Download Expression of Granulocyte Colony-stimulating Factor Receptor

[CANCER RESEARCH 58. 794-800.
February 15. I998|
Expression of Granulocyte Colony-stimulating Factor Receptor Correlates with
Prognosis in Oral and Mesopharyngeal Carcinoma1
Hideaki Tsuzuki, Shigeharu Fujieda,2 Hiroshi Sunaga, Ichiro Noda, and Hitoshi Saito
Department of Otarhinoluryngolagy.
Fukui Medical University, Matsuoka-cho,
Yashida-gun, Fukui 910-11. Japan
ABSTRACT
Granulocyte colony-stimulating factor receptors (G-CSFRs) have
been observed on the surface of not only hematopoietic cells but also
several cancer cells. The stimulation of G-CSF has been demonstrated
to induce proliferation and activation of G-CSFR-positive cells. In this
study, we investigated the expression of G-CSFR on the surface of
tumor cells and G-CSF production in oral and mesopharyngeal squamous cell carcinoma (SCO by an immunohistochemical approach. Of
58 oral and mesopharyngeal SCCs, 31 cases (53.4%) and 36 cases
(62.1%) were positive for G-CSFR and G-CSF, respectively. There was
no association between G-CSFR expression and G-CSF staining. In the
group positive for G-CSFR expression, relapse was significantly more
likely after primary treatment (/' = 0.0069), whereas there was no
association between G-CSFR expression and age, sex, tumor size,
lymph node metastasis, and clinical stage. Also, the G-CSFR-positive
groups had a significantly lower disease-free and overall survival
rate than the G-CSFR-negative groups i/' = 0.0172 and 0.0188, respec
tively). However, none of the clinical markers correlated significantly
with G-CSF staining, nor did the status of G-CSF production influence
the overall survival. The results imply that assessment of G-CSFR may
prove valuable in selecting patients with oral and mesopharyngeal SCC
for aggressive therapy.
INTRODUCTION
G-CSF,1 a glycoprotein growth factor, regulates the proliferation
and differentiation of granulocytic progenitor cells and functionally
activated mature neutrophils (1-3). Cells stimulated by G-CSF (espe
cially neutrophils) play an important role in the local host defense
response to infection or inflammatory disorders through their phago
cytosis, chemotaxis. and microbicidal activities (4, 5). However, a
recent study showed that the effect of G-CSF was not limited to bone
marrow cells or to hematopoietic deviation. Several malignant tumors,
including hepatocarcinoma (6), bladder carcinoma (7), SCC of the
oral cavity (8, 9), and malignant mesothelioma (10), have been dem
onstrated to secrete large amounts of G-CSF.
Various effects of G-CSF are triggered by the binding of G-CSF to
its receptor on the surface of the cells (11-17). Not only hematopoietic
cells but also several cancer cells, including colon adenocarcinoma
(18), small cell lung carcinoma (19), and bladder carcinoma (20, 21),
have functional receptors for G-CSF and stimulation via G-CSFRinduced proliferation of tumor cells. Autocrine growth of transitional
cell carcinoma of the bladder by G-CSF has been demonstrated,
suggesting that G-CSF-producing cancers with G-CSFR would have a
poor prognosis (21).
In this study, we investigated the expression of G-CSFR on the
surface of tumor cells and G-CSF production in oral and mesopha
ryngeal SCC by using an immunohistochemical approach. Also, we
analyzed the correlation between clinicopathological factors of the
patient and expression of G-CSFR or G-CSF production.
MATERIALS
AND METHODS
Patients and Sample Collection. We studied 58 patients with oral and
mesopharyngeal SCC who had undergone tumor-curative resections in the
Department of Otorhinolaryngology, Fukui Medical University from 1984 to
1996. The mean age of the patients was 63.5 years (median age, 65.0 years).
According to the general rules for head and neck cancer (tumor-node-metas
tasis classification), they were classified as 7 stage I cases (12%), 11 stage II
cases (19%), 13 stage III cases (22%), and 27 stage IV cases (47%). Seventy
percent of the patients (41 cases) were followed-up for 5 years, and the rest
were followed-up for at least I year. Ten patients died from other diseases.
