA) for 30 minutes. The sections
then were reacted with 3,3*-diaminobenzidine tetrahydrochloride (Dojin Chemicals, Tokyo, Japan) solution with 0.01% (weight per volume) hydrogen peroxide for 2 to 5 minutes at room temperature and counterstained with hematoxylin. Control staining was
done by incubation with normal control serum rather
than primary antibodies.
The specimens were histologically examined and
classified into two groups according to the degree of
stained cells as follows: c-erb B-2 expression positive:
marked cell membrane or cytoplasmic staining (Fig.
W: Cancer
c-erb B-2 of Cervical Cancer/Nakano et al.
515
FIGURE 1. (a) Immunohistochemical staining for c-erb B-2 oncoprotein with counter nuclear staining (1200). (b) Control staining with counter nuclear
staining (1200).
fraction of tumors separately. The mitotic index, specific for the pMI, can express the relative cell cycle
speed. To obtain pMI, mitotic index first was calculated by counting more than 3000 cancer cells in 3
fields of a specimen on 1400 magnification microscopic photography. pMI can be calculated by the mitotic index divided by Ki-GF, which was described in
detail elsewhere14:
pMI Å mitotic cell number of cycling cells/
total cycling cells Å Mitotic index/Ki-GF
FIGURE 2. Immunohistochemical staining for Ki-67 antigen (1200).
1a), negative: no cell membrane or cytoplasmic staining (Fig. 1b). Ki-67 antigen expression: Ki-67 positive
cells were determined as cancer cells whose nucleus
was positive with Ki-67 substance (Fig. 2) Ki-GF was
calculated as the percentage of Ki-67 positive cancer
cells by counting more than 1000 cancer cells of 3
fields of a specimen on 1400 magnification microscopy. To rule out the possibility of bias, the specimens
were classified into two groups without referring to
any clinical information.
pMI Calculation
The mitotic index of tumor cells in vitro is usually
proportional to the cell proliferating speed, but the
index of tumor cells in vivo is biased by the presence
of quiescent cells whose population is larger than the
cycling cell population in many cancers.14,21,22 Hence,
it is important to assess proliferation speed and growth
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Clinical Evaluation
In analysis of clinical data, patients were divided into
two groups according to CerbB-OPE positivity and values of Ki-GF and pMI. The cutoff values of Ki-GF and
pMI were determined retrospectively and statistically
when the statistical differences became maximum
(33% and 5.3%, respectively).
Statistical Analysis
Correlation between various cell proliferation parameters and clinical parameters were analyzed by the student’s t test. Cumulative survival rates were statistically analyzed by Peto’s log rank test.23
RESULTS
The c-erb B-2 oncoprotein was observed on the cell
membrane and cytoplasms of cancer cells (Fig. 1a).
Ki-67 antigen was observed in the nuclei of cancer
cells. Ki-67 positive cells were present dispersedly in
cancer nests (Fig. 2).
The positivity of CerbB-OPE, Ki-GF and pMI were
analyzed according to stage and histologic subtype
(Table 2). The positivity of CerbB-OPE was 42.4%, and
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CANCER February 1, 1997 / Volume 79 / Number 3
TABLE 2
c-erb B-2 Oncoprotein Expression, Ki-67 Immunohistochemistry, and Proliferating Cell Population According
to Stage, Histologic Subtype, and Tumor Volume
c-erb B-2
Stages
I
II
III
IV
Histology
Keratinizing
Nonkeratinizing
Small cell
Large cell
Tumor volume
Small
Medium
Large
Total
Patient
no.
No.
%
Ki-67
% / SD
pMI
% / SD
2
13
42
7
0
3
18
6
0
23.1
42.9
85.7
69.6 / 16.2
30 / 6
33.2 / 17.5
21.9 / 8.2
1.4 / 1.3
2.4 / 1.3
2.8 / 3
2.9 / 1.3
10
7
70
31.6 / 21.1
2.6 / 1.5
14
40
4
16
28.6
40
35.4 / 15.7
26.2 / 16.4
1.9 / 1.8
6.9 / 2.8
G
12
33
19
64
4
12
11
27
33.3
36.4
57.9
42.2
42.1 / 18.2
31.7 / 17
28.1 / 14.9
33 / 17
0.9 / 0.8
2.1 / 1.8
4.7 / 3.5
2.7 / 2.5
G
a
a
a
SD: standard deviation; pMI: mitotic index of proliferating cell population.
a
P õ 0.05.
