RESEARC H Open Access
Prognostic significance of Oct4 and Sox2
expression in hypopharyngeal squamous cell
carcinoma
Nan Ge
1,2†
, Huan-Xin Lin
1,2†
, Xiang-Sheng Xiao
1,3†
, Ling Guo
1,4
, Hui-Min Xu
1,2
, Xin Wang
1,2
, Ting Jin
1,2
, Xiu-Yu Cai
1,2
,
Yi Liang
1
, Wei-Han Hu
1,2*
, Tiebang Kang
1*
Abstract
Background: Oct4 and Sox2 are two major transcription factors related to the stem cell self-rene wal and
differentiation. The aim of this study was to examine the association between Oct4 and Sox2 expression levels with
both the clinicopathological characteristics and prognoses of patients with hypopharyngeal squamous cell

involvement rather than distant metastasis [6].
The cancer stem cell (CSC) hypothesis posits that
tumors may be initiated and maintained by a subset of
cells that maintain or acquire stem-cell properties and
that each tumor contains a small subpopulation of cells
that have the ability to differentiate into multiple cell
lineages and self-renew [7,8]. Indeed, cancer stem cells or
cancer stem-like cells have been identified in several solid
tumor ty pes such as breast cancer an d colon can cer
* Correspondence: ;
† Contributed equally
1
State Key Laboratory of Oncology in South China, Cancer Center of Sun
Yat-Sen University, Guangzhou 510060, China
Full list of author information is available at the end of the article
Ge et al. Journal of Translational Medicine 2010, 8:94
/>© 2010 Ge et al; licensee BioMed Central Ltd. This is an Open Access a rticle distributed under the terms of the Creative Commons
Attribution License (http://creativecom mons.org/license s/by/2.0), which permits unrestricted use, distribution, an d reproduction in
any medium, provided the original work is properly cited.
[9,10]. This subpopulation is closely associated not only
with carcinogenesis, but also with recurrence and metas-
tasis of tumors [7]. However, there is no sufficient evi-
dence for putative cancer stem cells in hypopharyngeal
cancer, and this may be important to elucidate carcino-
genesis, to analyze prognosis, and to establish new thera-
peutic approaches for this cancer type.
Oct4 is a major member of the POU domain transcrip-
tion factors, which are required for the self-renewal char-
acteristics and differentiation potential of pluripotent
embryonic stem and germ cells [11,12]. Recent data show

Health Organization classification of tumors: pathology
and genetics of head and neck tumors [23].
Immunohistochemistry (IHC) staining
Immunohistochemistry was performed on 4-μm-thick
routinely processed paraffin sections. Oct4 was detected
using a rabbit polyclonal anti-Oct4a antibody (Cell sig-
naling, #2890, UK, dilution 1:100). Sox-2 was detected
using a rabb it polyclonal anti-Sox antibody (Cell signal-
ing, #3579, UK, dilution 1:100). A total of 85 formalin-
fixed, paraffin-embedded hypopharyngeal squamous cell
carcinoma tissue samples were dried overnight at 56°C.
After deparaffinization and rehydration, sections were
heat-pretreated in a citrate buffer (92°C in microwave
oven) and incubated in 3% H
2
O
2
to block endogenous
peroxidase activity. Then the sections were examined by
immunostaining using the primary antibodies overnight
at 4°C in a humidity chamber. The avidin-biotin
Table 1 The expressions of Oct4 and Sox2 and their
relationships with clinicopathological characteristics
Features No.
patients
OCT4 P
a
SOX2 P
a
High Low High Low