None of the patients had been treated with either chemotherapy or irradiation
therapy before the surgery. Patients with oral and mesopharyngeal SCC had
been treated with standard therapy based on the disease stage. Briefly, patients
at stages I and II underwent only tumor resection, whereas those at stages III
and IV underwent tumor resection, standard neck dissection, and irradiation
(60 Gy) after surgery. One doctor (H. Sa.) conducted all of the surgeries.
Surgically resected SCC tissues were quickly fixed in 10% buffered formal
dehyde for 24 h and embedded in paraffin.
Immunohistochemical Staining. Immunohistochemical staining was
performed to detect G-CSFR and G-CSF in SCC tissues using the tradi
tional avidin-biotin-peroxidase complex technique. Paraffin-embedded
blocks were cut to 5-/¿m-thickspecimens and deparaffinized by ethanol.
After washing in distilled water and rinsing with PBS (pH 7.4), inhibition
of endogenous peroxidase activity was accomplished by incubation in 0.3%
H,O; solution dissolved in absolute methanol at room temperature for 15
min. All specimens were washed in distilled water, rinsed with PBS, and
incubated with normal sheep serum (DAKO LSAB kit; DAKO, CarpinterÃ-a,
CA) for 5 min at room temperature to block the background absorption of
antiserum; then they were incubated with mouse antihuman G-CSFR mAb
(LMM741 clone; PharMingen, San Diego, CA; diluted 1:50: G-CSFR
mAb. Ref. 16) or rabbit anti-rG-CSF polyclonal Ab (Chugai Pharmaceu
tical Co., Tokyo, Japan; diluted 1:200; aG-CSF Ab) at 4°Covernight. All
specimens were treated with goat antimouse biotinylated IgG (DAKO) or
goat antirabbit biotinylated IgG. Specimens were then rinsed with PBS and
allowed to react with the avidin-biotin-peroxidase complex for 40 min at
room temperature. After rinsing with PBS, peroxidase color visualization
was carried out with 3,3'-diaminobenzidine tetrahydrochloride solution
(DAB; Dojin, Kumamoto, Japan; 30 mg dissolved in 150 ml of PBS and
added to 10 fil of 30% HjO, solution). Before staining with aG-CSF Ab,
specimens were subjected to microwave irradiation for 10 min in distilled
water to enhance immunohistochemical staining (22). Nuclear counterstaining was carried out with Harris hematoxylin for 30 s before mounting.
Evaluation of Specimens. Formicroscopeanalysisof G-CSFRandG-CSF
staining, we selected 5 high-powered fields, with each field containing more
than 200 tumor cells, and counted both the number of positive and the total
number of cancer cells. In total, at least 1000 tumor cells were counted. We
calculated the average of 10 readings showing the percentage of G-CSFR- or
G-CSF-positive cells and expressed them as the G-CSFR score or the G-CSF
score, respectively. Both G-CSFR and G-CSF stainings were scored independ
ently by two doctors (H. T. and H. Su.) in a coded manner (without knowledge
of the clinical parameters and outcomes). The plus (+ ) shows that over 20%
of the tumor cells were positive. Infiltrating neutrophils in the section were
used as a positive control of the stain for the G-CSFR mAb, and mouse IgG or
Received 8/25/97; accepted 12/19/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grant-in-Aid 07457397 (to H. Sa.) from the Ministry of
Education, Science, Sports and Culture, Japan.
: To whom requests for reprints should be addressed. Phone: 81-776-61-3111, exten
sion 2398; Fax: 81-776-61-8118; E-mail: [email protected].
1 The abbreviations used are: G-CSF, granulocyte colony-stimulating factor; G-CSFR,
G-CSF receptor; ru-CSF, recombinant G-CSF; SCC. squamous cell carcinoma; mAb,
monoclonal antibody: Ab. antibody; HGF. hepatocyle growth factor; CSF-1, macrophage
colony-stimulating factor.
