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Loss of PTEN Expression as a Prognostic Marker for Tongue Cancer
Janet I. Lee, MD;
Jean-Charles Soria, MD;
Khaled A. Hassan, MD;
Adel K. El-Naggar, MD, PhD;
Ximing Tang, MD, PhD;
Diane D. Liu, MS;
Waun Ki Hong, MD;
Li Mao, MD
Arch Otolaryngol Head Neck Surg. 2001;127:1441-1445.
ABSTRACT
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Background Abnormalities of PTEN, a candidate tumor suppressor
gene located at 10q23.3, play an important role in the tumorigenesis of multiple
tumor types.
Objectives To investigate the expression of PTEN and its clinical implication in
squamous cell carcinoma of the tongue.
Design Retrospective analysis of PTEN protein expression in archived primary
oral tongue tumor samples.
Setting Academic center.
Patients and Methods PTEN expression was determined by immunohistochemical analysis in tissue
samples from 41 patients with stage II, III, and IV squamous cell carcinoma
of the tongue. All the patients underwent curative surgical treatment with
a median follow-up of 81 months. The Kaplan-Meier method was used for survival
analysis. Multivariate analysis was performed according to the Cox proportional
hazards model.
Results Lack of staining for PTEN was demonstrated in 12 (29%) of the 41 tumors.
Patients whose tumors lacked PTEN expression had a significantly shorter overall
survival time (P = .03) and event-free survival time
(P = .01) than those patients with positive PTEN
expression. Multivariate regression analysis demonstrated that PTEN expression
is an independent predictor of poor outcome when compared with tumor stage
and nodal status.
Conclusions Although genetic alterations of the PTEN gene
are rare in head and neck squamous cell carcinoma, loss of PTEN is not an uncommon event in squamous cell carcinoma of the tongue.
Lack of PTEN expression may be an independent prognostic indicator for clinical
outcome in patients with this tumor type.
INTRODUCTION
HEAD AND NECK squamous cell carcinoma (HNSCC) accounts for 3% to 5%
of all malignancies in Western countries, with cancer of the oral cavity accounting
for 30% of these cancers. In the United States alone, it was estimated that
30 200 new cases of oral cancer would be diagnosed in 2000, with an estimated
7800 deaths.1 Of these, an estimated 6900 new
cases of oral tongue cancers would be diagnosed in 2000, with an estimated
1700 deaths.1 While for early-stage tumors
excellent cure rates can usually be achieved, the 5-year survival rate for
advanced-stage disease is only 40% to 60%, with little improvement during
the last 2 decades.2 To further improve the
survival rate of these patients, new biomarkers for understanding tumor biology
and its prognostic value are crucial for future management.
PTEN/MMAC1/TEP1 (phosphatase and tensin homolog
deleted on chromosome TEN), located at 10q23.3, is a tumor suppressor gene
that encodes a dual-specificity phosphatase with lipid and protein phosphatase
activity.3 Germline mutations of PTEN are found in patients with Cowden syndrome, a familial syndrome
associated with a predisposition for multiple benign hamartomas, and malignant
breast and thyroid neoplasms.3 Somatic mutation
or deletion of PTEN has been reported in a variety
of tumor types, including glioblastoma, melanoma, breast, prostate, and endometrial
carcinomas.4-8
Genetic analysis of PTEN in head and neck cancers
has demonstrated alterations in PTEN in 5% to 10%
of tumors, suggesting that PTEN may play a role in
head and neck tumorigenesis.9-10
Because alternative mechanisms may also inactivate gene function, such as
promoter hypermethylation, alternative splicing of pre–messenger RNA,
and posttranslational modification, the actual frequency of PTEN abnormalities in HNSCC may be underestimated. However, to date,
few analyses of PTEN protein expression in HNSCC have been performed, and
little is known about epigenetic or posttranslational mechanisms that could
participate in PTEN inactivation.