Treatment Type
e
L+N 7 5 2 - 5 2 -
L+N+R 4 1 3 3 1
L+N+C 4 1 3 3 1
L+N+R+C 6 0 6 2 4
N+R 2 0 2 1 1
N+R+C 6 2 4 4 2
R31221
R + C 21 2 19 20 1
C 20 1 19 17 3
No treatment or
tracheotomy
12 1 11 10 2
a
Chi-square test.
b
Patients were divided according to the median values of age.
c
SCCA, Squamous Cell Carcinoma Antigen.
d
TSGF, Tumor Supplied Group of Factor.
e
L = Laryngectomy, N = Neck dissection, R = Radiotherapy, C =
Chemotherapy.
Ge et al. Journal of Translational Medicine 2010, 8:94
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technique was applied using DAB for visualization and
hematoxylin for nuclear countersta ining. Negative con-
trols were prepared by omitting the primary antibody.

theintervalfromtheonsetoftreatmenttothedateof
rec urrence. Recurrence was defined as local tissue inva-
sion by the primary tumor or regional lymph no de
involvement. Disease-free survival (DFS) was defined as
the interval between the onset of treatment and the date
when recurrence or metastasis was diagnosed. OS, LRC
and DFS probabilities were estimated by the Kaplan-
Meier method and the signific ance of differe nces were
assessed by the log-rank test. A P -value < 0.05 was con-
sidered statistical ly significant, and a P-value < 0.0 1 was
considered strongly statistically significance.
Results
Clinicopathological features
Table 1 presents a summary of sex, age, tumor stage,
histological grade, and the status of SCCA (Squamous
Cell Carcinoma Antigen) as well as TSGF (Tumor
Supplied Group of Factor). In this study, there were 85
hypopharyngeal carcinoma p atients consisting o f 84
males and 1 female. The median age was 60 years
(range: 37-82 years). According to the 6th Edition of
the International Union Against Cancer (UICC) TNM
classification system, there were 7 S tage II patients,
24 Stage III patients, and 54 Stage IV patients, as
shown in Table 2. Recurrences were confirmed by his-
topathology or visual examination and were found to
have occurred in 72 patients. The median time to
Table 2 The relationships between clinicopathological
variables and immunohistochemical features with the
overall survival
Variables No.

positive 66 63.6 18.2 15.2 0.103 2.662
negative 19 68.4 36.8 31.6
TNM Stage
I~II 7 100 28.5 28.5 0.678 0.173
III~IV 78 61.5 21.8 17.9
Oct4
e
High Expression 14 85.7 71.4 57.1 0.000 15.661
Low Expression 71 60.6 12.7 11.3
Sox2
e
High Expression 67 59.7 23.9 19.4 0.683 0.166
Low Expression 18 83.3 16.7 16.7
Oct4 & Sox2 0.000 17.991
Both high 13 88.2 76.9 61.5
Either high 55 54.5 10.9 9.1
Both low 17 82.4 17.6 17.6
a
Log-rank test.
b
Patients were divided according to the median values of age.
c
SCCA, Squamous Cell Carcinoma Antigen.
d
TSGF, Tumor Supplied Group of Factor.
e
Two-sided log rank test with an overall sample size of 85 subjects (of which
71 are in group.
1 and 14 are in group 2) achieves 84% power at a 0.0500 significance level to
detect a difference of 0.5870 between 0.1270 and 0.7140–the proportions

related with the histological grade (p =0.03),asin
Table 1.
Association with prognosis
Univariate analyses showed no significant association
between OS, DFS or LRC and T stage, cervical lymph
node metastasis, TNM stage, age, or histological grade
(Table 2, 3). Patients with high Oct4 expression had a
significantly better prognosis, including longer survival
(p = 0.000) and lower recurrence rate (p = 0.000). Even
though Sox2 expression showed no association with
prognosis, the highest overall survival rate was doc u-
mented in the high Oct4 expression/high Sox2 expres-
sion subgrou p. The 5-years overall survival rate was
61.5% for this group (p = 0.000) (Fig. 2).
Figure 1 Expr ession of Oct4 and Sox2. Immunohistochemical s taining for Oct4 and Sox2 expression in hypopharyngeal squamous cell
carcinoma. Brown grains represent a positive signal (3, 3-diaminobenzidine staining). The positive expression site of Oct4 and Sox2 was mainly
localized in the nucleus of tumor cells. (A) High Oct4 expression in tumor cells, (B) low Oct4 expression in tumor cells, (C) high Sox2 expression
in tumor cells, and (D) low Sox2 expression in tumor cells.
Ge et al. Journal of Translational Medicine 2010, 8:94
/>Page 4 of 7
Multivariate analysis
A multivariate s urvival analysis was performed with the
Cox regression model for each predictor of prognosis to
calculate odds ratios, as well as 95% confidence interval s.
The model was simp lified in a stepwise fashion by
removing variables that had a p value ≥0.05. Only three
variables remained statistically significant as independent
predictors of OS and LRC in the multivariate analysis.
Also, because the varia ble Oct4 & Sox2 expression con-
sisted of Oct4 expression and Sox2 expression, this vari-