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G-CSFR AND PROGNOSIS
*£
B
Fig. 1. H&E staining (A) and immunohistochemical staining of G-CSFR (ß)in a G-CSFRpositive oral SCC. Cytoplasmic and plasma mem
branes of the tumor cells were positively stained
(G-CSFR score, 95%; X200). H&E staining (O
and lack of G-CSFR staining (D; G-CSFR score,
5%| in a different oral SCC than that shown in A.
Infiltrating neutrophils (E) served as a positive con
trol for G-CSFR staining (X2000).
•
c
rabbit serum was used as a negative control instead of primary Ab for G-CSFR
or G-CSF, respectively.
survival rate of the patients was determined and plotted according to the
Kaplan-Meier test. The Macintosh personal computer system (Stat View
Statistical Analyses. The clinical characteristics of the patients in relation
to G-CSFR and G-CSF were analyzed by log-rank (Mantel-Cox) tests. The
software; Abacus Concepts, Inc., Berkeley, CA) was used for all statistical
analyses.
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G-CSFR AND PROGNOSIS
Fig. 2. A, H&E staining of cancerous and noncancerous areas in oral SCC. B and D, noncancerous areas; C and £,cancerous areas. Noncancerous area B was negative for G-CSFR.
Cancerous area C was positively stained for G-CSFR (G-CSFR score, 53%; X200). Noncancerous area D was positive for G-CSF. Cancerous area £was also positively stained for
G-CSF (G-CSF score, 78%; X200).
RESULTS
1.0
Expression of G-CSFR Correlated Significantly with the Prog
nosis. Using G-CSFR mAb, 58 formalin-fixed paraffin-embedded
oral and mesopharyngeal
>
SCC specimens were stained with standard
h--
- - G-CSFR (-)
(+)
0.8
3
l
£ 0.6
Table 1 Association betH-een clinicopathological
factors and expression of G-CSFR
0.4
0.2
(nNo.61.8
31)%506050486057555257553859574373387841groupingNegative
27)%504050524043454843456241435727622159P0.38490.46800.88240.79240.663
(nNo.64.8
0 •
0
SD)GenderMaleFemaleTumor
Age (mean ±
12.81912411)98161546516(primary)25619121516G-CSFR=
±
13.11984M66131435811198720423=
±
10
20
30
40
50
60
Months
B
sizeTlT2T3T4Lymph
LO
w 0.8metastasisPositiveNegativeClinical
node
I
- - G-CSFR (-)
(+)
0.6-
U)
stageI11111IVPostoperative
I
0.4
10.36170.00690.0066
O 0.2
treatmentIrradiationNoneRecurrenceYesNoDeath
0
10
20
30
40
50
60
Months
diseaseYesNoPositive
from
Fig. 3. Disease-free survival curves (A) and overall survival curves (B) for patients with
oral and mesopharyngeal SCC. The disease-free survival and overall survival of 31
patients with G-CSFR-positive cancer cells were significantly shorter than those of 27
patients with G-CSFR-negative tumors (/> = 0.0172 and 0.0188, respectively).
796
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O-CSFR AND PROGNOSIS
Fig. 4. H&E staining (.Aland immunnhistochemical
staining of G-CSF (B) in a G-CSF-positive oral SC'C. In the cytoplasm and plasma membrane »Ithe iunior trii, immunoreaclivity
for G-CSF was observed (G-CSF score, 90%; X200). H&E staining (O and lack of G-CSF staining (O) (G-CSF score, 7%; X200) in a different oral SCC than that shown in A.
and noncancerous areas. Cancer cells were positive for G-CSFR
staining (G-CSFR score, 53%; Fig. 1C). However, noncancerous
areas were negative (Fig. IB}. Nine percent of these samples (3 of 33)
showed positive staining for G-CSFR in the normal adjacent area.
Both normal squamous epithelium and fibroblast were positively
stained.
The association between clinical factors and expression of G-CSFR
is shown in Table 1. There was no association between G-CSFR
expression and age, sex, tumor size, lymph node metastasis, or clinical
stage. The G-CSFR score did not correlate with these five clinical
factors (data not shown).