FIGURE 3. Correlation between c-erb B-2 oncoprotein expression and
Ki-67 growth fraction (P õ 0.01).
FIGURE 4. Correlation between c-erb B-2 oncoprotein expression and
mitotic index of proliferating cell population (P õ 0.05).
it was observed to increase significantly with advancing stage (P õ 0.05). No significant difference was observed among histologic subtypes. The mean Ki-GF
and pMI were 36% and 2.5%, respectively. There was
no significant difference between Ki-GF and pMI
among stages.
The correlations of CerbB-OPE expression with KiGF and pMI are shown in Figures 3 and 4. The mean
Ki-GF of patients with CerbB-OPE was 26.2%, signifi-
cantly lower than the 38.3% of those without CerbBOPE (P õ 0.01). The mean pMI of patients with CerbBOPE was 3.7%, significantly higher than the 2% of
those without CerbB-OPE (P õ 0.05).
Disease free survival rates according to CerbB-OPE
are shown in Figure 5. The 5-year survival rates for
CerbB-OPE positive and negative patients were 44.4%
and 74.8%, respectively, indicating that the positive
patients showed significantly poorer survival than the
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c-erb B-2 of Cervical Cancer/Nakano et al.
FIGURE 5. Disease free survival rates according to c-erb B-2 oncoprotein expression for all and Stage III patients. The survival curves were
significantly different between the positive and negative groups (P õ 0.05).
517
CerbB-OPE positive patients was 37.3%, significantly
higher than the 8.3% rate of negative patients. The
difference was also significant when Stage III patients
were selectively analyzed. The difference in metastatic
rates between the two groups was not significant (data
not shown).
Figure 7a shows the disease free survival rates and
cumulative recurrence rates according to Ki-GF status.
The 5-year survival rates for patients with a Ki-67 index
of 33% or greater were significantly better than those
with a Ki-67 index below 33% (87.5% vs. 44.7%; P õ
0.01). The 5-year recurrence rates for the patients with
a Ki-67 index of 33% or greater were significantly lower
than those for patients with a Ki-67 index below 33%
(8.7% vs. 30.4%; P õ 0.05). Similar significant trends
were also observed in Stage III patients.
Figure 8a shows the disease free survival rates and
cumulative recurrence rates according to pMI status.
The 5-year survival rates for patients with a pMI of
3.5% or greater were significantly lower than those for
patients with a pMI of below 3.5% (0% vs. 81.8%; P õ
0.001). The 5-year recurrence rates for the patients
with a pMI of 3.5% or greater were significantly higher
than for those with a pMI below 3.5% (62.5% vs. 9.5%;
P õ 0.001). The difference was also significant when
Stage III patients were selectively analyzed.
DISCUSSION
FIGURE 6. Cumulative recurrence rates according to c-erb B-2 oncoprotein expression (CerbB-OPE) for all and Stage III patients. (
) and
(rrrr) indicate recurrence rate curves of all patients with and without
CerbB-OPE, respectively. (
) and (– – – –) indicate those of Stage
III patients with and without CerbB-OPE. The recurrence curves were
significantly different between the positive and negative groups (P õ 0.05).
negative ones (P õ 0.01). A similar trend was also observed in Stage III patients, whose 5-year survival rates
for those positive and negative for CerbB-OPE were
44.4% and 69%, respectively (P õ 0.05). Cumulative
recurrence rates according to CerbB-OP expression are
shown in Figure 6. The 5-year recurrence rates of
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CerbB-OPE has been reported to be associated with
tumor malignancy in various cancers, particularly in
breast carcinoma, for which many investigators have
confirmed the poorer prognosis of the patients with
CerbB-OPE24,25 Hale et al.12 and the authors11 have reported that CerbB-OPE showed significantly poorer
prognosis in patients with squamous cell carcinoma
of the cervix. The current study confirmed this with
the recent materials. The positivity of CerbB-OPE in
the current study was 42%, higher than the 19% of the
previous study in which older specimens of cervical
carcinoma were utilized. The different positivity may
be due to lower antigenicity of older specimens or
different fixation methods between formalin for the
former study and paraformaldehide for the current
one. Hale et al. also reported a positivity rate of 42%.12
Hence, the authors are inclined to conclude that approximately 40% of the cervical squamous cell carcinoma of the cervix expressed c-erb B-2 oncoprotein.
The positivity increased with advancing stage, which
was similar to other reports. Therefore, CerbB-OPE is
considered to be an important indicator of the malignant nature of the disease, as well as a strong prognostic factor for patients with various carcinomas.