T Stage 0.437 0.605 0.567 0.328
Cervical lymph node metastasis 0.050 3.832 0.106 2.610
TNM Stage 0.877 0.024 0.934 0.007
Oct4 expression 0.000 22.275 0.000 20.405
Sox2 expression 0.680 0.170 0.383 0.761
Oct4 & Sox2 expression 0.000 26.331 0.000 22.101
a
Log-rank test.
b
Patients were divided according to the median values of age.
c
SCCA, Squamous Cell Carcinoma Antigen.
d
TSGF, Tumor Supplied Group of Factor.
Figure 2 The Kaplan-Meier survival curves . Kaplan-Meier curves for overall survival rates according to (A) Oct4 expression status, (B) Sox2
expression status, (C) combined expression status of Oct4/Sox2 and loco-regional control rates according to (D) Oct4 expression status in
hypopharyngeal squamous cell carcinoma. Statistical differences were calculated through log-rank comparisons.
Ge et al. Journal of Translational Medicine 2010, 8:94
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which confers not only the capacity of self-re newal but
also of differentiation and maturation [7,8]. This subpopu-
lation of cancer cells may be similar to stem cells or stem-
like cells. Oct4 and Sox2 have been proven to be two
major transcription factors that can render an adult cell
capable of being reprogrammed to become a pluripotent
stem cell [12,24,25]. In addition, the expression of Oct4 or
Sox2 has been reported in the cancer stem-like cells and is
related to a cancer patient’s prognosis. Taken together,
Oct4 or Sox2 might play an important role in carcinogen-
esis and tumor progression and may be used as an indica-

roles of Oct4 and Sox2 in hypopharyngeal squ amous cell
carcinoma still require further investigation.
Conclusion
Currently, clinical TNM stage is insufficient to predict
prognoses of patients with hypopharyngeal s quamous
cell carcinoma, patients of the same clinical stage often
show different clinical course. In this study we demon-
strate that Oct4 ex pression is an independen t predictive
factor for patients with hypopharyngeal squamous cell
carcinoma, suggesting that Oct4 expression may be a
useful indicator for p redicting the prognosis o f hypo-
pharyngeal squamous cell carcinoma.
Acknowledgements
We thank Dr. Rong-Zhen Luo, Dr. Ma-Yan Huang and Dr. Mei Li for
immunohistochemical analysis. This work was supported by the Natural
Science Foundation of Guangdong Province, P. R. China, (To: WHH, No.
9151008901000223) 985 funding from Sun Yat-sun University (To: TK).
Author details
1
State Key Laboratory of Oncology in South China, Cancer Center of Sun
Yat-Sen University, Guangzhou 510060, China.
2
Department of Radiation
Oncology, Cancer Center of Sun Yat-Sen University, Guangzhou 510060,
China.
3
Department of Breast Oncology, Cancer Center of Sun Yat-Sen
University, Guangzhou 510060, China.
4
Department of Nasopharyngeal

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Table 4 Multivariate analysis
Variables OS LRC
Odds
ratio
95%CI
a
P
b
Odds
ratio
95%CI
a
P
b
Oct4
expression
0.296 0.129-
0.679
0.004 0.183 0.040-
0.475
0.001
Sox2

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