To determine PTEN expression in patients with squamous cell carcinoma
(SCC) of the tongue and whether it can be used as a prognostic marker, we
examined immunohistochemically the expression pattern of PTEN in 41 patients
with stage II, III, and IV SCC of the tongue. We then analyzed PTEN expression
status with clinical parameters to determine whether it has any prognostic
significance in this homogeneous population. We found that loss of PTEN expression
occurred in 12 (29%) of 41 tumors and that this loss of PTEN is an independent
adverse prognostic factor for both overall and event-free survival.
MATERIALS AND METHODS
STUDY POPULATION
Specimens of oral tongue SCC were obtained from archived tissue samples
of surgically resected, pathologic, stage II, III, and IV tumors from 41 patients
treated at The University of Texas M. D. Anderson Cancer Center, Houston,
from 1991 to 1994 under a prospective oral tongue surgical pathology protocol.
All patients were treated by surgery and received a median of 81 months of
follow-up care after surgical treatment. Survival data were available for
all patients; the minimum length of follow-up care was 10 months. The study
population consisted of 25 men and 16 women. The mean age of patients was
56.2 years (SD, 11.2 years).
IMMUNOHISTOCHEMICAL STAINING FOR PTEN PROTEIN
Paraffin-embedded, 4-µm thick tissue sections from all 41 primary
tumors were stained for the PTEN protein using a primary rabbit polyclonal
anti-PTEN antibody (Zymed Laboratories, San Francisco, Calif). Deparaffinization
of all sections was performed through a series of xylene baths, and rehydration
was performed through graded alcohols. The sections were then immersed in
methanol containing 0.3% hydrogen peroxidase for 20 minutes to block the endogenous
peroxidase activity and were incubated in 2.5% blocking serum to reduce nonspecific
binding. Sections were incubated overnight at 4°C with primary anti-PTEN
antiserum (1:100). The sections were then processed using standard avidin-biotin
immunohistochemical analysis according to the manufacturer's recommendations
(Vector Laboratories, Burlingame, Calif). Diaminobenzidine was used as a chromogen,
and commercial hematoxylin was used for counterstaining. Adjacent normal-appearing
epithelium within the tissue sections served as a positive internal control.
Representative areas of each tissue section were selected, and cells
were counted in at least 4 fields (original magnification x200). Immunohistochemical
staining was classified into 3 groups as previously reported6:
increased or equal staining intensity compared with the corresponding normal
tissue, decreased staining intensity, and absence of staining. All slides
were evaluated and scored independently by 2 investigators (J.I.L. and J.-C.S.)
who were blinded to the clinical information pertaining to the subjects.
STATISTICAL ANALYSIS
Overall survival and event-free survival curves were estimated by the
Kaplan-Meier method, and the resulting curves were compared using the log-rank
test. Event-free survival time was calculated from the date of surgery to
relapse or death. Two-sided Fisher exact test or the  2 test
was used to analyze the association between 2 categorical variables. P<.05 was considered statistically significant. Multivariate
analysis was performed according to the Cox proportional hazards model.
RESULTS
In the areas of normal squamous epithelium, staining for the PTEN protein
was present and served as a positive internal control. The staining pattern
was more prominent in the stratum spinosum and in areas of keratin differentiation,
with minimal or no staining in the basal and parabasal cells (Figure 1A). The staining was cytoplasmic. The intensity of staining
in the normal-appearing epithelium could be classified as moderate to strong.
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Figure 1. Immunohistochemical staining patterns
of PTEN in squamous cell carcinoma of the tongue (primary rabbit polyclonal
anti-PTEN antibody and hematoxylin counterstaining, original magnification
x200). A, Normal stratified squamous epithelium with prominent PTEN
staining in the stratum spinosum and stratum superficiale and negative PTEN
staining in the underlying carcinoma cells. B, Heterogeneous staining pattern
with positive PTEN staining in the well-differentiated areas. C, Well-differentiated
tumor with negative PTEN staining. D, Well-differentiated tumor with strongly
positive PTEN staining.