ABC methods. Cytoplasm and plasma membrane of tumor cells were
positively stained (Fig. 1, A and B), but partial heterogeneity of
staining was observed in each of these cases. The mean G-CSFR score
in this study was 41.9 ± 37.5% (mean ± SD; median, 27.9%).
Positive expression of G-CSFR, ranging from 25.1-100% with a
mean ±SD of 72.8 ±22.6%, was found in 31 of 58 cases (53.4%).
A specimen of oral SCC demonstrated to be negative for G-CSFR
staining is shown in Fig. 1, C and D. Neutrophils that had infiltrated
the section stained positive for G-CSFR in all specimens (Fig. IE).
Thirty-three of 58 samples had histologically normal adjacent tissue.
The specimen of oral SCC shown in Fig. 2A included both cancerous
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G-CSFR AND PROGNOSIS
G-CSFR expression was significantly associated with recurrence of
the disease (P = 0.0069). In 26 of 58 patients, primary cancer
treatment failed. Nineteen of these 26 patients with recurrence (73%)
had G-CSFR-positive tumors, whereas the remaining 7 (27%) had
G-CSFR-negative tumors. In the patients with recurrent disease, the
average G-CSFR score was 55.2 ±35.3%, whereas in patients with
out recurrence, it was 31.0 ±36.2% (P = 0.0134). Seventeen of 26
patients with recurrence were examined again for G-CSFR expression
at the time of recurrence. Eleven of 17 cases (64%) stained positively
both times (initial and recurrent). Ten of these patients died of the
disease. Three cases (18%) that did not stain for G-CSFR initially
were found to have become positive for G-CSFR expression at the
time of recurrence. All three patients died of the disease. Two cases
(12%) that initially stained positive for G-CSFR were later negative
for G-CSFR staining; one of these patients is alive. Only one patient
(6%) showed no expression of G-CSFR at the initial and recurrent
times, and this patient is still alive.
Also, a significant association was found between G-CSFR expres
sion and the number of deaths from the disease. Nineteen patients died
of the disease in this series of patients, and 15 of these 19 patients
(78%) had G-CSFR-positive cancer cells (P = 0.0066). The average
G-CSFR score of patients who died of the disease was significantly
higher than that of well-controlled patients (56.2 ± 35.5 versus
34.9 ±36.8%, mean ±SD; P = 0.0412).
Fig. 3 shows disease-free and overall survival curves stratified by
G-CSFR status. The disease-free and overall survival rates of both
G-CSFR-positive and -negative groups were calculated using the
Kaplan-Meier method. There was a significant difference in the dis
ease-free survival rate in favor of the G-CSFR-negative groups
(P = 0.0172; Fig. 3A). There was also a significant difference in the
overall survival rate between G-CSFR-positive and -negative groups
(P = 0.0188; Fig. 3ß). Although 72.8% of the G-CSFR-negative
group survived 5 years, only 44.0% of G-CSFR-positive patients
survived for that period.
The administration of rG-CSF to patients seems to be an important
factor in the contribution of G-CSFR to survival. A total of two
Table 2 Clinicopathological
features of the study population in relation to C-CSF
staining
=No.63.6 (n
=No.62.5 (n
Table 3 Muli/variate
factorsGender
Prognostic
Cox proportional
hazard analysis
CI"0.43-2.890.72-5.740.96-7.731.04-10.180.54-4
ratio1.1122.0292.7193.2581.54195%
male)Tumor
(female or
T^/T4)Lymph
size (T!/T2 or
+)G-CSFR
node metastasis (—or
+)G-CSF (- or
(- or +)Hazards
1 CI, confidence interval.
patients were treated with rG-CSF for the leukocytopenia induced by
chemotherapy. In one of these patients, the recurrent tumor positively
expressed G-CSFR, whereas in the other, there was no expression of
G-CSFR.
No Correlation of G-CSF Staining with the Prognosis. The
same 58 oral and mesopharyngeal SCC specimens that were examined
for G-CSFR expression were also stained with aG-CSF Ab (Fig. 4).
