p27kip1 and

Asian Cyclin
J Oral D1 in Oral
Maxillofac SurgSquamous Cell Carcinoma
2004;16:34-37.
ORIGINAL RESEARCH

Correlation of p27kip1 and Cyclin D1 Proteins with Growth

Activity in Grade 1 Oral Squamous Cell Carcinoma in
Myanmar: a Preliminary Study

Shwe Toe,1 Aye Aye Thike,2 Ba Myint,3 Aye Aye Myint,2 Minoru Takagi4
1
Department of Oral Medicine, Institute of Dental Medicine, Mandalay, 2 Department of Pathology,
Institute of Medicine (2), Yangon, 3Department of Oral Medicine, Institute of Dental Medicine, Yangon,
Myanmar, and 4Department of Molecular Pathology, Division of Oral Health Sciences, Graduate School,
Tokyo Medical and Dental University, Japan

Abstract
Objective: To identify the cell cycle-related proteins in World Health Organization grade 1 oral squamous
cell carcinoma and to correlate the immunoreactivity of these proteins with clinicopathological parameters.
Patients and Methods: 24 patients with oral squamous cell carcinoma, were enrolled. P27kip1 and cyclin D1
expression in the tumours were determined by immunohistochemical staining of paraffin sections. Brown
staining of tumour cell nuclei was regarded as positive immunoreactivity.
Results: p27kip1 and cyclin D1 immunoreactivity was observed in 13 (54%) and 9 (37%) patients, respectively.
Although p27kip1 expression in oral squamous cell carcinoma did not statistically correlate with clinico-
pathological parameters, cyclin D1 expression was significantly associated with mitotic count (p = 0.002).
P27kip1 immunoreactivity was found to be inversely associated with cyclin D1 expression (p = 0.002; r = 0.54).
Conclusion: Cyclin D1 immunoreactivity may be useful as a proliferation marker for World Health
Organization grade 1 oral squamous cell carcinoma.

Key words: Cyclin D1, Oral, Squamous cell carcinoma

Introduction p53, p16 and p27 are prominent among the genes
Squamous cell carcinoma (SCC) of the head and neck that have been confirmed as being abnormally
is one of the most common cancers, with a global expressed in oral cancers.3-7
incidence of 500,000 cases per year.1 Oral cancer and
head and neck SCC appear as a consequence of multi- p27kip1 is a cyclin-dependent kinase inhibitor that
ple molecular genetic events in many chromosomes and negatively regulates cell proliferation.8 In mammalian
genes. The consequence of this chromosomal (genetic) cells, transition from the G1 phase into the S phase
damage is cell dysregulation with disruption in cell is governed by cyclin-dependent kinases (CDKs) and
signalling, the cell growth cycle, and/or mechanisms inhibited by CDK inhibitors.9 CDK inhibitors fall into
to repair cell damage or eliminate dysfunctional cells.2 2 families, inhibitor of kinase (INK) and cyclin
inhibitor protein/kinase inhibitor protein (CIP/KIP),
Dysregulation of the cell cycle is a frequent find- on the basis of sequence homology.10 p27 has been
ing in the development of oral cancer. The cell cycle- described as a negative regulator of G1 progression
associated oncogenes cyclin D1 and Murine Double and has been speculated to function as a possible
Minute-2 (MDM-2), and the tumour suppressor genes mediator of transforming growth factor β (TGF β)
-induced G1 arrest.11
Correspondence:
Shwe Toe, No. 32/A Kokine Swimming Club Lane, Sayasan Road, Cyclin D1 is an unstable protein essential for G1
Bahan Township, Yangon, Myanmar.
E-mail: [email protected] progression, through complex formation with CDK4

