Cyclin D1 overexpression and poor clinical outcomes in Taiwanese oral cavity squamous cell carcinoma.

Abstract

Cyclin D1 gene regulates cell cycle and plays an important role in the tumorigenesis of human cancers. The association between cyclin D1, clinicopathologic parameters and prognosis in oral cavity squamous cell carcinoma (OSCC) is inconclusive.
A total of 264 male OSCCs were examined for cyclin D1 protein expression using immunohistochemistry (IHC). The expression levels of cyclin D1 were defined as overexpression when more than 10% of tumor cells displayed nuclear staining with moderate to strong intensity.
Overexpression of cyclin D1 was found in 97 (36.7%) OSCCs. Cyclin D1 protein overexpression was significantly associated with lymph node metastasis (P = 0.002), tumor cell differentiation (P = 0.031) and tumor stage (P = 0.051), but not associated with age onset, cigarette smoking, alcohol drinking, or areca quid chewing. Overexpression of cyclin D1 was also significantly associated with poor clinical outcomes in terms of disease-free survival (DFS, P = 0.002) and overall survival (OS, P < 0.001). The effects of cyclin D1 protein overexpression on DFS (hazard ratio (HR) = 1.540; 95% confidence interval (CI), 1.068 - 2.222) and OS (HR = 1.702; 95% CI, 1.168 - 2.480) were still existed after adjusting for clinicopathological parameters (such as age, primary tumor status, tumor cell differentiation, and lymph node metastasis) using logistic multivariate analysis.
Cyclin D1 protein worked as an independent prognostic factor and can be as a biomarker for the aggressiveness of OSCC.

Full text

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RESEARCHOpen Access
Cyclin D1 overexpression and poor clinical
outcomes in Taiwanese oral cavity squamous cell
carcinoma
Shiang-Fu Huang1†, Sou-De Cheng2†, Wen-Yu Chuang3, I-How Chen1, Chun-Ta Liao1, Hung-Ming Wang4and
Ling-Ling Hsieh5*
Abstract
Background: Cyclin D1 gene regulates cell cycle and plays an important role in the tumorigenesis of human
cancers. The association between cyclin D1, clinicopathologic parameters and prognosis in oral cavity squamous
cell carcinoma (OSCC) is inconclusive.
Methods: A total of 264 male OSCCs were examined for cyclin D1 protein expression using immunohistochemistry
(IHC). The expression levels of cyclin D1 were defined as overexpression when more than 10% of tumor cells
displayed nuclear staining with moderate to strong intensity.
Results: Overexpression of cyclin D1 was found in 97 (36.7%) OSCCs. Cyclin D1 protein overexpression was
significantly associated with lymph node metastasis (P = 0.002), tumor cell differentiation (P = 0.031) and tumor
stage (P = 0.051), but not associated with age onset, cigarette smoking, alcohol drinking, or areca quid chewing.
Overexpression of cyclin D1 was also significantly associated with poor clinical outcomes in terms of disease-free
survival (DFS, P = 0.002) and overall survival (OS, P < 0.001). The effects of cyclin D1 protein overexpression on DFS
(hazard ratio (HR) = 1.540; 95% confidence interval (CI), 1.068 - 2.222) and OS (HR = 1.702; 95% CI, 1.168 - 2.480)
were still existed after adjusting for clinicopathological paremeters (such as age, primary tumor status, tumor cell
differentiation, and lymph node metastasis) using logistic multivariate analysis.
Conclusion: Cyclin D1 protein worked as an independent prognostic factor and can be as a biomarker for the
aggressiveness of OSCC.
Keywords: oral squamous cell carcinoma, lymph node metastasis, cyclin D1
Background
In Taiwan, oral cancer is the fourth most common can-
cer in men [1]. Epidemiologic studies have shown that
environment and personal habits, particularly tobacco
use, areca quid (AQ) chewing and alcohol abuse, are
major etiologic factors in the induction and progression
of this disease. About two thirds of oral cancers were
occurred in oral cavity. The primary treatment for oral
cavity squamous cell carcinoma (OSCC) is radical sur-
gery with or without post-operative chemoradiation [2].