The mean G-CSF score was 53.5 ±35.6% (mean ±SD; median,
62.9%). Thirty-six cases (62%) were found to have a G-CSF score of
more than 20% (from 23.0-100%) and determined to stain positively
for G-CSF. The clinicopathological features of the study population in
relation to G-CSF staining of cancer cells are shown in Table 2. None
of the clinical markers correlated significantly with G-CSF staining or
G-CSF score (data not shown). The Kaplan-Meier analysis showed
that there was no difference in the status of G-CSF staining
(P = 0.7735). The survival rate for 5 years was 54.9 and 60.8% in the
positive and negative group for G-CSF staining, respectively.
Association of G-CSFR Expression and G-CSF Staining. The
association of G-CSFR expression and G-CSF staining in the tumor
area was examined. Twenty-one specimens (36%) stained positively
for both G-CSFR and G-CSF, 10 cases (17%) were G-CSFR positive
and G-CSF negative, 15 cases (26%) were G-CSFR negative and
G-CSF positive, and 12 cases (21%) were both G-CSFR and G-CSF
negative. No significant association between G-CSFR expression and
G-CSF staining was found by the x2 test (P = 0.3400). In addition, no
correlation was found between the G-CSFR and the G-CSF score
using the Spearman rank correlation coefficient test (r = 0.245;
P = 0.067). We also evaluated G-CSF staining in the tissue surround
ing the cancer cells. The areas shown in Fig. 2, D and E, are the same
as those in Fig. 2, B and C, respectively. Because both cancerous and
noncancerous areas stained positively for G-CSF (Fig. 2, D and E), 49
of 58 samples (84%) showed some positive G-CSF staining of normal
No signif
icant relationship was found between G-CSFR expression in cancer
cells and the G-CSF status of the whole section, including adjacent
9.31573676148324131931012715=
±
SD)GenderMaleFemaleTumor
Age (mean ±
14.8231351588152149914(primary)251116201224G-CSF36)%61656371535752725782M52577862636362stainingNegative
±
tissue, using the x2 test (P = 0.8503).
si/eTlT2T3T4Lymph
metastasisPositiveNegativeClinical
node
stageIIIIIIIVPostoperative
treatmentIrradiationNoneRecurrenceYesNoDeath
22)%393537294743482843183148432138373738P0.75690.73860.70260.10440.33740.14400.94020.9050
squamous epithelium and fibroblast in the adjacent tissue.
Next, we focused on the 19 patients who died from disease and the
association between G-CSFR and total G-CSF staining. Six of 15
G-CSFR-positive patients (40%) were negative for G-CSF, whereas 3
of 4 G-CSFR-negative patients (75%) were G-CSF positive. These
results indicate that G-CSFR expression on the tumor cells is com
pletely independent of G-CSF status in cancer tissue.
Relative Risks Contributing to Survival Time. The prognostic
value of G-CSFR expression was examined by multivariate analysis
using the Cox proportional hazard model. G-CSFR expression was
significant as an independent prognostic indicator for overall survival,
followed by lymph node metastasis (Table 3). The risk ratio of death
was 3.258 among patients who were G-CSFR positive versus those
who were G-CSFR negative (P = 0.0422).
DISCUSSION
diseaseYesNoPositive
from
In this study, we demonstrated that there was a significant associ
ation between G-CSFR expression in cancer cells and a poor prog-
798
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G-CSFR AND PROGNOSIS
nosis in patients with oral and mesopharyngeal SCC. Expression of
G-CSFR is also associated with a possibility for relapse of the disease,
although the physiological functions of G-CSFR on the surface of
patients with carcinoma expressing G-CSFR, we feel that care should
be taken in the clinical use of rG-CSF in patients with G-CSFRpositive cancer. The expression of G-CSFR in oral and mesopharyn
cancer cells remains unclear.