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Most specimens had areas of normal epithelium adjacent to carcinoma,
with the normal epithelium having positive expression and the carcinoma having
variable expression levels. PTEN staining among the tumor specimens was negative,
diffusely weak, or a heterogeneous pattern of variable intensity. Among the
heterogeneously stained specimens, staining was prominent in the well-differentiated
areas.
According to our scoring criteria, loss of PTEN expression was noted
in 12 (29%) of the 41 SCC tongue specimens. Weak expression was seen in 25
(61%) of the tumors, and strongly positive expression was seen in 4 (10%)
of the tumors. Table 1 shows the
relationships between the expression of PTEN and the clinicopathologic factors.
The frequency of PTEN expression did not differ significantly by sex. A precise
evaluation of tobacco and alcohol use was not available in a subset of our
patients; therefore, analysis of PTEN expression in relation to tobacco use
or alcohol consumption was not performed. There was no significant relationship
between PTEN expression and the histologic grade of the tumor, the clinical
stage of the disease, or nodal involvement. Positive PTEN expression was more
likely to be present in patients younger than 50 years (P = .03). There was, however, no significant correlation with age and
overall survival or event-free survival.
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PTEN Status and Clinicopathologic Characteristics of Tumor Samples
From 41 Patients With Squamous Cell Carcinoma of the Tongue*
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We also investigated the relationship between PTEN expression and patient's
survival. Figure 2A shows a comparison
of the Kaplan-Meier overall survival curves, which demonstrates that patients
whose tumors were PTEN negative had a significantly shorter survival time
than those patients whose tumors were PTEN positive (P
= .03 by log-rank test). At 5 years, only 4 (33%) of 12 patients whose tumors
had negative PTEN expression were alive compared with 19 (65%) of the 29 patients
whose tumors had positive PTEN expression. When event-free survival time was
analyzed, patients with negative PTEN expression also showed a significantly
worse prognosis than did patients with positive PTEN expression (P = .01). Of the 12 patients whose tumors were PTEN negative, 9 (75%)
had recurrence, metastases, or death at follow-up, whereas 12 (41%) of the
29 patients whose tumors were PTEN positive had recurrence, metastases, or
death (P = .01) (Figure 2B). As expected, clinical stage (II vs III and IV) and nodal
status (N0 vs N1-3) were statistically associated with poor outcome (data
not shown). These well-established clinical markers were then compared with
PTEN expression status using multivariate analysis. Given the moderate patient
sample size and number of events in this study, multivariate analysis could
only be performed on 2 covariates at a time. When PTEN status and stage of
disease were analyzed in relation to event-free survival time, PTEN continued
to be a statistically significant marker (P = .03).
When the same analysis was performed with PTEN status and nodal status in
the model, PTEN remained statistically significant (P
= .02).
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Figure 2. A, Overall survival curves of
patients with squamous cell carcinoma of the tongue according to PTEN expression.
B, Event-free survival curves of patients with squamous cell carcinoma of
the tongue according to PTEN expression.
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COMMENT
PTEN is a tumor suppressor gene that encodes
a dual-specificity lipid and protein phosphatase enzyme. Its major substrate
is phosphatidylinositol-3,4,5-triphosphate (PIP-3), a direct product of phosphoinositol-3-kinase
activity.11 This substrate mediates growth
factor–induced activation of intracellular signaling, notably through
serine-threonine kinase Akt (also known as Akt1, RAC1, or PKB), which promotes
cell survival and proliferation. In actively proliferating cells with elevated
PIP-3 levels, the Akt complex is activated through phosphorylation, and the
role of PTEN is to maintain low levels of PIP-3. This is supported by studies
that show that induction of apoptosis by low levels of PIP-3 and phosphorylated
Akt has been associated with high levels of PTEN.12
Conversely, loss of PTEN expression results in increased Akt activity and
continued cell survival and cell proliferation. In glioma, breast, and prostate
cell lines, PTEN has been shown to mediate G1 cell-cycle arrest and/or apoptosis
through the suppression of the phosphoinositol-3-kinase-Akt pathway.12 PTEN, therefore, seems to play an important role
in the modulation of cell cycle progression and/or apoptosis.