34 Asian J Oral Maxillofac Surg Vol 16, No 1, 2004

 Toe, Thike, Myint

and retinoblastoma protein (pRb) binding.12 Cyclin D1 Statistical Analysis

gene amplification and protein overexpression have Findings were analysed using the Statistical Package
been found in both breast cancer and head and neck for the Social Sciences for Windows, Version 8.0. The
SCC. Deregulation of expression of the cell cycle relationship between p27kip1 and cyclin D1 immuno-
regulator, cyclin D1, may be responsible for rapid reactivity and the parameters of age, mitotic count,
proliferation of SCC of the head and neck.13 The degree of necrosis, lymphocytic infiltrates, and
purpose of this study was to identify the cell cycle- vascular invasion were tested using chi-square and
related proteins in World Health Organization (WHO) Fisher’s exact tests.
grade 1 oral SCC in Myanmar and to correlate the
immunoreactivity of those proteins with clinico- Results
pathological parameters. Of the 24 patients in this study, 14 were men and 10
were women, aged from 39 to 79 years (mean age,
Patients and Methods 54 years) [Table 1]. Of the 24 patients in the series,
The study group comprised 24 patients with well necrosis of tumour cells was detected in 8 (33%).
differentiated WHO grade 1 oral SCC in Myanmar. Minute foci of necrosis were found in 6 tumours
Histological parameters were assessed on 4 mm whereas 2 tumours showed moderate necrosis.
thick sections stained with hematoxylin and eosin Lymphocytic infiltrates were observed in the stroma
from representative tumour blocks after formalin of 20 tumours (83%). Vascular invasion was absent
fixation, routine processing, and paraffin wax em- in 23 tumours (96%), while only 1 tumour showed
bedding. Mitotic count, vascular invasion, lympho- vascular emboli. Thirteen tumours (54%) expressed
cytic infiltrates, and degree of necrosis were studied. p27kip1 protein, while 9 (37%) showed cyclin D1
Mitotic count was analysed by counting mitotic expression (Figure 1).
activities per 10 consecutive high-power fields in
the field diameter 0.05 mm. The degree of necrosis p27kip1 expression did not statistically correlate with
was assessed as extensive, moderate, minute foci, clinicopathological parameters. However, cyclin D1
or absent.
Parameters Number of patients (%)
Immunohistochemistry Age (years; mean, 54 years)
Immunoreactivity for p 27kip1 (using the primary <54 years 12 (50)
>54 years 12 (50)
antibody Clone/Klon SX 53 G8 Dako, at 1:50
Sex
dilution) and cyclin D1 (using the primary antibody Male 14 (58)
Clone/Klon DCS-6 Dako, at 1:50 dilution) were Female 10 (42)
assessed on 4 mm sections cut from formalin-fixed, Necrosis
Absent 16 (67)
paraffin-embedded blocks of tumours, using the Present 8 (33)
labelled streptavidin-biotin method with antigen Lymphocytic infiltrates
retrieval by way of microwaving. Immunohisto- Absent 4 (4)
chemical staining was considered positive only when Present 20 (83)

there was nuclear reactivity. The proportion of Vascular invasion

Absent 23 (96)
positive nuclei was categorised as negative in those Present 1 (4)
with positivity in 0% to 25% of cells. If more than Mitosis
25% of tumour cells showed brown nuclear staining, Absent 15 (62)
Present 9 (38)
the tumour was regarded as positive immunoreactvity.
p27kip1 expression
Immunoreactivity in the basal cell layer was used as Positive 13 (54)
an internal positive control. Negative controls were Negative 11 (46)
included for each sample in which primary antibodies Cyclin D1 expression
were omitted. Immunohistochemical findings were Positive 9 (37)
Negative 15 (63)
analysed by 2 independent investigators and dis- Table 1. Clinicopathological parameters and the expression
cordant results were reviewed together. of p27kip1 and cyclin D1.