G-CSFR expression has been frequently observed not only in
cancer cell lines (18-20) but also on human transitional carcinoma
cells of the bladder (21, 23). G-CSFR-positive cancer cells derived
from a clinical specimen have been shown to proliferate in vitro by
spontaneous G-CSF production by cancer cells or by the addition of
exogenous G-CSF. This proliferation was blocked by anti-G-CSF Ab
(21, 23). These results suggest that G-CSF and its receptor act as a
paracrine and/or autocrine loop mechanism for the proliferation of
tumor cells. This concept was demonstrated by a gene transfection
model in which a human osteosarcoma cell line transfected by retroviral infection to produce human G-CSF became autostimulatory in
vitro and grew easily in vivo (24). However, we showed that there was
no association between G-CSFR expression and G-CSF production in
geal SCC may be a prognostic factor, and effective measures includ
ing aggressive therapy against the disease after primary surgery
should be considered.
ACKNOWLEDGMENTS
We thank Drs. T. Saito, G. Tsuda, N. Tanaka, C. Sugimoto, M. Seki, T. Ito,
and S. Noriki for their critical review of this work. We also thank Kyowa
Hakko Kogyo Co. Ltd. and Chugai Pharmaceutical Co. Ltd. for supplying the
Abs.
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oral mesopharyngeal SCC, suggesting that cancer cells do not neces
sarily produce G-CSF by themselves. G-CSF is usually produced by
normal fibroblasts and tissue adjacent to the cancer (25, 26). Actually,
the serum levels of endogenous G-CSF were significantly elevated in
patients with lung cancer compared with healthy people (27). Exog
enous G-CSF immediately up-regulated G-CSFR expression in leu
kemia cells (28). Studies of whether G-CSF stimulates proliferation
and enhances the expression of G-CSFR in oral and mesopharyngeal
carcinoma cells are under way.
Human G-CSFR has five isoforms differing in the cytoplasmic
domain, arising from alternative RNA splicing (11, 29, 30). The
membrane-bound receptor isoforms are speculated to differentially
regulate proliferative and other signals (i.e., mature signals; Ref. 30).
A portion of the cytoplasmic domain of G-CSFR has been demon
strated to be indispensable to the transduction of the G-CSF signal
(12). A point mutation in the region coding for the cytoplasmic
domain of the G-CSFR gene was shown to be involved in a reduced
or abnormal response to G-CSF (31). Thus, we speculate that the
signal via G-CSFR has functions other than proliferation in oral and
mesopharyngeal SCC. The most likely function of the G-CSFR signal
is enhancement of cancer cell invasiveness. Several growth factors,
CSF-1, transforming growth factor ß,epidermal growth factor, HGF,
and basic fibroblast growth factor, enhanced the invasiveness and
metastatic activity of cancer cells (32-43). In the invasive model,
CSF-1 and HGF require CSF-1 receptor and HGF receptor to function
on the surface of cancer cells, respectively (32, 35, 40). The expres
sion of CSF-1 receptor has been associated with adverse clinicopathological prognostic variables in ovarian carcinoma (32). We have
evidence that exogenous G-CSF enhanced the invasive potential of
head and neck SCC cell lines through the elevation of metalloproteinase produced by cancer cells (44).4 Also, G-CSF promoted inva
sion by lung cancer cell lines (45). Thus, we hypothesize that the poor
prognosis of G-CSFR-positive cancer patients results mainly from
enhanced invasiveness triggered via the G-CSFR signal.
rG-CSF is being used more and more frequently to treat patients
with head and neck cancer after chemotherapy in an attempt to reduce
bacterial/fungal infections during neutropenia and/or to administer
high-dose chemotherapy (4, 5). The present study provides evidence
that the poor prognosis of patients with oral and mesopharyngeal
carcinoma is related to the expression of G-CSFR on the surface of
cancer cells. Although we have no data as of yet to determine whether
exogenous G-CSF induces tumor cell proliferation in vivo in the
4 I. Noda. S. Fujieda, H. Tsuzuki. H. Sunaga. N. Tanaka. T. Otsubo, and H. Saito.
G-CSF enhances the invasive potential of human head and neck carcinoma cell lines,
submitted for publication.
799
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O-CSFR AND PROGNOSIS
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Expression of Granulocyte Colony-stimulating Factor Receptor
Correlates with Prognosis in Oral and Mesopharyngeal
Carcinoma
Hideaki Tsuzuki, Shigeharu Fujieda, Hiroshi Sunaga, et al.
Cancer Res 1998;58:794-800.
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