However, for patients with tumors at advanced stage,
their prognoses are usually discouraging. If we can better
understand the characteristics of OSCCs, this may ulti-
mately help clinicians to provide OSCC patients with
more appropriate treatment.
The cyclin D1 gene (CCND1) located on chromosome
11q13 is a positive regulator of the cell cycle. It encodes a
nuclear protein that forms complexes with cyclin-depen-
dent kinases 4 and 6, which phosphorylate and inactivate
the retinoblastoma protein (pRb). Inactivation of pRb
allows cell cycle progression from G1 to S phase [3].
Although it has been shown that increased expression of
cyclin D1 caused potential for growth advantage and
enhances tumorigenesis [4,5], the role of cyclin D1 as a
prognostic marker in OSCC remains controversial. Over-
expression of cyclin D1, was reported to be associated
* Correspondence: llhsieh@mail.cgu.edu.tw
† Contributed equally
5Department of Public Health, Chang Gung University, Tao-Yuan, Taiwan
Full list of author information is available at the end of the article
Huang et al. World Journal of Surgical Oncology 2012, 10:40
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WORLD JOURNAL OF
SURGICAL ONCOLOGY
© 2012 Huang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
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with recurrence and shortened overall survival in oper-
able cases of squamous cell carcinoma of head and neck
(SCCHN) [6-8]. However, a study of 45 patients with a
significantly higher proportion of oral carcinoma found
no significant correlation between overexpression of
cyclin D1 and any of the clinicopathological parameters
studied [9]. Therefore, this study was designed to investi-
gate the correlation of cyclin D1 expression with clinico-
pathologic parameters and disease outcome in 264
Taiwanese male OSCC patients.
Materials and methods
Patients and clinical diagnosis
This study was approved by the Institutional Review
Board of Chang Gung Memorial Hospital. Two hundred
and sixty-four male Taiwanese oral cancer patients
received radical surgery prior to any treatments during
March 1999 and December 2005 at Chang Gung Memor-
ial Hospital, Lin-Kuo, were recruited for participation in
this study. All patients gave informed consent for partici-
pation and were interviewed uniformly before surgery by
a well-trained interviewer. The questionnaire used in the
interview sought detailed information on general demo-
graphy as well as current and past cigarette smoking,
alcohol drinking and AQ chewing habits. If the patients
had ever smoked cigarette, chewed AQ and drank alcohol
on a regular basis (at least once a week for 1 year) were
classified as tobacco, AQ and alcohol users, respectively.
For each patient, clinical histological parameters (ie, pT
classification including skin and bone invasion, differen-
tiation, nodal-status, lymph node extracapsular spread
(ECS), and perineural invasion) were reviewed by the
pathologist and scored according to the recommenda-
tions for the reporting of specimens containing oral cav-
ity and oropharynx neoplasms by the Association of
Directors of Anatomic and Surgical Pathology [10].
Immunohistochemical analysis
Immunohistochemical staining for cyclin D1 protein was
performed as described previously [11]. Briefly, the paraf-
fin embedded tumor sections (5 μm) were deparaffinized,
retrieved with heat in 10 mM citrate buffer (pH 6.0) and
treated with 3% hydrogen peroxide to remove endogen-
ous peroxidase activity. Anti-cyclin D1 monoclonal anti-
body SP4 (1:200) (Lab Vision, Fremont, CA) was used as
the primary antibody and the NovoLink™ Polymer
Detection System (Novocastra Laboratories Ltd., Newcas-
tle Upon Tyne, UK) was used as the detection system.
The slides were then counterstained with hematoxylin
and coverslipped with Permount and examined for the
extent and intensity of nuclear and non-nuclear staining
in tumor cells and for background staining by the pathol-
ogist (WYC) in a blind manner. In the present study,
when more than 10% of cells displayed nuclear staining
[7,12,13] and intensity scores of moderate and strong,
then there was considered to be cyclin D1 overexpression
(Figure 1).