Recently, frequent genetic alterations and loss of expression of the PTEN gene were demonstrated in several malignant neoplasms.4-8
The studies of PTEN in HNSCC have focused exclusively
on searching for mutations or deletions of the gene, with little emphasis
on abnormalities at the protein level. In HNSCC, homozygous deletions of PTEN were reported in 2 of 19 patients, and mutations were
detected in 1 of 19 patients.10 Two subsequent
studies, however, have failed to demonstrate homozygous deletion or mutation
of PTEN in the HNSCC samples studied, in both cell
lines and primary tumors.13-14
Using immunohistochemical analysis of SCC of the tongue from 41 patients,
we show that the rate of PTEN inactivation at the protein level may be more
frequent than that identified at the genetic level. The loss of PTEN expression
in 29% of patients with SCC of the tongue in this study suggests that abrogations
of PTEN function may occur through multiple mechanisms. Loss of PTEN expression
may be explained by decreased protein synthesis, elevated protein degradation
or turnover, or other posttranslational modifications. Another possible mechanism
is the epigenetic inactivation of the gene through hypermethylation of the
promoter region.15-16 Indeed,
inactivation of other tumor suppressor genes by methylation has been previously
reported in HNSCC.17-18
Regarding the 41 specimens of invasive SCC of the tongue, we demonstrated
that loss of PTEN expression is not a rare event, since it occurred in 12
(41%) patients. The survival time analysis revealed a significant correlation
between loss of PTEN expression and overall survival time (P = .03) and event-free survival (P = .01).
When compared with other well-established clinical prognostic factors, such
as nodal involvement or stage of disease, using multivariate analysis for
event-free survival, the prognostic value of PTEN was retained. This suggests
that the tumors with loss of PTEN may reflect a more aggressive biological
behavior and that PTEN may serve as a potential new prognostic marker and
interventional tool in the management of SCC of the tongue. The loss of PTEN
in multiple tumor types has been linked to advanced disease,19
and a correlation between loss of PTEN and poor patient outcome has been reported
in gliomas.20 To our knowledge, this is the
first report that correlates loss of PTEN expression with poor patient outcome
in HNSCC. The fact that all of the patients had tongue cancer and were treated
at a single institution with lengthy follow-up care after surgery increased
the statistical power of our study.
In summary, we have demonstrated that loss of PTEN is not a rare event
in SCC of the tongue and bears an independent prognostic value, therefore
suggesting that PTEN plays an important role in tongue tumorigenesis and progression
of disease.
AUTHOR INFORMATION
Accepted for publication July 25, 2001.
This study was supported in part by Cancer Center grant P30 CA 16620
(to M. D. Anderson Cancer Center), Tobacco Research Fund from State of Texas
(to M. D. Anderson Cancer Center), and Fondation de France, Assistance Publique-Hôpitaux
de Paris, Paris, France (Dr Soria), and a Lilly Fondation, Paris, grant (Dr
Soria). Dr Hong is an American Cancer Society Clinical Research Professor.
Corresponding author and reprints: Li Mao, MD, Molecular Biology
Laboratory at the Department of Thoracic/Head and Neck Medical Oncology, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston,
TX 77030 (e-mail: lmao{at}notes.mdacc.tmc.edu).
From the Bobby R. Alford Department of Otorhinolaryngology and Communicative
Sciences, Baylor College of Medicine, Houston (Dr Lee); and the Molecular
Biology Laboratory, Departments of Thoracic/Head and Neck Medical Oncology
(Drs Soria, Hassan, Tang, Hong, and Mao and Ms Liu) and Pathology (Dr El-Naggar),
The University of Texas M. D. Anderson Cancer Center, Houston.
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