In addition to the c-erb B-2 oncogene, c-myc and
H-ras oncogenes have also been studied in relation to
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CANCER February 1, 1997 / Volume 79 / Number 3
FIGURE 7. (a and b) Disease free survival and recurrence rates according to Ki-67 growth fraction respectively. (
) and (rrrr) indicate survival
curves or recurrence curves of all patients. (
) and (– – –) indicate survival curves or recurrence curves of Stage III patients. The survival and
recurrence curves were significantly different between the positive and negative groups (P õ 0.01).
FIGURE 8.
(a and b) Disease free survival and recurrence rates according to mitotic index of proliferating cell population (pMI), respectively.
) and (rrr) indicate survival curves or recurrence curves of all patients. (
) and (– – – –) indicate survival curves or recurrence curves
(
of Stage III patients. The survival and recurrence curves were significantly different between the positive and negative groups (P õ 0.01).
tumor progression, and a positive prognostic value
was reported.26,27 However, a possible correlation between these oncogenes and local control in radiation
therapy has not been evaluated. Hence the reasons for
the poor prognosis of these oncogene expressions
have not been fully understood in terms of cellular
biology. The current study demonstrated that tumors
with CerbB-OPE showed higher local recurrence after
radiation therapy. In other words, the c-erb B-2 oncogene was mainly associated with radiation resistance
of the tumor.
The current study suggests that patients with a
lower growth fraction as measured by Ki-67 antibody
possessed higher local recurrence and consequently
they showed poor outcome. This may be due to G0
cells, which are the major component of tumors with
a low growth fraction, being resistant to radiation.28,29
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Growth fraction and cell cycle time are independent major tumor proliferation characteristics. The
mitotic index, the labeling index with thymidine or
bromodeoxyuridine incorporation, and the potential
doubling time can be used to assess gross proliferation
activity together with these two factors, but cannot be
derived from these two proliferative activities separately.14 The mitotic index of the proliferating cell population (pMI) is suggested to be correlated with the
cell cycle speed of the proliferative cell population.14
In the current study, high pMI indicated a significantly
poor prognosis or poor local control. Hence, the faster
cell production rate or repopulation of the critical tumor cell population suggested by the high pMI may
be one of the major causes of recurrence.
The c-erb B-2 oncoprotein is believed to be a receptor-like tyrosine kinase similar to EGF receptor.1,2
W: Cancer
c-erb B-2 of Cervical Cancer/Nakano et al.
In addition, an interaction between EGF and c-erb B2 oncogene product has been reported.30 By using
monoclonal antibodies and chimeric neuproteins, the
c-erb B-2 oncoprotein can be stimulated to transmit
growth regulatory biochemical signals.2,31 Thus, c-erb
B-2 oncogene is believed to play a role in cell proliferation. However, there has been little clinical data to
assess the correlation between c-erb B-2 oncogene
amplification and cell cycle parameters.
The current study demonstrated that tumors with
CerbB-OPE had lower Ki-GF and higher pMI than
those without CerbB-OPE. Hence tumors with CerbBOPE are comprised of larger G0 populations and also
have faster cell proliferation. It was reported that ligands or antibodies specific for c-erb B-2 oncoprotein
might modulate the cell cycle or the differentiation of
tumor cells.2,31
Therefore, the reason for the higher local recurrence of tumors with CerbB-OPE in the current study
may be due to higher radiation resistance of the tumor
cells with CerbB-OPE, because tumors with CerbBOPE are comprised of larger G0 populations that are
rather radioresistant.28,29 The poor prognosis with
lower Ki-GF for the patients with cervical carcinoma
of the current study supports this possibility. Another
possible explanation may be that the higher proliferative activity of the tumor with CerbB-OPE increases
the repopulation of tumor cells during irradiation, and
thereby reduces the radiation curability. The higher
pMI of tumors with CerbB-OPE suggests that the tumors have a faster cell cycle time and are highly proliferative, which decreases the chance of local control
with radiation therapy. In fact, recent radiobiological
studies have demonstrated that tumor cell repopulation during radiation therapy causes the loss of local
control with increasing cell clonogens.13,32,33 Further in
vivo studies in terms of cellular biology will be required to evaluate these possibilities.
In conclusion, it is suggested that c-erb B-2 oncogene overexpression in cervical carcinoma is associated with the tumor cell proliferation status and affects
the local control probability of radiation therapy and
long term prognosis.
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