Statistical analysis
Statistical analysis was performed using the SPSS statis-
tical package (SPSS, Chicago, IL). The correlations
between the cyclin D1 status and age, TNM stage, cigar-
ette smoking, alcohol drinking, and AQ chewing was
examined by c2test or Fisher’s exact test. Survival
curves were constructed by the Kaplan-Meier method
and the curves were compared using the log-rank test.
The Cox regression model was applied to adjust simul-
taneously all potential prognostic variables including
age, primary tumor status, tumor cell differentiation,
and lymph node metastasis. A two-sided value of p <
0.05 was considered statistically significant.
Results
Patient characteristics
The clinicopathological features of the 264 OSCC male
patients who took part in this study are listed in Table
1. The major primary sites were the bucca (40.2%, 106/
264) and the tongue (37.9%, 100/264). Overall, 85.2%
(225/264) of the patients were cigarette smokers, 50.4%
(133/264) were alcohol drinkers and 86.0% (227/264)
were AQ chewers. The primary treatment for these 264
patients was surgery and 124 (47.0%) and 59 (22.3%) of
the patients undergoing additional radiation therapy and
chemoradiotherapy, respectively. The median follow-up
was 46.5 months.
Prognostic implications of cyclin D1 protein expression
Overexpression of cyclin D1 was found in 97 (36.7%)
OSCCs. As shown in Table 2, cyclin D1 overexpression
was more prevalent in tumors at advanced stage than
those at early stage (p = 0.051) and moderate/poor dif-
ferentiation than well differentiation (p = 0.031). More
specifically, tumors with characteristics of lymph node
metastasis and lymph node ECS had significantly higher
frequency of cyclin D1 overexpression than tumors
without those characteristics (p = 0.002). The frequency
of cyclin D1 overexpression was lower in buccal cancer
than other subsites (p = 0.007). On the other hand,
cyclin D1 protein overexpression was not associated
with age, primary tumor status, skin invasion, bone
invasion, perineural invasion, cigarette smoking, AQ
chewing, and alcohol drinking.
Disease-free and overall survival (DFS and OS) were
significantly worse in patients that were cyclin D1 over-
expressed in the tumors when compared with patients
that were cyclin D1 not overexpressed (Figure 2). To
assess the effect of other clinicopathological factors on
DFS and OS, a Cox model was carried out initially
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using univariate analysis. As shown in Table 3, primary
tumor status (p = 0.010), differentiation (p = 0.020), and
lymph node metastasis (p < 0.001) were significantly
associated with a poorer OS. Lymph node ECS (p <
0.001) was also significantly associated with a poorer
DFS. After adjusting for age, tumor cell differentiation,
primary tumor status and lymph node metastasis using
multivariate analysis, cyclin D1 overexpression was still
associated with a significantly increased hazard ratio for
DFS and OS, respectively, of 1.540 (95% CI, 1.068 -
2.222) and 1.702 (95% CI, 1.168 - 2.480) compared with
the patients with tumors without cyclin D1 overexpres-
sion (Table 4).
Discussion
The clinical outcome of OSCC patients is strongly influ-
enced by the stage of disease, particularly with positive
nodal metastasis or lymph node ECS [14]. In the present
study, we found that lymph node metastasis with posi-
tive lymph node ECS was the major determinant of
OSCC outcomes (Table 3 and 4) and overexpression of
cyclin D1 was significantly associated with lymph node
metastasis (Table 2). In addition, OSCC survival was sig-
nificantly influenced by the level of cyclin D1 expression
(Figure 2a and 2b) and the relationship still exists in the
multivariate analysis after adjusting for age and lymph
node metastasis (Table 4). Cyclin D1 overexpression fre-
quencies were reported ranging from 17.1% to 83.0% in
OSCC [7,8,12,13,15-19]. In the present study, 36.7% of
OSCCs showed overexpression of cyclin D1 protein.