Asian J Oral Maxillofac Surg Vol 16, No 1, 2004 35

p27kip1 and Cyclin D1 in Oral Squamous Cell Carcinoma

of proliferative fractions, as evaluated by proliferating

cell nuclear antigen or Ki-67 antigen, has been shown
to be associated with a high incidence of lymph
node metastases. Reduced expression of p27kip1 may
play an important role in an abnormal proliferation
through loss of cell cycle regulation.15 Decreased
p27kip1 expression significantly correlates with tumour
progression and predicts poor prognosis in carci-
nomas of the gall bladder.11 However, our study could
not demonstrate a significant correlation between the
expression of p27kip1 and clinicopathological para-
meters. This may be due to the small sample size. A
larger sample size in a follow-up study is necessary
to highlight the role of p27kip1 expression in oral SCC.

Cyclin D1 is frequently expressed in oral SCC

and this expression is related to the grade of the
tumour. High-grade tumours (moderately or poorly
differentiated SCC) are often more positive for cyclin
D1 than low-grade tumours (well differentiated
SCC).16 Cyclin D1 mapped to 11q13, regulates the
cell cycle by involvement in control of the transit from
G1 to S phase.17 Overexpression of cyclin D1 appears
in many oral cancers leading to increased proliferation
rate and premature transition through the gap-1 DNA
synthesis checkpoint. 12 Increased proliferation
Figure 1. Positive cyclin D1 expression in oral squamous cell activity may be predicted for those patients showing
carcinoma. (Immunoperoxidase stain x 100.)
positive expression of cyclin D1.17 This study revealed
immunoreactivity was significantly associated with the expression of cyclin D1 in 9 of 24 tumours. In
mitotic count (p = 0.002) and inversely correlated addition, cyclin D1 expression was significantly
with p27kip1 immunoexpression (p = 0.002; r = 0.54). associated with mitotic count (p < 0.05) Increased
mitotic count reflects increased proliferative activity
Discussion of a tumour and may lead to an increased chance of
The issue of the use of molecular markers to determine recurrence and poor outcome. The study pointed out
prognosis and to potentially direct therapy for head that cyclin D1 can be used as a predictor of
and neck cancer is a research topic currently being proliferation activity of oral SCC.
extensively investigated. The proliferation and pro-
gression of cancer may be caused by abnormalities of An inverse correlation between cyclin D1 and
various positive and negative cell cycle regulators.13,14 p 27kip1 expression was found in human gastric
cancers.18 This study also demonstrated an inverse
p27kipl is a cyclin-dependent kinase inhibitor and relationship between cyclin D1 expression and p27kip1
is thought to be an independent prognostic indicator expression (p < 0.05; r = 0.54). This finding is also
for numerous human cancers including breast, colon, expected in oral cancers.
stomach, prostate, non-small cell lung, endocrine,
ovarian, and oral cavity cancers.12 This study was a preliminary study of the
biological characteristics of oral SCC in Myanmar
The loss of p27kip1 protein is also closely asso- with a limited sample size and no survival analysis.
ciated with metastasis in oral SCC.11 The presence Further studies with larger sample sizes and survival

36 Asian J Oral Maxillofac Surg Vol 16, No 1, 2004

 Toe, Thike, Myint

analysis are needed to search for biological charac- epithelia, precancerous lesions, and carcinoma of
teristics and the role of cell cycle-related proteins in gall bladder: association with cancer progression
carcinogenesis of oral SCC. and prognosis. Hepatology 200;31:1068-1072.
9. Peter M, Herskowitz I. Joining the complex:
Conclusion cyclin-dependent kinase inhibitory proteins and
It was concluded that cyclin D1 immunoreactivity the cell cycle. Cell 1994;79:181-184.
can be used as a proliferation marker in WHO grade 10. Sherr CJ, Roberts JM. Inhibitors of mammalian
1 oral SCC and may have a role in routine evaluation G1 cyclin-dependent kinase. Genes Dev 1995; 9:
of oral cancers. 1149-1163.
11. Polyak K, Kato JY, Solomon MJ, Sherr CJ,
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