The reason for such variation is uncertain, but may
reflect mainly differences in the primary antibody used
and scoring systems applied. In the present study, a rab-
bit monoclonal antibody SP4 raised against a synthetic
peptide from C-terminus of human cyclin D1 was used,
which is deemed to be specific to cyclin D1 [20]. Gown
et al. [21] have reported that a normalized scoring
method that subtracts the score for non-neoplastic cells
from that for tumor cells reduces the false-positive rate
from 31% to 5% in assessing HER2 IHC results for
AB
CD
Figure 1 Immunohistochemistry staining patterns of cyclin D1 in tissues from oral squamous cell carcinoma (400×). A. Tumor-normal
adjacent with weak intensity, B. Tumor with weak intensity, C. Tumor with moderate intensity, and D. Tumor with strong intensity.
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breast cancer. We found that most normal basal epithe-
lial cells demonstrated cyclin D1 equivocal weak nuclear
staining or background staining in the present series.
Therefore, we defined that samples with more than 10%
of the cells with moderate and strong cyclin D1 intensity
were as having a state of overexpression.
Cyclin D1 is one of the key proteins involved in cell
cycle control and is essential for G1 to S transition.
Cyclin D1 interacts with cyclin-dependent kinase 4/6
and forms a complex that inactivates pRB through phos-
phorylation, allowing passage through the restriction
point and progression through the G1 phase [22]. Dys-
regulation of cyclin D1 expression or function contri-
butes to the loss of normal cell cycle control during
tumorigenesis. Our results indicated the cyclin D1 over-
expression was an early event in the tumorigenesis of
OSCC.
Previous studies have found a correlation between
cyclin D1 overexpression and the presence of regional
lymph node metastases in several human tumors,
including head and neck SCC [7,19,23]. Our results
indicated that the odds ratio of nodal metastases in
tumors that overexpressed cyclin D1 was 2.482 (95% CI,
1.484 - 4.149) times of those without overexpression.
This suggests that cyclin D1 overexpression may be
related to local invasiveness and a more aggressive clini-
cal behavior of OSCC. Lymph node metastasis could
thus be the pathway that patients with cyclin D1 overex-
pression had poor prognosis. To clarify the mechanism
of lymph node spread of tumor cells via cyclin D1
Table 1 Characteristics of the 264 OSCC patients
Characteristic
Age (year)
Mean ± SD
Range
Site of primary tumor [No. of patients (%)]
Tongue
Bucca
Others*
Pathologic stage [No. of patients (%)]
Stage I
Stage II
Stage III
Stage IV
AQ chewing [No. of patients (%)]
Yes
No
Cigarette smoking [No. of patients (%)]
Yes
No
Alcohol drinking [No. of patients (%)]
Yes
No
49.33 ± 11.01
26-78
100 (37.9)
106 (40.2)
58 (22.0)
18 (6.8)
56 (21.2)
53 (20.1)
137 (51.9)
227 (86.0)
37 (14.0)
225 (85.2)
39 (14.8)
133 (50.4)
131 (49.6)
SD: standard deviation; AQ: areca quid. *Including mouth floor (n = 10), lip
(n = 6), alveolar ridge (n = 26), hard palate (n = 5) and retromolar trigone
(n = 11).
Table 2 The associations between cyclin D1
overexpression and clinicopathological parameters
(N = 264)
Cyclin D1 protein
overexpression
No
[N (%)]
Yes
[N (%)]
p value
Age
< 50 yrs (n = 151)
≥ 50 yrs (n = 113)
Subsites
Tongue (n = 100)
Bucca (n = 106)
Others (n = 58)
Tumor stage
Early (n = 76)
Advanced (n = 188)
Primary tumor
T1/T2 (n = 129)
T3/T4 (n = 135)
Differentiation
Well (n = 107)
Moderate/poor (n = 157)
Tumor depth
< 10 mm (n = 95)
≥ 10 mm (n = 169)
Lymph node metastasis
LN (-); ECS (-) (n = 138)
LN (+); ECS (-) (n = 55)
LN (+); ECS (+) (n = 71)
Skin invasion
Yes (n = 28)
No (n = 236)
Bone invasion
Yes (n = 67)
No (n = 197)
Perineural invasion
Yes (n = 72)
No (n = 192)
AQ chewing
Yes (n = 227)
No (n = 37)
Cigarette smoking
Yes (n = 225)
No (n = 39)
Alcohol drinking
Yes (n = 133)
No (n = 131)
93 (61.6)
74 (65.5)
58 (38.4)
39 (34.5)
0.516
52 (52.0)
81 (76.4)
34 (58.6)
48 (48.0)
25 (23.6)
24 (41.4)
0.007
55 (72.4)
112 (59.6)
21 (27.6)
76 (40.4)
0.051
77 (59.7)
90 (66.7)
52 (40.3)
45 (33.3)
0.240
76 (71.0)
91 (58.0)
31 (29.0)
66 (42.0)
0.031
66 (69.5)
101 (59.8)
29 (30.5)
68 (40.2)
0.116
101 (73.2)
30 (54.5)
36 (50.7)
37 (26.8)
25 (45.5)
35 (49.3)
0.002
0.001*
22 (78.6)
145 (61.4)
6 (21.4)
91 (38.6)
0.075
42 (62.7)
125 (63.5)
25 (37.3)
72 (36.5)
0.911
39 (54.2)
128 (66.7)
33 (45.8)
64 (33.3)
0.061
147 (64.8)
20 (54.1)
80 (35.2)
17 (45.9)
0.210
147 (65.3)
20 (51.3)
78 (34.7)
19 (48.7)
0.093
83 (62.4)
84 (64.1)
50 (37.6)
47 (35.9)
0.772
LN: lymph node metastasis; ECS: extracapsular spread; AQ: areca quid. * c2
trend test.
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overexpression, further study is necessary to explore
cyclin D1-dependent molecules which would play an
important role in the tumor invasion and metastasis.
In our series, moderately and poorly differentiated
tumors demonstrated a significantly higher prevalence
of cyclin D1 overexpression than well-differentiated
tumors (p = 0.031). The relationship was consistent with
previous reports [7,13,18]. However, the reason for this
relationship remains unclear. Both in vitro and in vivo
studies have found that cyclin D1 overexpression can
inhibit the differentiation of myoblasts [9,24] and intest-
inal epithelial cells [25], thus raising the possibility that
cyclin D1 overexpression may play a role in the inhibi-
tion of tumor cell differentiation in some cell types [7].
Overexpression of cyclin D1 was also associated with
reduced DFS and OS. This is in agreement with several
AB
Figure 2 Kaplan-Meier curves for disease-free survival (DFS) and overall survival (OS) based on analysis of cyclin D1 protein
overexpression. (A) Cyclin D1 overexpression was significantly associated with disease-free survival. (B) Cyclin D1 overexpression was
significantly associated with overall survival.
Table 3 Univariate regression model of prognostic covariates in 264 patients with oral cavity squamous cell carcinoma
regarding disease-free and overall survival
DFS
CharacteristicHR (95% CI)
OS
P valueHR (95% CI)P value
Age (years old)
< 50
≥ 50
Differentiation
Well
Moderate/poor
Primary tumor
T1/T2
T3/T4
Nodal status
(-) metastasis, (-) ECS
(+) metastasis, (-) ECS
(+) metastasis, (+) ECS
Overexpression of cyclin D1 protein
No
Yes
11
1.038 (0.731 - 1.472) 0.8361.118 (0.779 - 1.605)0.545
11
1.232 (0.864 - 1.758)0.2501.571 (1.074 - 2.300)
0.020
11
1.301 (0.918 - 1.843)0.1381.617 (1.123 - 2.328)
0.010
11
1.042 (0.639 - 1.699)
2.550 (1.726 - 3.765)
0.870
< 0.001
1.349 (0.813 - 2.238)
3.391 (2.265 - 5.077)
0.247
< 0.001
11
1.710 (1.202 - 2.434)
0.002
1.970 (1.371 - 2.831)
< 0.001
DFS: disease-free survival; OS: overall survival; HR: hazard ratio; CI: confidence interval; ECS: extracapsular spread.
Huang et al. World Journal of Surgical Oncology 2012, 10:40
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